BOOK OF ABSTRACTS - Geyseco

BOOK OF 

ABSTRACTS 

www.isc2010.eu 

SOCIEDAD ESPAÑOLA DE 

CROMATOGRAFIA 

Y TÉCNICAS AFINES

EXHIBITORS Y SPONSORS 

Agilent Technologies 

AB Sciex 

Broker 

Bischoff Chromatography 

Chromsword 

Csisp-Centro Superior de Investigación en Salud Pública 

Dionex-Vertex 

Grupo Biomaster-Gerstel 

Hitc 

Icsa Instrumentos Científicos sa 

International Labmate Ltd 

Knauer 

Lcgc Europe 

Leco instrumentos 

Methrom-Gomensoro 

Millipore sas 

Micrux Fluidic 

Perkin Elmer 

Scharlab-Bischoff 

Shimadzu Europa Gmbh 

Sigma-Aldrich 

Termo-Fisher Scientific 

Waters 

SYMPOSIUM PARTNERS 

Generalitat Valenciana 

Ministerio de Ciencia e Innovación 

Secyta (Sociedad Española de Cromatografía y Técnicas Afines) 

Universitat de València 

2 ISC 2010 - 28TH INTERNATIONAL SYMPOSIUM ON CHROMATOGRAPHY - SEPTEMBER 12-16, 2010 - VALENCIA CONFERENCE CENTRE, SPAIN

SCIENTIFIC COMMITTEE 

Damià Barceló | IDAEA-CSIC | Barcelona, Spain 

Bogulaw Buszewski | Nicolaus Copernicus University | Torun, Poland 

Alejandro Cifuentes | IFI-CSIC | Madrid, Spain 

Marie-Claire Hennion | ESPCI | Paris, France 

José Carlos Díez Masa | IQOG-CSIC | Madrid, Spain 

Attila Felinger | University of Pécs | Hungary 

Amadeo R. Fernandez-Alba | University of Almeria | Spain 

Maria Teresa Galcerán | University of Barcelona | Spain 

Maria José González Carlos | IQOC-CSIC | Madrid, Spain 

Tyge Greibrokk | University of Oslo | Norway 

Uwe Karst | University of Münster | Germany 

John Lough | University of Sunderland | UK 

Yolanda Picó | University of Valencia | Spain 

CHAIRMAN 

Joan O. Grimalt | IDAEA-CSIC | Barcelona, Spain 

ORGANIZING COMMITTE 

Ana Agüera | U. Almería 

Coral Barbas | U. San Pablo-CEU | Madrid 

Maria Teresa Domènech | U. Politécnica de Valencia 

Carmen Dorronsoro | U. Basque Country | Donostia-San Sebastián 

Francesc Farrè-Rius | CSIC Residence of Researchers | Barcelona 

Pilar Fernandez | IDAEA-CSIC | Barcelona 

Ana María García-Campaña | U. Granada 

Elena Ibáñez | IFI-CSIC | Madrid 

Begoña Jiménez | IQOG-CSIC | Madrid 

Miguel Ángel Pérez | Bruker (Chemical Analysis Division) | Madrid 

Lourdes Ramos | IQOG-CSIC | Madrid 

José Luís Rubio | CIDE-CSIC | Valencia 

Josep Maria Sangenís | Thermo Fisher Scientific | Barcelona 

Francisco J. Santos | U. Barcelona 

Mercedes Torre | U. Alcalá 

Francesc Ventura | Agbar | Barcelona 

Vicent Yusà | CSISP - Valencia 

Rosa Maria Marcé | U. Rovira i Virgili | Tarragona 

CHAIRWOMAN 

Yolanda Picó | U. Valencia 

ISC 2010 - 28TH INTERNATIONAL SYMPOSIUM ON CHROMATOGRAPHY - SEPTEMBER 12-16, 2010 - VALENCIA CONFERENCE CENTRE, SPAIN 

3

SPONSORS 

CORPORATE SUPPORTERS 

GOLD SPONSOR 

SILVER SPONSOR 

BRONZE SPONSOR 


SPONSORS 

EXHIBITORS 

SPONSORS 

SYMPOSIUM PARTNERS 


5

OVERVIEW 

OF 

PRESENTATION

OVERVIEW OF PRESENTATION 

VENDOR SEMINARS 

VENDOR SEMINAR 1 

SHIMADZU EUROPA GMBH 

MODERN MULTIDIMENSIONAL TECHNOLOGIES: MDGC AND GCXGC(QMS) 


WATERS 

NEW TOOLS FOR EFFICIENT METHOD DEVELOPMENT. NEW CSH STATIONARY PHASES AND FUSION MD SOFTWARE 

56 

57 


TERMO FISHER SCIENTIFIC 

NEW ADVANCES IN QUATERNARY ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 

58 


DIONEX CORPORATION 

THE TRUE BENEFITS OF UHPLC FOR EVERYONE 

59 


HITC CHROMATOGRAPHIC SOLUTIONS 

A NEW CONCEPT OF TOF-MS SYSTEM FOR FAST GC AND GCxGC ANALYSIS 

60 


BRUKER CHEMICAL ANALYSIS DIVISION. 

RAPID AND ROBUST MULTI-RESIDUE PESTICIDE ANALYSES; TARGETED AND NON-TARGETED APPROACHES 

61 


AGILENT TECHNOLOGIES 

RAISING THE BAR IN ANALYTICAL CHROMATOGRAPHY 

62 


AB SCIEX 

SIMULTANEOUS COMPOUND QUANTIFICATION AND IDENTIFICATION USING HIGH RESOLUTION MS: THE AB SCIEX TRIPLETOF 

5600 SYSTEM 

63 


MEHTROM 

AUTOMATED ION CHROMATOGRAPHIC DETERMINATIONS OVER SIX ORDERS OF MAGNITUDES 

64 


LECO 

APPLICATION OF GC- AND GCXGC-TOFMS FOR FOOD ANALYSIS 

65 


PERKIN ELMER 

PERKIN ELMER INITIATIVES IN ENVIRONMENTAL AND FOOD ANALYSIS 

66 


BISCHOFF CHROMATOGRAPHY 

SPEED, SELECTIVITY AND RESOLUTION, KEY ASPECTS FOR A SEPARATION IN MODERN LIQUID CHROMATOGRAPHY 

67 


7


PLENARY SESSIONS 

PS1 RECENT CONTRIBUTIONS OF CHROMATOGRAPHY TO THE UNDERSTANDING OF DRUG METABOLISM 

Iam D. Wilson 

PS2 SEPARATION SCIENCE, KEY TO SUCCESSFUL BIOPHARMACEUTICALS 

Georges Guiochon and Lois Ann Beaver 

PS3 FACETS OF SPECIFIC DETECTION IN CHROMATOGRAPHY AND ELECTROPHORESIS: 

PROGRESS IN SPECIATION ANALYSIS 

Ryszard Lobinski 

PS4 PROGRESS IN THE OMICS ANALYSIS OF FOODS: FOODOMICS 

Alejandro Cifuentes 

PS5 NEW FINDINGS IN HIGH SPEED, HIGH TEMPERATURE AND HIGH PRESSURE CHROMATOGRAPHY 

Jean-Luc Veuthey 

PS6 ELECTROMIGRATION METHODS FOR THE SEPARATION OF BACTERIA. EFFECT OF CHARGE DISTRIBUTION 

Boguslaw Buszewski 

PS7 CAPILLARY AND FREE-FLOW ELECTROPHORESIS APPLIED TO ANALYSIS, PURIFICATION AND CHARACTERIZATION OF 

BIOLOGICALLY ACTIVE PEPTIDES 

Vaclav Kasicka 

PS8 CHALLENGES AND FUTURE TRENDS PF PESTICIDE FOOD RESIDUE CONTROL IN EUROPE 

Amadeo R. Fernandez-Alba 

PS9 CHIRAL ANALYSIS: A CHALLENGING ISSUE FOR MINIATURIZED TECHNIQUES 

Salvatore Fanali 

69 

70 

71 

72 

73 

74 

75 

76 

77 

8 

ISC 2010 - 28TH INTERNATIONAL SYMPOSIUM ON CHROMATOGRAPHY - SEPTEMBER 12-16, 2010 - VALENCIA CONFERENCE CENTRE, SPAIN


ORAL SESSIONS 

S1 CLINIC AND PHARMACEUTICAL APPLICATIONS 

S1-01 ELECTROCHEMISTRY COUPLED ONLINE TO LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRY AS A COMPLEMENTARY 

TOOL IN DRUG METABOLISM STUDIES 

Vielhaber T., Baumann A., Jahn S., Melles D., Lohmann W., Karst U. 

University of Münster 

S1-02 APPLICATION OF AN EXPERIMENTAL DESIGN TO OPTIMIZE THE METABOLIC PROFILES OF CINITAPRIDE BY UPLC 

Marquez H. 1 , Albertí J. 1 , Salvá M. 1 , Saurina J. 2 , Sentellas S. 1 

1 

Almirall SA 

2 

University of Barcelona 

S1-03 IN VITRO CAPTURING OF VARIOUS LIPOPHILIC ILLICIT DRUGS BY LIPID DISPERSIONS STUDIED BY ELECTROKINETIC 

CAPILLARY CHROMATOGRAPHY AND FLUORESCENCE POLARIZATION 

Lokajová J. 1 , Pukkila J. 2 , Holopainen J.M. 1 , Wiedmer S. 1 

1 

University of Helsinki 

2 

Finnish Police, National Bureau of Investigation 

S1-04 SEPARATION OF ANTIPARKINSON DRUGS BY CAPILLARY ELECTROPHORESIS COUPLED WITH AMPEROMETRIC DETECTION BY 

BORON DOPED DIAMOND ELECTRODE 

Zhou L. 1 , Corcoran C. 1 , Luong HT J. 2 , Glennon D. J. 1 

1 

University College Cork 

2 

National Research Council Canada 

79 

80 

81 

82 

83 

S2 ENVIRONMENT 

S2-01 CHARACTERIZATION AND DETERMINATION OF MIXTURES OF FATTY ALCOHOL ETHOXYLATES AND ALKYLETHER SULFATES BY 

SPE, DERIVATIZATION WITH PHTHALIC ANHYDRIDE AND HPLC-UV 

Ripoll-Seguer L., Beneito-Cambra M., Herrero-Martínez J.M., Simó-Alfonso E.F., Ramis-Ramos G. 

University of València 

S2-02 EVALUATION OF ENVIRONMENTAL TOBACCO SMOKE CONTAMINATION AND ITS EFFECT ON PASSIVE SMOKERS 

Alonso M., Besal E., Godayol A., Anticó E., Sanchez Navarro J.M. 

University of Girona 

S2-03 IDENTIFICATION AND QUANTITATION OF EXPLOSIVE RESIDUES WITH UHPLC/MS 

Jiang G., Guazzotti S., Preston K., Elmashni D. 

Thermo Fisher Scientific 

S2-04 FAST LC SEPARATIONS OF TRIAZINE HERBICIDES AT ELEVATED TEMPERATURE 

Guazzotti S., Jiang G. 


84 

85 

86 

87 

88 

S3 LIQUID CHROMATOGRAPHY 

S3-01 SELECTIVE SAMPLE PRETREATMENT USING APTAMER-BASED EXTRACTION SORBENTS AND COMPARISON TO 

IMMUNOSORBENTS AND MOLECULARLY IMPRINTED POLYMERS 

Hennion M.C. - Madru B. - Thibert V. - Chapuis-Hugon F. - Pichon V. 

ESPCI , Paris 

S3-02 SUPERCRITICAL CO2 PREPARATION AND CHARACTERIZATION OF PHENYL AND FLUORO-PHENYL STATIONARY PHASES FOR 

USE IN LIQUID CHROMATOGRAPHY 

Ashu-Arrah A.B. 1 , Omamogho O.J. 1 , Yeman H. 2 , Albert K. 2 , Glennon D.J. 1 

1 


2 

University of Tübingen 

S3-03 THE APPLICATION OF DYNAMIC TEMPERATURE PROFILES AND GRADIENTS FROM A PELTIER ARRAY BASED PLATFORM TO 

THE FABRICATION AND APPLICATION OF CAPILLARY HPLC COLUMNS 

Collins D. 1 , Nesterenko E. 1 , Connolly D. 1 , Macka M. 2 , Brabazon D. 1 , Paull B. 1 

1 

Dublin City University 

2 

University of Tasmania 

S3-04 ESTIMATION OF THE FLUOROPHILIC-LIPOPHILIC-HYDROPHILIC BALANCE OF FLUORINE-CONTAINING SOLVENTS BY MEANS OF 

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 

Nakajima Y., Fukuhara K., Kakiuchi T., Okamoto H., Yamamoto K. 

Asahi Glass Co., Ltd. 

89 

90 

91 

92 

93 


9

OVERVIEW OF PRESENTATION - ORAL SESSIONS 

S4 HYPHENATED TECHNIQUES 

S4-01 ON LINE DERIVATISATION IN ON-LINE COUPLED NPLC-GC USING THE THROUGH OVEN TRANSFER ADSORPTION DESORPTION 

(TOTAD) INTERFACE: APPLICATION TO THE ANALYSIS OF TOTAL STEROLS IN EDIBLE OILS 

Toledano R.M., Cortés J.M., Aragón A., Vázquez A., Villén J. 

University of Castilla-La Mancha 

S4-02 ANALYSIS OF MELAMINE-FORMALDEHYDE CONDENSATES AND METHYLATED MELAMINES IN REACTION MIXTURES BY CZE-MS 

Himmelsbach M., Vo T.D.T., Schwarzinger C., Buchberger W., Klampfl C.W. 

Johannes Kepler University of Linz 

S4-03 SCIENCE BASED CALIBRATION FOR THE EXTRACTION OF ‘ANALYTE-SPECIFIC’ CHROMATOGRAMS IN LC 

Kuligowski J., Quintás G., de la Guardia M. 

University of Valencia 

S4-04 POTENTIAL OF COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY COUPLED TO A VERY FAST QUADRUPOLE 

MASS SPECTROMETER (20000 AMU/SEC) 

Purcaro G., Quinto Tranchida P., Dugo P., Dugo G., Mondello L. 

University of Messina 

94 

95 

96 

97 

98 


S5-01 APPLICATION OF STIR BAR SORPTIVE EXTRACTION FOLLOWED BY COMPREHENSIVE TWO-DIMENSIONAL GAS 

CHROMATOGRAPHY-TIME-OF-FLIGHT MASS SPECTROMETRY FOR THE DETERMINATION OF TARGET AND NON-TARGET 

ORGANIC MICRO-CONTAMINANTS IN RIVER WATER 

Gómez Ramos M.J. 1 , Herrera S. 1 , Solé D. 1 , Fernández-Alba A.R. 2 

1 

Fundación IMDEA Agua 2 Almería University 

S5-02 EVALUATION OF GLYPHOSATE IN SUPERFICIAL AND DRINKING WATERS OF AN AGRICULTURAL AREA OF RIO DE JANEIRO STATE, 

BRAZIL, BY HPLC-FLUORESCENCE 

Olivier B.C. 1 , Pereira Netto A.D. 2 

1 

National Institute of Technology, Brazil 

2 

Federal Fluminense University, Brazil 

S5-03 HIGH PERFORMANCE LIGAND EXCHANGE CHROMATOGRAPHY AND FOURIER TRANSFORM MASS SPECTROMETRY — A 

COMBINATION TOWARD THE CHARACTERIZATION OF SULFUR AROMATICS IN ARABIAN HEAVY CRUDE OIL AND ITS DISTILLED 

FRACTIONS 

Panda S.K., Hajji A.A., Mueller H., Koseoglu O.R. 

Saudi Aramco 

99 

100 

101 

102 

S6 FOOD QUALITY AND SAFETY 

S6-01 INFANT EXPOSURE OF PERFLUORINATED COMPOUNDS: LEVELS IN BREAST MILK AND COMMERCIAL BABY FOOD 

Farré M. 1 , Llorca M. 1 , Picó Y. 2 , López-Teijón M. 3 , Alvarez J. 3 , Barceló D. 1 

1 

IDAEA-CSIC 

2 


3 

Instituto Marqués 

S6-02 DEVELOPMENT AND APPLICATION OF A HPLC-UV-ESI-MS METHOD FOR THE SIMULTANEOUS DETERMINATION OF UREA 

BY-PRODUCTS IN COMMERCIAL UREA 

Ferreira C.G. 1 , Albuquerque F.C. 1 , Pereira Netto A.D. 2 

1 

PETROBRAS-CENPES 

2 

Federal Fluminense University 

S6-03 NANO-LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY TO STUDY THE IN VITRO ABSORPTION AND METABOLIC 

CONVERSION OF OLIVE OIL POLYPHENOLS IN HUMAN BREAST CANCER CELLS 

García-Villalba R. 1 , Carrasco-Pancorbo A. 1 , Menéndez J.A. 2 , Segura- Carretero A. 1 , Fernández-Gutiérrez A. 1 

1 

University of Granada 

2 

Catalan Institute of Oncology, Barcelona 

103 

104 

105 

106 

10 



S7 FUNDAMENTALS OF CHROMATOGRAPHY 

S7-01 METHOD DEVELOPMENT IN LIQUID CHROMATOGRAPHY USING TEMPERATURE GRADIENTS 

Wiese S. 1 , Teutenberg T. 1 , Schmidt T.C. 2 

1 

Institute of Energy and Environmental Technology-Germany 

2 

University of Duisburg-Essen 

S7-02 SIMULTANEOUS EFFECT OF PH, TEMPERATURE AND MOBILE PHASE COMPOSITION IN THE CHROMATOGRAPHIC RETENTION 

OF IONIZABLE COMPOUNDS 

Agrafiotou P., Ráfols C., Castells C., Bosch E., Rosés M. 


S7-03 DETERMINATION OF PERSISTENT ORGANIC POLLUTANTS IN FISH BY GAS CHROMATOGRAPHY–MS/MS 

Muñoz G., Pineda L., Serrahima E., Centrich F. 

Laboratori Agència Salut Pública de Barcelona 

107 

108 

109 

110 

S8 EXTRACTION 

S8-01 IONIC LIQUIDS IN MICROEXTRACTION TECHNIQUES COMBINED WITH CHROMATOGRAPHIC SEPARATIONS 

Cárdenas S., Lucena R., Aguilera-Herrador E., Cruz-Vera M., Valcárcel M. 

University of Cordoba 

S8-02 NOVEL UNBREAKABLE SOLID PHASE MICROEXTRACTION FIBER BY CHEMICALLY BONDED CARBON NANOTUBES ON MODIFIED 

GOLD SUBSTRATE 

Bagheri H., Ayazi Z., Sistani H., Aghakhani A. 

Sharif University of Technology, Chemistry 

S8-03 STIR MEMBRANE IN MICROSOLID PHASE EXTRACTION AND LIQUID PHASE MICROEXTRACTION APPROACHES COMBINED WITH 

GAS CHROMATOGRAPHY 

Lucena R., Alcudia-león M.C., Cárdenas S., Valcárcel M. 


S9 SPECIATION ANALYSIS 

S9-01 INTERACTION OF THIOPHILIC ELEMENT SPECIES WITH PROTEINS 

Karst U. 


S9-02 ANALYSIS OF LABILE METAL COMPLEXES IN PLANTS BY HILIC-ESI/MS 

Köster J., von Wiren N., Weber G. 

Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Metabolomics 

S9-03 SPECIES ANALYSIS OF PLATINUM BASED CYTOSTATIC DRUGS AND BIOLOGICALLY RELEVANT THIOLS 

Brauckmann C., Karst U. 

Institute of Inorganic and Analytical Chemistry 


S9-04 SPECIATION OF GADOLINIUM-BASED MRI CONTRAST MEDIA IN WASTEWATER TREATMENT PLANTS BY HILIC/ICP-MS 

Telgmann L., Künnemeyer J., Karst U. 




S10-01 CHROMATOGRAPHIC METHODS FOR THE ANALYSIS OF POLYCYCLIC AROMATIC SULFUR HETEROCYCLES 

Nocun M., Andersson J.T. 

Institute of Inorganic and Analytical Chemistry, University of Münster 

S10-02 POLYCHLORINATED DIBENZO-P-DIOXINS, DIBENZOFURANS AND BIPHENYLS IN GENERAL POPULATION LIVING NEAR AN 

URBAN WASTE TREATMENT PLANT IN MATARÓ 

Parera J. 1 , Serra-Prat M. 2 , Moreno V. 2 , Martrat M.G. 1 , Palomera E. 2 , Abalos M. 1 , Rivera J. 1 , Abad E. 1 

1 

IDÆA-CSIC, Dioxins Laboratory, Mass Spectrometry Laboratory, Barcelona 

2 

Hospital of Mataró, Consorci Sanitari del Maresme, Research Unit, Barcelona 

S10-03 LC-MS/MS STUDY OF DISTRIBUTION OF PHARMACEUTICALS IN WATER, SUSPENDED SOLIDS AND SEDIMENTS OF THE 

EBRO RIVER 

Silva B.F. 1 , Jelic A. 1 , Lopez R. 1 , Mozeto A.A. 2 , Petrovic M. 1 , Barcelo D. 1 

1 

IDÆA-CSIC, Department of Envinromental Chemistry, Barcelona 

2 

UFSCar, Departamento de Química - Lab. Biogequímica, University of Sao Carlos 

111 

112 

113 

114 

115 

116 

117 

118 

119 

120 

121 

122 

123 


11


S10-04 DERIVATIZATION AND FRAGMENTATION PROPERTIES OF THE TRIMETHYLSILYL (OXIME) ETHER/ESTER DERIVATIVES OF 

STEROIDS, BY GAS CHROMATOGRAPHY MASS-SPECTROMETRY: STEROID POLLUTANTS IN WASTEWATERS 

Andrási N., Helenkár A., Vasanits-Zsigrai A., Záray Gy., Molnár-Perl I. 

Eötvös Loránd University, Institute of Chemistry 

124 


S11-01 ALKYLATED POROUS GRAPHITIC CARBON FOR LIQUID CHROMATOGRAPHY 

Jensen D., Wiest L.A., Dadson A., Vail M.A., Linford M.R. 

Brigham Young University, Chemistry and Biochemistry of USA 

S11-02 CAPILLARY ELECTROPHORESIS PROCEDURE FOR THE SIMULTANEOUS ANALYSIS AND STOICHIOMETRY DETERMINATION OF 

A VINCA ALKALOID AND ITS COUNTER-ION BY USING DUAL-OPPOSITE END INJECTION AND CONTACTLESS CONDUCTIVITY 

DETECTION: APPLICATION TO CATHARANTHINE SULFAT 

Lopez C. 1 , Nehme R. 1 , Claude B. 1 , Morin P. 1 , Penna R. 2 , Max J.P. 2 , Filaquier J.P. 2 , Ribet J.P. 2 

1 

ICOA - University of Orléans 

2 

Institut de Recherche Pierre Fabre, Analytical chemistry, Boulogne Billanncourt 

S11-03 OPTIMIZATION OF SIMPLE IN SITU DERIVATIZATION REACTIONS FOR IBUPROFEN GAS CHROMATOGRAPHY ANALYSIS USING 

HEADSPACE GENERATION 

Casas Ferreira A.M., Fernández Laespada M.E., Pérez Pavón J.L., Moreno Cordero B. 

University of Salamanca 

125 

126 

127 

128 


S12-01 NEW LC PUMP TECHNOLOGY TO IMPROVE PERFORMANCE UNDER ALL OPERATING CONDITIONS 

Preiswerk T. 1 , Guazzotti S. 2 , Boehm G. 1 

1 

Thermo Fisher Scientific, SID-Switzerland 

2 

Thermo Fisher Scientific, SID-USA 

S12-02 ADVANCES IN CAPILLARY ION CHROMATOGRAPHY SYSTEMS USING ON-LINE ELECTROLYTIC ELUENT GENERATION AND 

SUPPRESSED CONDUCTIVITY DETECTION 

Divan K., Liu Y., Divan K. 

Dionex Corporation 

S12-03 QUALITY BY DESIGN LC METHODS DEVELOPMENT USING ORTHOGONAL STATIONARY PHASES 

Zhe Yin, Fountain K., Morrison D., Bosch G. 

Waters Corporation 

129 

130 

131 

132 


S13-01 FAST CHROMATOGRAPHY WITH SUB-2 µM PARTICLE-SIZE COLUMNS IN THE ANALYSIS OF HIGH-COMPLEX MATRICES: 

REALITY OR UTOPIA? VALIDATION OF A RAPID RESOLUTION LC-MS/MS METHOD FOR THE ANALYSIS OF MARINE TOXINS 

de la Iglesia P., Barber E., Giménez G., Diogène J. 

Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Sant Carles de la Ràpita 

S13-02 DEVELOPMENT AND APPLICATION OF AN IMMUNOAFFINITY COLUMN FOR THE SOLID-PHASE EXTRACTION OF 

PYRACLOSTROBIN RESIDUES FROM FRUIT JUICES 

Esteve Turrillas F.A. 1 , Mercader J.V. 1 , Agulló C. 2 , Abad-Somovilla A. 2 Abad-Fuentes A. 1 

1 

Instituto de Agroquímica y Tecnología de Alimentos - CSIC, Valencia 

2 

University of València, Department of Organic Chemistry 

S13-03 DETERMINATION OF ISOTHIOCYANATES AND INTACT GLUCOSINOLATES IN FOODSTUFF USING GC-MS AND HPLC-MS 

Kuebler K., Paetzold R., 

Institute of Nutritional Science, Professorship of Food Sciences-Germany 

S13-04 ASSESSMENT OF THE EFFECT OF NON-VOLATILE WINE MATRIX ON THE VOLATILITY OF TYPICAL WINE AROMA COMPOUNDS 

BY HS-SPME-GC-MS ANALYSIS 

Rodríguez-Bencomo J.J., Muñoz-González C., Andujar-Ortiz I., Martín-Alvarez P.J., Moreno-Arribas M.V., Del Pozo-Bayón M. A. 

Instituto de Investigación en Ciencias de la Alimentación (CIAL) (CSIC-UAM), Madrid 

133 

134 

135 

136 

137 

12 




S14-01 DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS USING MICRO EXTRACTION BY PACKED SORBENT 

Lezsak G. 1 , Eke Z. 1 , Rikker T. 2 , Torkos K. 1 

1 

Eötvös Loránd University Institute of Chemistry 

2 

WESSLING International Research and Educational Centre, Hungary 

S14-02 SPATIAL DISTRIBUTION AND SOURCES OF PERFLUOROCHEMICALS IN THE NW MEDITERRANEAN COASTAL WATERS 

(CATALONIA, SPAIN) 

Sánchez-Avila J., Meyer J., Lacorte S. 

IDÆA-CSIC, Barcelona 

S14-03 DEVELOPMENT OF A QSRR MODEL FOR IDENTIFICATION OF UNKNOWNS IN ENVIRONMENTAL SAMPLES 

Ulrich N., Schymanski E., Brack W. 

Helmholtz Centre for Environmental Research-UFZ, LeipzigÆ 

138 

139 

140 

141 

S15 NEW CHROMATOGRAPHIC APPLICATIONS FOR BIOMARKERS 

S15-01 LC-MS METABOLIC FINGERPRINTING OF SECRETOME FROM HUMAN ATHEROTHROMBOTIC ANEURYSMS 

Ciborowski M. 1,2 ; Martin-Ventura J.L. 3,4 , Meilhac O. 5 , Michel J-B. 5 , Tuñon J.3,4, Egido J. 3,4 , Barbas C. 1 

1 

Medical University of Bialystok 

2 

San Pablo CEU University, Madrid 

3 

Fundación Jiménez Díaz, Madrid 

4 

Autonomous University of Madrid 

5 

Inserm, U698, Paris 

S15-02 NOVEL DERIVATIZATION STRATEGIES FOR THE LC-ESI-MS MEASUREMENT OF RELEVANT BIOMARKERS 

Kretschmer A., Giera M., Eggink M., Wijtmans M., Lingeman H., Niessen W.M.A., Irth H. 

VU University Amsterdam 

142 

143 

144 

S16 GAS CHROMATOGRAPHY 

S16-01 THERMAL DESORPTION GAS CHROMATOGRAPHY–MASS SPECTROMETRY AS AN ENHANCED METHOD FOR THE 

QUANTIFICATION OF POLYCYCLIC AROMATIC HYDROCARBONS FROM AMBIENT AIR PARTICULATE MATTER 

Van Drooge L.B. 1 , Pérez-Ballesta P. 2 

1 

IDÆA-CSIC, Environmental Chemistry, Barcelona 

2 

JRC-Ispra, Air quality and transport 

S16-02 NEEDLE MICROEXSTRATION TRAPS: A POWERFUL, ROBUST AND SIMPLE TOOL FOR VOC ANALYSIS 

Alonso M., Muñoz S., Godayol A., Anticó E., Sanchez J.M. 


S16-03 IN-TUBE EXTRACTION OF VOLATILE ORGANIC COMPOUNDS FROM AQUEOUS SAMPLES: AN ECONOMICAL ALTERNATIVE TO 

PURGE AND TRAP ENRICHMENT 

Laaks J. 1 , Jochmann M.A. 1 , Schilling B. 2 , Schmidt T.C. 1 

1 

University Duisburg-Essen 

2 

BGB Analytik AG-Switzerland 

145 

146 

147 

148 

S17 FAST SEPARATIONS 

S17-01 DATA ACQUISITION AND DATA HANDLING IN FAST LIQUID CHROMATOGRAPHY 

Felinger A. 

University of Pécs 

S17-02 SEPARATION OF PARTICLES AND CELLS BY USING A HYDRODYNAMIC–ACOUSTIC CONTINUOUS SORTER (HACS) 

Hoyos M. 1 , Rarier C. 2 

1 

CNRS, UMR7636, PMMH, Paris 

2 

ESPCI, PMMH, Paris 

149 

150 

151 


13


S18 LIFE SCIENCES AND BIOANALYSIS 

S18-01 OPEN TUBULAR LC - ANY NEWS? 

Greibrokk T. 

University of Oslo 

S18-02 ACTIVITY ASSAY OF METHYLTHIOADENOSINE PHOSPHORYLASE IN TISSUE SPECIMENS BY MEANS OF STABLE ISOTOPE 

LABELED METHYLTHIOADENOSINE AND LC-MS/MS 

Stevens A.P. 1 , Kirovski G. 2 , Czech B. 2 , Dettmer K. 1 , Hellerbrand C. 2 , Bosserhoff A.K. 2 , Oefner P.J. 1 

1 

University Regensburg, Institute of Functional Genomics 

2 

University Hospital Regensburg, Internal Medicine I 

S18-03 SEPARATION OF PHOSPHO AND GLYCO PEPTIDES USING POROUS GRAPHITIC CARBON FOR THE PROTEOMIC STUDY OF 

ONCOLOGY PATIENTS 

Barattini V. 1 , Pereira L. 1 , Smith D. 2 , Griffiths J. 3 

1 


2 

Patterson Institute for Cancer Research, NA 

3 

The University of Manchester, NA 

S18-04 LITHOGRAPHICALLY DEFINED METAL SURFACES FOR BIOANALYTICAL APPLICATIONS 

Juskova P. 1 , Ostatna V. 2 , Palecek E. 2 , Foret F. 1 

1 

Institute of Analytical Chemistry, ASCR, v.v.i., Brno, Czech Republic 

2 

Institute of Biophysics, ASCR, v.v.i, Brno, Czech Republic 

152 

153 

154 

155 

156 


S19-01 DEVELOPMENT OF NEW STATIONARY PHASE FOR SHAPE SELECTIVE SEPARATION OF SELECTED CARBAMATE PESTICIDES 

Omamogho J.O. 1 , Crean C. 1 , Stack E.M. 1 , Zhou L. 1 , Yeman H. 2 , Albert K. 2 , Glennon J.D. 1 

1 


2 


S19-02 DEVELOPMENT OF A NOVEL POLAR COATING FOR STIR BAR SORPTIVE EXTRACTION TO DETERMINE POLAR 

PHARMACEUTICAL AND PERSONAL CARE PRODUCTS FROM WATER SAMPLES 

Bratkoswka D. 1 , Fontanals N. 1 , Cormack P.A.G. 2, Borrull F. 1 , Marce R.M. 1 

1 

Universitat Rovira i Virgili, Tarragona 

2 

University of Strathclyde 

S19-03 CHROMATOGRAPHIC ISOLATION OF ANTIFUNGAL STILBENES FROM SHOREA BELANGERAN, DIPTEROCARPACEAE 

Kusuma I.W. 1 , Tachibana S. 2 

1 

Mulawarman University, Faculty of Forestry/Wood Chemistry-Indonesia 

2 

Ehime University, Faculty of Agriculture/Applied Bioresources Sciences-Japan 

S19-04 DISTRIBUTION OF PERFLUORINATED COMPOUNDS IN SEAGULL EGGS (LARUS MICHAHELLIS) FROM THE IBERIC PENINSULA 

Vicente-Bobes J., Meyer J.I., Bertolero A., Viana P., Lacorte S. 

IDÆA -CSIC, Barcelona 

157 

158 

159 

160 

161 

S20 INNOVATIVE CHROMATOGRAPHIC APPLICATIONS 

S20-01 MICRO-DISPERSE SINTERED NANO-DIAMONDS: A NEW VERSATILE STATIONARY PHASE FOR HPLC 

Nesterenko P. 

University of Tasmania, Australian Centre for Research on Separation Science (ACROSS) 

S20-02 GCXGC WITH CAPILLARY FLOW MODULATION FOR THE SEPARATION OF SEVERAL FAME´S ISOMERS IN BROCCOLI LEAVES 

Manzano P. 1 , Bernal J.L. 1 , Diego J.C. 1 , Arnaiz E. 1 , Bernal J. 2 

1 

University of Valladolid 

2 

Institute of Industrial Fermentations (CSIC), Madrid 

S20-03 MULTIDIMENSIONAL HPLC+GC-MS: EXTENDED USE FOR NON-VOLATILE AND POLAR COMPOUNDS USING ON-LINE 

DERIVATIZATION 

Sarrión N., Gibert J.M., Alcón M.J. 

KONIK-Tech S.A., Sant Cugat del Vallès 

S20-04 HYPERCROSSLINKED POLYMERS: ADVANCES AND PERSPECTIVES OF APPLICATION IN ADSORPTION TECHNOLOGIES AND 

CHROMATOGRAPHY 

Davankov V.A., Tsyurupa M.P. 

Institute of Organo-Element Compounds, Russian Academy of Sciences, Moscow 

162 

163 

164 

165 

166 

14 



S21 ELECTRODRIVEN SEPARATIONS 

S21-01 CAPILLARY ELECTROPHORESIS AS A NEW METHOD FOR THE SEPARATION OF POLYCYCLIC AROMATIC SULFUR 

HETEROCYCLES IN DIESEL FUELS 

Nolte T., Andersson J.T. 

University of Münster, Institute of Inorganic and Analytical Chemistry 

S21-02 ELECTROKINETIC ACCUMULATION FOR ON-LINE PRECONCENTRATION OF WEAK ACIDS IN CAPILLARY ELECTROPHORESIS 

Petr J., Maier V., Ranc V., Znaleziona J., Ginterova P., Sevcik J. 

Palacky University in Olomouc 

S21-03 AN INTEGRATED ON-CHIP SIRTUIN ASSAY 

Beyreiss R. 1 , Ohla S. 1 , Fan Y. 2 , Scriba G.K.E. 2 , Belder D. 1 

1 

University of Leipzig 

2 

University of Jena 

167 

168 

169 

170 


S22-01 COMPARATIVE STUDY OF TWO DIFFERENT LC-QLIT-MS/MS OPERATION MODES APPLIED TO THE ANALYSIS OF PSYCHOACTIVE 

STIMULATORY AND TRANQUILIZING DRUGS IN WASTEWATERS BY DIRECT INJECTION 

Martinez Bueno M.J. 1 , Uclés S .1 , Hernando M.D. 2 , Agüera A. 1 , Fernandez-Alba A.R. 1 

1 

Universidad de Almeria 

2 

Universidad de Alcalá 

S22-02 NEW STANDARDISED METHODS FOR THE BIOMONITORING OF PRIORITY PERSISTENT ORGANIC POLLUTANTS IN GULL EGGS 

Lacorte S .1 , Santos J. 2 , Abad E .1 , Olmos J. 2 , Meyer J. 1 , Abalos M. 1 , Morales L. 1 , Antunes P. 3 , Viana P. 3 , Bertolero A. 1 

1 

IDÆ-CSIC, Environmental Chemistry, Barcelona 

2 

University of Barcelona, Analytical Chemistry 

3 

Ministerio do Ambiente 

S22-03 NOVEL PRECONCENTRATION TECHNIQUES FOR ENHANCING SENSITIVITY IN THE DETERMINATION OF NON-STEROIDAL 

ANTIINFLAMMATORY DRUGS IN ENVIRONMENTAL SAMPLES BY CAPILLARY ELECTROPHORESIS 

Maijó I., Botello I., Borrull F., Aguilar C., Calull M. 


171 

172 

173 

174 


S23-01 ON-CHIP LC-MS ANALYSIS OF COCAINE FROM HAIR AND URINE 

Thibert V., Chapuis-Hughon F., Pichon V. 

UMR PECSA7195 CNRS - UPMC - ESPCI Paris 

S23-02 APPLICATION OF ULTRA-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY COMBINED WITH HIGH RESOLUTION MASS 

SPECTROMETRY ON SCREENING OF VETERINARY DRUG RESIDUES IN MILK AND POWDERED-BASED MILK SAMPLES 

Romero-González R., Aguilera-Luiz M.M., Plaza-Bolaños P., Garrido-Frenich A., Martinez Vidal J.L. 

Almeria University of Almeria 

S23-03 LIQUID CHROMATOGRAPHY- ISOTOPE RATIO MASS SPECTROMETRY: OPPORTUNITIES AND CHALLENGES FOR A NEW HYPHENATION 

Schmidt T.C., Kujawinski D.M., Zhang L., Jochmann M.A. 


175 

176 

177 

178 


15


S24 LIFE SCIENCES / BIOANALYSIS 

S24-01 DETERMINATION RESVERATROL AND RELATED POLYPHENOLS IN ARMENIAN WINES BY HPLC/DAD 

Avakyan A.P., Gabrielyan E.S., Khachvankyan, G.Yu. 

Scientific Center of drug and medical technology expertise, Expert analytical laboratory-Armenia 

S24-02 VERSATILE, EASY AND RAPID ATMOSPHERIC MONITOR (VERAM) FOR THE PASSIVE SAMPLING OF VOLATILE ORGANIC 

COMPOUNDS IN AIR 

Ly-Verdú S., Esteve-Turrillas F.A., Pastor A., De la Guardia M. 


S24-03 NEW TRENDS ON PORTABLE INSTRUMENTATION FOR MICROCHIP CAPILLARY ELECTROPHORESIS 

Pozo-Ayuso D.F., Fernández-la-Villa A., Castaño-Alvarez M. 

Micrux Technologies, Research and Development 

179 

180 

181 

182 


S25-01 ADVANTAGES OF MONOLITHIC OVER PARTICULATE COLUMNS FOR SCREENING ANALYSIS OF ORGANIC POLLUTANTS BY 

IN-TUBE SOLID-PHASE MICROEXTRACTION COUPLED TO CAPILLARY LIQUID CHROMATOGRAPHY 

Moliner-Martinez Y., Carracedo-Marzo R., Molins-Legua C., Verdú-Andrés J., Herráez-Hernández R., Campins-Falcó P. 


S25-02 CONTINUOUS FLOW INTEGRATIVE SAMPLER (CFIS). AN NEW AND FUTURE ALTERNATIVE TO COMPLETE CONTROL THE 

WATER POLLUTION 

Llorca J. 

LABAQUA, Chromatography, Murcia 

S25-03 HYPHENATED µLC-SERS SYSTEM USING A NOVEL SILVER-QUANTUM DOTS “SPONGE” NANOCOMPOSITE 

Carrillo-Carrión C. 1 , Simonet Suau B.M. 1 , Valcárcel M. 1 , Lendl B. 2 

1 

University of Córdoba 

2 

Vienna University of Technology 

S25-04 MONITORING OF ABRUPT CLIMATE CHANGE WITH CHROMATOGRAPHYC METHODS FOR MEASUREMENT OF PAST SEA 

SURFACE TEMPERATURES AND DEEP WATER FLOWS 

Rama O., Martrat B., Grimalt J.O. 

IDÆA-CSIC, Environmental Chemistry 

183 

184 

185 

186 

187 


S26-01 METHOD DEVELOPMENT AND VALIDATION OF A LC-MS/MS METHOD FOR PESTICIDE RESIDUES ANALYSIS IN FRUIT JUICES 

BY DIRECT INJECTION 

Ferrer C., Martínez-Bueno M.J., Lozano A., Fernandez-Alba A.R. 

University of Almería 

S26-02 DETERMINATION OF PAHS IN MEZCAL BY SOLID PHASE MICROEXTRACTION FOLLOWED BY GAS CHROMATOGRAPHY- 

MASS SPECTROMETRY 

Alonso-Villegas R. 

Universidad de Castilla-La Mancha 

S26-03 COMPARISON OF UV-VIS AND TANDEM-MS DETECTION OF SUDAN DYES AFTER RRLC SEPARATION 

Ochs S.M., Santos A.M.S., Pereira Netto A.D. 


188 

189 

190 

191 

16 




S27-01 PREPARATION OF MONOLITHS IN GLASS/SILICON MICROFLUIDIC CHIPS AS STATIONARY PHASES AND FRITS FOR COLUMN 

PACKING 

Vázquez M., Collins D., Nesterenko E., Brabazon D., Paull B. 


S27-02 A NEW FAST WAY OF SCREENING MULTIPLE SOLVENTS AS ELUENTS FOR CHIRAL HPLC: APPLICATION FOR RACEMATE 

SEPARATION ON IMMOBILIZED POLYSACCHARIDE PHASES 

Duchateau A.L.L. Boesten J.M.M., Thomas-Verweij A. 

DSM Pharma Chemicals 

S27-03 DETERMINATION OF PROLINE ANALOGUES IN PLANTS SUBMITTED TO DROUGHT STRESS BY LIQUID CHROMATOGRAPHY 

AND MASS SPECTROMETRY 

Guignard C., Lefèvre I., Legay S., Evers D., Hoffmann L. 

Public Research Center Gabriel Lippmann, Luxembourg 

S27-04 TITANIUM DIOXIDE (TIO2) MONOLITHIC SUPPORTS FOR LIQUID CHROMATOGRAPHY AND SAMPLE TREATMENT 

Abi Jaoudé M., Randon J. 

University of Lyon, CNRS-UCBL 

192 

193 

194 

195 

196 


S28-01 NEW NANOSTRUCTURED MATERIALS FOR HPLC AND TLC 

Linford M.R. 1 , Wiest L. 1 , Jensen D. 1 , Kanyal S.S. 1 , Hung D. 1 , Dadson A. 2 , Vail M.A. 2 , Davis R.C. 1 , Vanfleet R. 1 

1 

Brigham Young University 

2 

US Synthetic 

S28-02 AN EFFICIENT APPROACH FOR ACCURATE GRADIENT ELUTION IN NANO-LC 

Palma P. 1 , Cappiello A. 1 , Famiglini G. 1 , Bergna M. 2 , Siviero A. 2 , Mantegazza A. 2 

1 

University of Urbino 

2 

DANI Instruments SpA 

S28-03 CORE-SHELL STATIONARY PHASES USING GRAPHITE CORES WITH POROUS NANODIAMOND SHELLS FOR HIGH PH 

REVERSED-PHASE HPLC 

Wiest L.A. 1 , Jensen D.S. 1 , Hung D. 1 , Olsen R. 1 , Vail M.A. 2 , Dadson A. 2 , Linford M.R. 1 

1 


2 

US Synthetic Corporation 

S28-04 NOVEL MICROFABRICATED BINDER FREE THIN LAYER CHROMATOGRAPHY PLATES ASSEMBLED USING CARBON 

NANOTUBES 

Jensen D.S. 1 , Singh S. 1 , Song J. 1 , Vanfleet R. 1 , Davis R. 1 , Vail M.A. 2 , Dadson A. 2 , Linford M.R. 1 

1 


2 


197 

198 

199 

200 

201 


S29-01 ASSESSMENT OF CHIRAL STATIONARY PHASES FOR SUITABILITY FOR COMBINED ENANTIOMERIC IMPURITY / RELATED 

SUBSTANCES ASSAYS 

Perera R.W.H., Lough W.J. 

University of Sunderland 

S29-02 POLYSACCHARIDE-TYPE CHIRAL SELECTORS: THE IMPACT OF CHLORINE-CONTAINING GROUPS ON ENANTIOSELECTIVITY 

IN POLAR ORGANIC SOLVENT CHROMATOGRAPHY 

Ates H., Mangelings D., Vander Heyden Y. 

Vrije Universiteit Brussel 

S29-03 ENANTIOSEPARATION IN CHROMATOGRAPHIC LIQUID-LIQUID SYSTEMS 

Pérez A.M., Rubio N., Pérez E., Minguillón C. 

Parc Cientific de Barcelona / University of Barcelona 

S29-04 POTENTIAL OF CAPILLARY ELECTROPHORESIS FOR ESTIMATION OF HUMIC SUBSTANCES PHYSICO-CHEMICAL 

PROPERTIES 

d’Orlyé F., Reiller P.E. 

CEA, CE Saclay 

202 

203 

204 

205 

206 


17



S30-01 DETERMINATION OF CARBONYL COMPOUNDS BY RRLC-UV IN THE ATMOSPHERE OF NITERÓI CITY, BRAZIL 

Ochs S.M. 1 , Albuquerque F.C. 2 , Pontes Massa M.C.G. 2 , Pereira Netto A.D. 1 

1 


2 

PETROBRAS-CENPES, Leopoldo Américo Miguez de Mello Research and Development Center 

S30-02 FAST LIQUID CHROMATOGRAPHY-QUADRUPOLE-LINEAR ION TRAP MASS SPECTROMETRY FOR THE ANALYSIS OF 

PHARMACEUTICALS IN WATER RESOURCES 

Huerta-Fontela M. 1 , Galceran M.T. 1 , Ventura F. 2 

1 


2 

AGBAR, Analytical-Organic Chemistry 

S30-03 PESTICIDE ANALYSIS IN ENVIRONMENTAL WATERS BY DIRECT INJECTION IN AN ADVANCED LC-MS/MS SYSTEM 

Pareja L. 1 , Martínez-Bueno M.J. 2 , Cesio V. 1 , Heinzen H. 1 , Fernandez-Alba A.R. 2 

1 

Universidad de la República, Cátedra de Farmacognosia y Productos Naturales 

2 


S30-04 EVALUATION OF EMPORE® DISKS VS POWDER RESINS AS RECEIVING PHASE FOR MONITORING OF HYDROPHILIC 

WATERBORNE MICROPOLLUTANTS IN WATERS BY PASSIVE SAMPLING AND UPLC-MS/MS 

de la Cal A. 1 , Dávila T.J. 2 , Céspedes R. 3 , Ventura F. 1 

1 

SGAB S.A. Laboratory of Organic Chemistry, Barcelona 

2 

Cetaqua, Laboratory of Organic Chemistry, Barcelona 

3 

SGAB and Cetaqua, Environmental Health R & D, Barcelona 

207 

208 

209 

210 

211 

S31 MULTIDIMENSIONAL CHROMATOGRAPHY 

S31-01 SAMPLE SOLVENT STRENGTH AND VISCOUS FINGERING EFFECTS IN TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY 

Martin M. 1 , Mishra M. 2 , De Wit A. 3 , Grivel C. 4 , Heinisch S. 4 

1 


2 

IIT Ropar, Indian Institute of Technolgy Ropar-India 

3 

Free University of Brussels 

4 

University of Lyon 

S31-02 A COMPREHENSIVE STUDY ON THE OPTIMIZATION OF TWO-DIMENSIONAL CHROMATOGRAPHIC SYSTEMS CONSIDERING 

LOSSES IN THEORETICAL PEAK CAPACITY IN FIRST- AND SECOND-DIMENSIONS 

Vivó-Truyols G. 1 , van der Waal S. 2 , Schoenmakers P.J. 1 

1 

University of Amsterdam 

2 

DSM Resolve 

S31-03 COMPREHENSIVE TWO-DIMENSIONAL GC–FID AND GC–TOF MS WITH MULTIPLE COLUMNS IN THE SECOND DIMENSION: 

MATCHED FLOW CONDITIONS 

de Koning S. 1 , Janssen H-G. 2,3 , Peroni D. 3 ,Cochran J. 4 , van Egmond W. 5 

1 

LECO European Technical Centre 

2 

Unilever Research and Development, Advanced Measurement and Data Modeling 

3 


4 

Restek, New Business and Technology 

5 

NLISIS 

S31-04 ADEQUATE CRITERIA FOR THE DEVELOPMENT OF ROBUST COMPREHENSIVE TWO-DIMENSIONAL CHROMATOGRAPHY METHODS 

Vanbel P.F., Vivo-Truyols G., Schoenmakers P.J. 


212 

213 

214 

215 

216 

18 



S32 FORENSICS AND ENVIRONMENTAL RESEARCH 

S32-01 SIMULTANEOUS SEPARATION AND DETECTION OF ILLICIT DRUGS AND THEIR SALT FORMS USING LC/MS 

Guazzotti S., Jiang G., Preston K., Elmashni D. 


S32-02 OPTIMISATION OF SOLID PHASE EXTRACTION FOR THE ANALYSIS OF BENZODIAZAPINES FROM PLASMA 

Edge A. 1 , Hui Y. 2 , Liddicoat T. 1 , Pereira L. 1 

1 


2 

Kings Colleague London 

S32-03 OBJECTIVE ODOUR MEASUREMENT OF RECYCLED PAPERBOARD INTENDED FOR CONFECTIONERY FOOD PACKAGING, 

USING HS-SPME-GC-MS AND MS-BASED ELECTRONIC NOSE TECHNOLOGY 

Van Caelenberg T., Dirinck P. 

Catholic University College Ghent, K.U. 

Leuven Association 

S32-04 THE SCARCE CONSOLIDER PROJECT ON IBERIAN RIVER BASINS 

Navarro-Ortega A. 1 , Sabater S. 2 , Muñoz I. 3 , Sanchez-Vila X. 4 , Conde C. 5 , Picó Y. 6 , La-Roca F. 7 , Blasco J. 8 , Elosegi A. 9 , Schuhmacher M. 10 , Batalla R. 11 

Francés F. 12 , Barceló D. 1 

1 


2 

ICRA, Ecology of fluvial ecosystems, Girona 

3 

University of Barcelona, Flumen, ecology of rivers and reservoirs 

4 

Technical University of Barcelona, Hydrogeology 

5 

Technical University of Madrid, Numerical Techniques in earth Sciences 

6 

University of Valencia, Food and Environmental Safety 

7 

Technical University of Madrid, Economy 

8 

ICMAN-CSIC, Ecology and ecotoxicology of estuarine ecosystems, Cadiz 

9 

University of the Basque Country, River Ecohydrology 

10 

University Rovira i Virgili, Tecnatox, Tarragona 

11 

University of Lleida, Fluvial Geomorphology 

12 

Technical University of Valencia, Hydraulic engineering and environment 

217 

218 

219 

220 

221 


S33-01 RP-HPLC ANALYSIS OF FUROSINE AND ACID-SOLUBLE ß-LACTOGLOBULIN TO ASSESS THE HEAT LOAD OF EXTENDED 

SHELF LIFE (ESL) MILK IN AUSTRIA 

Mayer H.K., Raba B., Meier J., Schmid A. 

BOKU University of Natural Resources and Applied Life Sciences 

S33-02 COMPREHENSIVE GCXGC(QMS) PESTICIDE ANALYIS PREPARED WITH QUECHERS: QUALITATIVE AND QUANTITATIVE 

ANALYSIS WITH AN ULTRA FAST QUADRUPOL MASS SPECTROMETER 

Baier H.U. 

Shimadzu Europa GmbH 

S33-03 COMPARISON OF LIQUID-LIQUID EXTRACTION AND SOLID PHASE EXTRACTION FOR THE GC-MS ANALYSIS OF PHENOLIC 

ACIDS RESULTING FROM THE DEGRADATION OF WINE POLYPHENOLS BY FAECAL MICROBIOTA 

Muñoz-González C., Del Pozo-Bayón M.A., Rodríguez-Bencomo J.J., Martín-Alvarez P.J., Cueva C., Bartolomé B., Moreno-Arribas M.V. 


222 

223 

224 

225 


S34-01 VARIOUS STRATEGIES INVOLVING ON-LINE ELECTROKINETIC SAMPLE PRECONCENTRATION STEPS FOR CAPILLARY 

ELECTROPHORESIS ANALYSIS OF INORGANIC IONS AT TRACE-LEVEL IN REAL MATRICES 

Delaunay N. 1 , Sarazin C. 2 , Ghasemi M. 1 , Varenne A. 1 , Costanza C. 2 , Eudes V. 2 , Gareil P. 1 

1 

Chimie Paristech (PECSA), Paris 

2 

Central Laboratory of the Prefecture de Police, Paris 

S34-02 CE-TOF OPTIMIZATION FOR A METABOLOMIC APPROACH TO STUDY THE NUTRACEUTICAL EFFECT OF CYSTOSEIRA 

EXTRACTS ON DIABETIC RATS 

Moraes E.P., Rupérez F.J., Barbas C. 

University CEU San Pablo, Madrid 

S34-03 SIMPLE AND RAPID CAPILLARY ELECTROPHORESIS METHOD FOR THE CHARACTERIZATION AND SEPARATION OF CDSE 

QUANTUM DOTS 

Carrillo-Carrión C. 1 , Moliner-MartÌnez Y. 2 , Simonet Suau B.M. 1 , Valcárcel M. 1 

1 


2 


226 

227 

228 

229 


19


S35 EXTRACTION 

S35-01 POROUS LAYER OPEN TUBULAR COLUMNS FOR SOLID PHASE MICRO-EXTRACTION 

Nesterenko E., Yavorsky A., Yavorska O., Paull B. 


S35-02 ELECTRODEPOSITION OF SILICA SOL-GEL ON METALLIC SUBSTRATE AS A NEW APPROACH TOWARDS UNBREAKABLE 

FIBERS IN SOLID PHASE MICROEXTRACTION 

Bagheri H., Sistani H., Ayazi Z. 

Sharif University of Technology 

S35-03 POLYPYRROLE-POLYAMIDE ELECTROSPUN-BASED SORBENT FOR MICRO-SOLID PHASE EXTRACTION 

Bagheri H., Aghakhani A., Akbari, Ayazi Z. 


230 

231 

232 

233 


S36-01 AUTOMATED IMMUNOAFFINITY CAPILLARY ELECTROPHORESIS BASED ON MAGNETIC BEADS FOR THE DETERMINATION 

OF ALPHA-1 ACID GLYCOPROTEIN ISOFORMS PROFILE TO FACILITATE ITS USE AS BIOMARKER OF VASCULAR DISEASES 

Morales-Cid G. 1 , Puerta A. 1 , Díez-Masa J.C. 1 , Martín-Alvarez P.J. 1 , Martín-Ventura J.L. 2 , Barbas C. 3 , Tuñón J. 2 , Egido J. 2 , de Frutos M. 1 

1 

CSIC, Institute of Organic Chemistry, Madrid 

2 

IIS-Fundación Jiménez, Autonomous University of Madrid 

3 


S36-02 RAPID ANALYSIS OF WHEY PROTEINS USING MICROCHIP CAPILLARY ELECTROPHORESIS 

González Crevillén A., Barrios Romero M., de la Puerta A., de Frutos M., Diez-Masa J.C. 

Institute of Organic Chemistry, Madrid 

S36-03 FLUORESCENT DERIVATIZATION THROUGH THE AMINO GROUPS ALLOWS CE-LIF ANALYSIS OF PROSTATE-SPECIFIC 

ANTIGEN (PSA) GLYCOFORMS 

Garrido-Medina R., de Frutos M., Diez-Masa J.C. 

Institute of Organic Chemistry (CSIC), Madrid 

234 

235 

236 

237 

20 



POSTER SESSIONS 

P01 FUNDAMENTALS OF CHROMATOGRAPHY 

P01-001 PEAK HALF-WIDTH PLOTS TO STUDY PEAK PERFORMANCE OF BASIC DRUGS IN SURFACTANT-MEDIATED LIQUID 


García-Álvarez-Coque M.C. 1 , Ruiz-Ángel M.J. 1 , Carda-Broch S. 2 

1 


2 

University Jaume I, Castelló de la Plana 

P01-002 ACETONITRILE AND ETHANOL: TWO BELITTLED MODIFIERS IN MICELLAR LIQUID CHROMATOGRAPHY 

Torres-Lapasió J.R. 1 , Ruiz-Ángel M.J., Carda-Broch S. 2 , García-Álvarez-Coque M.C. 1 

1 


2 


P01-003 OPTIMAL EXPERIMENTAL DESIGNS IN RPLC AT VARIABLE PH BASED ON ERROR SURFACES AND GENETIC ALGORITHMS 

Torres-Lapasió J.R., Baeza-Baeza J.J., Pous-Torres S., García-Álvarez-Coque M.C. 


P01-004 GLOBAL PARAMETERS ACCOUNTING FOR PEAK BROADENING AND SKEWNESS 

Baeza-Baeza J.J., Pous-Torres S., Torres-Lapasió J.R., García-Álvarez-Coque M.C. 


P01-005 APPROACHES TO ESTIMATE THE RETENTION TIME IN CHROMATOGRAPHY 

Baeza-Baeza J.J., García-Álvarez-Coque M.C., Torres-Lapasió J.R. 


P01-006 CHANGES IN A SURFACTANT-MEDIATED CHROMATOGRAPHIC SYSTEM UPON ADDITION OF SHORT CHAIN ALCOHOLS AND 

ACETONITRILE 

Ruiz-Ángel M.J. 1 , Carda-Broch S. 2 , García-Álvarez-Coque M.C. 1 

1 


2 


P01-007 PERFORMANCE OF IONIC LIQUIDS IN REVERSED-PHASE LIQUID CHROMATOGRAPHY OF BASIC DRUGS 

Ruiz-Ángel M.J., Fernández-Navarro J.J., García-Álvarez-Coque M.C. 


P01-008 DETERMINATION OF THE HYDRPHOBICITY (LOGPO/W) OF ORGANIC BASES THROUGH A NEW CHROMATOGRAPHIC METHOD 

Pallicer J.M., Sales J., Rosés M., Ràfols C., Bosch E. 


P01-009 A WIDELY-APPLICABLE SYSTEM FOR STRUCTURE-BASED CHROMATOGRAPHIC RETENTION TIME PREDICTION 

Cimpan G., Kolovanov E., Vazhentsev A., Japertas P., McBrien M. 

ACD/Labs, UK 

P01-010 DENTIN - PROTEOMIC ANALYSIS OF HUMAN TOOTH BY HPLC-MS/MS 

Eckhardt A.¹, Pataridis S.¹, Sedlakova P.¹, Lacinova K.¹, Zmatlikova Z.², Miksik, I.¹ 

¹ Institute of Physiology, Academy of Sciences of the Czech Republic, Dpt. Analysis of Biologically Important Compounds 

² Institute of Analytical Chemistry, University of Pardubice 

P01-011 IMMUNOGLOBULIN G PURIFICATION WITH PROTEIN A IMMOBILIZED BIOCOMPATIBLE MICROPOROUS MEMBRANES 

Uzun L., Türkmen D., Karakoç V., Yavuz H., Çelik H., Denizli A. 

Hacettepe University 

239 

240 

241 

242 

243 

244 

245 

246 

247 

248 

249 

250 

P02 GAS CHROMATOGRAPHY 

P02-001 IONIC LIQUID BASED HEADSPACE SOLID-PHASE MICROEXTRACTION-GAS CHROMATOGRAPHY FOR POLAR ORGANIC 

COMPOUNDS 

Ruiz-Ángel M.J. 1 , Carda-Broch S. 2 , Armstrong D.W.3, Berthod A. 4 

1 


2 


3 

University of Texas-Arlington 

4 


P02-002 CHARACTERIZATION OF RHODIOLA EXTRACTS BY MS AND GC-MS 

Rodríguez-Sánchez S. 1 , Díaz P. 2 , Sanz M.L. 1 , Sanz J. 1 , Martínez-Castro I. 1 

1 

Instituto de Química Orgánica General, CSIC, Madrid 

2 

Technical University of Madrid 

251 

252 

253 


21

OVERVIEW OF PRESENTATION - POSTER SESSIONS 

P02-003 NEW ATMOSPHERIC PRESSURE CHEMICAL IONIZATION SOURCE FOR PESTICIDE RESIDUES SCREENING BY GC-QTOF MS 

Portolés, T. 1 , Sancho J.V. 1 , Hernández F .1 , Newton A. 2 , Hancock P. 2 

1 


2 

Water Corporation 

P02-004 ORGANIC CHARACTERIZATION OF MURAL PAINTINGS FROM SPANISH ARCHEOLOGICAL SITES 

Rosales-Conrado N., Leon-González M.E., Pérez-Arribas L.V., Navarro-Villoslada F., Vidal-Cabeza L., Báez-Aglio M.I. 

Complutense University of Madrid 

P02-005 DETERMINATION OF CURRENTLY USED PESTICIDES (CUPS) IN FINE AIRBORNE PARTICULATE MATTER USING MICROWAVE- 

ASSISTED EXTRACTION AND GAS CHROMATOGRAPHY COUPLED TO TRIPLE QUADRUPOLE MASS SPECTROMETRY 

Coscollà C. 1 , Yusà V. 1 , Castillo M. 1 , Pastor A. 2 

1 

Public Health Research Center (CSISP) 

2 


P02-006 GAS CHROMATOGRAPHIC DETERMINATION OF PHYTOSTEROLS AND THEIR OXIDATION PRODUCTS IN ENRICHED FRUIT BEVERAGES 

Alemany-Costa L., González-Larena M., García-Llatas G., Alegría A., Barberá R., Lagarda M.J. 


P02-007 A SIMPLE PARALLEL GC COLUMN SCREENING SYSTEM 

Schafer W. 1 , Pirzada Z. 1 , Hamilton S., Welch C.J. 1 

1 

Merck, Early Development Analytical Research 

2 

MSD, Early Development Analytical Research 

P02-008 ANALYSIS OF RESIDUAL SOLVENTS USING GC/FID WITH HEADSPACE AND A CYANOPROPYLPHENYL POLYSILOXANE PHASE 

Khan A., Pereira L., Faulkner W., Barattini V. 


P02-009 DETERMINATION OF SUSPECTED ALLERGENS IN COSMETIC PRODUCTS BY HEADSPACE-PROGRAMMED TEMPERATURE 

VAPORIZATION-FAST GAS CHROMATOGRAPHY-QUADRUPOLE MASS SPECTROMETRY 

del Nogal Sánchez M., Pérez Pavón, J. L., Moreno Cordero B. 


P02-010 DETERMINATION OF BENZENE AND 22 ALKYLBENZENES IN SOIL WITH THERMAL DESORPTION-GAS CHROMATOGRAPHY-MASS 

SPECTROMETRY 

Kramarics Á. 1 , Szekeres Z. 1 , Eke Zs. 1 , Rikker T. 2 , Torkos K. 1 

1 

Eötvös Loránd University 

2 

WESSLING International Research and Educational Center 

P02-011 INNOVATIVE USE OF IONIC LIQUIDS FOR CAPILLARY GC 

Sidisky L.M. 1 , Buchanan M. 1 , Stenerson K. 1 , Ferrari R. 2 

1 

Supelco, Research & Development-USA 

2 

Sigma-Aldrich, Sales & Marketing-Italy 

254 

255 

256 

257 

258 

259 

260 

261 

262 

P02-012 DETERMINATION OF PESTICIDE RESIDUES IN FOODS OF PLANT ORIGIN USING FUSED CORE RP AMIDE HPLC, MS/MS AND 

DISPERSIVE SPE - QUECHERS-METHOD 

Belotti E. 1 , Meni L. 1 , Ruggeri M. 1 , Ferrari R. 2 

1 

Water&Life Entratico (BG)-Italy 

2 


P02-013 DETERMINATION OF TRICHLOROANISOLE IN CORK PLANKS BY HS-SPME-GC/MS 

Martínez-Cañas M.A., Yuste-Córdoba F.J., 

Instituto del Corcho, la Madera y el Carbón Vegetal. Junta de Extremadura, Tecnología y Calidad-Spain 

P02-014 COMPARISON OF STIR BAR SORPTIVE EXTRACTION AND SOLID PHASE EXTRACTION FOR THE DETERMINATION OF POLYCYCLIC 

AROMATIC HYDROCARBONS IN WASTEWATER BY GAS CHROMATOGRAPHY COUPLED TO TANDEM MASS SPECTROMETRY 

Barco Bonilla N. 1 , Romero-Gonzalez R. 1 , Plaza-Bolaños P. 1 , Fernández-Moreno J.L. 2 , Garrido-Frenich A. 1 , Martinez Vidal J.L 1 

1 

Almeria University 

2 

Laboratorio Analítico Bioclínico LAB, Almeria 

P02-015 VOLATILE COMPOUNDS AS MARKERS OF LA MANCHA D.O. RED WINES ACCORDING TO THE AGEING PERIOD BY GC/MS 

Castro-Vázquez L., Calvo E., Alañón E., Diaz-Maroto M.C., Pérez-Coello M.S. 

University of Castilla la Mancha 

P02-016 ROLE OF GC-APCI-MAXIS MS IN FOOD METABOLOMICS: DETERMINATION OF POLYPHENOLS FROM EXTRA-VIRGIN OLIVE OIL 

García-Villalba R. 1 , Pacchiarotta T. 2 , Carrasco-Pancorbo A. 1 , Segura-Carretero A. 1 , Fernández-Gutiérrez A. 1 , Deelder A.M. 2 

Mayboroda O.A. 2 

1 


2 

Leiden University Medical Center 

263 

264 

265 

266 

267 

22 



P03 LIQUID CHROMATOGRAPHY 

P03-001 QUANTITATIVE DETECTION OF CARCINOID TUMOR MARKERS BY HPLC–MS 

Jambor E., Patko B., Bona A., Maasz G., Mark L, Ohmacht R, Chemistry-Hungary 

University of Pecs-Hungary 

P03-002 SIMULTANEOUS DETERMINATION OF ENALAPRIL AND LERCANIDIPINE BY HPLC-DAD TECHNIQUE IN PHARMACEUTICAL 

DOSAGE FORMS 

Gumustas M. 1 , Sanli S. 2 , Sanli, N. 2 , Ozkan S.A. 1 

1 

Ankara University-Turkey 

2 

Hitit University-Turkey 

P03-003 DEVELOPMENT AND VALIDATION OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) METHOD TO DETERMINE 

VANCOMYCIN IN PLASMA AND PULMONARY TISSUE 

Guerrero L., Martínez-Olondris P., Rigol M., Esquinas C., Piñer R., Esperatti M., Luque N., Liapikou M., Li Bassi G., Torres A., Soy D. 

Hospital Clinic Barcelona 

P03-004 ADVANTAGES OF MCROEMULSIONS AS MOBILE PHASES IN HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 

Pashkova E.B., Pirogov A.V., Shpigun O.A., 

Moscow State University 

P03-005 DEVELOPMENT OF A STABILITY INDICATING HPLC METHOD FOR MELOXICAM, ITS RELATED SUBSTANCE 2-AMINO-5-METHYL 

THIAZOLE AND FORMULATORY ADJUVANTS IN AN AMPOULE DOSAGE FORM USING RETENTION MODELING 

Mokhtar H., Elian M. 

Medical union Pharmaceuticals Co.-Egypt 

P03-006 HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY: RETENTION STUDIES ON SUGAR BASED POLAR STATIONARY 

PHASES 

Schuster G., Hüser T., Lindner W. 

University of Vienna 

P03-007 DETERMINATION OF NITROSAMINES IN WATER AT THE NG/L LEVELS BY LIQUID CHROMATOGRAPHY COUPLED TO TANDEM 


Ripollés C., Pitarch E., Sancho J.V., López F.J., Hernández F. 

Research Institute for Pesticides and Water, University Jaume I, Castelló de la Plana 

P03-008 RP-CHIRAL LC-METHOD DEVELOPMENT WITH THE QUALITY BY DESIGN PRINCIPLES 

Vogel-da Silva F. 1 , Galushko S. 2 

1 

Novartis Pharma AG-Switzerland 

2 

ChromSword, Method Development-Germany 

268 

269 

270 

271 

272 

273 

274 

275 

276 

P03-009 DEVELOPMENT AND VALIDATION OF MICELLAR LIQUID CHROMATOGRAPHIC METHODS FOR THE DETERMINATION OF 

ANTIBIOTICS IN DIFFERENT MATRICES 

Rambla-Alegre M., Esteve-Romero J., Carda-Broch S. 


P03-010 CATECHOLAMINE DETERMINATION IN PHEOCHROMOCITOMA PATIENTS BY LIQUID CHROMATOGRAPHY 

Esteve-Romero J. 1 , Carda-Broch S. 1 , Rambla-Alegre M. 1 , Bose D. 2 , Durgbanshi A. 2 , Agrawal N. 2 , Raviolo M.A. 1 , Ochoa-Aranda E. 3 

1 

Uiversitat Jaume I 

2 

Dr.H.S.Gour University 

3 

Hospital Provincial de Castellón 

P03-011 DETERMINATION OF BIOGENIC AMINES IN WINE 

Esteve-Romero J. 1 , Carda-Broch S. 1 , Rambla-Alegre M. 1 , Peris-Vicente J. 1 , Chin-Chen M.L. 1 , Bose D. 2 , Raviolo M.A. 1 , Agrawal N. 2 

1 


2 


P03-012 MICELLAR LIQUID CHROMATOGRAPHY DETERMINATION OF QUINOLONES IN FOOD SAMPLES WITH FLUORESCENCE 

DETECTION 

Rambla-Alegre M., Collado-Sánchez M.A., Esteve-Romero J., Carda-Broch S. 

Universitat Jaume I, Castelló de la Plana 

P03-013 VALIDATION OF A LIQUID CHROMATOGRAPHIC PROCEDURE FOR THE DETERMINATION OF FIVE QUINOLONES IN FISH ACCORDING 

WITH REGULATION 2002/657/EC 

Rambla-Alegre M. 1 , Peris-Vicente J. 2 , Esteve-Romero J. 1 , Carda-Broch S. 2 

1 


2 

Química Analítica, QFA, Universitat Jaume I, Castelló, Spain 

277 

278 

279 

280 

281 


23


P03-014 STABILITY STUDIES OF 3’-AZIDO-3’-DEOXY-5’-O-OXALYL-N-LEUCINETHYMIDINE IN AQUEOUS SOLUTIONS AND SIMULATED 

GASTRIC AND INTESTINAL FLUID 

Raviolo M.A. 1 , Esteve-Romero J. 2 , Briñón M.C. 1 , Carda-Broch S. 2 , Rambla-Alegre M. 2 , Bose D. 3 

1 

Universidad Nacional Córdoba 

2 


3 


P03-015 ANALYSIS OF HYDROXYTYROSOL IN OLIVE EXTRACT SAMPLES BY LIQUID CHROMATOGRAPHY USING A SURFACTANT-MEDIATED 

MOBILE PHASE 

M. Rambla-Alegre M. 1 , Marco-Peiró S. 1 , Peris-Vicente J. 1 , Beltran-Martinavarro B. 1 , Collado-Sanchez M.A. 1, Carda-Broch S. 1 , 

Esteve-Romero J. 1 , Bose D. 2 

1 


2 


P03-016 TERNARY ISOCRATIC MOBILE PHASE OPTIMIZATION USING A PEAK WIDTH PREDICTION MODEL 

Kawabe T. 1 , Tomitsuka T. 2 , Takemura A. 2 , Kishi N. 2 , Toyo’oka T. 1 

1 

School of Pharmaceutical Sciences, University of Shizuoka 

2 

Daiichi Sankyo Co., Ltd., Analytical & Quality Evaluation Research Laboratories 

P03-017 RP-HPLC DETERMINATION OF HYDROPHOBIC PROPERTIES OF NOVEL SUBSTITUTED BENZOTHIAZOLE DERIVATIVES 

Oktabec Z. 1 , Imramovsky A. 2 , Pejchal V. 2 , Jampilek J. 1 

1 

Zentiva, k. s./ Faculty of Pharmacy, Charles University, Development-Czech Republic 

2 

Faculty of Chemistry and Chemical Technology, University of Pardubice 

P03-018 HPLC EVALUATION OF THE FUROSINE CONTENT IN INFANT FORMULAS DURING THEIR SHELF-LIFE 

Salcedo J. 1 , Lacomba R. 1 , Alegría A. 1 , Barberá R. 1 , Vasallo I. 2 , Lagarda M.J. 1 , 

1 


2 

Hero Institute for Infant Nutrition, R&D 

P03-019 HPLC DETERMINATION OF SIALIC ACID (NEU5AC AND NEU5GC) CONTENTS IN INFANT FORMULAS 

Lacomba R. 1 , Salcedo J. 1 , Alegria A. 1 , Barberá R. 1 , Matencio E. 2 , Lagarda M.J. 1 

1 


2 


282 

283 

284 

285 

286 

287 

P03-020 OPTIMIZATION OF LC METHODS FOR THE SIMULTANEOUS DETERMINATION OF BISOPROLOL AND HYDROCHLOROTHIAZIDE IN 

THEIR PHARMACEUTICAL DOSAGE FORMS 

Gumustas M., Bozal B., Dogan-Topal B., Ozkan S.A., Uslu B. 


P03-021 OPTIMISATION OF A LIQUID CHROMATOGRAPHIC METHOD FOR THE SEPARATION OF HYDROXYLATED POLYCHLORINATED 

BIPHENYLS ON A POLAR-EMBEDDED STATIONARY PHASE APPLYING EXPERIMENTAL DESIGN PROCEDURES 

Galindo-Iranzo P. 1 , Quintanilla-López J.E. 2 , Lebrón-Aguilar R. 1 , Gómara B. 2 

1 

Institute of Physical Chemistry ‘Rocasolano’ (CSIC), Madrid 

2 

Institute of General Organic Chemistry (CSIC), Madrid 

288 

289 

P03-022 APPLICABILITY OF SOLID PHASE EXTRACTION WITH MICELLAR DESORPTION (SPE-MD) COMBINED WITH HPLC TO THE 

DETERMINATION OF FLUOROQUINOLONES IN HOSPITAL AND MUNICIPAL WASTEWATERS 

Montesdeoca-Esponda S., Sosa-Ferrera Z., Santana-Rodríguez J.J. 

University of Las Palmas de Gran Canaria 

P03-023 SIMULTANEOUS DETERMINATION OF ENDOCRINE DISRUPTING CHEMICALS IN WASTEWATER TREATMENT PLANT SLUDGES BY 

MICROWAVE ASSISTED EXTRACTION AND LC-ESI-MS/MS 

Vega-Morales T., Sosa-Ferrera Z., Santana-Rodríguez J.J. 

Universidad de Las Palmas de Gran Canaria 

P03-024 CHARACTERISATION OF THE LIQUID CHROMATOGRAPHIC BEHAVIOUR OF HYDROXYLATED POLICHLORINATED BIPHENYLS ON 

AN AMIDE-TYPE STATIONARY PHASE 

Quintanilla-López J.E. 1 , Galindo-Iranzo P. 2 , Gómara B. 1 , Lebrón-Aguilar R. 2 

1 


2 


P03-025 A NEW CHROMATOGRAPHIC MODEL TO PREDICT THE RETENTION OF IONIZABLE ANALYTES UNDER GRADIENT ELUTION RP-HPLC 

Andrés A., Téllez A., Rosés M., Bosch E. 

Faculty Chemistry University of Barcelona 

P03-026 HOP’S (HUMULUS LUPULUS SP) PRENYLATED FLAVONOIDS EXTRACTED BY PLE AND ANALYZED BY LC-ESI-MS/MS 

Gil-Ramírez A., Mendiola J.A., Marín F.R., Ibáñez E. 

Instituto de Investigación Ciencias de la Alimentación CIAL-CSIC 

290 

291 

292 

293 

294 

24 



P03-027 HOW CLEAN ARE YOUR VIALS AND CLOSURES ? 

Pereira L., Edge A., Shick L., King B., 


P03-028 CHARACTERIZATION OF INDUSTRIAL ENZYMES BY HPLC OF THE INTACT PROTEINS AND THEIR TRYPTIC DIGESTS USING A 

POLYMERIC MONOLITHIC COLUMN 

Beneito-Cambra M. 1 , Herrero-Martínez J.M. 1 , Ramis-Ramos G. 1 , Lindner W. 2 , Lämmerhofer M. 2 

1 


2 


P03-029 NOVEL POLYSTYRENE-DIVINYLBENZENE ANION EXCHANGERS FOR ION CHROMATOGRAPHY 

Zatirakha A.V., Smolenkov A.D., Shpigun O.A. 

Lomonosov Moscow State University 

P03-030 THE EFFECT OF HYDROTHERMAL TREATMENT ON COLUMN PERFORMANCE FOR MONOLITHIC SILICA CAPILLARY COLUMNS 

Hara T., Mascotto S., Weidmann C., Heuser S., Smarsly B. 

Justus Liebig University of Giessen-Germany 

P03-031 GREEN CHROMATOGRAPHY DETERMINATION OF PARACETAMOL, PROPYPHENAZON, CAFFEINE, AND P-AMINOPHENOL IN 

PHARMACEUTICAL PREPARATIONS 

Brabcová I., Šatínsky D., Solich P. 

Charles University in Prague Faculty of Pharmacy in Hradec Králové 

P03-032 APPLICATION OF AN HPLC METHOD FOR A RAPID DETERMINATION OF -TOCOPHEROL, ERGOCALCIFEROL AND CHOLECALCIFEROL 

IN DIFFERENT LIQUID FOODS AND CEREALS 

Barba F.J., Esteve M.J., Frigola A., 


P03-033 ANALYSIS OF CHEMICAL MARKERS IN MEAT AND MEAT PRODUCTS BY HILIC 

Mora L. 1 , Hernández-Cázares A. 1 , Aristoy M-C. 1 , Toldrá F. 1 , Reig M. 2 

1 

Instituto de Agroquímica y Tecnología de Alimentos, Ciencia de la Carne, Valencia 

2 

Technical University of Valencia 

P03-034 HOW THE DISSOLUTION SOLVENT AFFECTS PEAK SHAPES IN HYDROPHILIC INTERACTION CHROMATOGRAPHY? 

Josephine R. 1 , Guillarme D. 1 , McCalley D. 2 , Rudaz S. 1 , Veuthey J-L. 1 

1 

University of Geneva 

2 

University of the West of England 

P03-035 DEVELOPMENT OF A VALIDATED LC METHOD FOR THE SIMULTANEOUS DETERMINATION OF AMLODIPINE AND PERINDOPRIL 

IN THEIR COMMERCIAL DOSAGE FORMS 

Gumustas M., Ozkan S.A., 

Ankara University Faculty of Pharmacy 

P03-036 INVESTIGATION OF THE SELECTIVE DEPLETION OF PHOSPHOLIPID INTERFERENCES UTILIZING HYBRID-SPE TECHNOLOGY 

FOR LC-MS 

Buckendahl K. 1 , Aurand C.R. 2 , Brandes H. 2 , Bell D.S. 3 , Gutierrez P. 4 , Ferrari R. 5 

1 

Sigma-Aldrich, Sales & Marketing 

2 

Supelco, Research & Development 

3 


4 

Sigma-Aldrich, Sales & Marketing-Spain 

5 


P03-037 MOBILE PHASE CONSIDERATIONS FOR IMPROVED LC-MS AMENABLE PEPTIDE SEPARATIONS 

Brandes H.K. 1 , Claus J.E. 1 , Aurand C. 1 , Bell, David S. 1 , Gutierrez P. 2 , Ferrari R. 3 

1 


2 


3 


P03-038 PURIFICATION OF ANGIOTENSIN-CONVERTING-ENZYME FROM PIG LUNG 

Eisele T., Stressler T., Kranz B., Fischer L. 

University Hohenheim, Biotechnology-Germany 

P03-039 PURIFICATION OF A PROLINE SPECIFIC EXOPEPTIDASE FROM LACTOBACILLUS HELVETICUS 

Stressler T., Eisele T., Kranz B., Fischer L. 

University Hohenheim, Biotechnology-Germany 

P03-040 RP-HPLC OF MALONDIALDEHYDE IN BLOOD PLASMA OF PATIENTS WITH ACUTE MYOCARDIAL INFARCTION 

Tomandl J., Pes O., Parenica J. 

Masaryk University-Czech Republic 

295 

296 

297 

298 

299 

300 

301 

303 

304 

305 

306 

307 

308 

309 


25


P03-041 APPLYING QUALITY BY DESIGN CONCEPTS TO CHROMATOGRAPHIC METHOD DEVELOPMENT 

Ponzio T., Sinclair T., Cimpan G. 

ACD/Labs, D 

P03-042 HPLC DETERMINATION OF CAFFEINE AND PARAXANTHINE IN SALIVA AS A METHOD FOR CYP1A2 METABOLIC ACTIVITY 

ASSESSMENT IN HUMANS 

Jurica J., Zendulka O., Tomandl J. 


P03-043 INFLUENCE OF EXTRACTION VARIABLES ON FATTY ACID COMPOSITION AND TRIACYLGLYCEROL PROFILE OF DURIAN SEED GUM 

Hamed Mirhosseini 

Faculty of Food Science and Technology, Universiti Putra Malaysia 

P03-044 MULTI-MYCOTOXIN ANALYSIS IN EGGS USING A BASED QUECHERS EXTRACTION PROCEDURE AND ULTRA-HIGH-PRESSURE 

LIQUID CHROMATOGRAPHY COUPLED TO TRIPLE QUADRUPOLE MASS SPECTROMETRY 

Gómez Perez M.L., Romero-Gonzalez R., Garrido Frenich A., Martinez Vidal J.L. 

University of Almeria 

P03-045 EVALUATION OF POROUS AND CORE-SHELL PARTICLES FOR USE IN LC-MS BIOANALYSIS 

Butchart K. 1 , Woodruff M. 1, Saunders K. 2 

1 

Pfizer, Sandwich, Kent 

2 

Fortis Technologies Ltd- UK 

P03-046 RESOLUTION VERSUS EFFICIENCY OF COMPLEX MIXTURES IN UHPLC 

Butchart K., Woodruff M. 


P03-047 WHAT SHOULD WE RELY ON IN UHPLC? ARE WE REALLY BEING MORE PRODUCTIVE? 

Woodruff M., Butchart K. 


P03-048 APPLICATIONS OF A NEW HILIC STATIONARY PHASE 



P03-049 INVESTIGATION ON THE ANALYSIS AND QUANTIFICATION OF FULLERENES IN REAL AEROSOL SAMPLES 

Sanchis J., Berrojalbiz N., Caballero G., Dachs J., Farre M., Barceló D. 

IDÆA-CSIC, Departament de Química Ambiental, Barcelona 

P03-050 IN VITRO TRANSPORT EVALUATION OF CHITOOLIGOSACCHARIDES MIXTURES THROUGH INTESTINAL EPITHELIA BY SEC-HPLC 

Fernandes J.C. 1 , Cardelle-Cobas A. 2 , Pintado M. 1 , Corzo N. 2 

1 

Escola de Biotecnologia e Quimica Fina. Universidade Católica Portuguesa 

2 

Instituto de Fermentaciones Industriales (CSIC), Madrid 

P03-051 TEMPERATURE ASSISTED HILIC SEPARATION OF NUCLEOSIDE TRIPHOSPHATES 

Wilson S., Johnsen E., Odsbu I., Lundanes E. 


P03-052 DEVELOPMENT OF A STABILITY INDICATING METHOD FOR SIMVASTATIN USING A QBD APPROACH WITH EXPERIMENTAL DESIGN 

Alden P.G., Potts W., Moore D., Yurach D. 

Waters Corporation, Pharmaceutical Market Development-USA 

P03-053 REFERENCE METHOD FOR THE ABSOLUTE QUANTIFICATION OF TROPONIN I IN SERUM USING HIGH PERFORMANCE LIQUID 

CHROMATOGRAPHY AND TRIPLE QUADRUPOLE MASS SPECTROMETRY 

Dullnig V. 1 , Kobold U. 2 , Hallermayer K. 2 , Huber C.G. 1 

1 

University of Salzburg- Austria 

2 

Roche Diagnostics, Penzberg-Germany 

P03-054 NUCLEOTIDE AND SUGAR NUCLEOTIDE ANALYSIS BY LC-ESI-MS ON PRETREATED POROUS GRAPHITIC CARBON 

Pabst M., Grass J., Léonard R., Altmann F. 

University of Natural Resources and Applied Life Sciences, Department of Chemistry-Austria 

P03-055 DEVELOPMENT OF A LOW COST, ANALYTICAL PROCESS TO EXTRACT, SEPARATE AND DETERMINE EFAVIRENZ AND 

RIFAMPICIN PLASMA CONCENTRATIONS IN HIV/TB CO-INFECTED PATIENTS 

Fox D. 1 , O’Connor R. 2 3 , Mallon P. 4 , McMahon G. 1 

1 

School of Chemical Sciences, Dublin City University. 

2 

National Institute of Cellular Biotechnology, Dublin City University 

3 

School of Nursing, Dublin City University 

4 

Mater Misericordiae University Hospital, Dublin 

310 

311 

312 

313 

314 

315 

316 

317 

318 

319 

320 

321 

322 

323 

324 

26 



P04 ELECTRODRIVEN SEPARATIONS 

P04-001 IDENTIFICATION OF ENZYMES IN DETERGENTS BY CAPILLARY ELECTROPHORESIS 

Beneito-Cambra M., Herrero-Martínez J.M., Ramis-Ramos G. 


P04-002 DEVELOPMENT OF A CAPILLARY ELECTROPHORESIS METHOD FOR THE DETERMINATION OF POLYPHENOLS IN WINES 

Hernández Cassou S. 1 , Oliver R. 2 , Saurina J. 1 

1 


2 

Polytechnic University of Catalonia 

P04-003 CHARACTERIZATION OF WINES THROUGH THE ELECTROPHORETIC PROFILES OF POLYPHENOLS 


1 


2 


P04-004 CATIONIC COPOLYMERS AS PHYSICALLY ADSORBED COATINGS FOR CAPILLARY ELECTROPHORESIS: CONNECTIONS BETWEEN 

STRUCTURE AND PERFORMANCE 

Bernal J. 1 , Herrero M. 1 , Velasco D. 2 , Elvira C. 2 , Cifuentes A. 1 

1 


2 

Institute of Science and Technology of Polymers (CSIC), Madrid 

325 

326 

327 

328 

329 

P04-005 SEPARATION OF PHENOTYPICALLY INDISTINGUISHABLE CANDIDA SPECIES, ORTHOPSILOSIS, METAPSILOSIS AND 

PARAPSILOSIS, BY CAPILLARY ELECTROMIGRATION TECHNIQUES 

Horká M. 1 , Ružicka F. 2 , Kubesová A. 1,2 , Šlais K. 1 

1 

Institute of Analytical Chemistry of the ASCR, v.v.i., Brno, Czech Republic 

2 

Masaryk University Brno, Czech Republic 

330 

P04-006 APPLICATION OF AFFINITY CAPILLARY ELECTROPHORESIS AND DENSITY FUNCTIONAL THEORY TO INVESTIGATION OF 

HEXAARYLBENZENE-BASED RECEPTOR INTERACTIONS WITH ALKALI METAL AND AMMONIUM IONS IN METHANOL 

Ehala S. 1 , Toman P. 2 , Rathore R. 3 , Makrlik E. 4 , Kasicka V. 1 

1 

Institute of Organic Chemistry and Biochemistry AS CR, v.v.i. 

2 

Institute of Macromolecular Chemistry AS CR, v.v.i. 

3 

Marquette University 

4 

University of West Bohemia 

P04-007 PARTIAL FILLING ELECTROKINETIC CAPILLARY CHROMATOGRAPHY – A USEFUL TOOL FOR INTERACTION STUDIES BETWEEN 

LIPID BILAYER DISKS AND PHARMACEUTICALS 

Vainikka K. 1 , Reijmar K. 2 , Edwards K. 2 , Riekkola M-L. 1 

1 

Univeristy of Helsinki 

2 

Uppsala University 

P04-008 COMPARISON OF ALKYL ACRYLATE MONOLITHIC COLUMNS FOR CAPILLARY ELECTROCHROMATOGRAPHY USING CHEMICAL 

INITIATION 

Ten-Doménech I., Bernabé-Zafón V., Simó-Alfonso E.F., Herrero-Martínez J.M. 


P04-009 DETERMINATION OF MUSCLE RELAXANTS PANCURONIUM AND VECURONIUM BY CAPILLARY ELECTROPHORESIS WITH 

CAPACITIVELY COUPLED CONTACTLESS CONDUCTIVITY DETECTION 

Petru K., Siroka J., Luzova V., Jac P., Polasek M. 

Faculty of Pharmacy, Charles University-Czech Republic 

P04-010 PREPARATION AND CHARACTERIZATION OF OCTADECYL ACRYLATE MONOLITHS FOR CAPILLARY ELECTROCHROMATOGRAPHY 

BY THERMAL, CHEMICAL AND PHOTOCHEMICAL INITIATION 

Ten-Doménech I., Escrig-Doménech A., Bernabé-Zafón V., Simó-Alfonso E.F., Herrero-Martínez J.M. 


P04-011 PREPARATION AND CHARACTERIZATION OF POLY(LAURYLMETHACRYLATE) MONOLITHS WITH SILVER NANOPARTICLES BY 

PHOTOPOLYMERIZATION FOR CAPILLARY ELECTROCHROMATOGRAPHY 

Navarro-Pascual-Ahuir M., Lerma-García M.J., Simó-Alfonso E.F., Ramis-Ramos G., Herrero-Martínez J.M. 


P04-012 ANALYSIS, PURIFICATION AND CHIRAL SEPARATION OF BETA-ALANYL-D,L-TYROSINE AND ITS DERIVATIVES BY CAPILLARY 

AND FREE-FLOW ZONE ELECTROPHORESIS 

Sazelova P., Solinova V., Schimperkova T., Masova A., Jiracek J., Kasicka V., 

Institute of Organic Chemistry and Biochemistry AS CR, v.v.i.-Czech Republic 

331 

332 

333 

334 

335 

336 

337 


27


P04-013 CAPILLARY ELECTROPHORETIC ASSAY OF FLAVONOIDS AND PHENOLIC ACIDS IN PROPOLIS USING COMPLEX FORMATION 

WITH TUNGSTATE AND ON-LINE PRECONCENTRATION 

Siroka J., Petru K., Jac P., Polasek M. 

Charles University, Faculty of Pharmacy 

P04-014 DETERMINATION OF HISTAMINE, PHENYLETHYLAMINE AND TYRAMINE IN RED WINES USING ON-LINE COUPLED CAPILLARY 

ISOTACHOPHORESIS CAPILLARY ZONE ELECTROPHORESIS WITH PHOTOMETRIC DETECTION 

Ginterova P. 1 , Marak J. 2 , Stanova A. 2 , Kaniansky D. 2 , Maier V. 1 , Sevcik J. 1 

1 

Palacký university in Olomouc 

2 

Comenius University in Bratislava 

P04-016 STABILITY OF LINEAR POLYACRYLAMIDE COATED CAPILLARIES IN ACIDIC MEDIA IN THE PRESENCE OF ORGANIC MODIFIERS 

AND SURFACTANTS 

Anres P., Delaunay N., Gareil P. 

Chimie Paristech 

P04-017 METABOLOMIC STUDY OF HUMAN HT29 COLON CANCER CELLS BY CE-MS. A COMPARATIVE STUDY OF METABOLITE 

PURIFICATION STRATEGIES 

Ibáñez C. 1 , Simó S. 1 , Rocamora L. 2 , Gomez A. 2 , Ferragut J.A. 2 , Cifuentes A. 1 

1 

Institute of Industrial Fermentations, Food Characterization, Madrid 

2 

Universidad Miguel Hernandez, Alacant 

338 

339 

340 

341 

P04-018 DEVELOPMENT AND OPTIMIZATION OF PEPTIDES DERIVATIZATION THROUGH THIOL GROUPS. USE IN CHEMILUMINESCENCE 

DETECTION IN CAPILLARY ELECTROPHORESIS 

Regueiro-Vilar O., Puerta A., Uriel C., Gómez A., de Frutos M., Diez-Masa J.C. 

Institute of Organic Chemistry (C.S.I.C.) 

342 

P05 SUPERCRITICAL FLUID CHROMATOGRAPHY 

P05-001 STUDY OF SEVERAL POLYSACCHARIDE BASED CHIRAL STATIONARY PHASES FOR THE ENANTIOSELECTIVE SEPARATION OF 

AN ACETAMIDE INTERMEDIATE BY USING SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) 

Toribio L., Nozal Mª J, Bernal J.L., Diego,J.C., Martin Mª T. 


343 

344 

P06 MULTIDIMENSIONAL CHROMATOGRAPHY 

P06-001 DEVELOPMENT OF A TWO-DIMENSIONAL COMPREHENSIVE HPLC-SYSTEM FOR THE ANALYSIS OF COMPLEX SAMPLES 

Haun J. 1 , Teutenberg T. 1 , Schmidt T.C. 2 

1 

Institute of Energy and Environmental Technology, R&D-Germany 

2 


P06-002 COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY—TIME-OF-FLIGHT MASS SPECTROMETRY FOR THE ANALYSIS 

OF ANTHROPOGENIC AND NATURALLY-PRODUCED ORGANOBROMINATED COMPOUNDS IN BLUEFIN TUNA FROM THE 

MEDITERRANEAN SEA 

Pena-Abaurrea M. 1 , Covaci A. 2 , Ramos L. 1 

1 

Institute of Organic Chemistry (IQOG-CSIC), Madrid 

2 

University of Antwerp 

P06-003 PRESSURIZED HOT WATER EXTRACTION, SUPERCRITICAL FLUID EXTRACTION AND GC X GC-TOFMS IN THE ANALYSIS OF 

FATTY ACIDS IN BROCCOLI FLORETS 

Bernal J. 1 , Arnaiz E. 2 , Bernal J.L. 2 , Toribio L. 2 , Kronholm J., Ruiz-Jiménez J. 3 , Hartonen K. 3 , Riekkola M.L. 3 

1 


2 

I.U.CINQUIMA, University of Valladolid 

3 


P06-004 ANALYSIS OF CHLOROPARAFFINS USING COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY 

Hofmeister S., de Koning S. 

LECO European Technical Centre-Germany 

P06-005 APPLICATION OF MULTI-DIMENSIONAL CHROMATOGRAPHY TO THE SEPARATION AND IDENTIFICATION OF THE COMPONENTS 

OF FRESHWATER AND SEAWATER DERIVED DISSOLVED ORGANIC MATTER (DOM) 

Sandron S., Nesterenko E., Kelleher B., Paull B. 


P06-006 TWO-DIMENSIONAL ION CHROMATOGRAPHY OF CATIONS 

Mc Gillicuddy N. 1 , Nesterenko E. 1 , Paull B. 1 , Nesterenko P.N. 2 

1 


2 


345 

346 

347 

348 

349 

350 

351 

28 



P06-007 PROBABILITY OF FAILURE OF THE WATERSHED ALGORITHM FOR PEAK DETECTION IN COMPREHENSIVE TWO-DIMENSIONAL 


Vivó-Truyols G. 1 , Janssen H.G. 2 

1 


2 

Unilever Research and Development Vlaardingen, Advanced Measurement and Data Modelling 

352 

P06-008 METABOLOMICS OF SMALL SAMPLES: MINIATURIZED SAMPLE-PREPARATION FOR GC×GC-TOFMS BASED METABOLITE 

ANALYSIS 

Kay L. 1 , Erban A. 2 , de Koning S. 1 , Kopka J. 2 

1 


2 

Max Planck Institut für Molekulare Pflanzenphysiologie , Willmitzer 

P06-009 INTER-LABORATORY REPEATABILITY OF RAPID GC-TOFMS PLANT METABOLITE PROFILING AND GC-TOFMS SPATIAL 

METABOLITE ANALYSIS DURING MELON FRUIT DEVELOPMENT 

Kay L. 1 , Allwood W. 2 , de Koning S. 1 , Goodacre R. 2 

1 


2 

University of Manchester 

P06-010 METABOLOMICS OF THE HYDROGEN PRODUCING ALGAE CHLAMYDOMONAS REINHARDTII USING GCXGC-TOFMS 

Keck M. 1 , Kay L. 2 , de Koning S. 2 

1 

University of Bielefeld 

2 


353 

354 

355 

P07 HYPHENATED TECHNIQUES 

P07-001 DEVELOPMENT OF A NOVEL HYPHENATION SYSTEM BASED ON LIQUID CHROMATOGRAPHY, ISOTOPE RATIO MASS 

SPECTROMETRY AND RAMAN SPECTROSCOPY 

Wiese S. 1 , Teutenberg T. 1 , Jochmann M.A. 2 , Kujawinski D.M. 2 , Zhang L. 2 , Fischer B. 3 , Bettermann H. 3 

1 

Institute of Energy and Environmental Technology, R&D 

2 


3 

University of Heinrich-Heine 

P07-002 DETERMINATION OF PRODUCTS OF HEMI (CELLULOSE) DEGRADATION BY GC/MS, PYROLYSIS-GC/MS AND CE/MS 

Bogolitsyna A. 1 , Borgards A. 2 , Rosenau T. 1 , Potthast A. 1 

1 

University of Natural Resources and Applied Life Sciences, Vienna 

2 

Lenzing AG, Research and Development-Austria 

356 

357 

358 

P07-003 ANALYSIS OF SYNTHETIC PHOSPHODIESTERASE INHIBITORS IN COUNTERFEIT PRODUCTS BY DATA DIRECTED LC/MS/MS, 

ACCURATE MASS MS/MS AND LC-UV 

Jones M.D., Twohig M. 

Waters Corporation-USA 

P07-004 DEVELOPMENT OF A DIRECT COUPLED HIGH-TEMPERATURE HPLC FOR THE FAST DETERMINATION OF ENZYMATIC 

REGULATION IN HOUSE DUST COMPONENTS USING AN ENZYMATIC ASSAY IN COMBINATION WITH MASS SPECTROMETRY 

Oeste K. 1 , Portner C. 1 , Teutenberg T. 1 , Letzel T. 2 

1 


2 

TU München, Competence Pool Weihenstephan 

359 

360 

P07-005 ANALYSIS OF WAX ESTERS IN EDIBLE OILS BY AUTOMATED ON-LINE LC-GC COUPLING USING THE THROUGH OVEN TRANSFER 

ADSORPTION DESORPTION (TOTAD) INTERFACE 

Aragon A., Toledano R.M., Cortés J.M., Villén J., Vázquez A. 


P07-006 AUTOMATED ANALYSIS OF WAX ESTER IN OLIVE BY ON-LINE NPLC-GC USING THE TOTAD INTERFACE: APPLICATION TO THE 

STUDY OF THE WAX ESTER CONTENT IN MONOVARIETY OLIVE OILS 

Lozano Y., Cortés J.M., Toledano R.M., Aragon A., Vázquez A., Villén J. 


P07-007 ANALYSIS OF FULLERENE NANOMATERIALS BY LIQUID CHROMATOGRAPHY/ATMOSPHERIC PRESSURE IONIZATION- 

ENHANCED RESOLUTION TANDEM MASS SPECTROMETRY 

Núñez O. 1 , Gallart-Ayala H. 1 , Martins C.P.B. 2 , Moyano E. 1 , Galceran M.T. 1 

1 


2 


P07-008 UHPLC-MS/MS FOR THE ANALYSIS OF PHOTOINITIATORS IN PACKAGED FOOD: EVALUATION OF API SOURCES 

Gallart-Ayala H. 1 , Núñez O. 1 , Martins C.P.B. 2 , Moyano E. 1 , Galceran M.T. 1 

1 


2 


361 

362 

363 

364 


29


P07-009 STABILITY-INDICATING ANALYSIS OF THE ACETYLCHOLINESTERASE INHIBITORS (-) HUPERZINE A, TACRINE AND GALANTAMINE 

Marques L.A., Maada I., Giera M., Kool J., de Kanter F., Lingeman H., Niessen W.M.A., Irth H. 


P07-010 THE USE OF RAPID AND PRECISE ELUTION PLATE AS EXTRACTION STEP FOLLOWED AS A PART OF HYPHENATED 

EXTRACTION METHOD BY HIG 

Snoblova M. 1 , Plaza M. 2 , Lojkova L. 1 , Valentova E. 1 , Vlcek J. 1 , Klejdus B. 1 

1 

Mendel University in Brno 

2 


P07-011 SOLID PHASE MICRO EXTRACTION (SPME) OF OPIATES FROM URINE COUPLING WITH DESI-MS/MS DETECTION 

Kennedy J.H. 1 , Aurand C. 2 , Shirey R. 2 , Bell D.S. 2 , Wiseman J.M. 1 , Laughlin B. 1 , Sweeney B. 3, Gutierrez P. 4 , Ferrari R. 5 

1 

Prosolia, Inc, Research & Development-USA 

2 


3 

AIT Laboratories, Research & Development-USA 

4 


5 


P07-012 SIMULTANEOUS ANALYSIS OF CHLOROPHENOLS, NITROPHENOLS, ALKYLPHENOLS AND CRESOLS IN AGRICULTURAL SOILS BY 

GC-QQQ-MS/MS APPLYING A QUECHERS-BASED EXTRACTION APPROACH 

Padilla-Sánchez J.A., Plaza-Bolaños P., Romero-Gonzalez R., Garrido-Frenich A., Martínez Vidal J.L. 


P07-013 FAST AND SENSITIVE METHOD FOR THE ANALYSIS OF ESTROGENS IN WATER 

Lucci P., Núñez O., Moyano E., Galceran M.T. 


365 

366 

367 

368 

369 

P08 COLUMN TECHNOLOGY 

P08-001 MICROBORE COLUMNS: A CONTRIBUTION TO GREEN CHEMISTRY 

Espuelas C. 1 , Ruth K. 2 , Hartmann T. 2 , Wille A. 2 

1 

Gomensoro S.A., Ion Chromatography-Spain 

2 

Metrohm International Headquarters-Switzerland 

P08-002 ON THE DEVIATIONS IN RETENTION TIMES AT INCREASING FLOW-RATE WITH CHROMOLITH COLUMNS 

García-Álvarez-Coque M.C., Pous-Torres S., Torres-Lapasió J.R., Ruiz-Ángel M.J. 


P08-003 POLYETHYLENEIMINE-FUNCTIONALIZED METAL OXIDE MATERIALS FOR CAPILLARY LIQUID CHROMATOGRAPHY AND 

CAPILLARY ELECTROCHROMATOGRAPHY 

Bourdin D. 1 , Smått J-H. 2 , Sakeye M. 2 , Ruiz-Jiménez J. 1 , Baños-Pérez C. 1 , D’Orazio G. 3 , Kopperi M. 1 , Wiedmer S.K. 1 , Riekkola M-L. 1 , Fanali S. 3 

1 


2 

Åbo Akademi University 

3 

Institute of Chemical Methodologies, National Council of Research, Rome, Italy 

P08-004 EFFECT OF MONOMER MIXTURE COMPOSITION ON SEPARATION OF SMALL MOLECULES USING POLY(DIVINYLBENZENE-CO- 

ETHYLVINYLBENZENE-CO-2-HYDROXYETHYL METHACRYLATE) MONOLITHIC ROD COLUMNS 

Smirnov K.N., Dyatchkov I.A., Telnov M.V., Pirogov A.V., Shpigun O.A., 


P08-005 A DEVELOPMENT AND APPLICATION STUDY FOR ANION EXCHANGE+CATION EXCHANGE+NORMAL PHASE+REVERSED 

PHASE MULTI-MODE ODS COLUMN 

Yazawa I. 

Imtakt Corporation 

P08-006 COMPARISON OF FUSED CORE AND CONVENTIONAL PARTICLE HPLC COLUMNS FOR THE ANALYSIS OF ISOFLAVONES 

Manchón N. 1 , D’Arrigo M. 1 , García-Lafuente A. 1 , Villares A. 1 , Guillamón E. 1 , Martínez J.A. 2 , Rostagno M.A. 1 

1 

Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid 

2 

University of Navarra 

P08-007 A NEW RANGE OF HIGHLY RETENTIVE REVERSED-PHASE PACKINGS FOR LC AND LC/MS 

Faulkner W., Gartland J., Pereira L., Milton D. 


P08-008 EVALUATION OF TWO NEW IONIC LIQUIDS AS HIGH STABILITY AND SELECTIVE STATIONARY PHASES IN GAS CHROMATOGRAPHY 

González Álvarez J., Blanco Gomis D., Arias Abrodo P., Díaz Llorente D., Busto E., Ríos Lombardía N., Gotor Fernández V., Gutiérrez 

Álvarez M.D. 

University of Oviedo 

370 

371 

372 

373 

374 

375 

376 

377 

378 

30 



P08-009 USE OF SCANNING CONTACTLESS CONDUCTIVITY DETECTION (SC4D) FOR THE VISUALISATION OF STATIONARY PHASE 

GRADIENTS IN CAPILLARY POLYMERIC MONOLITHIC ROD COLUMNS 

Currivan S., Connolly d., Paull B. 


P08-010 GOLD NANO-PARTICLE MODIFIED MONOLITHIC CAPILLARY COLUMNS FOR USE IN SELECTIVE MICRO-EXTRACTION OF THIOL 

GROUP CONTAINING COMPOUNDS 

Danilevics U. 1 , Walsh Z. 1 , Collins D. 1 , Nesterenko E.P. 1 , Paull B. 1 , Macka M. 2 

1 


2 


P08-012 HIGH CAPACITY GOLD NANO-PARTICLE MODIFIED MONOLITHIC STATIONARY PHASES FOR FLOW-THROUGH CATALYSIS OF 

SELECTED REDOX REACTIONS 

Floris P., Connolly D., Alwael H., Paull B. 


P08-013 PREPARATION AND CHARACTERISATION OF C-60 FULLERENE-MODIFIED GRAPHITISED-CARBON MONOLITHS FOR 

CHROMATOGRAPHIC APPLICATIONS 

He X., Paull B. 


P08-015 CORE-SHELL STATIONARY PHASES USING ZIRCONIA CORES WITH POROUS NANODIAMOND SHELLS FOR REVERSED-PHASE HPLC 

Wiest L.A. 1 , Jensen D.S. 1 , Olsen R. 1 , Vail M.A. 2, Dadson A. 2 , Linford M.R. 1 

1 

Brigham Young University-Unites States 

2 

US Synthetic Corporation, Research and Development 

379 

380 

381 

382 

383 

P09 NANOTECHNOLOGY 

P09-001 ABSORPTION ENHANCEMENT ACHIEVED BY NANOSIZED ACTIVE PHARMACEUTICAL INGREDIENTS – PAMPA/HPLC EXPERIMENTS 

Rezacova A. 1 , Grunwaldova V. 1 , Oktabec Z. 1,2 , Kral V. 1,3 

1 

Zentiva k.s., Development-Czech Republic 

2 

Faculty of Pharmacy, Charles University 

3 

Institute of Chemical Technology 

P09-002 CAN TEMPERATURE PLAY A ROLE ON NANO-LC COLUMN PACKING? 

Capriotti F., Palma P., Leonardis I., Famiglini G., Cappiello A. 

Università di Urbino, DiGeoTeCA-Italy 

P09-003 GAS CHROMATOGRAPHY-MASS SPECTROMETRY DETERMINATION OF UV-FILTERS BY MAGNETIC ASSISTED CHEMICAL 

EXTRACTION WITH NANOPARTICLES IN ENVIRONMENTAL WATERS 

Román Falcó I.P. 1 , Chisvert A. 2 , Salvador A. 2 , Canals Hernández A. 1 

1 

Universidad de Alicante, Dpto. Química Analítica, Nutrición y Bromatología 

2 

Universidad de Valencia, Dpto. Química Analítca 

384 

385 

386 

387 

P10 POLYMERS 

P10-001 DETERMINATION OF HALOGENS BY COMBUSTION ION CHROMATOGRAPHY 

Espinosa M.1, Emmenegger C.2, Wille A.2 

1 Gomensoro S.A. 

2 Metrohm International Headquarters-Switzerland 

P10-002 CHARACTERIZATION AND DETERMINATION OF POLYVINYLALCOHOL BY NON-EQUILIBRIUM CAPILLARY ELECTROPHORESIS OF 

POLYMER-DYE EQUILIBRIUM MIXTURES 

Carrasco-Correa E.J., Beneito-Cambra M., Herrero-Martínez J.M., Ramis-Ramos G. 


P10-003 DETERMINATION OF FLUOROQUINOLONE ANTIMICROBIALS IN DRINKING AND AQUACULTURE WATER SAMPLES BY 

AUTOMATED ON-LINE 

Rodriguez E., Navarro-Villoslada F., Benito-Peña E., Moreno-Bondi M.C., Marazuela M.D. 


P10-004 CHARACTERIZATION OF CELLULOSE TREATED WITH ACETIC ACID VAPOUR –HAVING A LOOK INTO CLOSED OLD BOOKS 

Kostic M., Böhmdorfer S., Henniges U., Bogolitsyna A., Potthast A. 


P10-005 IDENTIFICATION OF SURFACTANTS IN MODERN PAINTS BY USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

Osete-Cortina L., Doménech-Carbó M.T., Silva M.F. 

Technical University of Valencia, Instituto de Restauración del Patrimonio 

388 

389 

390 

391 

392 

393 


31


P11 ENANTHIOSEPARATIONS 

P11-001 COMPARATIVE ENANTIOSEPARATIONS OF PHARMACEUTICALS IN CAPILLARY ELECTROCHROMATOGRAPHY ON 

POLYSACCHARIDE-BASED CHIRAL STATIONARY PHASES CONTAINING SELECTORS WITH OR WITHOUT CHLORINATED 

DERIVATIVES 

Hendrickx A. 1 , Mangelings D. 1 , Chankvetadze B. 2 , Vander Heyden Y. 1 

1 


2 

Tbilisi State University 

P11-002 OPTIMIZATION OF DIRECT CHIRAL LC FOR ENANTIOMERIC SEPARATION OF ARYLPHENOXYPROPIONIC AND SAFENER 

HERBICIDES USING EXPERIMENTAL DESIGN METHODOLOGIES 

Magro-Moral J., Guillén-Casla V., Rosales-Conrado N., Pérez-Arribas L.V., León-González M.E., Polo-Díez L.M. 


P11-003 SEPARATION OF CIS-BIFENTHRIN ENANTIOMERS BY CYCLODEXTRIN MODIFIED MICELLAR ELECTROKINETIC CHROMATOGRAPHY. 

QUANTITATIVE ANALYSIS IN A COMMERCIAL INSECTICIDE FORMULATION 

Pérez-Fernández V., García M.A., Marina M.L. 

University of Alcalá 

P11-004 CHIRAL SEPARATION OF METALAXYL AND BENALAXYL FUNGICIDES BY CD-EKC FOR THEIR SIMULTANEOUS DETERMINATION 

AND QUANTITATION WITH FOLPET IN COMMERCIAL FORMULATIONS. DETERMINATION OF ENANTIOMERIC IMPURITIES 



P11-005 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ENANTIOSEPARATION OF AMINONAPHTHOL ANALOGS ON 

POLYSACCHARIDE-BASED CHIRAL STATIONARY PHASES 

Peter A., Aranyi A., Ilisz I., Pataj Z., Szatmári I., Fülöp F. 

University of Szeged-Hungary 

P11-006 STEREOSELECTIVE DETERMINATION OF BUFURALOL, 1’-OXOBUFURALOL AND 1’-HYDROXYBUFURALOL IN RAT LIVER 

MICROSOMAL FRACTION USING HOLLOW FIBER LIQUID PHASE MICROEXTRACTION AND HPLC 

Barth T., Simões R.A., Calixto L.A., Okano L.T., Pupo, M.T., Bonato P. S. 

Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo 

P11-007 STEREOSELECTIVE HPLC DETERMINATION OF ISRADIPINE AND ITS MAIN METABOLITE IN RAT LIVER MICROSOMAL FRACTION 

USING HOLLOW-FIBER LIQUID-PHASE MICROEXTRACTION AS SAMPLE PREPARATION 

Simões R.A., Bonato P.S. 


P11-008 ENANTIOSELECTIVE ANALYSIS OF ZOPICLONE AND ITS METABOLITES BY LIQUID CHROMATOGRAPHY/TANDEM MASS 

SPECTROMETRY 

Tonon M.A., Jabor V.A.P., Bonato P.S. 

Faculty of Pharmaceutical Sciences of Ribeirão Preto 

P11-009 ANALYSIS OF CHIRAL COMPOUNDS USING ONLINE HYPHENATION OF MASS SPECTROMETRY AND ACHIRAL 

ELECTROPHORETIC SEPARATION 

Ranc V., Knob R., Petr J., Sevcik J., Maier V. 

Palacky University Olomouc 

P11-010 STUDY ON THE ENANTIOMERIC SEPARATION OF METHYL-SULFONYL METABOLITES OF PCBS 

Castro-Puyana M., Fernández M.A., González M.J., Gómara B. 


P11-011 DETERMINATION OF CHIRAL ALPHA-HYDROXYCARBOXYLIC ACID IN FOOD BY CAPILLARY ELECTROPHORESIS WITH 

INDIRECT UV DETECTION 

Maier V., Znaleziona J., Ranc V., Petr J., Sevcik J. 

Palacký University, Faculty of Science 

P11-012 REVERSAL OF ENANTIOMER MIGRATION ORDER USING POLYPYRROLE COATED CAPILLARIES 

Knob R., Znaleziona J., .Ginterova P., Ranc V., Petr J., Maier V., Sevcik J. 

Faculty of Science, Palacky University in Olomouc 

P11-013 DETERMINATION OF D,L-LACTIC ACID IN SERUM FROM DIABETIC PATIENTS BY CE-ESI-MS 

Znaleziona J., Chrastina J., Ranc V., Petr J., Ginterova P., Sevcik J., Maier V. 


P11-014 THE PILOT STUDY OF USING BOROMYCIN AS CHIRAL SELECTOR IN CAPILARY ELECTROPHORESIS AND DIRECT INJECTION 

MASS SPECTROMETRY – A COMPARATIVE STUDY 

Maier V., Ranc V., Svidrnoch M., Petr J., Sevcik J. 


394 

395 

396 

397 

398 

399 

400 

401 

402 

403 

404 

405 

406 

407 

408 

32 



P11-015 COMPARATIVE CHIRAL HPLC METHODS FOR β-BLOCKERS USING DIFFERENT TYPES OF CHIRAL STATIONARY PHASES IN 

NORMAL PHASE AND POLAR PHASE ELUTION MODE 

Morante-Zarcero S., Sierra I. 

E.S.C.E.T., Universidad Rey Juan Carlos 

P11-016 GAS CHROMATOGRAPHY FOR SEPARATIONS OF CHIRAL COMPOUNDS USED IN PHARMACEUTICAL DEVELOPMENT 

PROJECTS 

Laniewski K. 

AstraZeneca R&D, Pharmaceutical Development, Department of Analytical Science-Sweden 

409 

410 

P12 FAST SEPARATIONS 

P12-001 DETERMINATION OF 1,3-OCTANEDIOLS VIA 1,3-DIOXANES BY SOLID-PHASE MICROEXTRACTION AND HIGH-SPEED GAS 


Díaz Llorente D. 1 , Arias Abrodo P. 1 , Dapena de la Fuente E. 2 , Mangas Alonso J.J. 2 , Gutiérrez Álvarez M.D. 1 , Blanco Gomis D. 1 

1 


2 

SERIDA-Spain 

P12-002 MICRO COLUMN HPLC WITH DETECTION AT NANOSTRUCTURE 

Orinak A. 1 , Skantarova L. 2 , Orinakova R. 1 , Talian I. 2 , Fedorkova A. 2 

1 

University of P.J.Safarik in Kosice-Slovakia 

2 

Comenius University in Bratislava-Slovakia 

P12-003 DETERMINATION OF BENZIMIDAZOLE COMPOUNDS IN FOOD BY UHPLC-MS/MS 

Martínez-Villalba A., Moyano E., Galceran M.T. 


P12-004 ULTRA-FAST ANALYSIS OF ISOFLAVONES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING FUSED CORE 

COLUMN TECHNOLOGY 


1 


2 

Universidad de Navarra, Dpto. Fisiología y Nutrición 

411 

412 

413 

414 

415 

P12-005 MULTISYRINGE CHROMATOGRAPHIC SEPARATION (MSC) OF COPPER, CHROMIUM, AND NICKEL ON A DYNAMICALLY-COATED 

MONOLITHIC COLUMN WITH CHEMILUMINESCENCE DETECTION 

Diaz G. 1 , Horstkotte B. 2 , Maya F. 1 , Sanchez A.T. 2 , Duarte C.M. 2 , Cerdà V.1 

1 

University of the Balearic Islands 

2 

IMEDEA, Department of Global Change Research 

P12-006 VERY FAST AND EFFICIENT SIZE-BASED SEPARATIONS OF POLYMERS AT ULTRA-HIGH PRESSURES 

Uliyanchenko E. 1 , Schoenmakers P.J. 1 , van der Waal S. 2 

1 

University of Amsterdam, Analytical-Chemistry Group, Van ‘t Hoff Institute for Molecular Sciences (HIMS) 

2 

DSM Resolve 

P12-007 COMBINING UHPLC WITH ADVANCED CHROMATOGRAPHIC TECHNIQUES FOR SELECT FOOD & BEVERAGE APPLICATIONS 

Steiner F., Fehrenbach T., Schmidt C., McLeod F., Martin M.M. 

Dionex Corporation, Product Marketing-Germany 

416 

417 

418 

P13 NEW DETECTION METHODS 

P13-001 NEW ELECTROCHEMICAL DETECTOR FOR HPLC BASED IN SCREEN-PRINTED ELECTRODES: POLYPHENOLS ANALYSIS, ONE 

APLICATION 

Hevia D. 1 , Fanjul P. 2 , Hernandez D. 2 , Gómez-Cordovés C. 1 

1 

Instituto de Ciencia y Tecnología de los Alimentos y Nutrición (ICTAN)-CSIC 

2 

Dropsens S.L. 

P13-002 INVESTIGATION OF AN ENANTIOSELECTIVE SEPARATION PROCESS WITH SUSPENDED STATE HR/MAS NMR SPECTROSCOPY 

Yeman H., Albert K. 


419 

420 

421 


33


P14 SPECIATION ANALYSIS 

P14-001 THE ALTERATION OF FRACTIONATION OF TRACE ELEMENTS SPECIES DUE TO BREAD MAKING 

Mestek O., Kominkova J., Koplik R., Santrucek J. 

Institute of Chemical Technology Prague 

P14-002 DETERMINATION OF MERCURY SPECIES IN FOOD BY LIQUID CHROMATOGRAPHY - INDUCTIVELY COUPLED PLASMA MASS 

SPECTROMETRY 

Koplik R., Mestek O., Kominkova J. 


422 

423 

424 

P15 FOOD QUALITY AND SAFETY 

P15-001 VALIDATION OF A CONFIRMATORY METHOD FOR DETERMINATION OF THYREOSTATS IN PIG URINE BY LC-MS/MS 

Taka T., Baras M.C., Bet Z.F.C. 

Lanagro-SP/Ministry of Agriculture-Brazil 

P15-002 DETERMINATION OF POLYPHENOLS IN WINES BY LIQUID CHROMATOGRAPHY WITH SPECTROPHOTOMETRIC AND FLUORIMETRIC 

DETECTION. APPLICATION TO THE CHARACTERIZATION OF WINES 

Hernández Cassou S. 1 , Oliver R. 2 , Checa A. 1 , Saurina J. 1 

1 


2 


P15-003 GC-MS ANALYSIS OF CANNED FOOD PRODUCTS FOR BISPHENOL A 

Cao X-L. 

Health Canada, Food Research Division 

P15-004 UNCERTAINTY STUDY OF A MULTI-RESIDUE METHOD TO DETERMINE ORGANOCHLORINE PESTICIDES AND POLYCHLORINATED 

BIPHENYLS IN BOVINE FAT BY GAS CHROMATOGRAPHY - ELECTRON CAPTURE DETECTION 

Olivares I.R.B., Antunes Lopes F., Cordeiro Maia Saglioni E., Lopes Modro A., Hoffmann Kowalski C. 

National Laboratory of Ministry of Agriculture-Brazil 

P15-005 VOLATILE CHARACTERIZATION OF MONOFLORAL AND MULTIFLORAL TYPES OF HONEYBEE-COLLECTED POLLENS BY GC-MS. 

INFLUENCE OF INDUSTRIAL PROCESSING IN THEIR VOLATILE FLAVOR CONSTITUENTS 

Domínguez-Valhondo D. 1 , García-Parra J. 1 , Bohoyo-Gil D. 1 , Hernández M.T. 1 , Bernalte M.J. 2 , Ayuso M.C. 2 , González-Gómez D. 1 

1 

Insituto Tecnológico Agroalimentario (INTAEX) 

2 

University of Extremadura 

P15-006 ODOUR CHARACTERISATION OF DIFFERENT SPECIES/ORIGINS OF CURED VANILLA BEANS BY MEANS OF HS-SPME-GC-MS 

AND MS-BASED ELECTRONIC NOSE TECHNOLOGY 


Catholic University College Ghent 

P15-007 EXTRACTION OF SELECTED FLAVONOIDS FROM VARIOUS BERRIES BY PRESSURIZED SOLVENTS 

Hohnova B. 1 , Stavikova L. 1 , Karasek P. 1 , Vespalcova M. 2 

1 

Institute of Analytical Chemistry of the ASCR, v. v. i. 

2 

Brno University of Technology 

P15-008 EXTRACTION AND IDENTIFICATION OF FLAVONOIDS IN BRANCHES AND LEAVES OF DIFFERENT VARIETIES OF SAMBUCUS NIGRA 

Stavikova L. 1 , Grulichova H. 2 , Hohnova B. 1 , Karasek P. 1 , Vespalcova M. 2 

1 


2 


P15-009 CHARACTERIZATION OF NOVEL GALACTOOLIGOSACCHARIDES DERIVED FROM LACTULOSE 

Hernández-Hernández O. 1 , Moreno F.J. 2 , Montilla A. 2 , Clemente A. 3 , Olano A. 2 , Sanz M.L. 1 

1 

Instituto de Quimica Organica General (CSIC), Madrid 

2 

Instituto de Fermentaciones Industriales-CIAL (CSIC), Madrid 

3 

Estación Experimental del Zaidín (CSIC), Granada 

425 

426 

427 

428 

429 

430 

431 

432 

433 

434 

P15-010 DETERMINATION OF INOSITOLS AND OTHER LOW MOLECULAR WEIGHT CARBOHYDRATES IN DIFFERENT VEGETABLES 

EXTRACTS USING GC-MS 

Hernández-Hernández O., Ruiz-Aceituno L., Martínez-Castro I., Sanz M. L. 


P15-011 A NOVEL MULTIPLEX PCR-CGE-LIF METHOD FOR THE ANALYSIS OF GENETICALLY MODIFIED YEASTS IN WINE SAMPLES 

Leon C. 1 , García-Cañas V. 1 , Gonzalez R. 2 , Cifuentes A. 1 

1 


2 

Institute of wine and grapevine science (CSIC) 

435 

436 

34 



P15-012 SURVEY OF BISPHENOL A IN PLASTIC BOTTLES BY GAS CHROMATOGRAPHY TANDEM MASS SPECTROMETRY 

Mansilha C. 1 , Rocha S. 2 , Pinho C. 2 , Domingues V. 3 , Rebelo H. 1 , Gameiro P. 2 

1 

Instituto Nacional de Saúde Dr. Ricardo Jorge-Portugal 

2 

Requimte, Faculdade de Ciências Universidade Porto 

3 

Reqimte, Instituto Superior de Engenharia do Porto, Química 

P15-013 SEPARATION AND IDENTIFICATION OF TRITERPENES FROM PISTACIA LENTISCUS L. RESIN 

Nicholson T., Gradl M., Metzger M., Albert K. 

University of Tuebingen-Germany 

P15-014 DEVELOPMENT OF AN EXTRACTION AND PURIFICATION PROCEDURE FOR THE OBTAINMENT OF INOSITOL ENRICHED 

FRACTIONS FROM PINE NUTS 

Ruiz-Aceituno L., Rodríguez-Sánchez S. , Ramos L., Martínez-Castro I., Sanz M.L. 

Instituto de Química Orgánica General (CSIC), Madrid 

P15-015 FIRST ULTRA-PERFORMANCE LC BASED STRATEGY FOR PROFILING INTACT PROTEINS IN COMPLEX MATRICES. 

APPLICATION TO THE EVALUATION OF THE PERFORMANCE OF OLIVE (OLEA EUROPAEA L.) STONE PROTEINS FOR CULTIVAR 

FINGERPRINTING 

Esteve C. 1 , del Río C. 2 , Marina M.L. 1 , Concepción García M. 1 

1 


2 

IFAPA , Centro Alameda del Obispo 

P15-016 SEPARATION OF PROTEINS FROM OLIVE OIL BY CAPILLARY ELECTROPHORESIS. APPLICATION TO THE DIFFERENTIATION OF 

MONOVARIETAL OLIVE OILS 

Montealegre C., Marina M.L., García-Ruiz C. 


P15-017 A COMBINATION OF A SIMPLE EXTRACTION METHOD AND CAPILLARY ELECTROPHORESIS APPROACH FOR THE 

SEPARATION OF OLIVE PROTEINS 



P15-018 DEVELOPMENT OF A CLEAN-UP PROCEDURE TO EXTRACT PROTEINS FROM THE OLIVE PULP. APPLICATION OF OLIVE PULP 

PROTEINS AS GENETIC FINGERPRINTERS IN THE OLIVE CROP 

Esteve C. 1 , del Río C. 2 , Marina M.L. 1 , García M.C. 1 

1 


2 


P15-019 STUDY OF VOLATILES PRODUCED BY PSEUDOMONAS SPP. ISOLATED FROM CORK USING HS-SPME-GC-MS 

Godayol A., Trias R., Prat C., Bañeras L., Anticó E. 


P15-020 INSECTICIDES EXTRACTION FROM BANANA LEAVES USING A MODIFIED QUECHERS METHOD 

González-Curbelo M.A., Hernández-Borges J., Ravelo-Pérez L.M., Rodríguez-Delgado M.A. 

Universidad de La Laguna (ULL) 

P15-021 VALIDATION OF A GC-MS METHOD FOR THE SIMULTANEOUS DETERMINATION OF EIGTH TRICHOTHECENES IN BARLEY SAMPLES 

Ibáñez-Vea M., Lizarraga E., González-Peñas E. 


P15-022 VALIDATION OF A UHPLC-FLD METHOD FOR THE SIMULTANEOUS DETERMINATION OF AFLATOXINS, OCHRATOXIN A AND 

ZEARALENONE IN BREAKFAST CEREALS 

Ibáñez-Vea M., Martínez R., González-Peñas E., Lizarraga E. 


P15-023 ANALYSIS OF 3-MCPD ESTERS IN EDIBLE OILS USING LARGE VOLUME INJECTION COUPLED TO COMPREHENSIVE GC×GC–TOF MS 

de Koning S. 1 , Zelinková Z. 2,3 , Hrnčiřík K. 2 , Janssen H-G. 2,4 

1 


2 

Unilever Research and Development Vlaardingen-the Netherlands 

3 


4 


P15-024 DETAILED CIS/TRANS FATTY ACID ANALYSIS OF EDIBLE OILS AND FATS USING COMPREHENSIVE GC×GC–FI 

de Koning S. 1 , Steenbergen H. 2 , Janssen H-G. 2,3 

1 


2 


3 


437 

438 

439 

440 

441 

442 

443 

444 

445 

446 

447 

448 

449 


35


P15-025 USE OF LINEAR DISCRIMINANT ANALYSIS AND STEROL PROFILES ESTABLISHED BY HPLC-MS TO PREDICT THE GENETIC 

VARIETY OF EXTRA VIRGIN OLIVE OILS PRODUCED AT LA COMUNIDAD VALENCIANA, SPAIN 

Lerma-García M.J. 1 , Concha-Herrera V. 2 , Herrero-Martínez J.M. 1 , Simó-Alfonso E.F. 1 

1 


2 

Autonomous University of Zacatecas 

P15-026 DEVELOPMENT AND COMPARISON OF HPLC METHODS FOR THE ANALYSIS OF PREBIOTIC OLIGOSACCHARIDES 

Brokl M., Hernández-Hernández O., Soria A.C., Sanz M.L. 


P15-027 METHACRYLATE ESTER-BASED MONOLITHIC COLUMNS FOR NANO-LC SEPARATION OF TOCOPHEROLS AND TOCOTRIENOLS 

IN VEGETABLE OILS 

Lerma-García M.J. 1 , Cerretani L. 2 , Herrero-Martínez J.M. 1 , Bendini A. 2 , Simó-Alfonso E.F. 1 

1 


2 

University of Bologna 

P15-028 FEASIBILITY OF ELECTROMIGRATION AND GAS CHROMATOGRAPHIC TECHNIQUES FOR ASSESSING GLYCOXIDATION OF PROTEINS 

Amigo-Benavent M., Montilla A., del Castillo M.D. 

Instituto de Investigación en Ciencias de Alimentación , Bioactividad y Análisis de Alimentos 

P15-029 ANALYSIS OF SOYBEAN BOWMAN BIRK INHIBITOR BY CAPILLARY ELECTROPHORESIS 

Amigo-Benavent M. 1 , Bravo L. 2 , del Castillo M.D. 1 

1 


2 

Instituto de Ciencia y Tecnología de los Alimentos y Nutrición, Metabolismo y Nutrición 

450 

451 

452 

453 

454 

P15-030 USE OF TRIGLYCERIDE PROFILES ESTABLISHED BY HPLC WITH UV-VIS DETECTION TO PREDICT THE BOTANICAL ORIGIN OF 

VEGETABLE OILS 

Lerma-García M.J. 1 , Lusardi R. 2 , Chiavaro E. 2 , Cerretani L. 3 , Ramis-Ramos G. 1 , Simó-Alfonso E.F. 1 

1 


2 

University of Parma 

3 


P15-031 USE OF UPLC-MS TO DETERMINE STEROL CONTENTS IN VEGETABLE OILS FROM DIFFERENT BOTANICAL ORIGINS 

Lerma-García M.J. 1 , Simó-Alfonso E.F. 1 , Méndez A. 2 , Lliberia J.L. 2 , Herrero-Martínez J.M. 1 

1 


2 

Waters Cromatografía S.A 

P15-032 PREDICTION OF THE GENETIC VARIETY OF EXTRA VIRGIN OLIVE OILS FROM LA COMUNITAT VALENCIANA, SPAIN, BY USING 

STEROL PROFILES ESTABLISHED BY UPLC-MS 


1 


2 


P15-033 DETERMINATION OF 15+1 EU POLYCYCLIC AROMATIC HYDROCARBONS IN TOASTED CEREALS OF THE CANARY ISLANDS 

(“GOFIOS”) BY HPLC UTILIZING IONIC LIQUIDS AGGREGATES AS EFFECTIVE EXTRACTION SOLVENTS 

Germán-Hernández M., Pino V., Ayala J.H., Afonso A.M. 

University of La Laguna 

P15-034 USE OF DISPERSIVE LIQUID-LIQUID MICROEXTRACTION AS SAMPLE TREATMENT IN THE DETERMINATION OF CARBAMATES 

IN FRUIT JUICES BY SWEPING-MICELLAR ELECTROKINETIC CHROMATOGRAPHY 

Moreno-González D., García-Campaña A.M., Gámiz-Gracia L., Bosque-Sendra J.M. 


P15-035 ANALYSIS OF OLIGOSACCHARIDES IN BEER VIA HPLC USING DIFFERENT STATIONARY PHASES 

Salplachta J., Flodrova D., Bobalova J., Cmelik R., 


P15-036 CAPILLARY ELECTROPHORESIS OF FREE FATTY ACIDS BY INDIRECT UV DETECTION: APPLICATION TO THE CLASSIFICATION 

OF VEGETABLE OILS ACCORDING TO THEIR BOTANICAL ORIGIN 

Escrig-Doménech A., Lerma-García M.J., Simó-Alfonso E.F., Herrero-Martínez J.M. 


P15-037 ACRYLATE ESTER-BASED MONOLITHIC COLUMNS FOR CAPILLARY ELECTROCHROMATOGRAPHY SEPARATION OF 

TRIACYLGLYCEROLS IN VEGETABLE OILS 

Medina-Escrivà L., Vergara-Barberán M., Lerma-García M.J., Simó-Alfonso E.F., Herrero-Martínez J.M. 


455 

456 

457 

458 

459 

460 

461 

462 

36 



P15-038 ANALYSIS OF GLYCATED SODIUM CASEINATE PROTEINS BY MALDI-MS AND LC-ESI-MS 

Corzo-Martinez M. 1 , Galindo-Iranzo P. 2 , Lebrón-Aguilar R. 2 , Villamiel M. 1 , Moreno F.J. 1 

1 


2 

Institute of Physical Chemistry “Rocasolano” (CSIC), Madrid 

P15-039 STUDY OF THE CO-OCCURRENCE OF SIX OCHRATOXINS IN WINE 

Remiro R., González-Peñas E., Lizarraga E. 


P15-040 CZE ANALYSIS OF COFFEE EXTRACTS POSSESSING ANTIOXIDANT AND ANTIBACTERIAL CAPACITIES 

Amigo-Benavent M., Mingo E., Martínez-Rodríguez A., del Castillo M.D. 

Instituto de Investigación en Ciencias de Alimentación, Microbiología y Biotecnología de los Alimentos 

P15-041 HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF STILBENES (CIS/TRANS-RESVERATROL AND CIS/ 

TRANS-PICEID) IN HONEY 

Nozal MªJ. 1 , Martin MªT. 1 , Bernal J.L. 1 , Soto E. 1 , Bernal J. 2 

1 


2 

Institute of Industrial Fermentations(CSIC), Madrid 

463 

464 

465 

466 

P15-042 DETERMINATION OF TRYPTOPHAN, KYNURENINE, KYNURENIC AND XANTHURENIC ACIDS IN HONEY BY LIQUID 

CHROMATOGRAPHY (DAD, FLD AND APCI-MS) 

Nozal MªJ., Martin MªT., Toribio L., Soto E., Moncadas MªJ. 


P15-043 IDENTIFICATION OF BIOACTIVE PEPTIDES IN HYPOALLERGENIC INFANT MILK FORMULAS BY CAPILLARY 

ELECTROPHORESIS-MASS SPECTROMETRY 

Giménez E. , Català-Clariana S., Benavente F., Barbosa J., Sanz-Nebot V. 

University of Barcelona, Department of Analytical Chemistry. Nutrition and Food Safety Research Institute 

P15-044 INFLUENCE OF VARIOUS STATIONARY PHASES ON HPLC SEPARATION OF GLYCATED INTACT BARLEY PROTEINS 

Flodrova D., Smetalova D., Salplachta J., . Bobalova J. 


P15-045 CHARACTERIZATION OF ANTIOXIDANTS AND NEOANTIOXIDANTS PRESENT AFTER SUBCRITICAL WATER EXTRACTION OF 

ALGAE AND PLANTS 

Plaza M. 1 , Amigo-Benavent M. 1 , Ibáñez E. 1, del Castillo M.D. 1 , Herrero M. 2 

1 


2 


P15-046 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) ANALYSIS OF FREE AMINO ACIDS AND BIOGENIC AMINES IN 

DAIRY PRODUCTS 

Mayer H.K., Fiechter G., Fischer E. 

BOKU – University of Natural Resources and Applied Life Sciences Vienna 

P15-047 EFFECTS OF HIGH PRESSURE PROCESSING ON FATTY ACID PROFILE IN VEGETABLES BEVERAGES 

Barba F.J., Esteve M.J., Frigola A. 


P15-048 CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITY OF PRESSURIZED LIQUID EXTRACTS FROM ROMANIAN AROMATIC 

PLANTS 

Miron T.L. 1 , Plaza M. 2 , Ibáñez E. 2 , Herrero M. 3 

1 

Faculty of Food Science and Engineering/”Dunarea de Jos” University, Department of Bioengineering–Romania 

2 


3 


P15-050 LC-MS; PROFILING AND QUALITY CONFIRMATION OF TEA SAMPLES FROM CAMELLIA SINENSIS & ASPALATHUS LINEARIS 

Tahrani A. 

University Heidelberg 

P15-051 SPME-GAS CHROMATOGRAPHY FOR LIPID OXIDATION EVALUATION DURING SHELF-LIFE OF INFANT FORMULAS 

García-Llatas G. 1 , Alegría A. 1 , Barberá R. 1 , Vasallo I. 2 , Lagarda M.J. 1 

1 


2 

Hero Institute for Infant Nutrition 

P15-052 SENSITIVE METHOD FOR DETERMINATION OF MARINE BIOTOXINS IN FEEDING BIVALVES MOLLUSCS BY ULTRA-PERFORMANCE 

LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY 

Roca M., Carbonell E., Castillo M., Mancebo M.T. 

Centro Superior de Investigación en Salud Pública-Valencia 

467 

468 

469 

470 

471 

472 

473 

474 

475 

476 


37


P15-053 DETERMINATION OF MELATONIN IN WINE AND PLANT EXTRACTS BY CAPILLARY ELECTROCHROMATOGRAPHY WITH 

IMMOBILIZED CARBOXYLIC MULTI-WALLED CARBON NANOTUBES AS STATIONARY PHASE 

Stege P.W. 1 , Sombra L.L. 1 , Wiedmer S.K. 2 , Messina G.A. 1 , Silva M.F. 3 

1 

University of San Luis. 

2 


3 

National University of Cuyo 

P15-054 LIQUID CHROMATOGRAPHY FOR THE SCREENING OF THIOURACYLS AND CORTICOSTEROIDS IN FARM ANIMALS 

Reig M. 1 , Toldrá F. 2 

1 

Instituto de Ingeniería de Alimentos para el Desarrollo (UPV), Technical University of Valencia 

2 

Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Valencia 

P15-055 DETERMINATION OF PESTICIDE RESIDUES IN ANIMAL FAT USING GAS CHROMATOGRAPHY 

Castillo M., Gonzalez C., Miralles A. 


P15-056 DETERMINATION OF POLYPHENOLS IN WINE BY CAPILLARY ZONE ELECTROPHORESIS 

Franquet H., Núñez O., Saurina X., Hernández S., Puignou L. 


P15-057 FATTY ACID CONTENTS IN FRESH AND PROCESSED FISH CONSUMMED IN VALENCIA (SPAIN) 

Ubillús F. 1 , Lagarda M.J. 2 , Farré R. 2 

1 

University of Piura (Perú) 

2 


P15-058 EFFECT OF PH ON CHROMATOGRAPHIC RETENTION OF MEAT POLAR COMPOUNDS USING FOUR DIFFERENT HILIC 

STATIONARY PHASES 

Aristoy M-C. 1 , Mora L. 1 , Toldrá F. 1 , Reig M. 2 

1 

Instituto de Agroquímica y Tecnología de Alimentos 

2 


P15-059 EVALUATION OF PHYTOESTROGEN CONTENT OF SOY-BASED NUTRITIONAL SUPPLEMENTS VIA ULTRA PERFORMANCE 

LIQUID CHROMATOGRAPHY 

Fiechter G., Raba B., Jungmayr A., Mayer H.K. 

BOKU–University of Natural Resources and Applied Life Sciences Vienna 

P15-060 DETERMINATION OF PESTICIDE RESIDUES IN APPLE BY GC/MS AND LC/MS 

Banic Simicic J., Stankov V., Marošanovic B. 

SP Laboratory, Instrumental analysis-Serbia 

P15-061 “SOYMILK” IN RELATION TO COW MILK 

Raba B., Fiechter G., Schreiner M., Mayer H.K. 


P15-062 RAPID HPLC METHOD FOR THE SCREENING OF CARBADOX IN FEED AND MEAT 

Batlle N. 1 , Reig M. 2 , Aristoy M-C. 1 , Toldrá F. 1 

1 


2 


P15-063 DETERMINATION OF COCCIDIOSTATS IN MILK BY LC-MS-MS 

Nász S., Eke Z. 


P15-064 EXTRACTION OF BIOACTIVE COMPOUNDS FROM BY-PRODUCTS OF THE OLIVE OIL INDUSTRY 

Temirzoda T.N. 1 , Plaza M. 1 , Segura-Carretero A. 2 , Herrero M. 3 , Ibáñez E. 4 

1 

Instituto de Fermentaciones Industriales-CSIC-Spain 

2 


3 


P15-065 EFFECT OF SEVERAL DOPANTS ON THE DETERMINATION OF CAROTENOIDS BY LIQUID CHROMATOGRAPHY/ ATMOSPHERIC- 

PRESSURE PHOTOIONIZATION/MASS SPECTROMETRY 

Rivera S., Vilaró F., Canela R. 

University of Lleida 

P15-066 A GC-MS METHOD FOR THE EVALUATION OF 3-DEOXYGLUCOSONE AND GLUCOSONE CONTENTS IN STORED HONEYS AND 

ARTISANAL MUSTS SYRUPS 

Ruiz-Matute A.I. 1 , Hernández-Hernández O. 1 , Castro-Vázquez L. 2 , Sanz M.L. 1 , Martínez-Castro I. 1 

1 

Instituto de Quimica Organica General(CSIC), Madrid 

2 

University of Castilla La Mancha 

477 

478 

479 

480 

481 

482 

483 

484 

485 

486 

488 

489 

490 

491 

38 



P15-067 LC-DAD-ESI-TOF MS METHOD FOR THE DETERMINATION OF PHENOLIC METABOLITES AND SOME OTHER POLAR 

CONSTITUENTS FROM AVOCADO (PERSEA AMERICANA) 

Hurtado-Fernández E., Carrasco-Pancorbo A., Fernández-Gutiérrez A. 


P15-068 PROFILING PHENOLIC ACIDS FROM AVOCADO (PERSEA AMERICANA) WITH A SENSITIVE CE-UV METHOD 



P15-069 DEVELOPMENT AND OPTIMIZATION OF A METHOD FOR DEOXYNIVALENOL DETERMINATION IN PAPRIKA USING LIQUID 

CHROMATOGRAPHY-TRIPLE QUADRUPOLE-MASS SPECTROMETRY 

Valle-Algarra F.M., Mateo E.M., Mateo R., Gimeno-Adelantado J.V., Jimenez M. 


P15-070 DETERMINATION OF HT-2 TOXIN AND T-2 TOXIN IN PAPRIKA BY CAPILLARY GAS CHROMATOGRAPHY WITH ELECTRON 

CAPTURE DETECTION 



P15-071 NITRATES IN BABY FOOD WITH FRUITS OR VEGETABLES BY ION CHROMATOGRAPHY 

Stankov V., Marosanovic B., Bognar A. 

SP Laboratory, Instrumental analysis Dpt-Serbia 

P15-072 MINERAL CONTENTS OF THE RESTAURANT FOODS AND PROCESSED FOODS IN SOUTH KOREA 

Kang S-H., Kim K-C., Kim J-B., Kim D-H., Aum H-S., Jin S-N., Yoon M-H., Lee J-B., Park Y-B. 

Gyeonggi-Do Institute of Health & Environment, Health Research Planning Team 

P15-073 TOTAL SUGAR CONTENTS OF THE RESTAURANT FOODS AND PROCESSED FOODS IN SOUTH KOREA 

Yong-Bae P., Suk-Ho K., Ki-Cheol K., Jung-Boem K., Dae-Hwan K., Hee-Suk A., Sun-Nam J., Mi-Hye Y., Jong-Bok L., 


P15-074 EFFECTS OF ELECTRON-BEAM IRRADIATION ON THE FORMATION OF CHOLESTEROL OXIDE PRODUCTS IN RAW PORK LOIN 

BY GC-MS. 

Lozada-Castro J.J. 1 , Santos-Delgado MªJ. 2 , Polo-Díez L.M. 2 

1 

Nariño University, Chemistry Sciences-Colombia 

2 


P15-076 DETERMINATION OF TRIGONELLINE IN SEEDS AND VEGETABLE OILS BY CAPILLARY ELECTROPHORESIS AS A NOVEL 

MARKER FOR THE DETECTION OF ADULTERATIONS IN OLIVE OILS 

Sánchez-Hernández L. 1 , Puchalska P. 2 , García-Ruiz C. 1 , Crego A.L. 1 , Marina M.L. 1 

1 

University of Alcala 

2 

Warsaw University, Department of Chemistry 

492 

493 

494 

495 

496 

497 

498 

499 

500 

P15-077 A CE-ITMS2 METHODOLOGY FOR THE DETERMINATION OF NON-PROTEIN AMINO ACIDS IN VEGETABLE OILS AS NOVEL 

MARKERS FOR THE DETECTION OF ADULTERATIONS IN OLIVE OILS 

Sánchez-Hernández L., Crego A.L., Marina M.L. 


P15-078 CHARACTERIZATION OF BIRCH EXTRACTS BY CHROMATOGRAPHIC AND MASS SPECTROMETRIC TECHNIQUES 

Soria A.C., Rodríguez-Sánchez S., Sanz M.L., Sanz J., Martínez-Castro I. 

Inst. Química Orgánica General (CSIC), Madrid 

P15-079 DETERMINATION OF ANTICARCINOGENIC PROTEINS IN SOYBEAN 

Anta-Saiz L., Marina MªL., García MªC. 


P15-080 DETERMINATION OF FLUOROQUINOLONES OF VETERINARY USE IN FOODS BY CAPILLARY HPLC WITH LASER INDUCED 

FLUORESCENCE DETECTION. COMPARISON OF METHODOLOGIES FOR SAMPLE TREATMENT 

Lombardo-Agüí M., Hernández-Mesa M., Gámiz-Gracia L., Cruces-Blanco C., García-Campaña A.M. 


P15-081 DETERMINATION OF SOYMETIDE IN DIFFERENT SOYBEAN DERIVED FOODS BY CAPILLARY-HPLC 

Domínguez-Vega E. 1 , Kotkowska O. 2 , García MªC. 1 , Crego A.L. 1 , Marina MªL. 1 

1 


2 

University of Warsaw 

501 

502 

503 

504 

505 


39


P15-082 HIGH INTENSITY ULTRASONIC ASSISTED ENZYMATIC DIGESTION AND CAPILLARY-HPLC FOR THE PEPTIDE MAPPING OF 

SOYBEAN PROTEINS 

Domínguez-Vega E., García MªC., Crego A.L., Marina MªL. 


P15-083 DETERMINATION OF CHLORAMPHENICOL RELATED COMPOUNDS IN FOOD MATRICES BY FAST LIQUID CHROMATOGRAPHY 

TANDEM MASS SPECTROMETRY 

Alechaga E., Moyano E., Galceran M.T. 


P15-084 PHENOLIC COMPOUNDS IN WHEAT CULTIVAR GRAINS 

Rodríguez Rodríguez E. 1 , Díaz Romero C. 1 , Afonso Morales D. 2 , Hernández Rodríguez L. 1 

1 


2 

Exmo. Cabildo Insular de Tenerife, Agricultural Biodiversity Conservation Center from Tenerife 

P15-085 EVALUATION OF RELEVANT TOF-MS PARAMETERS IN LC-MS SCREENING FULL SCAN METHODS FOR PESTICIDE RESIDUE ANALYSIS 

Mezcua M., Malato O., Lozano A., Agüera A., Fernandez-Alba A.R. 


P15-086 LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY FOR PERFORMING CONFIRMATORY ANALYSIS OF ANTBACTERILAS 

IN ANIMAL-FOOD PRODUCTS 

Blasco C., Masia A., Morillas F., Pico Y. 


P15-087 CHARACTERIZATION OF 4-O-β-D- GLUCOSIDE OF P-COUMARIC ACID IN MUSTS OF. CV ALBARIñO (VITIS VINíFERA L) 

Zamuz S., Vilanova M., Masa A. 

Mision Bioloxica de Galicia (CSIC) 

P15-088 RAPID METHODOLOGY FOR THE DETECTION OF OLIVE OIL ADULTERATIONS WITH SEED OILS USING FLOW INJECTION 

ELECTROSPRAY IONIZATION WITH TRIPLE QUADRUPOLE MASS SPECTROMETRY 

Sánchez-Hernández L., Castro-Rubio F., Nozal L., Novella J.L., Marina MªL., Crego A.L. 


P15-089 DETERMINATION OF WATER-SOLUBLE VITAMINS IN HONEY USING TWO RP-HPLCMETHODS: WITHOUT AND WITH CTAB IN 

THE MOBILE PHASE 

León Ruiz V. 1 , Vera S. 2 , González-Porto A.V. 1 , San Andrés M.P. 2 

1 

Junta de Comunidades de Castilla-La Mancha, Centro Agrario de Marchamalo 

2 


P15-090 SAMPLE PREPARATION METHOD DEVELOPMENT OF MELAMINE AND CYANURIC ACID IN SOME STOCK FARM PRODUCTS FOR 

THE ISOTOPE DILUTION GC-MS 

OH CH. 1 , Kim SH. 1 , Cheon SH. 1 , Yoon JE. 1 , Hahm JH. 1 , Lee HJ. 1 , Roh JS. 2 , Kim KS. 3 , Park JW. 4 , Woon JH. 4 

1 

Semyung University-South Korea 

2 

HAITAI Confectionery & Foods Co., Ltd.-Safety Guarantee Institute-South Korea 

3 

Chosun University, Dept. of Food & Nutrition-South Korea 

4 

National Veterinary Research & Quarnatine Service, NVRQS-South Korea 

P15-091 QUALITY INDICATORS IN CARROTS BLANCHED BY ULTRASOUND TECHNIQUES 

Gamboa-Santos J. 1 , Montilla A. 1 , Soria A.C. 2 , Villamiel M. 1 

1 

Instituto de Investigación en Ciencias de Alimentación (CIAL) 

2 

Instituto de Química Orgánica General (IQOG), Madrid 

P15-092 PRODUCTION OF AFLATOXINS BY ASPERGILLUS PARASITICUS CECT 2681 ON RICE STERILIZED BY TWO DIFFERENT METHODS 

Cuadrench-Tripiana A., Navajas H., Agut M., Comellas L. 

Ramón Llull University, Barcelona 

P15-093 GCXGC-ITD-MS CHIRAL ANALYSIS OF FLAVOURS 

Guadayol M. 1 , Vendrell E. 1 , Viscasillas C. 1 , Caixach J. 2 , Collgrós F. 1 

1 

Dallant, S.A., Functional Food Research Department 

2 

IDÆA-CSIC, Mass Spectrometry Laboratory, Barcelona 

506 

507 

508 

509 

510 

511 

512 

513 

514 

515 

516 

517 

P15-094 OBTENTION AND CHARACTERIZATION OF GLYCOSYLATED DERIVATIVES OF LOW MOLECULAR WEIGHT CHITOSAN THROUGH 

AMIDE FORMATION 

Ruiz-Matute A.I., Cardelle-Cobas A., Montilla A., Olano A., Corzo N. 

Instituto de Investigación en Ciencias de Alimentación CIAL (CSIC), Madrid 

518 

40 



P15-095 FORMATION OF CHITOSAN DERIVATIVES BY REDUCTIVE ALKYLATION WITH MODIFIED FUNCTIONAL PROPERTIES 

Cardelle-Cobas A., Ruiz-Matute A.I., García-Bermejo A.B., Montilla A., Corzo N., Olano A. 


P15-096 USE OF DIFFERENT ANALYTICAL TECHNIQUES TO EVALUATE CHITOSAN-CARBOHYDRATE COMPLEXES FORMATION VIA 

MAILLARD REACTION 

García-Bermejo A.B., Cardelle-Cobas A., Ruiz-Matute A.I., Olano A., Corzo N., 


P15-097 SEPARATION OF PROANTHOCYANIDINS FROM FOOD SUPPLEMENTS USING DIFFERENT HPLC COLUMNS 

Smrke S., Vovk I., Simonovska B. 

National Institute of Chemistry-Slovenia 

P15-098 TLC-MS METHOD FOR DETECTION OF NEOXANTHIN, VIOLAXANTHIN, LUTEIN AND BETA-CAROTENE 

Cernelic K. 1,2 , Simonovska B. 2 , Vovk I. 1,2 , Albreht A. 2 

1 

EN-FIST Centre of Excellence, Ljubljana 

2 

National Institute of Chemistry, Laboratory for Food Chemistry-Slovenia 

P15-099 TLC-MS DETECTION OF LECITHIN AND PROANTHOCYANIDINS IN CHOCOLATE 

Glavnik V., Simonovska B., Vovk I., Albreht A. 


P15-100 RAPID ANALYSIS OF 5-NITROIMIDAZOLES AND THEIR HYDROXY METABOLITES IN ANIMAL TISSUE BY LC-MS/MS 

León N., Igualada C., Moragues F., Roca M. 

Conselleria Sanitat, Public Health Research Center (CSISP), Valencia 

P15-101 DETERMINATION OF PBDES IN FISH BY GAS CHROMATOGAPHY-TRIPLE QUADRUPOLE MASS SPECTROMETRY AND 

ESTIMATION OF THE DIETARY INTAKE IN THE POPULATION OF VALENCIA (SPAIN) 

Pardo O. 1 , Beser M.I. 2 , Yusa V. 2 , Beltran J. 3 

1 

CSISP, Food Safety 

2 

CSISP, Public Health Laboratory 

3 

Univesity Jaume I, Castelló de la Plana 

P15-102 DETERMINATION OF OCHRATOXIN A BY CAPILLARY HPLC WITH LASER INDUCED FLUORESCENCE DETECTION USING 

DISPERSIVE LIQUID-LIQUID MICROEXTRATION 

Arroyo-Manzanares N., Gámiz-Gracia L., García-Campaña A.M. 


P15-103 APLICATION OF PRE-COLUMN DERIVATIZATION WITH 2,3-NAPHTALENEDIALDEHYDE AND RP-HPLC-FL FOR THE ANALYSIS 

OF GLUTATHIONE AND ITS PRECURSOR G-GLUTAMYL-CYSTEINE IN WINES AND MODEL WINES SUPPLEMENTED WITH 

OENOLOGICAL INACTIVE YEAST PREPARATIONS 

Andujar-Ortiz I., Rodríguez-Bencomo J.J., Moreno-Arribas, M.V., Del Pozo-Bayón M.A. 

Instituto de Investigación en Ciencias de la Alimentación (CIAL) (CSIC-UAM), Biotecnología y Microbiología 

519 

520 

521 

522 

523 

524 

525 

526 

527 

P16 CLINICS AND PHARMACEUTICS 

P16-001 SUITABILITY OF MICELLAR LIQUID CHROMATOGRAPHY FOR DOPING CONTROL 


1 


2 


P16-002 DETERMINATION OF ETHOXYDOL IN PLASMA BY MICROEMULSION LIQUID CHROMATOGRAPHY 

Pirogov A.V., Pashkova E.B., Shpigun O.A., 


P16-003 ADME(T) PARAMETERS OF FUSED MANNICH KETONE AND ISOCHROMANONE MOLECULAR LIBRARIES COLLECTED BY 

SEPARATION METHODS 

Huszar M. 1 , Varga A. 1 , Horvath A. 1 , Vantus T. 1 , Lorand T. 2 , Agocs A. 2 , Keri G. 1 , Idei M. 1 

1 

Semmelweis University, Hungary 

2 


P16-004 MULTIMARKERS SCREENING OF VARIOUS BODY FLUIDS FOR MONITORING OXIDATIVE STRESS DISEASE 

Syslova K. 1 , Kacer P. 1 , Kuzma M. 2 , Vlckova S. 3 , Lebedova J. 3 , Fenclova Z. 3 , Pelclova D. 3 

1 

Institute of Chemical Technology-ICT Prague-Czech Republic 

2 

Institute of Microbiology, Laboratory of Molecular Structure Characterization-Czech Republic 

3 

1st Medical Faculty, Charles University-Czech Republic 

528 

529 

530 

531 

532 


41


P16-005 DEVELOPMENT AND VALIDATION OF A RAPID HPLC METHOD FOR RELATED SUBSTANCES OF LASW1338 IN A FUSED-CORE 

COLUMN. IMPORTANCE OF THE GRADIENT DWELL VOLUME 

Carrera F., Serra C., Julià M., Ebri I., Dulsat J.F. 

Laboratorios Almirall, Analysis R&D 

P16-006 ADJUSTING SELECTIVITY WITH COLUMN CHEMISTRY IN LC/MS 

Pereira L., Faulkner W., Barattini V., Milton D. 

Thermo Fisher Scientific, Chromatography Consumables & Speciality Products 

P16-007 SELECTIVE SOLID PHASE EXTRACTION OF COCAINE FROM BIOLOGICAL SAMPLES USING MOLECULARLY IMPRINTED POLYMERS 


UMR PECSA 7195 CNRS - UPMC - ESPCI Paris Tech 

P16-008 HILIC CHROMATOGRAPHY A TOOL FOR PHARMACEUTICAL QUALITY CONTROL OF POLAR COMPOUNDS 

Rupérez F.J., Fernández-Fidalgo C., Martínez-Alcázar M.P., Barbas C., García A. 

Universidad CEU-San Pablo, CEMBIO 

P16-009 DETERMINATION OF ACIDITY CONSTANTS BY THE CE INTERNAL STANDARD METHOD: ACIDIC INTERNAL STANDARDS 

Cabot J.M., Fuguet E., Ràfols C., Bosch, E., Rosés, M., 

University of Barcelona, Química Analítica 

P16-010 CHARACTERIZATION OF VACCINE ADJUVANT COMPONENTS BY MS AND HPLC-MS 

Cotte J.F., Sonnery S., Martial F., Dubayle J., Dalençon F., Talaga P., Haensler J., Adam O. 

Sanofi Pasteur, Research Department 

P16-011 SIMULTANEOUS DETERMINATION OF DEXPHANTENOL, MEPYRAMINE MALEATE AND LIDOCAINE HYDROCHLORIDE IN 

COMBINED PHARMACEUTICAL GELS BY CAPILLARY ELECTROPHORESIS 

Basmakci G., Satana H.E., Yarimkaya S., Ertas N., Goger N. 

Gazi University, Analytical Chemistry 

P16-012 ADVANCES IN STATIONARY PHASE CHEMISTRY FOR LC METHODS DEVELOPMENT 


Waters Corporation, Europe 

P16-013 HYDROPHYLIC LIPOPHYLIC INTERACTION CHROMATOGRAPHY (HILIC) OF CITICOLINE USING A SILICA COLUMN 

Guermouche S. 

USTHB 

P16-014 ENDOCRINE DISRUPTOR CHEMICALS (EDCS) IN BIOMEDICAL DEVICES: GAS CHROMATOGRAPHIC-MASS SPECTROMETRIC 

METHOD FOR THE DETERMINATION OF BISPHENOL A AND PHTHALATE ESTERS IN NASOGASTRIC TUBES AND FEEDING 

SYRINGES 

Vela F. 1 , Ballesteros O. 1 , Zafra-Gomez A. 1 , Jiménez-Díaz I. 1 , Navalón A. 1 , Fernández M.F. 1,2 , Olea N. 1,2 , Vílchez J.L. 1 

1 


2 

San Cecilio University Hospital 

P16-015 DETERMINATION OF VITAMINS A-ACETATE, D2 A E-ACETATE IN OINTMENT SAMPLES BY HPLC USING MONOLITHIC COLUMN 

Zakova P., Sklenarova H., Solich P. 


P16-016 COMBINATION OF MONOLITHS AND MODERN TECHNOLOGIES FOR FAST HPLC ANALYSIS IN CLINICAL RESEARCH 

Krcmova L. 1 , Solichova D. 1 , Kasparova M. 1 , Plisek J. 1 , Melichar B. 1,2 , Sobotka L. 1 , Solich P. 3 

1 

Teaching Hospital-Czech Republic 

2 

Palacký University Medical School 

3 

Faculty of Pharmacy-Czech Republic 

P16-017 TROUBLESHOOTING OF SIMULTANEOUS DETERMINATION OF NEOPTERIN, CREATININE, KYNURENINE AND TRYPTOPHAN IN 

VARIOUS HUMAN BIOLOGICAL FLUIDS 


1 


2 


3 


533 

534 

535 

536 

537 

538 

539 

540 

541 

542 

543 

544 

545 

P16-018 INVESTIGATIONS OF THE INTERACTIONS BETWEEN METALS AND IODINE IN PATHOLOGICAL AND HEALTHY HUMAN THYROID 

GLANDS USING ION CHROMATOGRAPHY 

Blazewicz A., Orlicz-Szczesna G., Randhawa R., Dolliver W., Sivsammye S., Deol A. 

Medical University of Lublin 

546 

42 



P16-019 CAPILLARY ELECTROPHORETIC DETERMINATION OF TIMOLOL MALEATE AND DORZOLAMIDE HCL IN EYE DROPS 

Caglayan M.G., Palabiyik I.M., Onur F. 

Ankara University, Faculty of Pharmacy 

P16-020 INVESTIGATION OF THE INTERACTION OF MERCUROCHROME® CONSTITUENTS WITH PROTEINS USING LIQUID 

CHROMATOGRAPHY/MASS SPECTROMETRY 

Wilken A. 

Institute for Inorganic and Analytical Chemistry 

P16-021 A QBD WITH DESIGN OF EXPERIMENTS APPROACH TO THE DEVELOPMENT OF A CHROMATOGRAPHIC METHOD FOR THE 

SEPARATION OF IMPURITIES IN VANCOMYCIN 

Plankeele J.M. 

Waters European Headquarters, UPLC 

P16-022 MULTIVARIATE OPTIMIZATION OF THE EXPERIMENTAL CONDITIONS IN HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC 

METHOD USING RESPONSE SURFACE METHODOLOGY 

Cigdem Aybaba, Ismail Murat Palabiyik, Mehmet Gokhan Caglayan, Feyyaz Onur 


P16-023 MOLECULARLY IMPRINTED SOLID PHASE EXTRACTION COUPLED TO MICELLAR ELECTROKINETIC CHROMATOGRAPHY FOR 

THE DETERMINATION OF DIGOXIN AND DIGITOXIN IN CLINICAL SAMPLES 

Guijarro M. 1 , Paniagua G. 2 , Fernández P. 2 , Crego A.L. 1 , Marina M.L. 1 

1 


2 

National University of Distance Education 

547 

548 

549 

550 

551 

P16-024 STUDY OF THE INTERACTION OF EPHEDRINE ENANTIOMERS WITH NATIVE CYCLODEXTRINS BY CAPILLARY 

ELECTROPHORESIS AND NMR 

Domínguez-Vega E. 1 , Crego A.L. 1 , Marina M.L. 1 , Salgado A. 2 , Scriba G.K.E. 3 , Chankvetadze B. 4 

1 

University of Alcalá-Spain 

2 

Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 

3 


4 

Tbilisi State University-Georgia 

P16-025 IDENTIFICATION OF UNKNOWN DEGRADATION PRODUCTS IN NEW PHARMACEUTICAL DOSAGE FORMS OF PARACETAMOL 

BY LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY 

Pérez I., Castro-Rubio F., Nozal L., Novella J.L., Crego A.L 


P16-026 CHARACTERIZATION OF PHENOLIC PROFILE IN FOUR ROSMARINUS OFFICINALIS EXTRACTS AND DETERMINATION OF 

THEIR ANTIOXIDANT CAPACITY 

Borrás Linares I., Herrero M., Ibánez E., Segura Carretero A., Fernández Gutiérrez A. 



P16-027 CHARACTERIZATION OF PHENOLIC COMPOUNDS BY HPLC-ESI-TOF-MS IN THREE DIFFERENT PEPPER VARIETIES. UPDATING 

AND IMPROVING FOOD COMPOSITION TABLES 

Morales Soto A., Segura Carretero A., Fernández Gutiérrez A. 


P16-028 COMPARISON OF DIFFERENT METHODOLOGIES FOR THE EVALUATION OF BINDING OF ANTIHISTAMINES TO HUMAN SERUM 

ALBUMIN BY CAPILLARY ELECTROPHORESIS 

Martinez Gomez M.A. 1 , Sagrado S. 1,2 ,Villanueva Camañas R.M. 1 , Medina Hernandez M.J. 1 

1 


2 


P16-029 ENANTIOMERIC RPLC SEPARATIONS OF CHIRAL LOCAL ANESTHETICS USING POLYSACCHARIDE BASED CHIRAL 


Escuder Gilabert L., Sagrado S. 1,2 ,Villanueva Camañas R.M. 1 , Medina Hernandez M.J. 1 

1 


2 


P16-030 ELECTROKINETIC CHROMATOGRAPHY AS AN ANALYTICAL APPROACH TO EVALUATE THE ENANTIOSELECTIVE BINDING OF 

FLUOXETINE TO HUMAN SERUM ALBUMIN 

Asensi Bernardi L., Martín Biosca Y., Sagrado S., Medina-Hernández M.J. 


552 

553 

554 

555 

556 

557 

558 


43


P16-031 ENANTIOSELECTIVITY STUDY IN BINDING OF QUINUPRAMINE TO HUMAN SERUM ALBUMIN BY ULTRAFILTRATION AND 

CAPILLARY ELECTROPHORESIS USING EXPERIMENTAL DESIGNS 

Martinez Gomez M.A. 1 , Villanueva Camañas R.M. 1 , Sagrado S. 1,2 , Medina Hernandez M.J. 1 

1 


2 


P16-032 COMPARISON BETWEEN RPLC USING POLYSACCHARIDE BASED CHIRAL STATIONARY PHASES AND CAPILLARY 

ELECTROPHORESIS WITH CYCLODEXTRINS FOR THE CHIRAL SEPARATION OF BASIC DRUGS IN AQUEOUS SAMPLES 



P16-033 COMPARISON OF DART-MS AND GC-MS CAPABILITIES IN ANALYSIS OF MINT ESSENTIAL OIL SAMPLES 

Chernetsova E.S., Goryainov S.V., Ovcharov M.V., Bochkov P.O., Khomyakov Y.Y. 

People’s Friendship University of Russia 

P16-034 STABILITY OF PHARMACOKINETIC STUDIES DATA OBTAINED USING HPLC/MS/MS SYSTEMS WITH TRIPLE QUADRUPOLE 

MASS SPECTROMETERS 

Chernetsova E.S. 1 , Ovcharov M.V. 1 , Bochkov P.O. 1 , Kovaleva A.S. 2 , Koryakova A.G. 2 

1 


2 

Chemical Diversity Research Institute 

P16-035 UTLC-MS AND TLC-MS OF SINGLE PEPTIDES OF ACE INHIBITORS 

Vovk I. 1,2 , Popovic G. 3 , Simonovska B. 2 , Agbaba D. 3 , Albreht A. 2 

1 

EN-FIST Centre of Excellence-Slovenia 

2 


3 

Faculty of Pharmacy, Belgrade-Serbia 

P16-036 DISTRIBUTION OF METOPROLOL IN HUMAN AUTOPSY MATERIAL 

Oertel R., Pietsch J., Arenz N., Goltz L., Kirch W. 

TU Dresden 

P16-037 STUDY ON A METABOLIC PROFILE GENERATED BY CYTOCHROME P450 2D6 INSIDE THE SEPARATION CAPILLARY 

Zeisbergerová M., Mádr A., Glatz Z. 

Masaryk University, Faculty of Science 

P16-038 VALIDATION OF LC–UV AND LC–MS METHODS FOR DETERMINATION OF TORASEMIDE AND ITS IMPURITIES IN TABLETS 

Jovic Z. 1 , Zivanovic Lj. 2 , Radisic M. 1 , Malesevic M. 1 

1 

Medicines and Medical Devices Agency of Serbia, National Control Laboratory 

2 

Faculty of Pharmacy, Institute of Pharmaceutical Chemistry and Drug Analysis 

P16-039 VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF ZOLPIDEM TARTRATE AND ITS DEGRADATION 

PRODUCTS IN TABLETS 

Jovic Z. 1 , Zivanovic Lj. 2 , Malesevic M. 1 

1 


2 


559 

560 

561 

562 

563 

564 

565 

566 

567 

P17 LIFE SCIENCES AND BIOANALYSIS 

P17-001 LC-MS IDENTIFICATION OF MYCOLIC ACIDS AND THEIR METABOLITES AS BIOMARKERS FOR ANCIENT TUBERCULOSIS 

Bona A., Boros Major A., Maasz G., Jambor E., Mark L. 


P17-002 PROTEIN SEPARATION WITH A NEW HIGH RESOLUTION GLASS COLUMN 

Fuchs M. 

Wissenschaftliche Gerätebau Dr.Ing. H.Knauer GmbH-Germany 

P17-003 SEPARATION AND DETERMINATION OF ELEVEN PTERIDINES AND LUMAZINES IN HUMAN URINE BY LIQUID CHROMATOGRAPHY 

USING DIODE ARRAY AND FLUORESCENCE SERIAL DETECTION 

Cañada-Cañada F., Mancha de LLanos A., Espinosa-Mansilla A., Muñoz de la Peña A. 


P17-004 TRANS-RESVERATROL AND TRANS-PICEID IN HUNGARIAN WINES 

Boros Major A., Bona A., Jambor E., Montsko G., Ohmacht R., Mark L. 


568 

569 

570 

571 

572 

44 



P17-005 SEPARATION AND CHARACTERIZATION OF ALPHA 2-3 AND ALPHA 2-6 ISOMERIC SIALYLATED O-GLYCOPEPTIDES FROM 

PROTEOLYTICALLY DIGESTED CASEINOMACROPEPTIDE USING HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY 

(HILIC) - TANDEM MASS SPECTROMETRY 

Hernandez-Hernandez O. 1 , Lebrón-Aguilar R. 2 , Quintanilla-Lopez J. 2 , Sanz M.L. 1 , Moreno F.J. 3 

1 


2 

Instituto de Química-Física “Rocasolano”, Madrid 

3 

Instituto de Fermentaciones Industriales-CIAL, Madrid 

573 

P17-006 DEVELOPMENT AND VALIDATION OF AN HPLC METHOD FOR THE ANALYSIS OF A NEW NITROSYL RUTHENIUM COMPLEX, 

CIS-[RUCL(BPY)2NO](PF6)2, NITRIC OXIDE DONOR IN RAT LIVER MICROSOME 

Sesso T.A.C., Simões R.A., Santana R.S., de Oliveira A.R.M. 

University of São Paulo 

P17-007 QUANTIFICATION OF PHENOLIC ANTIOXIDANTS IN RAT CEREBROSPINAL FLUID BY GC–MS AFTER ORAL ADMINISTRATION 

OF COMPOUNDS 

Zafra Gómez A. 1 , Luzón-Toro B. 2 , Jiménez-Díaz I. 1 , Ballesteros O. 1 , Navalon A. 1 

1 


2 

Hospital “Virgen del Rocío” 

P17-008 DETERMINATION OF BISPHENOL A AND ITS CHLORINATED DERIVATIVES IN PLACENTAL TISSUE SAMPLES BY LC-MS/MS 

Zafra Gómez A., Jiménez-Díaz I., Navea N., Ballesteros O., Navalón A., Fernández M.F., Olea N., Vilchez J.L. 

1 


2 


P17-009 SUITABLE METHOD FOR THE DETERMINATION OF ROSIGLITAZONE AND ITS MAIN METABOLITES IN RAT LIVER MICROSOMAL 

FRACTION EMPLOYNG HOLLOW-FIBER LIQUID-PHASE MICROEXTRACTION (HF-LPME) 

Calixto L.A., Bonato P.S. 


P17-010 VALIDATION OF A QUANTITATIVE GC METHODOLOGY FOR PHYTOSTEROLS DETERMINATION IN SERUM 

Vidal C., García-Llatas G., Lagarda M.J., Alegría A., Barberá R. 


P17-011 DETERMINATION OF 2-THIOTHIAZOLIDINE-4-CARBOXYLIC ACID IN URINE BY HPLC 

Torrado S., Rosell M.G., Garrote P., Guardino X. 

Instituto Nacional de Seguridad e Higiene en el Trabajo, Centro Nacional de Condiciones de Trabajo, Barcelona 

P17-012 METABOLOMIC APPROACH TO THE NUTRACEUTICAL EFFECT OF ROSEMARY EXTRACT IN DIABETIC CHILDREN 

Balderas C. 1 , Villaseñor A. 1, García A. 1 , Rupérez F. 1 , Ibañez E. 2 , Señorans J. 3 , Guerrero-Fernández J. 4 , González I. 4 , Gracia-Bouthelier R. 4 , 

Barbas C. 1 

1 

University of San Pablo CEU, Madrid 

2 

Instituto de Fermentaciones Industriales, CSIC, Madrid 

3 


4 

Hospital La Paz, Madrid 

P17-013 QUANTIFICATION OF GLUTAMATE UPTAKE IN BRAIN BY CE-LIF 

Lorenzo MªP., Valladolid I., Merino B., Ruiz-Gayo M., Fernández-Alfonso M., Cano V., Barbas C., García A. 

University San Pablo CEU, Madrid 

P17-014 SEPARATION OF BETA-LACTOGLOBULIN A AND BETA-LACTOGLOBULIN B BY HIGH PERFORMANCE GRADIENT 

CHROMATOFOCUSING – MASS SPECTROMETRY 

Albreht A., Vovk I., Simonovska B. 


P17-015 ANALYSIS OF BIOMOLECULES BY UPLC-SEC AND UPLC-IEX 

Hong P., Fountain K., Morrison D., Bosch G. 


P17-016 CHARACTERIZING CFEX KNOCKOUT MICE URINE BY CE-UV 

Villaseñor A. 1 , Holmes E. 2 , Barbas C. 1 , Unwin R. 3 

1 


2 

Imperial College 

3 

University College London 

574 

575 

576 

577 

578 

579 

580 

581 

582 

583 

584 

P17-018 RAPID VITAMIN A AND E SAMPLE PREPARATION AND LIQUID CHROMATOGRAPHY QUANTIFICATION PROCEDURE FOR 

HUMAN BREAST MILK INVESTIGATION 

Kasparova M., Plisek J., Krcmova L., Hronek M., Zadak, Z., Solichova D. 

Charles University Medical School & Teaching Hospital-Czech Republic 

585 


45


P17-020 DEVELOPMENT OF HILIC UHPLC-FD METHOD FOR DETERMINATION OF NEOPTERIN, BIOPTERIN AND ITS DERIVATES IN URINE 

USING SOLID PHASE EXTRACTION AS THE SAMPLE PREPARATION TECHNIQUE 

Vlcková H. 1 , Plíšek J. 2 , Nováková L. 1 , Solich P. 1 

1 

Charles University-Czech Republic 

2 

Teaching Hospital in Hradec Králové-Czech Republic 

586 

P17-021 COMPREHENSIVE GCXGC, A VALUABLE TECHNIQUE FOR THE SCREENING OF BIOLOGICAL ACTIVE CHEMICAL COMPOUNDS IN 

POLYPORE SAMPLES 

Gilart-Alzuria N., Wiikinkoski E., Mantyla S., Ruiz-Jiménez J., Hartonen K., Riekkola M.L., Wiedmer S. 


P17-022 HILIC–UPLC-BASED METHOD FOR GENOMIC DNA METHYLATION MEASUREMENT 

Sotgia S., Zinellu A., Pisanu E., Murgia L., Gaspa L., Deiana L., Carru C. 

University of Sassari 

P17-023 MICRO-SOLID PHASE EXTRACTION AND SELECTIVE ENRICHMENT OF GLYCOPROTEINS USING LECTIN MODIFIED GOLD 

NANO-PARTICLES ON A MONOLITHIC SUPPORT 

Alwael H., Connolly D., Clarke P., Thompson R., O’Connor B., Twamley B., Paull B. 


P17-024 DETERMINATION OF CATECHOLAMINES USING MIXED-MODE REVERSED-PHASE AND CATION-EXCHANGE HIGH-PERFORMANCE 


Tsunoda M. 

University of Tokyo 

P17-025 URINE FINGERPRINTING WITH CE-MS REVEALS ROSMARINUS OFFICINALIS EXTRACT EFFECT ON DIABETIC RATS 

Godzien J. 1,2 , Moraes E. 1,3 , Rupérez F.J. 1 , Barbas C. 1 

1 

University San Pablo-CEU 

2 

The John Paul II Catholic University of Lublin 

3 

University of Sao Paulo 

P17-026 INVESTIGATION OF THE CHROMATOGRAPHIC BEHAVIOR OF BARE TITANIA IN HYDROPHILIC INTERACTION LIQUID 

CHROMATOGRAPHY (HILIC) 

Abi jaoudé M., El Debs R., Randon J. 


P17-027 EVALUATION OF THE BIO-FUNCTIONALIZATION OF POROUS MONOLITHS DEDICATED TO IMMUNO-PRECONCENTRATION 

AND SYNTHESIZED IN MICROSCALE CAPILLARY FORMAT 

Chamieh J. 1 , Faye C. 2 , Vandenabeele-Trambouze O. 2 , Moreau T. 2 , Faure K. 1 , Dugas V. 1 , Demesmay C. 1 , Randon J. 1 

1 


2 

University of Montpellier II 

P17-028 STUDY OF RETENTION BEHAVIOUR AND MASS SPECTROMETRY COMPATIBILITY IN ZWITTERIONIC HYDROPHILIC INTERACTION 

LIQUID CHROMATOGRAPHY FOR THE SEPARATION OF MODIFIED NUCLEOSIDES 

Rodríguez-Gonzalo E., García-Gómez D., Carabias-Martínez R. 


P17-029 DETERMINATION OF TETRAHYDROFURAN AND KETONES IN URINE BY HEADSPACE TECHNIQUE 

Prado Burguete C. 1 , Marín Carrasco P. 2 , Alcaraz Rodríguez J. 1 , Periago Jiménez F. 1 

1 

Instituto de Seguridad y Salud Laboral-España 

2 

University of Murcia 

P17-030 DEVELOPMENT AND OPTIMIZATION OF THE CONDITIONS FOR THE APPLICATION OF HYDROPHOBIC INTERACTION 

CHROMATOGRAPHY (HIC) FOR THE PURIFICATION OF F(AB)’2 FRAGMENTS 

Seijo D., Barbi L., Arathoon R., Gago-Martínez A. 

University of Vigo 

P17-031 A SOLID-PHASE EXTRACTION LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY ANALYTICAL METHOD FOR THE 

DETERMINATION OF 2-HYDROXY-4-METHOXYBENZOPHENONE AND ITS METABOLITES IN BOTH HUMAN URINE AND SEMEN 

León Z., Chisvert A., Tarazona I., Salvador A. 


P17-032 DEVELOPMENT OF INFORMATION RICH URINARY SIGNATURES IN HUMANS BY LC-MS FOR POTENTIAL ANTI-DOPING 

APPLICATIONS 

Kiss A., Flament-Waton M.M., Cren-Olivé C. 

Service Central d’Analyse, Biochemistry 

587 

588 

589 

590 

591 

592 

593 

594 

595 

596 

597 

598 

46 



P17-033 DETECTION OF TOREMIFENE ADMINISTRATION IN ANTIDOPING CONTROL ANALYSES 

Gómez C. 1 , Pozo O.J. 1 , Vila E. 2 , Salvador J.P. 2 , Marco M.P. 2 , Segura J. 1 , Ventura R. 1 

1 

IMIM-Hospital del Mar, Barclona 

2 

IQAC-CSIC, Applied Molecular Receptors Group, Barcelona 

P17-034 METABOLITE TARGET ANALYSIS OF CELL EXTRACT OF BACTERIUM PARACOCCUS DENITRIFICANS 

Musilova J., Foltova K., Glatz Z. 


599 

600 

P18 FORENSICS 

P18-001 LIQUID CHOMATOGRAPHY WITH DOUBLE DETECTION AS A MONITORING TOOL OF A NEW PROTOCOL FOR THE ISOLATION OF 

NITROCELLULOSE FROM GUNPOWDERS 

López-López M. 1 , Fernández de la Ossa M.A. 1 , Sáiz Galindo J. 1 , Ferrando J.L. 1,2 , Vega A. 1 , Torre M. 1 , García-Ruiz C. 1 

1 

University of Alcala, University Institute of Research in Police Sciences (IUICP) 

2 

Criminalistic Service of Guardia Civil 

P18-002 DETERMINATION OF NITROGEN CONTENT IN NITROCELULLOSE USED IN THE MANUFACTURE OF GUNPOWDERS BY ALKALINE 

HYDROLYSIS AND IONIC CHROMATOGRAPHY 

López-López M. 1 , Ramiro Alegre J.M. 1,2 , García-Ruiz C. 1 , Torre M. 1 

1 


2 


P18-003 DIPHENYLAMINE AND ITS DERIVATIVES AS PREDICTORS OF AGEING OF SINGLE AND DOUBLE BASED GUNPOWDERS BY 

MEANS OF HPLC 

López-López M. 1 , Ortiz R. 2 , Bravo J.C. 1,2 , Ferrando J.L. 1,2 , Velasco E. 2 , García-Ruiz C. 1 , Torre M. 1 

1 


2 


P18-004 DETERMINATION OF NITROCELLULOSE CONTAINED IN SMOKELESS PROPELLANTS BY CAPILLARY ELECTROPHORESIS 

Fernández de la Ossa M., Sáiz J., Torre M., García-Ruiz C. 


P18-005 DEVELOPMENT OF A NEW METHODOLOGY FOR THE DETERMINATION OF CELLULOSE BY CAPILLARY ELECTROPHORESIS 

Fernández de la Ossa M., Torre M., García-Ruiz C. 


P18-006 STUDY ON LOSSES OF VOLATILE COMPOUNDS OF EXPLOSIVES AND CROSS-CONTAMINATION AMONG EXPLOSIVE SAMPLES 

STORED IN POLYETHYLENE BAGS BY CROMATOGRAPHIC TECHNIQUES 

Sáiz J. 1 , Bravo J.C. 1,2 , Atoche J.C. 2 , Ferrando J.L. 1,2 , Torre M. 1 , García-Ruiz C. 1 

1 


2 


P18-007 DETERMINATION OF NITROGLYCOL IN DYNAMITE-LIKE EXPLOSIVES BY LIQUID CHROMATOGRAPHY 

Sáiz J. 1 , Bravo J.C. 1,2 , Velasco E. 2 , Torre M. 1 , García-Ruiz C. 1 

1 


2 


601 

602 

603 

604 

605 

606 

607 

608 

P18-008 SIMULTANEOUS CE ANALYSIS OF INORGANIC ANIONS AND CATIONS IN POST-BLAST RESIDUE EXTRACTS OF ACID-ALUMINUM 

MIXTURES 

Sarazin C. 1 , Delaunay N. 2 , Varenne A. 2, Costanza C. 1 , Eudes V. 1 , Gareil P. 2 

1 

Laboratoire Central de la Préfecture de Police de Paris-France 

2 

Chimie ParisTech(PECSA) 

P18-009 ANALYSIS OF INORGANIC CATIONS IN POST-BLAST RESIDUE EXTRACTS BY CAPILLARY ELECTROPHORESIS 

Sarazin C. 1 , Delaunay N. 2 , Costanza C. 1 , Eudes V. 1 , Gareil P. 2 

1 

Laboratoire Central de la Préfecture de Police de Paris 

2 


P18-010 DETERMINATION OF MESCALINE IN LOPHOPHORA WILLIAMSII PLANTS GROWN IN THE CULTURE USING CAPILLARY 

ELECTROPHORESIS WITH ELECTROSPRAY MASS SPECTROMETRY 

Svidrnoch M. 1 , Ranc V. 1 , Maier L. 2 , Sevcik J. 1 , Maier V. 1 

1 

Palacky University in Olomouc-Czech Republic 

2 


609 

610 

611 


47


P19 INDUSTRIAL PROCESSES 

P19-001 PILOT SCALE RECOVERY OF PHYCOCYANIN FROM SPIRULINA PLATENSIS USING EXPANDED BED ADSORPTION CHROMATOGRAPHY 

Bermejo R., Ramos A. 

Jaén University 

P19-002 ANALYSIS OF PYROLYSIS PRODUCTS FROM BIOMASS AND COAL BY COMPREHENSIVE TWO-DIMENSIONAL GAS-CHROMATOGRAPHY 

Rathsack P., Otto M. 

TU Bergakademie Freiberg 

P19-003 APPLICATION OF METHACRYLYC ACID-ETHYLENEGLYCOL DIMETHACRYLATE POLYMERIC SORBENT FOR THE REMOVAL OF 

ESTROGENS FROM WATER SAMPLES 

Gallego A., Bravo J.C., Garcinuño R.M., Fernández P., Durand J.S. 

National University of Distance Education, Madrid 

612 

613 

614 

615 

P20 ENVIRONMENT 

P20-001 BEHAVIOR OF COPLANAR AND NON-COPLANAR POLYCHLORINATED TERPHENYL (PCT) CONGENERS TOWARD STATIONARY 

PHASE OF COLUMN CHROMATOGRAPHY FOR DEVELOPMENT OF ANALYTICAL METHOD 

Wibowo A.H., Bahadir M., Vogt R., Wichmann H. 

Technical University of Braunschweig 

P20-002 DETERMINING THE ANHYDROSUGARS LEVOGLUCOSAN, MANNOSAN AND GALACTOSAN IN AEROSOLS 

Espuelas J. 1 , Kolb T. 2 , Bogenschütz G. 2 

1 

Gomensoro S.A.-Spain 

2 

Deutsche Metrohm GmbH & Co. KG 

616 

617 

618 

P20-003 DETERMINING TRACE LEVELS OF PERFLUORINATED COMPOUNDS IN WATER BY SUPPRESSED ION CHROMATOGRAPHY WITH 

INLINE MATRIX ELIMINATION 

Epalza A. 1 , Subramanian N.H. 2 , Manigandan P. 3 , Wille A. 4 

1 

Gomensoro S.A. 

2 

Metrohm USA-USA 

3 

Metrohm India-India 

4 

Metrohm International Headquaters-Switzerland 

P20-004 OCCURRENCE OF PERFLUORINATED COMPOUNDS IN SPANISH SEWAGE SLUDGE 

Navarro I., Sanz P., Martínez M.A. 

CIEMAT, Valencia 

P20-005 ODOR-CAUSING ORGANIC COMPOUNDS IN WASTEWATER TREATMENT PLANTS: EVALUATION OF HS-SPME AS 

CONCENTRATION TECHNIQUE 

Godayol A., Alonso M., Besalú E., Sanchez J.M., Anticó E. 

Universitat de Girona 

P20-006 DEVELOPMENT AND QUALITATIVE VALIDATION OF A WIDE SCOPE SCREENING OF EMERGING CONTAMINANTS IN NATURAL 

WATER AND WASTEWATER BY UHPLC-QTOF MS 

Ibáñez M., Díaz R., López F.J., Sancho J.V., Gracia E., Bijlsma L., Hernández F. 


P20-007 ANALYSES OF ENERGETIC MOLECULES TRACES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY / MASS SPECTROMETRY 

IN AQUEOUS MATRICES 

Legendre A, Bousquet M., Pin N., Hairault L. 

Commissariat à l’Energie Atomique, DXPL/SMEO/LPC 

P20-008 ASSESSMENT OF PESTICIDE CONTAMINATION IN SOIL AND WATER SAMPLES FROM NATURAL PARK OF L´ ALBUFERA USING 

LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY 

Masiá A. 1 , Vazquez-Roig P. 1 , BlascoC. 1 , Andreu V. 2 , Picó Y. 1 

1 

Faculty of Pharmacy, University of Valencia 

2 

Centro de investigaciones sobre desertificación–CIDE, Valencia 

P20-009 DETERMINATION OF TRICLOSAN AND METHYL TRICLOSAN IN ENVIRONMENTAL SOLID SAMPLES BY MATRIX SOLID-PHASE 

DISPERSION AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

Sánchez-Brunete C., Miguel E., Albero B., Tadeo J.L. 


P20-010 DISPERSIVE LIQUID-LIQUID MICROEXTRACTION COMBINED WITH NON AQUEOUS CAPILLARY ELECTROPHORESIS FOR THE 

DETERMINATION OF FLUOROQUINOLONE ANTIBIOTICS IN WATERS 

Herrera-Herrera A.V., Hernández-Borges J., Borges-Miquel T.M., Rodríguez-Delgado M.A. 


619 

620 

621 

622 

623 

624 

625 

626 

48 



P20-011 IONIC LIQUID-DISPERSIVE LIQUID-LIQUID MICROEXTRACTION WITH HPLC-FD FOR THE DETERMINATION OF A GROUP OF 

PESTICIDES AND METABOLITES IN SOILS 

Asensio-Ramos M., Hernández-Borges J., Herrera-Herrera A.V., Rodríguez-Delgado M.A. 


P20-012 EVALUATION OF A MODIFIED QUECHERS METHOD FOR THE EXTRACTION OF PESTICIDES FROM SOILS 

Asensio-Ramos M., Hernández-Borges J., Ravelo-Pérez L.M., González-Curbelo M.A., Rodríguez-Delgado M.A. 

University of La Laguna (ULL) 

P20-013 INVESTIGATION OF ORGANOPHOSPHATE ESTERS IN FRESH AND SEA WATER, BRINE AND SALT SAMPLES BY GC-TOF MS 

Nácher-Mestre J., Serrano R, Portolés T., Hernández F. 


P20-014 BEHAVIOUR OF PHARMACEUTICALS AND DRUGS OF ABUSE IN A DRINKING WATER TREATMENT PLANT USING CONVENTIONAL 

AND UF/RO TREATMENTS. 

Boleda M.R. 1 , Galceran M.T. 2 , Ventura F. 1 

1 

AGBAR. Aigües de Barcelona, Àrea Química Orgànica 

2 

University of Barcelona, Departament química analítica 

P20-015 OCCURRENCE OF POLYBROMINATED/CHLORINATED COPLANAR BIPHENYLS (CO-PXBS) IN HUMAN BREAST MILK FROM SPAIN 

Gomara B. 1 , Herrero L. 1 , Pacepavicius G. 2 , Alaee M. 2 , Gonzalez M.J. 1 

1 

IQOG (CSIC), Instrumental Analysis and Environmental Chemistry-Spain 

2 

Environment Canada, Water Science and Technology Directorate-Canada 

P20-016 CHARACTERIZATION OF ALKYLPOLYPHOSPHONATES BY HPLC USING A POROUS GRAPHITIC CARBON STATIONARY PHASE 

Carrasco-Correa E.J., Herrero-Martínez J.M., Ramis-Ramos G. 


P20-017 PARTITIONING OF PERFLUORINATED COMPOUNDS IN SEAWATER, SEDIMENT AND MUSSELS FROM THE CANTABRIC SEA 

(NORTH SPAIN) 

Gómez C., Vicente J., Porte C. Lacorte S. 


P20-018 DETERMINATION OF ANTIOXIDANTS IN NEW AND USED LUBRICANT OILS BY HEADSPACE-PROGRAMMED TEMPERATURE 

VAPORIZATION-GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

García Pinto C. 1 , del Nogal Sánchez M. 1 , Glanzer P. 2 , Pérez Pavón J.L. 1 , Moreno Cordero B. 1 

1 


2 


P20-019 HPLC-DAD-MS-MS MONITORING OF VETERINARY PHARMACEUTICALS IN WATER AFTER DEGRADATION BY FENTON PROCESS 

AND TOXICOLOGICAL EVALUATION 

Ašperger D., Djuric I., Vujevic D., Pelko S., Periša M., Babic S., Horvat A.J.M., Koprivanac N., Kaštelan-Macan M. 

Faculty of Chemical Engineering and Technology-Croatia 

P20-020 MEASUREMENT OF VOLATILE ORGANIC COMPOUNDS IN ALGIERS URBAN AREAS USING STEREOSELECTIVE GAS 


Ladji R. 1 , Yassaa N. 2 

1 

Centre for Scientific Research and Technology in Physico-chemical analysis, Enviromental Chemistry-ALGERIA 

2 

University of Sciences and Technology Houari Boumediene-Algeria 

627 

628 

629 

630 

631 

632 

633 

634 

635 

636 

P20-021 DETERMINATION OF ABUSE DRUGS AND METABOLITES IN WATER SAMPLES BY IN-LINE SOLID PHASE EXTRACTION IN 

CAPILLARY ELECTROPHORESIS 

Botello I., Borrull F., Calull M., Aguilar C. 

University Rovira i Virgili, Tarragona 

P20-022 ANALYSIS OF SHORT-CHAIN POLYCHLORINATED PARAFFINS IN BIOTA BY SELECTIVE PRESSURIZED LIQUID EXTRACTION AND 

GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

Olmos J.E., Santos F.J., Galceran M.T. 


P20-023 EVALUATING THE EFFICIENCY OF A NEW POLYMERIC IONIC LIQUID (POLY(VBHDIM-NTF2)) AS SORBENT COATING IN DIRECT 

IMMERSION MODE - SOLID-PHASE MICROEXTRACTION - GAS CHROMATOGRAPHY FOR THE DETERMINATION OF A GROUP 

OF ENDOCRINE DISRUPTING CHEMICALS 

López-Darias J. 1 , Pino V. 1 , Anderson J.L. 2 , Afonso A.M. 1 

1 


2 

The University of Toledo-USA 

637 

638 

639 


49


P20-024 APPLICATION OF PY-GC/MS TO STUDY CHANGES IN THE ORGANIC MATTER OF MACRO- AND MICROAGGREGATES OF A 

MEDITERRANEAN SOIL UPON HEATING 

Campo J. 1 , Cammeraat E. 2 , Andreu A. 1 , Rubio J.L. 1 

1 

Centro de Investigaciones sobre Desertificacion, Degradacion y conservacion de suelos, Valencia 

2 

Universiteit van Amsterdam 

P20-025 ANALYSIS OF SILOXANES IN WATER BY HEADSPACE-SOLID PHASE MICROEXTRACTION-GAS CHROMATOGRAPHY-MASS 

SPECTROMETRY 

Companioni E.Y., Santos F.J., Galceran M.T. 


P20-026 A SIMPLIFIED QUECHERS APPROACH FOR THE DETERMINATION OF THMS AND BTEX IN SOIL MATRICES BY FAST GAS 

CHROMATOGRAPHY WITH MASS SPECTROMETRY DETECTION 

Herrero Martín S., García Pinto C., Pérez Pavón J.L., Moreno Cordero B. 


P20-027 ANALYSIS OF ANTICOAGULANT RODENTICIDES IN WATER, SOIL AND MICROTUS ARVALIS TISSUES BY LIQUID CHROMATOGRAPHY 

Nozal M.J., Bernal J.L., Toribio L., Hernandez A., Martin M.T. 


P20-028 NEW METHOD FOR PREPARATIVE LIQUID CHROMATOGRAPHY FRACTIONATION OF CRUDE OIL SAMPLES USING A FILTRATION 

SYSTEM BY GRADIENT OF SILICA GEL 

Vicente M.A. 1 , Vicente A.R. 1 , Camacho C.F.B. 2 , Tamanqueira J.B. 1 , Lange R. 1 , Sad C.M.S. 1 , Neto R.R. 1 , Castro E.V.R. 1 , Medeiros E.F. 1 , Dias J.C.M. 3 

1 

Universidade Federal do Espirito Santo 

2 

Fundação Gorceix , CENPES P&D de Produção-Brazil 

3 

Petrobras, CENPES-Brasil 

P20-029 DETERMINATION OF VOLATILE HALOGENATED ORGANIC COMPOUNDS AND CHLOROBENZENES USING MODIFIED QUECHERS 

EXTRACTION AND GC-µECD ANALYSIS 

Moreno Cordero B., Casas Ferreira A.M., Fernández Laespada M.E., Pérez Pavón J.L. 


P20-030 COMPARATIVE STUDY OF THE ADSORPTION PERFORMANCE OF A MULTI-SORBENT BED (CARBOTRAP, CARBOPACK X, 

CARBOXEN 569) AND A RADIELLO DIFFUSIVE SAMPLER FOR THE ANALYSIS OF VOCS 

Gallego E. 1 , Roca F.X. 1 , Perales J.F. 1 , Guardino X. 2 , Rosell M.G. 2 

1 

Technical University of Catalonia 

2 

Instituto Nacional de Seguridad e Higiene en el Trabajo, Centro Nacional de Condiciones de Trabajo, Barcelona 

640 

641 

642 

643 

644 

645 

646 

P20-031 EVALUATION OF WORK-RELATED VOLATILE ORGANIC COMPOUNDS (VOCS) EXPOSITION IN AUTOMOTIVE PAINTING CABINS 

INDUSTRIAL CLEANING 

Rosell M.G. 1 , Carrasco A. 2 , Gallego E. 3 

1 

Instituto Nacional de Seguridad e Higiene en el Trabajo, Centro Nacional de Condiciones de Trabajo 

2 

ACCIONA Facility Services, Departamento de Seguridad y Salud de las Personas 

3 

LCMA-Technical University of Catalonia 

P20-032 SOLID-PHASE EXTRACTION ON-LINE COUPLED TO HYDROPHIIC INTERATION CHROMATOGRAPHY- MASS SPECTROMETRY 

TO DETERMINE POLAR DRUGS FROM WATER SAMPLES 

Fontanals N., Marcé R.M., Borrull F. 


P20-033 DEVELOPMENT OF A MULTI-RESIDUE METHODOLOGY FOR THE ANALYSIS OF HORMONAL STEROIDS AND VETERINARY 

COMPOUNDS TRACES IN SOIL 

Salvia M.V., Vulliet E., Wiest L., Baudot R., Cren-Olive C. 

Service Central d’Analyse (CNRS)-France 

P20-034 DETERMINATION OF 52 ORGANIC COMPOUNDS IN THE WHOLE WATER SAMPLE BY SPE-GC-MS CONSIDERING THE WATER 

FRAMEWORK DIRECTIVE 

Erger C. 1 , Balsaa P. 1 , Werres F. 1 , Schmidt T.C. 2 

1 

IWW Water Centre 

2 


P20-035 VALIDATION OF FAST AUTOMATED SPE AND ISOTOPE DILUTION-GC/MS ANALYSIS OF PRIORITY PESTICIDES IN WATER 

Planas C., Caixach J. 

IDÆA (CSIC)-Spain 

P20-036 EVALUATING THE CONTENT OF ALKYL- AND METHOXY-PHENOLIC COMPOUNDS IN BIOMASS SMOKE BY HEADSPACE-SINGLE- 

DROP-MICROEXTRACTION (HS-SDME) IN COMBINATION WITH HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 

Rincón A.A., Pino V., Ayala J.H., Afonso A.M.., González V. 


647 

648 

449 

650 

651 

652 

50 



P20-037 DETERMINATION OF VOLATILE ORGANIC COMPOUNDS IN WATER BY HEAD SPACE-SPME GC-MS/MS WITH TRIPLE 

QUADRUPOLE ANALYZER 

Cervera M.I., Beltrán J., Hernández F. 


P20-038 PESTICIDES IN WATER INTENDED FOR HUMAN CONSUMPTION BY UPLC-MS/MS AND GC-MS 

Gonzalez C., Castillo M., Carbonell E., Perez J.A. 

Centro Superior de Investigación en Salud Pública 

P20-039 OPTIMITATION OF SOME STEPS FOR THE ANALYSIS OF PER- AND POLYFLUORINATED ALKYL SUBSTANCES (PFAS) IN FINE 

AIRBONE PARTICULATE MATTER (PM 2.5) BY LC-MS/MS 

Beser M.I. 1 , Pardo O. 1 , Yusa V. 1 , Beltrán J. 2 

1 

Public Health Research Center (CSISP), Valencia 

2 


P20-040 MULTI RESIDUE ANALYSIS OF ENDOCRINE DISRUPTOR COMPOUNDS IN ENVIRONMENTAL SAMPLES USING LIQUID 

CHROMATOGRAPHY TANDEM MASS SPECTROMETRY 

Camilleri J., Vulliet E., Wiest L., Baudot R., Cren-Olivé C. 

Service Central d’Analyse du CNRS 

P20-041 DEVELOPMENT OF A RELIABLE AND SENSITIVE QUANTIFICATION METHOD FOR THE DETERMINATION OF POLAR 

PESTICIDES METABOLITES IN SURFACE WATER, GROUND WATER AND DRINKING WATER 

Kowal S. 1 , Balsaa P. 1 , Werres F. 1 , Schmidt T.C. 2 

1 

IWW Water Centre-Germany 

2 

University of Duisburg Essen 

P20-042 DEVELOPMENT OF A CAPILLARY CHELATION ION CHROMATOGRAPHY SYSTEM FOR APPLICATION WITHIN ENVIRONMENTAL 

MONITORING/TRANSITION METAL ANALYSIS 

Moyna A., Connolly D., Currivan S., Macka M., Paull B. 

Irish Separation Science Cluster, Dublin City University 

P20-043 RAPID ANALYSIS OF 52 PERSISTENT ORGANIC POLLUTANTS IN SOLIDS BY MEANS OF ULTRASONIC SOLVENT EXTRACTION 

AND GAS CHROMATOGRAPHY MASS/MASS SPECTROMETRY 

Martinez E., Llorca J. 

LABAQUA, Murcia 

P20-044 DETERMINATION OF ALKYL SULFATES IN MARINE SEDIMENTS BY LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY 

Fernández-Ramos C. 1 , Blanc R. 1 , Ballesteros O. 1 , Zafra-Gomez A. 1 , Navalón A. 1 , Crovetto G. 1 , Vílchez J.L. 1 , Verge C. 2 , Lopez I. 2 , De Ferrer J. 2 

1 


2 

Cepsa Química, Centro de Investigacion 

P20-045 CHEMICAL CHARACTERIZATION OF THE TARSAL SECRETION OF THE LOCUST S. GREGARIA 

Gradl M., Nicholson T., Schmitt C., Betz O., Albert K. 


P20-046 ENANTIOMER DETERMINATION OF M- AND P-IMAZAMETHABENZ METHYL ISOMERS IN POTATO SAMPLES BY TWO- 

DIMENSIONAL CHIRAL HPLC USING A PROTEIN BASED COLUMN 

Santos-Delgado M.J., Polo-Díez L.M. 


P20-047 SYNTHESIS OF STRONG ANION-EXCHANGE MONOLITHIC CAPILLARY COLUMN FOR THE MINIATURIZED ANALYSIS OF 

RADIOACTIVE SAMPLES 

Bruchet A. 1 , Dugas V. 1 , Goutelard F. 2 , Randon J. 1 

1 

University Claude Bernard-Lyon 

2 

CEA-Saclay 

P20-048 DETERMINATION OF PARABENS IN HOUSE DUST BY PRESSURISED HOT WATER EXTRACTION FOLLOWED BY STIR BAR 

SORBENT EXTRACTION AND THERMAL DESORPTION - GAS CHROMATOGRAPHY - MASS SPECTROMETRY ANALYSIS 

Ramírez N., Borrull F., Marcé R.M. 


P20-049 DETERMINATION OF ACIDIC DRUGS IN RIVER WATER SAMPLES BY IN-LINE-SOLID-PHASE EXTRACTION-CAPILLARY 

ELECTROPHORESIS 

Maijó I., Borrull F., Aguilar C., Calull M. 


653 

654 

655 

656 

657 

658 

659 

660 

661 

662 

663 

664 

665 


51


P20-050 ON-LINE ANALYSIS OF CARBONYL COMPOUNDS WITH DERIVATIZATION BY IN-TUBE SOLID-PHASE MICROEXTRACTION 

COUPLED TO CAPILLARY LIQUID CHROMATOGRAPHY 

Prieto-Blanco M.C. 1 , López-Mahía P. 1 , Campíns Falcó P. 2 

1 

University of Coruña 

2 


P20-051 COMPARISON OF BIOMARKER ASSEMBLAGE WITH POTENTIAL FOR PALEOCLIMATE EVOLUTION IN COASTAL PEATLANDS 

FROM ASTURIAS (NORTH SPAIN) 

López Días V., Blanco C.G., Borrego A.G. 

Instituto Nacional del Carbón, Oviedo 

P20-052 IDENTIFICATION OF ORGANIC COMPOUNDS FROM ARCHAEOLOGICAL SAMPLES BY MEANS OF IN-SITU THERMALLY ASSISTED 

PYROLYSIS-SILYLATION GC-MS 

Doménech-Carbó M.T. 1 , Osete-Cortina L. 1 , Ahmadi H. 2 

1 


2 

Art University of Esfahan-Iran 

P20-053 IDENTIFICATION OF MATERIALS EXTRACTED FROM CONTEMPORARY PAINTS AFTER SOLVENT CLEANING BY MEANS OF 

IN-SITU THERMALLY ASSISTED PYROLYSIS-SILYLATION GC-MS 

Silva M.F., Osete-Cortina L., Doménech-Carbó M.T. 


P20-054 ON-LINE COLUMN-SWITCHING FAST LC-MS/MS FOR THE ANALYSIS OF PERFLUORINATED ORGANIC COMPOUNDS IN WATER 

SAMPLES 

Esparza X., Núñez O., Moyano E., Galceran M.T. 


P20-055 LC-MS/MS FOR THE ANALYSIS OF PERFLUORINATED PHOSPONIC ACIDS IN WATER AND SEDIMENT SAMPLES 

Esparza X. 1 , Moyano E. 1 , Galceran M.T. 1 , van Leeuwen S.P.J. 2 

1 


2 

VU University 

P20-056 A NEW IN-VIAL CONFIGURATION FOR MEMBRANE ASSISTED LIQUID-LIQUID MICRO-EXTRACTION. APPLICATION TO THE 

DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN SEAWATER AND MARINE SEDIMENTS BY GC-MS 

March J.G. 1 , Moukhchan F. 2 , Cerdà V. 1 , Simonet Suau B.M. 3 

1 

University of Balearic Islands 

2 

University Abdelmalek Essaâdi-Marroco 

3 


P20-057 DEVELOPMENT OF DATA PROCESSING WORKFLOWS FOR THE AUTOMATED DETECTION OF TRANSFORMATION PRODUCTS 

OF EMERGING CONTAMINANTS IN THE ENVIRONMENT) 

García-Reyes J.F. 1 , Pérez-Parada A. 2 , Gómez-Ramos M.M. 2 , Fernández-Alba A.R. 2 , Robles-Molina J. 1 , Domínguez-Romero J.C. 1 , Gilbert- 

López B. 1 , Molina-Díaz A. 1 , Agüera A. 2 

1 

University of Jaén 

2 


P20-058 DETERMINATION OF PENICILLINS AND ITS MAIN DEGRADATION PRODUCTS IN INFLUENT AND EFFLUENT FROM A 

WASTEWATER TREATMENT PLANT BY LC-Q-TOF 

Pérez-Parada A. 1 , Heinzen H. 2 , Gómez-Ramos M.M. 1 , García-Reyes J.F. 3 , Agüera A. 1 , Fernández-Alba A.R. 1 , 

1 


2 

Universidad de la República, Pharmacognosy & Natural Products-Uruguay 

3 

Universidad de Jaén 

P20-059 LEVELS OF DRUGS OF ABUSE IN SUPERFICIAL WATERS OF THE OLIVA-PEGO MARSH (VALENCIA, SPAIN) 

Vazquez-Roig P. 1 , Blasco C. 1 , Andreu V. 2 , Picó Y. 1 

1 


2 

Centro de Investigaciones sobre Desertificación–CIDE, CSIC, Valencia 

P20-060 POLLUTION BY PHARMACEUTICALS IN A TYPICAL MEDITERRANEAN WETLAND ECOSYSTEM 


1 


2 


P20-061 HYPHENATED TECHNIQUES FOR THE ANALYSIS OF LINEAR ALKYLBENZENESULFONATES IN TECHNICAL FORMULATIONS 

Piechotta C. 1 , Schmidt S. 1, Nehls I. 1 , Godejohann M. 2 , Mügge C. 3 

1 

Federal Institute for Materials and Testing (BAM)-Germany 

2 

Bruker BioSpin GmbH 

3 

Humboldt-University Berlin 

666 

667 

668 

669 

670 

671 

672 

673 

674 

675 

676 

677 

52 



P20-062 DETERMINATION OF NITROMUSKS IN WATER SAMPLES BY DISPERSIVE LIQUID-LIQUID MICROEXTRACTION FOLLOWED BY 

GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

Chisvert A., Lopez-Nogueroles M., Salvador A., Carretero A. 


P20-063 MOLECULARLY IMPRINTED POLYMERIC FIBERS (MONOLITHS) FOR SOLID-PHASE MICROEXTRACTION FOR THE DETERMINATION 

OF TRIAZINES COUPLED WITH GAS CHROMATOGRAPHY 

Díaz-Álvarez M., Paniagua E., Turiel E., Martín-Esteban A. 

Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Medio Ambiente-Spain 

P20-064 MULTI-RESIDUE METHOD FOR THE ANALYSIS OF SYNTHETIC SURFACTANTS AND THEIR DEGRADATION METABOLITES IN 

AQUATIC SYSTEMS BY LIQUID CHROMATOGRAPHY – TIME-OF-FLIGHT – MASS SPECTROMETRY 

Lara-Martín P.A. 1 , Traverso-Soto J.M. 1 , Brownawell B.J. 2 , González-Mazo E. 1 

1 

University of Cádiz 

2 

Stony Brook University 

P20-065 MULTI-RESIDUE ROUTINE ANALYSIS OF 57 PESTICIDES BY GAS CHROMATOGRAPHY COUPLED WITH TIME OF FLIGHT MASS 

SPECTROMETRY IN HONEYBEE’S POLLENS 

Wiest L., Arnaudguilhem C., Giroud B., Buleté A., Fratta C. 

Service central d’analyse CNRS 

P20-066 ANALYSIS OF THE OCCURRENCE AND RISK ASSESSMENT OF PESTICIDES IN THE LLOBREGAT RIVER (NE, SPAIN) BY ONLINE 

SPE-LC(ESI)-MS/MS 

Köck-Schulmeyer M., Osorio V., Pérez S., de Alda M.L., Ginebreda A., Barceló D. 

CSIC, IDÆA-Spain, Barcelona 

P20-067 ANALYSIS OF 18 PERFLUORINATED COMPOUNDS IN SOILS AND BIOTA FROM TIERRA DEL FUEGO AND ANTARCTIC BY LC-MS/MS 

Llorca M. 1 , Farré M. 1 , Alonso B. 2 , Barceló D. 1 

1 


2 

Área Táctica-Spain 

P20-068 GEOGRAPHIC AND TROPHIC PATTERNS OF OCS IN PELAGIC SEABIRDS FROM THE NORTHEAST ATLANTIC: 

A MULTI-SPECIES/MULTI-LOCALITY APPROACH 

Roscales J.L. 1 , Muñoz-Arnanz J. 1 , González-Solís J. 2 , Jiménez B. 1 

1 

Institute of Organic Chemistry, CSIC, Madrid 

2 


P20-069 ENANTIORESOLUTION OF SOME TRIAZOLE AND RELATED CHIRAL PESTICIDES BY CAPILLARY ELECTROPHORESIS-PARTIAL 

FILLING TECHNIQUE USING HIGHLY SULFATED β-CYCLODEXTRIN AS CHIRAL SELECTOR 

Martin-Biosca Y. 1 , Villanueva-Camañas R.M. 1 , Moreno L. 1 , Sagrado S. 1,2 , Medina-Hernandez M.J. 1 

1 


2 


P20-070 NANO-LIQUID CHROMATOGRAPHY - NANOSPRAY - MASS SPECTROMETRY FOR THE DETECTION AND QUANTIFICATION OF 

TRACES OF ENDOCRINE DISRUPTORS IN BENTHIC INVERTEBRATES 

Bulete A., Baudot R., Wiest L., Cren-Olive C. 

USR 059 CNRS, Service central d’analyse-France 

P20-071 ANALYSIS OF METHANOL AND ETHANOL IN AUTOMOTIVE EXHAUST 

Cirera-Domènech E., Ribas Font C., Gotor Navarra G., Comellas Riera L., Broto-Puig F. 

Ramon Llull University, Barcelona 

P20-072 VOLATILE ORGANIC COMPOUNDS VARIATIONS IN URBAN AND INDUSTRIAL AREAS BY CONTINUOUS MONITORING 

Barrero M.A., Goikoetxea M., Cantón L. 

University of the Basque Country 

P20-074 OCCURRENCE AND ELIMINATION OF EMERGING CONTAMINANTS IN SEWAGE TREATMENT PLANTS. USE OF POLAR ORGANIC 

CHEMICAL INTEGRATIVE SAMPLERS (POCIS) VS GRAB SAMPLING METHODOLOGIES 

Dávila T.J., Céspedes R., Valle M.C., De la Cal A., Ventura F., Martin J. 

Barcelona´s Water Laboratory (AGBAR and CETAQUA) 

P20-075 EVALUATION OF THE SPATIAL VARIABILITY OF ATMOSPHERIC VOLATILE ORGANIC COMPOUNDS 

Goikoetxea M., Barrero M.A., Cantón L. 


P20-076 INTEGRATED ASSESSMENT OF EMERGING COMPOUNDS BY THE COMBINATION OF POCIS AND UPLC-QQLIT-MS. 

APPLICATION IN MEDITERRANEAN SEWAGE TREATMENT PLANTS 

Céspedes-Sánchez R., Dávila T.J., De la Cal A., Martin-Alonso J., Ventura F. 

Barcelona´s Water Laboratory (AGBAR and CETAQUA)–Spain 

678 

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53


P20-077 ANALYTICAL CHARACTERISATION OF MANNOSYLERYTHRITOL LIPID BIO SURFACTANTS PRODUCED BY BIOSYNTHESIS BASED 

ON FEEDSTOCK SOURCES FROM AGRO-FOOD INDUSTRY 

Onghena M. 1 , Wijnants M. 1 , Pico Y. 2 , Covaci A. 3 , Lemiere F. 3 

1 

Karel de Grote University College-Spain 

2 


3 


P20-078 EVALUATION OF SEQUENTIAL INJECTION CHROMATOGRAPHY FOR THE ANALYSIS OF ANTICOCCIDIAL AGENTS IN AQUEOUS 

MATRICES 

Maya F. 1 , Björklund E. 2 , Estela J.M. 1 , Cerdà V. 1 

1 


2 

University of Copenhagen 

P20-079 STUDY OF THE VOLATILE ORGANIC COMPOUNDS (VOCS) EMISSION FROM AN URBAN WASTE LANDFILL IN MALLORCA 

(SPAIN) BY TD-GC-MS. 

Rodriguez-navas Gonzalez C., Forteza R., Cerdà V. 


P20-080 COMPARATIVE STUDY USING LARGE-VOLUME INJECTION COUPLED-COLUMN REVERSED PHASE LIQUID CHROMATOGRAPHY 

WITH FLUORESCENCE DETECTION AND LIQUID CHROMATOGRAPHY-TIME-OF-FLIGHT MASS SPECTROMETRY IN THE 

DETERMINATION OF BETA-BLOCKERS IN GROUNDWATER 

Parrilla M.M., Martínez Galera M., Parrilla P. 


P20-081 MATRIX EFFECTS IN PESTICIDE RESIDUE ANALYSIS: THE BATTLE IS NOT YET OVER 

Fernandez-Alba A.R. 


P20-082 EFFECTIVENESS OF GC-MS/MS AND GC×GC TOFMS TECHNIQUES ON THE DETERMINATION OF CHLORINATED PESTICIDES 

FROM DRY MATERIALS 

Kemellár B. 1 , Gómez M.J. 2 , Martínez Uroz M.A. 3 , Fernández-Alba A.R. 3 

1 

Corvinus University of Budapest-Hungary 

2 

Fundación IMDEA Agua 

3 


P20-083 ELECTROCHEMICALLY SYNTHESIZED MOLECULARLY IMPRINTED POLYMER FILMS: NOVEL SORBENTS FOR SPME 

Mazzotta E. 1 , Malitesta C. 1 , Díaz-Álvarez M. 2 , Martin-Esteban A. 2 

1 

Università del Salento-Italy 

2 


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54 


VENDOR 

SEMINARS

VENDOR SEMINAR SHIMADZU EUROPA GmbH 

Monday, September 13 (Auditorium 2 – 12.30 – 13.30 h) 

MODERN MULTIDIMENSIONAL TECHNOLOGIES: MDGC AND GCXGC(QMS) 

H.-U. Baier | Shimadzu Europa GmbH, Albert-Hahn-Str. 6-10, D-47269, Germany 

1. COMPREHENSIVE GCXGC(QMS) PESTICIDE ANALYIS PREPARED WITH QUECHERS: QUALITATIVE AND QUANTITATIVE ANALYSIS 

WITH AN ULTRA FAST QUADRUPOL MASS SPECTROMETER (20000 AMU/SEC) 

For high sensitivity analysis of pesticides prepared by QuEChERS in routine work comprehensive GCxGC(qMS) 

measurements were done using the fastest quadrupol mass spectrometric detector worldwide, the new GCMS-QP2010 

Ultra. This MS allows up to 100 scans (spectra)/sec at a scanning speed of 20000 amu/sec. 

Comprehensive GCxGCMS with thermal modulation has a high separation power for complex matrices. In this 

technique analytes are continuously refocused between 2 columns and released in part to a second column. Here 

an RTX-1 30m, 025mm, 0.25 μm was coupled to a BPX-50 1m, 0.15 mm, 0.15 μm with a loop of 1.6 m. The ZOEX ZX2 

Modulator was used (ZOEX corporation, USA) which allows thermal modulation without the use of liquid nitrogen. 

Peak widths observed in GCxGC are in the range of 200 to 600 msec at the base , depending on the inner diameter 

of the second dimension column. As 10 data points/ peak is needed for quantitative work the detector needs to 

allows to aquire every 20-60 msec a data point (spectra) and this in full scan over the desired mass range. The 

modulation frequency was set to 8 sec which results in 3 peaks per compound. The mass spectrometric detector 

was run in full scan mode with a mass range from 80 – 390 amu and a sampling frequency of 50 scans/sec. This 

resulted in more than 15 data points across each modulated peak which ensured quantitative analysis with sufficient 

precision. For method validation a clean pesticide standard diluted in acetonitrile was measured and the image was 

compared to the data recorded with an apple matrix spiked with 54 seleted pesticides. The concentratio ns range 

was 0.01 to 0.2 mg/kg. The whole method covers more than 500 pesticides. The limit of detection was below 1 g/kg. 

The GCMS EI classical spectra allow a precise identification of the target compounds using a search algorithm including 

linear retention index filtering. 

2. FLAVOUR&FRAGRANCE ANALYSIS: EASY HEART CUT MDGC WITH MASS SPECTROMETRIC DETECTION IN 1ST AND 2ND DIMENSION 

In flavour and fragrance analysis co-elutions are often observed in one-dimensional gas chromatography. In 

order to separate those regions they are transferred into a second column using heart cut multidimensional gas 

chromatography GC/GCMS. A Carbowax column with 30 m, 0.25 mm i.D. and 0.25 μm film was coupled to a chiral 

RTβ DEX 30m, 0.25 mm, 0.25 μm in the second dimension in order to separate chiral compounds. To have also 

identification of the peaks in the first dimension an FID/MS splitting was realised. This was created by a capillary 

split connection to feed the effluent at the FID (1st dimension) partly into MS simultaneously (1/15 relative to 

FID). For this a desactivated fused silica tube (1m, 0.175 mm ID) was lead via the interface from the GC of the first 

dimension into the second dimensional GCMS. Both columns the split connection and the second dimensional 

column are mounted into the MS detector by using a special connector. Chiral compounds can be transferred to 

the second column in a subsequent run. In cut runs the FID/MS splitting transfer line is blocked by a small pressure 

increase of an auxiliary pressure unit which reverses the flow in the splitting line. Several cuts were done on 

commercial flavours. The identification was done using the library FFNSC1.3 dedicated to Flavour and Fragrance 

compounds with linear retention indices. 


VENDOR SEMINAR WATERS 

Monday, September 13 (Auditorium 3b – 12.30 – 13.30 h) 

NEW TOOLS FOR EFFICIENT METHOD DEVELOPMENT - NEW CSH STATIONARY PHASES AND FUSION MD SOFTWARE 

Godo Bosch | Business Development Team Manager, Chemistry Operations, Europe ; Waters European Headquarters 

Jean-Michel Plankeele | UPLC® Product Manager, Europe & India ; Waters European Headquarters 

Method developers employ several tools to achieve optimum separations for their compounds, including different 

columns, mobile phases, and software packages. Ideally, the optimum method is obtained using the minimum 

number of screening runs possible. The most common methods development approach is to screen multiple 

columns and mobile phases to cover as much of the selectivity space as possible prior to optimization, which can be 

performed manually or with software. 

In this talk, we investigate how the wide range of selectivity provided by new stationary phases benefits the 

automated LC methods development approach for the separation of various types of compounds. We will discuss the 

integration of Fusion Methods DevelopmentTM software with EmpowerTM Chromatography Data Software and how 

the automated process that can be designed according to Quality by Design (QbD) guidelines will ensure reliable 

and robust methods prior to validation. A UPLC® system was employed, which is ideal for method development and 

makes realistic the systematic screening of all selectivity parameters. The system’s ability to blend multiple solvents 

automatically substantially improves chromatographic flexibility and throughput. 


57

VENDOR SEMINAR THERMO FISHER SCIENTIFIC 

Tuesday, September 14 (Auditorium 2 – 12.00 – 13.00 h) 

NEW ADVANCES IN QUATERNARY ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 

Sergio Guazzotti | Global Product Marketing Manager Liquid Chromatography, Thermo Fisher Scientific 

Techniques for qualitative and quantitative analysis that offer improvements in productivity are currently in high 

demand. Ultra High Performance Liquid Chromatography (UHPLC) offers several advantages when compared 

to HPLC including shorter analysis times, excellent separation efficiencies, and increased sensitivity. In order to 

improve flexibility and performance, systems that can operate both under HPLC and UHPLC conditions have been 

designed. The main drawback of these systems is the loss of performance when operating outside of a specific set 

of conditions for which they have been optimized. Another limitation was the lack of quaternary capabilities since 

this requires low pressure mixing and in the past low pressure mixing demanded larger delay (dwell) volumes for 

optimum performance; this is no longer the case due to newly developed pump technology to be discussed in this 

presentation. This technology employees unique force sensors to modulate pumping efficiency based on assessed 

mobile phase compressibility, thereby enabling the delivery of accurate and reproducible gradients with extremely 

low delay volumes. The force sensors employed in these pumps measure the actual force that is needed to move 

each piston. This force sensor signal can be used to measure compressibility of the displaced solvent in real-time, 

therefore variations in compressibility due to changes in mobile phase composition, temperature, etc. are accounted 

and compensated during each individual piston stroke resulting in a true volumetric, virtually pulsation free flow from 

the pump over its complete dynamic working range. This outstanding flow and pressure stability eliminates the need 

for a pulse dampening device which increases the ease of use. This technological breakthrough results in increased 

accuracy, precision, and reliability in pump performance under all operating conditions, permitting the development 

of flexible HPLC/ UHPLC systems with outstanding characteristics. Details on the development and performance of 

these systems will be presented. 

ANALYSIS OF PACKAGING MATERIAL RESIDUES IN FOOD 

E. Moyano, M.T. Galceran | Dpt. Analytical Chemistry, University of Barcelona 

Food as a major xenobiotics and heavy metal exposure route to humans is nowadays intensively studied and 

some typical food contaminants such as pesticides, dioxins, PCBs, PBDEs, methylmercury, lead, arsenic, etc. are 

well characterized in food, with high public and regulatory awareness. In contrast, the role of food and beverage 

packaging as an additional source of contaminants has received much less attention, even though food packaging 

contributes significantly to human xenobiotic exposure (Grob et al., 2006 ). This scenario is now changing since food 

packaging is considered a large but underestimated source of chemical food contamination. 

Food packaging can interact with the packaged foodstuff and this interaction can lead to food contact material (FCM) 

compounds leaching from the packaging to the food (migration process). Compounds that can leach from plastic 

FCMs are starting substances used for the initial polymerization step, like monomers or catalysts, and additives that 

are included during the manufacturing process to achieve special material properties. Starting substances can leach 

either because of incomplete polymerization during the formation of the material, or because of material degradation 

over time. Moreover, some side-products from the complex polymerization can also be leached. 

This presentation will focus on the use of fast liquid chromatography and mass spectrometry analytical tools to 

help us in the analysis of these packaging residues in food even at very low concentration levels (below ppb). Fast 

and ultra-high efficiency liquid chromatography to short the analysis time have been coupled to tandem mass 

spectrometry working in low resolution or in high resolution when improving selectivity and sensitivity is necessary 

or when some extra confirmation is required to avoid false positives. As study cases, the analysis of bisphenol A 

(BPA) and bisphenol B (BPF) simultaneously with other bisphenol compounds, which are intended to substitute 

them in a near future, in soft drinks below µg/L level are shown. The residue analysis of metal cans coatings are also 

discussed and the determination of some bisphenol A diglycidyl-ethers (BADGEs) and bisphenol F diglycidyl-ethers 

(BFDGEs) at µg/Kg level were identified in canned food and beverages. Finally, a fast and selective LC-MS/MS for 

the analysis of 11 UV-ink photoinitiators is also shown to determine them at below ppb level in carton packaged 

foods and packing materials. 


VENDOR SEMINAR DIONEX CORPORATION 

Tuesday, September 14 (Room 1 + 2 – 12.00 – 13.00 h) 

THE TRUE BENEFITS OF UHPLC FOR EVERYONE 

Wulff Niedner, Markus Martin, John Cottam | Dionex Corporation 

We all recognize the benefits that UHPLC brings to laboratories – faster and better separations, and quicker results. 

However, not all laboratories have switched over to UHPLC technologies – the main reasons being lack of support 

for existing applications, and budgetary restrictions. Dionex is happy to announce that these restrictions no longer 

apply – our continued research and development in the UHPLC field have yielded a range of UHPLC modules that 

support all chromatography applications and that can be configured in a way that meet the budgetary requirements 

of typical laboratories. The UltiMate 3000 HPLC system offers UHPLC compatibility across all modules, ensuring 

maximum performance for all users and all laboratories. Covering flows from 20 nL/min to 10 mL/min and offering 

a wide range of pumping, sampling, and detection modules, the UltiMate 3000 series provides solutions for all your 

chromatography needs. 

• UHPLC design philosophy throughout the whole range of nano, standard analytical, and RSLC 

• 620 bar maximum pressure for basic and standard analytical systems set a new benchmark in HPLC 

• x2 Dual Systems form a unique platform for routine analysis, increased productivity solutions, and advanced 

chromatographic techniques 

• Controlled using Chromeleon® Chromatography Data System software – providing Intelligent Functionality and 

Operational Simplicity 

• Offered with Viper and nanoViper fitting systems – fingertight, zero dead volume connections even at UHPLC 

pressures 

This is the true meaning of UHPLC for everyone. If you want to find out more about how to UHPLC-enable your lab, 

come to our vendor seminar on Tuesday, September 14th at 12:00-13:00 at room 1+2. 


59

VENDOR SEMINAR HiTC CHROMATOGRAPHYC SOLUTIONS 

Tuesday, September 14 (Room 3 + 4 – 12.00 – 13.00 h) 

A NEW CONCEPT OF TOF-MS SYSTEM FOR FAST GC AND GCxGC ANALYSIS 

Alessandro Casilli | DANI Instruments SA, Contone, Switzerland 

Nowadays in the analytical field, demands in terms of analysis speed and higher separation power are continuously 

increasing. These trends have been successfully addressed in Gas Chromatography (GC), in the last decades, 

through continuous developments in both Fast GC and Comprehensive Two-dimensional GC (GCxGC). However, 

when positive identifications are required, Mass Spectrometry (MS) is the technology capable of providing structural 

information and positive identification of chromatographically separated compounds. 

In conventional 1D-GC analyses the most commonly applied mass analyzers are ion trap, magnetic sectors, 

quadrupole (qMS), and time of flight. Out of these the most suitable detector for Fast GC and GCxGC applications is 

the TOF-MS. The use of qMS detection for Fast GC and GCxGC applications is widely reported in literature, however, 

when performing quantitative analyses several shortcomings can be observed such as the slow acquisition speed, 

the reduced acquired mass range, and the generation of skewed spectra. 

Different Fast GC and GCxGC methods have been optimized using qMS systems, however, taking in consideration 

that minimum 15-20 points are required to correctly reconstruct a Gaussian peak shape, structural information are 

completely sacrificed when setting the acquisition system to monitor single ions. 

TOF-MS is the only technique able to handle very narrow peaks, provide unskewed mass spectra and thus deliver 

full spectral information, even at very low concentration levels, guaranteeing optimal analytical responses in both 

qualitative and quantitative terms. 

The present work regards the investigation of complex matrices using Fast GC and GCxGC properly supported 

by the Time of Flight MS technology. Adopting the fast gas chromatographic approach the analysis time has been 

drastically reduced, meanwhile the GCxGC has enormously increased the separation power, in both techniques, the 

time of flight mass spectrometer has guaranteed distinguishable signals for identifying and quantifying the peaks of 

interest. 


VENDOR SEMINAR BRUKER CHEMICAL ANALYSIS DIVISION 

Tuesday, September 14 (Auditorium 3b – 12.00 – 13.00 h) 


Patrick Jeanville, Ph.D. | Bruker Chemical Analysis, Fremont, CA 94538 

There are currently over 1,000 pesticides in use worldwide in the production of foodstuffs. The use of pesticides is 

required to meet growing consumer demand for food at reasonable prices, including food out of season. There is a 

significant risk to human health and the environment due to increased pesticide use, poor agricultural practices and 

illegal use. Most countries have adopted strict regulations governing pesticide usage; imposing Maximum Residue 

Limits (MRLs) for pesticides. These levels are set well below perceived safety levels and therefore require analytical 

techniques that are sensitive, selective and robust. Pesticide control laboratories are now under increased pressure 

to maximize the range of pesticides detected in a single run and in complex matrices. By developing a multi-residue 

method, laboratories can lower their cost per analysis and increase sample throughput, thereby maximizing returns 

from the investment in analytical instrumentation. 

Conventional multi-target screening methods for monitoring of contaminants like pesticides in food are usually 

based on triple-quadrupole mass spectrometers (GC/QqQ-MS and LC/QqQ-MS). Advantage of triple-quadrupole 

approaches are: high selectivity’s/specificities, low detection limits and the ability to meet quantitative requirements 

in complex sample matrices. Emerging techniques like GC-ToF and LC-ToF are gaining popularity as alternative 

approaches for residue screening methods. Herein we will present information detailing both approaches, and the 

pro’s and con’s afforded by each technique. 


61

VENDOR SEMINAR AGILENT TECHNOLOGIES 

Wednesday, September 15 (Auditorium 2, 11.30 – 12.30 h) 


Christian Gotenfels | Product Manager | Agilent Technologies Germany 

Taegen Clary | Product Manager | Agilent Technologies USA 

Malgorzata Sierocinska | Technical Marketing Specialist | Agilent Technologies Germany 

In this seminar we will discuss three distinct aspects of how innovative technologies can address today’s analytical 

challenges. 

In the first part we will introduce the new Agilent 1200 Infinity Series, which delivers next levels of productivity, 

data quality and UV sensitivity. The lecture will demonstrate that all Agilent Infinity systems are compatible with 

conventional HPLC methods, allow for seamless method transferability thus enabling to run established, validated 

methods unchanged with no adjustments in conditions. The LC performance if fully scalable while keeping method 

transferability. 

The 1200 Infinity Series includes the new Agilent 1220 Infinity LC, the new Agilent 1260 Infinity LC and the enhanced 

Agilent 1290 Infinity LC, with seamless integration of column technology, instrumentation and software. The 1220 

and 1260 Infinity LCs offer enhanced HPLC and RRLC performance and are standardized on 600 bar system 

pressure, 80 Hz data-acquisition speed and 2x or 10x higher UV sensitivity. The 1220 and 1260 Infinity LCs are 100 

percent compatible with HPLC methods. The power range of 1200 Infinity Series LC systems is fully compatible with 

the latest column technology, such as 600 bar Poroshell and Zorbax RRHT or 1200 bar Zorbax RRHD columns. 

The second part of the seminar addresses advances in HPLC-based characterization of Biologics. We will discuss 

the development of a new Bio-inert HPLC system which operates at 600bar, while having a completely metal free 

sample flow path. Data will be presented to show resolution and productivity improvements when used with the 

new Bio MAb, Bio IEX and Bio SEC HPLC columns. Overall these enhancements lead to significant improvements 

for monoclonal antibody charge isoform analysis and aggregation monitoring. Furthermore, the identification and 

monitoring of the glycosylation states of monoclonal antibodies is critical to the development and manufacturing of a 

successful therapeutic. Agilent’s newest HPLC chip technology allows for enzymatic removal and analysis of these 

glycans in under 15 minutes. An overview of how this technology works will also be presented. 

The third section covers the advancement of implementing Capillary Flow Technology (CFT) devices for gas 

chromatography systems. Agilent CFT allows users to easily make reliable, leak-free, in-oven capillary connections 

that can stand up to the temperature extremes of a modern GC. It will add a degree of freedom and flexibility to your 

current workflow plus save analysis cycle time. It finds great usability for GC and GC/MS applications supporting 

various functions such as heart-cutting, detector selection, backflushing of whole column or pre-column, flow 

splitting, GCxGC modulating and non-venting capabilities for GC/MS systems. 


VENDOR SEMINAR AB SCIEX 

Wednesday, September 15 (Auditorium 3b, 11.30 – 12.30 h) 

SIMULTANEOUS COMPOUND QUANTIFICATION AND IDENTIFICATION USING HIGH RESOLUTION MS: THE AB SCIEX TripleTOF 

5600 SYSTEM 

Antonio Serna Sanz | AB Sciex 

Increasing productivity in discovery and development continues to be a primary goal not only in pharmaceutical 

research but in many other scientific disciplines. One of the most promising approaches to improving productivity 

is combining quantitative and qualitative analysis in a single analytical run (Quant / Qual). Quantitative information 

can be obtained on the parent compound while simultaneously acquiring qualitative information on metabolites in a 

completely automated fashion. 

Triple quadrupole instruments have been the workhorse instrument for the quantitative portion of this application due 

to their excellent sensitivity and high throughput. Accurate mass instruments such as Time of Flight (TOF) and orbital 

trapping analyzers have been used for metabolite identification due to their high mass accuracy and resolution. 

Unfortunately, orbital trapping involves a compromise between resolution and speed. Traditional TOF technology 

has shown limitations in linearity and requires internal calibration to maintain mass accuracy. Modifying study 

designs, sample preparation procedures, or chromatography in order to accommodate instrument limitations is not 

acceptable. 

Here we introduce a new technology, The AB SCIEX TripleTOF 5600 System, which combines the best attributes 

of triple quadrupoles and accurate mass analyzers in a single instrument. The highly innovative design of the 

TripleTOF 5600 System, combines a proven high performance triple quadrupole front end with the Accelerator 

TOF Analyzer, a state of the art accurate mass analyzer with unprecedented performance and stability. This results 

in linearity and sensitivity of a high performance triple quadrupole, combined with speed, high mass accuracy (2 ppm 

or less) and high resolution (30,000) of an accurate mass instrument, even at low mass for small molecules. 


63

VENDOR SEMINAR METHROM 

Thursday, September 16 (Auditorium 2, 10.00 – 11.00 h) 

AUTOMATED ION CHROMATOGRAPHIC DETERMINATIONS OVER SIX ORDERS OF MAGNITUDES 

Thomas Hartmann | Metrohm International Headquarters 

Trace-analysis methods require contamination-free sample handling and accurate calibration graphs. In particular, 

the method calibration in the low-ppb and ppt range is critical, as standard solutions in this range can hardly be 

handled without introducing errors. Moreover, if the sample concentration is outside the calibration range, laborious 

and error-prone dilution or preconcentration is required. 

These drawbacks are overcome by inline coupling of the patented 800 Dosino and intelligent ion chromatography 

systems. The Dosino enables high-precision dosing of variable solution volumes into the sample loop or 

preconcentration column of the ion chromatograph. The Dosino can exactly dose and transfer down to 0.2 µL liquid. 

Without compromising the accuracy of the results, a whole range of new possibilities opens up for determinations 

over wide concentration ranges. By using only one analytical setup and without additional rinsing, samples 

containing both ultratraces and high concentrations can be analyzed. 

Furthermore, the Metrohm intelligent Pick-up Technique (MiPuT) and the Metrohm intelligent Preconcentration 

Technique with Matrix Elimination (MiPCT-ME) enable the user to work with low sample volumes and to remove 

interfering sample matrices. Combined with automated multipoint calibration, these techniques cover a measuring 

range of six orders of magnitude. 

The combination of intelligent software and hardware allows the system to compare results, take logical decisions 

and carry them out: if, for example, the concentration of the analyte in the sample exceeds that of the calibration 

range, the system, after the first injection, compares peak areas, calculates the appropriate injection volume and 

automatically reinjects the sample. 

The presented inline techniques reduce the workload, extend the measuring range to six orders of magnitude, are 

free of carryover effects and significantly improve results`accuracy and reproducibility. Applications from different 

fields will be presented and discussed with regard to flexibility, recovery and accuracy. 


VENDOR SEMINAR LECO 

Thursday, September 16 (Room 1 + 2, 10.00 – 11.00 h) 

Chairman: Rui Rocha | LECO Spain 

APPLICATION OF GC- AND GCXGC-TOFMS FOR FOOD ANALYSIS. 

Sonja Hofmeister | Application Chemist, LECO European Technical | Centre (E.T.C) 

NEW DEVELOPMENTS ON GC- AND GCXGC-TOFMS. (SHOWCASING THE PEGASUS AND THE TRUTOF.) 

Dr. Sjaak de Koning (Ph.D) | European Application Manager, LECO | European Technical Centre (E.T.C) 

LECO design and manufacture the TruTOF® and Pegasus® GC- and GC×GC- Time of Flight Mass Spectrometers 

(TOFMS). LECO also provide a single software platform thus complimenting the final instrument solution design. 

The LECO ChromaTOF® software platform continuously evolves side-by-side customer feature requests and new 

instrument developments and has done for over 13 years. Ultimately, the advantages include flexibility and ease 

of use, fully automated data deconvolution without data export, personalised and loadable operator workspaces, 

optimised features for GC×GC operation and data processing. Hence, an efficient and fully automated data 

acquisition and data processing GC- and GC×GC-TOFMS instrument solutions are available for every type of 

laboratory set-up. 

The technical seminar will firstly detail the use of GC- and GC×GC-TOFMS in food analysis. Co-elution of 

compounds of interest with large amounts of matriz constituents, sensitivity and system stability are the greatest 

challenges for the analysis of 3-MCPD-ester in edible oils & fats. Application of Pegasus® 4D GCxGCTOFMS using 

large volume injection techniques for their analysis in food will be presented. Using this new approach resulted in an 

efficient and more reliable method and achieved a more sensitive 3-MCPD ester analysis in edible oils and fats. 

Secondly, new technological features and developments by LECO will be presented. The new aspects covered will 

include both hardware and software developments including 

i. ChromaTOF controlled Variable GC×GC Modulation Times - within each GC×GC chromatographic run for a more 

specialized analysis. This maximizes resolution in both the first and second dimensions. 

ii. Traditional LN2 and Consumable Free (CF) GC×GC systems – explanation of hardware features and the 

ChromaTOF software control. 

iii. LECO-Fiehn Metabolomics Library - with over 1,100 spectra of 700 unique metabolomics for metabolite 

identification in your most complex samples. Fully integrated within ChromaTOF, the library works seamlessly with 

the software’s Library Search function to automatically search for analyte matches without the need for importing 

or exporting data. 

iv. Statistical Compare – a new option within the ChromaTOF software that enables the user to view statistical 

comparisons between and within data sets acquired using LECO’s TruTOF GC- ToF-MS, Pegasus GC ToF- 

MS and/or Pegasus 4D GC×GC- ToF-MS instruments. Comparisons are made as part of the data processing 

procedures in which groups of samples are divided into different subsets or classes. The Statistical Compare 

feature in ChromaTOF then aligns data according to the peak tables using both mass spectral information and 

analyte retention time. 


65

VENDOR SEMINAR PERKIN ELMER 

Thursday, September 16 (Room 3 + 4, 10.00 – 11.00 h) 

PERKINELMER INITIATIVES IN ENVIRONMENTAL AND FOOD ANALYSIS - NEW FLEXAR SQ 300MS: THE ULTIMATE MOLECULAR 

WEIGHT MACHINE ADVANCES IN HYPHENATIONTG-GCMS: THE MORE COMPLETE CHARACTERIZATION OF COMPLEX MATRICES 

Massimo Santoro | PerkinElmer, Shelton, USA 

PerkinElmer is providing laboratories with application-focused solutions that meet their regulatory, technology and 

operational requirements. PerkinElmer analytical solutions are critical in helping companies to monitor water and air 

pollution, soil contamination and many other aspects of environmental safety. PerkinElmer solutions are also used 

in food quality and food safety research and testing, (which encompasses authenticity, potency, manufacturing and 

contamination) and can directly impact people’s health and wellbeing. 

The technological advances in instrumentation that have been developed by PerkinElmer provide innovative 

solutions to support a number of application areas including food and environmental. 

- Designed for HPLC and UHPLC applications, the FlexarTM SQ 300 MS detector combines the most efficient 

chromatographic separation with fast MS detection capabilities, to attain a new level of sample insight through 

speed, confirmatory analysis and best-in-class sensitivity. 

Patented technologies are utilized in the FlexarTM SQ 300 MS detector to enable efficient and reliable ionization of 

compounds, in positive and negative mode, for complete sample information. 

The highest limits of detection sensitivity required in food or environmental assays are achieved thanks to a patented 

multi-stage ion path that brings an unsurpassed level of performance. The Collision Induced Dissociation (CID) 

technology for detailed structural information, provides reproducible confirmatory analysis. 

- Advances in hyphenation technology enable the analysis of complex matrices in a single analytical method. 

Combining TGA with gas chromatography mass spectrometry (GC/MS) allows for a more complete characterization 

of the material 

TGA analysis allows quantification of the weight loss of a material at specific temperatures. MS increases the power 

of the technique by providing the ability to identify the species evolved during thermal analysis. TG-GC/MS adds the 

additional capability of chromatographic separation of co evolved gases. The improved separation by the GC/MS 

makes data interpretation easier than TG-MS alone. TG-GC/MS facilitates the separation of fairly complex mixtures 

with the minimal sample preparation by using the TGA to volatilize the sample. 


VENDOR SEMINAR BISCHOFF CHROMATOGRAPHY 

Thursday, September 16 (Auditorium 3b, 10.00 – 11.00 h) 

“SPEED, SELECTIVITY AND RESOLUTION, KEY ASPECTS FOR A SEPARATION IN MODERN LIQUID CHROMATOGRAPHY” 

Dr. Stefan Lamotte | Bischoff Chromatography, Leonberg, Germany 

In this seminar different ways in method development or method optimization will be presented. Also the transfer 

of an existing method into a high speed method will be shown. Practical hints for the daily work in an HPLC lab are 

given in numerous examples. Software will be presented to calculate and to simulate separations under the influence 

of speed, selectivity and resolution. 


67

PLENARY 

SESSIONS


PS1 RECENT CONTRIBUTIONS OF CHROMATOGRAPHY TO THE UNDERSTANDING OF DRUG METABOLISM 

Prof. Ian D. Wilson * 

AstraZeneca, CPD & DMPK-UK 

* 

E-Mail: ian.wilson@astrazeneca.com 

Área: Liquid Chromatography 

Recent Contributions of Chromatography to the Understanding of Drug Metabolism Ian D Wilson CPD & DMPK, 

AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK. The application of LC coupled to 

mass spectrometry is now ubiquitous in the study of drug metabolism offering speed, sensitivity, specificity and 

high structural information content for both the discovery and development phases of drug metabolism studies. 

Indeed, it is difficult to imagine how modern drug discovery programs could function in the absence of HPLC, or 

UHPLC, -MS for drug metabolite detection and identification. Examples will be provided that illustrate the value of 

LC-MS in the investigation of in vitro studies for the detection of reactive metabolites and metabolite profiling, and 

in vivo drug metabolism in conventional, transgenic and chimeric animal models. As well as conventional LC-MSbased 

investigations the potential of more unusual techniques such as ICPMS that can, in particular circumstances, 

overcome some of the limitations of LC-MS for the quantification of unknown will be described. Lastly, potential 

future developments that might provide further advances that can be applied to the determination of the metabolic 

fate of drugs/xenobiotics will be highlighted. 


69


PS2 SEPARATION SCIENCE, KEY TO SUCCESSFUL BIOPHARMACEUTICALS 

Prof. Georges Guiochon * 

University of Tennessee, Chemistry-USA 

* 

E-Mail: guiochon@utk.edu 

Área: Liquid Chromatography 

Separation Science, Key to Successful Biopharmaceuticals Georges Guiochon (a) and Lois Ann Beaver (b) (a) 

Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA and Division of Chemical and 

Analytical Sciences, Oak Ridge National Laboratory. (b) LAB Enterprises, 4515 Willard Avenue, Chevy Chase, MD, 

20815-3669 As global demand increases for targeted effective therapies, biopharmaceuticals continue to grow 

in importance. At least 300 such products are in use. Initially, the excitement and focus was on their upstream 

manufacturing, using living organisms, but as yields increased, it became evident that chromatography and other 

downstream purification steps would play a major role in the safety of use and the economics of production. 

Changes in separation science approaches are inevitable as yields and costs continue to rise. The size, complexity 

and behavior of biomolecules are not compatible with the traditional chromatographic techniques used for the 

purification of small molecular weight compounds. The retention mechanisms necessary for the separation of 

biopharmaceuticals should be different from those used for the purification of small molecules. Avoidance of 

changes in molecular configuration requires care in the choice of the mobile phase. Soft molecular interactions 

rather than harsh adsorption seem necessary. The energy involved in these molecular interactions must be small 

to permit rapid equilibration between mobile and stationary phases. Due to the low molecular diffusivity of large 

molecules, mass transfer kinetics tend to be slow. The packing materials used must have large average pore sizes. 

All this explains the prevalence of polymer based materials over the classical silica-based supports. The issue 

of column packing comes into play. Engineers must learn how to gently but efficiently pack these soft particles. 

Separation and purification demands are further complicated by challenges presented by impurities associated with 

the living organisms used in production. This presentation will consider potential approaches to resolve these issues. 

70 



PS3 FACETS OF SPECIFIC DETECTION IN CHROMATOGRAPHY AND ELECTROPHORESIS: PROGRESS IN SPECIATION ANALYSIS 

Prof. Ryszard Lobinski * 

* 

E-Mail: ryszard.lobinski@univ-pau.fr 

Área: Hyphenated Technique 

The need for the determination of metal-containing environmental contaminants, metallodrug metabolites, and 

interest in the mechanism of action of trace elements in living systems resulted in a research field, referred to as 

speciation analysis [1]. Gas chromatographers were the first to appreciate the advantages of element selective 

detectors for the sensitive compound-specific analysis. Element-specific detectors allowed the elimination of the 

non-specific background and thus a considerable decrease in the detection limits. The technical solutions varied 

from the moderately selective electron capture detection and flame photometry to truly element-specific microwaveinduced 

plasma atomic emission detection (MIP AED) or isotope specific inductively coupled plasma mass 

spectrometry (ICP MS). The specificity was achieved by targeting a heteroelement, i.e. a metal, metalloid or nonmetal 

(e.g. sulphur or iodine). The reported detection limits in GC, LC, capillary electrophoresis or gel electrophoresis 

(by laser ablation ICP MS imaging) were at the femtogram levels [2]. The analysis was quantitative but was limited to 

known molecules for which standards were available; the identification was based on the retention time matching. 

The development of molecular MS in the 90ties raised hopes for the specific detection of molecules by means 

of their molecular mass. However, the complexity of real-world samples, in combination with the poor resolution 

of the typically used quadrupole analysers, resulted in the impossibility of reaching a true specificity, especially 

that signals from trace level molecules were eclipsed by the co-eluting compounds. This would change with the 

availability of tandem mass spectrometers operating in the multiple reaction monitoring (MRM) mode and of high 

resolution mass spectrometers. The possibility of the simultaneous monitoring of the parent ion and its fragment(s) 

offers an additional contribution to the selectivity and revolutionized the analysis for environmental contaminants, 

illicit substances, drug metabolites and biomarkers. Advances in the design of electrospray sources and triple quad 

analysers have resulted over the last decade in a dramatic decrease in detection limits making ESI QqQMS a viable 

alternative to ICP MS in speciation analysis. High resolution mass spectrometers, such as FT ICR MS or, FT orbital 

trap MS offer a possibility of the specific detection of molecules in GC or HPLC without their containing necessarily 

a heteroelement. The sub-ppm mass accuracy is specific to a given empiric formula. This mass accuracy and the 

rapidity of acquisition of tandem or even multistage mass spectra allow the unique confirmation of the compound’s 

identity. The high intrascan dynamic range assures a high sensitivity comparable with that of ICP MS. The lecture 

discusses the contribution, the advantages and limitations, and the development trends of various specific detection 

modes for the detection, identification and quantitative determination of heteroatom-containing biomolecules: 

contaminants, metabolites and proteins. 1. D.M. Templeton, F. Ariese, R. Cornelis, L.-G. Danielsson, H. Muntau, 

H.P. Van Leeuwen, R. Lobinski (2000) Pure and Applied Chemistry, 2000, 72,1453-1470. 2. J. Szpunar, R. Lobinski, 

Hyphenated Techniques in Speciation Analysis, Royal Society of Chemistry, Cambridge, 2003. 


71


PS4 PROGRESS IN THE OMICS ANALYSIS OF FOODS: FOODOMICS 

Prof. Alejandro Cifuentes * , Garcia-Cañas V., Simo C. Ibañez C., Herrero M., Ibañez E. 

CSIC, IFI 

* 

E-Mail: acifuentes@ifi.csic.es 

Área: Hyphenated Technique 

Nowadays, boundaries among the different research disciplines are becoming more and more diffuse giving rise 

to impressive possibilities in the emerging interdisciplinary areas. In food science and nutrition, this trend has 

given rise to the development of new methodologies in which advanced analytical methodologies are applied to 

investigate topics considered unapproachable few years ago. As a result, researchers in food science and nutrition 

are being pushed to move from classical methodologies to more advanced strategies usually borrowing methods well 

established in medical, pharmacological and/or biotechnology research. In this context, we have recently defined 

for the first time in a SCI journal the new field of Foodomics (A. Cifuentes, J. Chromatogr. A, 1216 (2009) 7109) as 

a discipline that studies the food and nutrition domains through the application of modern omics technologies. 

Thus, Foodomics would cover e.g., the development of new transgenic foods using molecular tools, the genomic/ 

transcriptomic/proteomic and/or metabolomic study of foods used for compounds profiling/authenticity and/or 

biomarkers detection, new investigations on food functions including functional foods and/or functional ingredients 

through in vivo assays and/or clinical trials, etc. Consequently, Nutrigenomics and Nutrigenetics approaches can 

be considered as subdisciplines of the more general Foodomics field. In this work, we will show the development of 

advanced analytical methodologies carried out by our group and their application to solve different problems in the 

new Foodomics field. 

72 



PS5 NEW FINDINGS IN HIGH SPEED, HIGH TEMPERATURE AND HIGH PRESSURE CHROMATOGRAPHY 

Prof. Jean-Luc Veuthey * , Guillarme D., Rudaz S. 

University of Geneva, School of pharmaceutical sciences 

* 

E-Mail: jean-luc.veuthey@unige.ch Tel. 

Área: Fast Separations 

In the last ten years, a strong development has emerged in Liquid Chromatography (LC) in terms of chromatographic 

supports and instrumentations to achieve ultra-fast and/or highly efficient separations [1]. In terms of 

instrumentation, systems compatible with pressures up to 1200 bar and able to work adequately with mobile phase 

temperatures beyond 100°C are now commercially available from several providers. In addition, the extra-column as 

well as gradient delay volumes have been drastically reduced to meet the requirements of fast-LC with short columns 

at elevated mobile phase flow rate. Regarding the chromatographic packing, two types of particle geometries 

present nowadays an obvious interest, namely the sub-2µm porous particles commercially available since 2004 

[2] (i.e. UHPLC) and the sub-3µm superficially porous particles made of a core shell surrounded by a thin layer of 

porous material and originally developed by J.J. Kirkland in the 90’s (i.e. fused-core or core-shell technologies) 

[3]. In the present contribution, these two types of promising chromatographic supports will be compared in terms 

of efficiency, peak capacity, generated pressure drop, loadability and selectivity. For this purpose, various small 

molecules, peptides as well as proteins will be considered as model analytes. During this evaluation, the mobile 

phase temperature was systematically considered as an additional parameter to further improve performance. 

[1] D. Guillarme, J. Ruta, S. Rudaz, J.L. Veuthey, New trends in fast and high resolution liquid chromatography: a 

critical comparison of existing approaches, Analytical and Bioanalytical Chemistry, 2010, 397: 1069-1082 [2] J.R. 

Mazzeo, U.D. Neue, M. Kele, R.S. Plumb, A new separation technique takes advantage of sub-2µm porous particles, 

Analytical chemistry, 2005, 77: 460A-467A [3] J.J. Kirkland, superficially porous silica microspheres for the fast highperformance 

liquid-chromatography of macromolecules, Analytical chemistry, 1992, 64: 1239-1245 


73


PS6 ELECTROMIGRATION METHODS FOR THE SEPARATION OF BACTERIA. EFFECT OF CHARGE DISTRIBUTION 

Prof. Boguslaw Buszewski * 

Nicolaus Copernicus Univeristy, Faculty of Chemistry, chair of Environmental Chemistry & Bioanalytics 

* 

E-Mail: bbusz@chem.umk.pl Tel. 

Área: Miscellaneus 

Microorganisms are the new “smart” particles that can change their surface properties based on interactions with 

each other and the surrounding environment. This change in surface characteristics dictate how bacterial cells 

may interact when forming biofilms or aggregates. On the other hand the fused silica capillary surface, similarly 

as a packing material particles in HPLC shows numerous types of interactions with the solvent and the solutes. In 

the case of biomolecules strong adhesion and aggregation effects are observed. Bacterial adhesion and surface 

colonization are highly correlated with physicochemical properties of microorganism surface (zeta potential). 

Different kind of biomolecules with different surface properties show different adhesion kinetics and affinity for the 

substrate. Interactions between molecules vary from weak association to strong electrostatic or covalent bonds. 

These interactions are unstable and this analytes can easily be removed from adsorbed surfaces by different 

solvents or by agitation. Nevertheless, when bioanalytes are in close proximity to a fused silica surface or coated 

capillary wall they can form short-range specific interactions, which are very stable. In this work physicochemical 

surface characteristics of bacteria were measured to establish their role in bacterial adhesion and aggregation 

on the basis on electrophoretic behavior of three selected bacteria species, namely H. pylori, S. aureus, E. coli. 

The number and the shape of peaks obtained on the electropherograms were connected with the zeta potential 

measurements and in-line microscope observation using specially designed CE – fluorescence stereomicroscope 

setup. These results suggest that the lower the zeta potential the higher number of smaller peaks were detected. 

The direct microscopic observation of electrophoretic movement proved the presence of many small aggregates 

originating from individual or clustered bacterial cells. Although the exact mechanisms are not completely 

understood, it is believed that extracellular proteins (e.g., outer membrane proteins, flagella, etc.), polysaccharides, 

and lipopolysaccharides play important role in strengthening bonds between biomolecules and the stationary phase 

and/or between other active molecules. 

74 



PS7 CAPILLARY AND FREE-FLOW ELECTROPHORESIS APPLIED TO ANALYSIS, PURIFICATION AND CHARACTERIZATION OF 

BIOLOGICALLY ACTIVE PEPTIDES 

Dr. Vaclav Kasicka * , Sazelova P., Solinova V., Koval D., Ehala S. 

Institute of Organic Chemistry & Biochemistry, Acad. Sci. CR, Electromigration methods-Czech Republic. 

* 

E- Mail: kasicka@uochb.cas.cz 

Área: Electrodriven Separations 

Capillary zone electrophoresis (CZE) is now widely used as a high-performance and high-sensitive separation 

technique for analysis and physico-chemical and biochemical characterization of peptides [1]. In this area, it has 

become a recognized counterpart and/or complement to high-performance liquid chromatography (HPLC). However, 

for preparative purposes, the application potential of CZE remains relatively limited, partially due to the more 

complicated conversion of analytical capillary systems into preparative ones, but mainly due to the low preparative 

capacity of the capillary systems (nanogram to microgram amount per run). Principle solution of this problem is 

to realize the electrophoretic separation in the continuous free-flow arrangement in the rectangular flow-through 

electrophoretic chamber [2, 3]. In this free-flow zone electrophoresis (FFZE) format, it is possible to increase the 

separation capacity up to 50-100 milligram of substance per hour. Since the CZE and FFZE formats are based on 

the same separation principle (zone electrophoresis) and both are performed in the carrierless medium with the 

same composition of the background electrolytes, a direct correlation exists between these two formats [4]. Based 

on this correlation, the procedure for conversion of analytical CZE separations to preparative FFZE separations 

has been developed and applications of this procedure for preparative separations of biologically active peptides 

(insulin derivatives and fragments, thymopentin, gonadotropin releasing hormones) will be demonstrated. First, CZE 

was used for analysis of these peptide preparations (crude or HPLC purified products) and suitable conditions for 

their analyses and for separation of their admixtures were developed in the capillary microscale minimizing time and 

material expenses for the development of optimized separation conditions. Then, the optimized CZE separations 

were converted to FFZE separations, and FFZE was used for preparative purification of these peptides. The high 

purity degree of peptides prepared by FFZE was confirmed by CZE and/or HPLC analyses. Carrierless media of both 

CZE and FFZE allow separation of peptides in mild conditions, under which their biological activity is preserved and 

their losses are minimized. The work was supported by the GACR, grant no. 203/08/1428, 203/09/0675, and by the 

ASCR, Research Project AV0Z40550506. Ms V. Liskova is thanked for her skilful technical assistance. [1] V. Kasicka. 

Recent advances in CE and CEC of peptides (2007-2009). Electrophoresis 2010, 31, 122-146. [2] V. Kasicka. From 

micro to macro: Conversion of capillary electrophoretic separations of biomolecules and bioparticles to preparative 

free-flow electrophoresis scale. Electrophoresis 2009, 30, S40-S52. [3] V. Kasicka, Analytical and preparative 

separations of peptides by capillary and free-flow zone electrophoresis, in H. Y. Aboul-Enein (Ed.), Analytical and 

preparative separation methods of biomacromolecules, Marcel Dekker, Inc., New York, 1999, pp. 39-97. [4] V. 

Kasicka, Z. Prusik, P. Sazelova, J. Jiráček, T. Barth, Theory of the correlation between capillary and free-flow zone 

electrophoresis and its use for the conversion of analytical capillary separations to continuous free-flow preparative 

processes. J. Chromatogr. A, 1998, 796, 211 220. 


75


PS8 CHALLENGES AND FUTURE TRENDS PF PESTICIDE FOOD RESIDUE CONTROL IN EUROPE 

Amadeo R. Fernandez-Alba * 

Head of the European reference Laboratory for Pesticde Residues in Fruits and Vegetables. University of Almería. 

* 

E-Mail: amadeo@ual.es 

The use of pesticides to control pests of crops, to eliminate parasites and insects of livestock, and to maintain 

hygienic conditions in production lines or during storage, has played a major role in expanding the availability of crop 

products at a reasonable price to the consumers. But the consequences can drive to unacceptable residue levels for 

human health. In Europe coordinated by the DG SANCO, important decisions have been taken during the last years, 

upgrading few regulations concerning pesticide residues. Those measures are accomplished by the support of the 

EFSA. In 2005, the EU MRL Regulation 396/2005 was introduced; it became effective on September 1, 2008. EEC 

396/2005 has the goal to harmonize all MRLs / tolerances within Europe; it gives a very high priority to consumer 

safety and, consequently to dietary risk assessment. In the regulation, the ALARA principle for MRLs is mentioned. 

For non-target crops and for actives being not registered in Europe, “default” MRLs are set. It also includes several 

annexes which are under permanent revision and update. In 2009, the European Parliament voted for the Registration 

Regulation 1107/2009 (“revision of 91/414”); it will become effective in June 2011. The new regulation includes several 

points as e.g. hazard-based cut-off criteria, zonal authorisations and conditions for comparative assessment. 

Consequence of that, main focus will be given to the impact on residue analytical methods. Such situation requires 

important efforts in harmonization and improvements of the food control laboratories. To help in that direction and 

supported by the DG SANCO four European Reference Laboratories were created on pesticide residues. The main 

aim of them is to coordinate and improve the official network of the pesticide control laboratories and to assist to 

the DG SANCO in all issues related with technical difficulties and trade problems consequence of those compounds. 

From 2006 to now the EURL has developed and coordinated important activities updating analytical methodologies 

and quality control guidelines according with the new exigencies and new intrumentation available especially based 

on gas or liquid chromatography coupled to mass spectrometry. In this work the main outcomes of those activities 

are presented and also are given the critical points detected to be developed in the near future. 

76 



PS9 CHIRAL ANALYSIS: A CHALLENGING ISSUE FOR MINIATURIZED TECHNIQUES 

Dr. Salvatore Fanali * 

Institute of Chemical Methodologies, Italian National Research Council-Italy 

* 

EMail: salvatore.fanali@imc.cnr.it 

Área: Enantioseparations 

Nano-liquid chromatography (nano-LC) is recent developed miniaturized technique of high-performance liquid 

chromatography (HPLC) with great potentiality in the analytical field. Since its introduction offered several 

advantages over conventional HPLC such as high efficiency, ability to work with minute sample sizes, low 

consumption of mobile and stationary phases, good compatibility with mass spectrometry (MS), etc.[1, 2]. Nano-LC 

has been widely investigated from a point of view of both theory and instrumentation including column preparation 

and detection [3]. The methodology allowed many significant applications in various fields such as proteomic, 

pharmaceutical, food, environmental, enantiomers separation etc. [4, 5]. The analysis of chiral compounds has been 

recently investigated because a challenging issue for nano-LC as well as for electrodriven miniaturized techniques. In 

this communication we present a short introduction of nano-LC focusing on the main advantages of this miniaturized 

method in solving analytical problems. Furthermore some selected applications concerning chiral separations of 

compounds of pharmaceutical, food and environmental interest will also be proposed. Comparison between nano- 

LC and electrodriven miniaturized techniques will also be presented. 

References: [1] Karlsson, K. E., Novotny, M., Anal. Chem. 1988, 60, 1662. [2] Fanali, S., D’Orazio, G., Lomsadze, K., 

Samakashvili, S., Chankvetadze, B., J. Chromatogr. A 2010, 1217, 1166. [3] Vissers, J. P. C., J. Chromatogr. A 1999, 

856, 117. [4] Ishihama, Y., J. Chromatogr. A 2005, 1067, 73. [5] Hernandez-Borges, J., Aturki, Z., Rocco, A., Fanali, S., 

J. Sep. Sci. 2007, 30, 1589. 


77

ORAL 

SESSIONS 

SESSIONS 







S7 FUNDAMENTALS OF CHROMATOGRAPHY 

S8 EXTRACTION 

S9 SPECIATION ANALYSIS 






S15 NEW CHROMATOGRAPHIC APPLICATIONS FOR BIOMARKERS 

S16 GAS CHROMATOGRAPHY 

S17 FAST SEPARATIONS 



S20 INNOVATIVE CHROMATOGRAPHIC APPLICATIONS 











S31 MULTIDIMENSIONAL CHROMATOGRAPHY 

S32 FORENSICS AND ENVIRONMENTAL RESEARCH 





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PHARMACEUTICAL 

S1APPLICATIONS 

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SESSIONS

ORAL SESSION 1 - CLINIC AND PHARMACEUTICAL APPLICATIONS 

S1-01 ELECTROCHEMISTRY COUPLED ONLINE TO LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRY AS A 

COMPLEMENTARY TOOL IN DRUG METABOLISM STUDIES 

Vielhaber T., Baumann A., Jahn S., Melles D., Lohmann W., Karst U. 


Corresponding author e-mail: uk@uni-muenster.de 

In the human body, the biotransformation of drugs can be described as a series of metabolic reactions, ideally 

transforming xenobiotics into less toxic and more polar compounds, which can be easily excreted from the human 

body. In many cases, the metabolic pathway is initiated by oxidation reactions (phase I metabolism), being catalysed 

by enzymes of the cytochrome P450 superfamily. To investigate the metabolites generated during the oxidative 

phase I metabolism, in vivo and in vitro experiments are widely applied. As reactive metabolites tend to undergo 

covalent binding to cellular macromolecules, the isolation and identification of the metabolites in these experiments 

is often hampered. Thus, a purely instrumental method has been developed using an electrochemical cell coupled 

online to HPLC-ToF/MS (EC-LC-MS). The EC-LC-MS system has been proven its utility in a number of metabolic 

studies, including the detection of reactive metabolites of drugs like acetaminophenone and amodiaquine (1). Recent 

instrumental developments have enlarged the application window for this technique. Using electrochemical thin layer 

cells with platinum or boron doped diamond (BDD) working electrodes enables the electrochemical generation of 

hydroxy radicals. Thus, for the first time aliphatic and allylic hydroxylation processes of drugs can been simulated in 

an online EC-LC-MS setup (2). The experimental EC-LC-MS set-up has been further extended by different detection 

techniques. Based on inductively coupled plasma mass spectrometry (ICP/MS) detection, providing low limits of 

detection and elementspecific quantification, iodine containing drugs like amiodarone (3) or arsenic containing drugs 

like melarprosol can be studied in an EC-LC-ICP/MS set-up. A simple set-up, consisting of the direct hyphenation of 

electrochemistry and mass spectrometry has been established as valuable tool in fast screening of oxidative lable 

sites in drugs. By plotting three-dimensional “mass voltammograms”, potential oxidation products can be predicted 

at an early stage of drug development. The detailed comparison between electrochemistry, conventional microsomal 

based approaches and in vivo experiments for different drug molecules has underlined the fact that electrochemistry 

is a valuable complementary tool to study oxidation product of pharmaceuticals. EC-LC-MS has the potential 

to predict metabolites which can not be foreseen in microsomal approaches due to either binding of reactive 

metabolites to cellular macromolecules or due to the different distribution of P450 enzymes in each organism. (1) 

Lohmann W., Karst U., Anal Bioanal Chem, 2008, 391, 79 (2) Baumann A., Schubert B., Oberacher H., Karst U., J 

Chromatogr A, 2009, 1216, 3192 (3) Lohmann W., Meermann M., Möller I., Scheffer A., Karst U., Anal Chem, 2008, 80, 

9769 

80 



S1-02 APPLICATION OF AN EXPERIMENTAL DESIGN TO OPTIMIZE THE METABOLIC PROFILES OF CINITAPRIDE BY UPLC 

Marquez H. 1 , Albertí J. 1 , Salvá M. 1 , Saurina J. 2 , Sentellas S. 1 

1 

Almirall SA 

2 


Corresponding author e-mail: sonia.sentellas@almirall.com 

The complete characterization of the metabolism of new drugs is important in order to predict possible adverse 

effects. This characterization includes the identification of the main metabolites and their possible relationship 

with toxic effects, the determination of the metabolic clearance, as well as the prediction of drug-drug interactions. 

Cinitapride (4-amino-N-[1-(3-cyclohexen-1-yl-methyl)-4-piperidinyl]-2-ethoxy-5-nitrobenzamide) is a gastrointestinal 

stimulant with prokinetic activity, developed and marketed in Spain and Mexico by Almirall, S.A. This compound is 

metabolised to three main metabolites and to a large number of minor metabolites, which made difficult to establish 

a good separation method. Indeed, the optimization of the separation when dealing with metabolic studies is usually 

difficult due to the wide variety of major and minor close metabolites that are formed. 

In this communication, the UPLC separation has been thoroughly optimized to reach the best resolution of the most 

significant metabolites with a minimum analysis time. In particular, the optimization criterion relies on a desirability 

function D = (d1 d2), being d1 and d2 the individual desirabilities of number of resolved peaks and analysis time, 

respectively. 

Experimental variables to be considered in the optimization study comprise flow rate, pH and the gradient profile. 

Flow rate and pH have been studied independently. The gradient profile, which consists of a MeCN ramp, has been 

optimized by means of a 2-factor grid design. In more detail, the initial and final percentages of MeCN in the mobile 

phase have been varied as follows: initial MeCN%, 5, 10 and 15%; final MeCN%, 20, 30 and 50%. In all cases, the 

ramp time has been set to 15 min. The optimal separation conditions finally chosen were: mobile phase A: Acetic 

acid/ammonium acetate (pH 6.5); mobile phase B: MeCN; with a linear gradient profile from 5 to 32 % B in 16 min 

with a flow rate of 350 µl/min. As a result, up to 24 peaks have been separated by UPLC in samples from metabolism 

studies, among them at least 14 corresponded to cinitapride metabolites. The method will be applied to establish 

the kinetic parameters of cinitapride metabolites in studies using human liver subcellular fractions and recombinant 

drug-metabolising enzymes. 


81


S1-03 IN VITRO CAPTURING OF VARIOUS LIPOPHILIC ILLICIT DRUGS BY LIPID DISPERSIONS STUDIED BY ELECTROKINETIC 

CAPILLARY CHROMATOGRAPHY AND FLUORESCENCE POLARIZATION 

Lokajová J. 1 , Pukkila J. 2 , Holopainen J.M. 1 , Wiedmer S. 1 

1 


2 

Finnish Police, National Bureau of Investigation 

Corresponding author e-mail: susanne.wiedmer@helsinki.fi 

Fatal drug overdoses are a cause for concern all over the world. We present here a lipid-based formulation, which 

has a strong affinity for some common illicit street drugs and can be used in the future as an in vivo lipid ‘sink’. In 

this study, in vitro interactions of nine lipophilic drugs and three lipid dispersions were determined by electrokinetic 

capillary chromatography and fluorescence polarization. Two lipid dispersions, zwitterionic 

1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and an anionic mixture of POPC and 

palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) were tested and compared with a commercial 

lipid dispersion Intralipid®, which has been successfully used for resuscitation of patients in cases of anesthetic 

overdoses. The interactions between dispersions and the drugs were quantified by means of retention factors and 

distribution constants, which makes the results highly comparable to those obtained from any other formulation 

of lipids. The results demonstrate stronger interaction between the drugs and the POPC/POPG artificial liposome 

dispersion than with the commercial Intralipid dispersion. Data on the interactions between some local anesthetics 

and lipid dispersions will be presented as well and we will demonstrate, that the liposome dispersion composed of 

POPC and POPG functions as a lipid ‘sink’ for efficient in vitro entrapment of various lipophilic drugs. 

82 



S1-04 SEPARATION OF ANTIPARKINSON DRUGS BY CAPILLARY ELECTROPHORESIS COUPLED WITH AMPEROMETRIC DETECTION 

BY BORON DOPED DIAMOND ELECTRODE 

Zhou L. 1 , Corcoran C. 1 , Luong HT J. 2 , Glennon D. J. 1 

1 


2 

National Research Council Canada 

Corresponding author e-mail: l.zhou@student.ucc.ie 

Anticholinergic agents, such as triesiphenydile, biperiden and orphenadrine, are used for the treatment of 

Parkinson’s disease. All these drugs have the ability to correct the relative excess of cholinergic activity which is 

thought to happen as a consequence of dopamine deficit. The drugs are administered orally at doses usually ranging 

from 2 to 12 mg/day. Side effects include nervousness, dizziness and gastro-intestinal problems, while cognitive 

processes can be impaired, especially in the elderly. The capillary electrophoresis coupled with amperometric 

detection can separate anticholinergic agents with neurotransmitters through human urine samples. Compared to 

UV detection, amperometric detection with a boron doped diamond electrode has been proved more sensitive and 

effective for the detection. Boron doped diamond electrode can give very attractive sensitivity down to 1 µM. A fused 

silica capillary (45 cm × 50 µm i.d.) was dynamically coated with poly (diallyldimethylammonium chloride) (PDDA) and 

employed for the separation of trihexyphenidyl, biperiden, diphenidol, dopamine, epinephrine, ascorbic acid and uric 

acid. The positively charged PDDA formed a stable coating which significantly improved the selectivity of the solutes 

via ionic interaction. The effects of separation voltage, buffer pH and concentration were investigated. Under the 

optimal stacking condition, the drugs were baseline separated within 20 min in 50 mM Tris-phosphate buffer (pH 9). 


83


ORAL 

SESSIONS

ORAL SESSION 2 - ENVIRONMENT 

S2-01 CHARACTERIZATION AND DETERMINATION OF MIXTURES OF FATTY ALCOHOL ETHOXYLATES AND ALKYLETHER SULFATES 

BY SPE, DERIVATIZATION WITH PHTHALIC ANHYDRIDE AND HPLC-UV 

Ripoll-Seguer L., Beneito-Cambra M., Herrero-Martínez J.M., Simó-Alfonso E.F., Ramis-Ramos G. 


Corresponding author e-mail: ramis@uv.es 

The important surfactant classes fatty alcohol ethoxylates (FAEs) and alkylether sulfates (AESs) are widely used in 

cleaners and body care products. FAEs consist of a long alkyl chain (C12-C18) bound to a polar chain of ethylene 

oxide (EO) units of variable length (up to 30) with terminal –OH group. AESs are obtained by esterification of 

FAEs with either sulfur trioxide or chlorosulfonic acid, thus ending with a sulfate group in substitution of the –OH 

group. Important characteristics of these surfactants, including viscosity, detergency, foam formation and skin 

compatibility, as well as their environmental impact, depend on the variable distributions of both the alkyl and 

EO chains. Thus, methods for their characterization and determination are required; however, owing to sample 

complexity, lack of chromophores, and wide polarity and volatility ranges of the oligomers, the analysis of FAEs, 

AESs and their mixtures is not easy. The unavailability of commercial standards constitutes an added difficulty of 

AESs analysis. In this work, a method for the characterization and determination of FAEs, AESs and their mixtures is 

described. Separation of the two surfactant families was first achieved in a 1:1 water-methanol medium by retaining 

AESs on a strong anionic exchanger (SAX) whereas most FAEs were eluted. After washing the SAX cartridge with 

1:1 water-methanol to remove all cations, the methanol percentage in the eluent was increased thus to breakdown 

the remaining mixed micelles, and to elute the hydrophobic fraction of FAEs without risk of AES elution. Then, a 

mixture of aqueous HCl and methanol was used to elute AESs. The excess acid in the eluate was neutralized with 

concentrated aqueous ammonia. The solvents were evaporated, the residues were dissolved in 1,4-dioxane and 

esterification of FAEs and transesterification of AESs with phthalic anhydride was performed. Separation of the 

derivatized oligomers was carried out by gradient elution on a C8 column with water/ acetonitrile in the presence 

of 0.1% acetic acid, using UV detection at 214 nm. Good resolution between both the hydrocarbon series and the 

successive oligomers within the series was achieved. Cross-contamination of FAEs with AESs, and vice-versa, was 

not observed. Both FAEs and AESs gave 100% esterification yields. Thus, an advantage of the proposed procedure 

is the possibility of using fatty alcohols and FAEs as calibration standards for the determination of AESs. Other 

common surfactants, including alkylether sulfonates and linear alkylbenzene sulfonates, did not interfere. The 

proposed method was successfully applied to the characterization and determination of FAEs and AESs in detergent 

bases and commercial products. 


85


S2-02 EVALUATION OF ENVIRONMENTAL TOBACCO SMOKE CONTAMINATION AND ITS EFFECT ON PASSIVE SMOKERS 

Alonso M., Besal E., Godayol A., Anticó E., Sanchez Navarro J.M. 


Corresponding author e-mail: juanma.sanchez@udg.edu 

Contamination by environmental tobacco smoke (ETS) in smoking premises and its effect on passive smokers 

have been evaluated. Sixty-seven samples were analyzed (41 smoking premises, 15 non-smoking premises and 11 

outdoors) and 11 variables were used for each sample (i.e., the content for nine volatile organic compounds, VOCs, 

plus temperature and relative humidity, RH). Principal component analysis (PCA) of the results was performed 

and it has been possible to differentiate between the three types of environments evaluated. Three factors were 

extracted for a cumulative variance of 83.7 %. Factor one (variance 58.0 %) contains toluene, ethylbenzene, m-,pxylene, 

o-xylene, and 2-ethyltoluene. Factor two (variance 16.6 %) contains benzene, 2,5-dimethylfuran, styrene, 

and benzaldehyde. Factor three (variance 9.1 %) contains temperature and RH. Scores plots show that smoking and 

non-smoking premises are separated using PC1 and PC2. Separation between non-smoking premises and outdoors 

are observed when axes PC1 and PC3 are plotted. Then, the three groups can be separated in a 3D plot (Figure 1). 

2,5-Dimethylfuran showed the best results as ETS contamination marker. This compound has never been detected 

in outdoor environments and has only been detected in one non-smoking indoor, but it has been detected in 39 

of the smoking indoors evaluated (95.1 %). A preliminary study has been developed to evaluate the possibility of 

using 2,5-dimethylfuran as a marker of passive smoking because this compound has been confirmed as a selective 

smoking breath biomarker [1]. It was continuously detected in the breath of non-smoker employees working in 

smoking premises after few hours of being in direct contact with ETS. These results open a new discussion forum 

about health policies in working environments in those countries were smoking is still allowed in hospitality industry. 

Figure 1. 3D-scores plot. red=smoking premises, yellow=non-smoking, blue=outdoors. 

References: [1]. Alonso, M; Castellanos, M.; Sanchez, J.M. Anal. Bioanal. Chem. 396 (2010), 2987-2995 

Acknoledgements: This study has been financed by the MICINN (Spanish Ministry of Education and Science), 

projects CTM2008-06847-C02-02/TECNO and CTQ2009-09370. 

86 



S2-03 IDENTIFICATION AND QUANTITATION OF EXPLOSIVE RESIDUES WITH UHPLC/MS 

Jiang G., Guazzotti S., Preston K., Elmashni D. 


Corresponding author e-mail: sergio.guazzotti@thermofisher.com 

Explosives are widely used for warfare, mining, and civil constructions. Explosive contaminated soils are found in 

firing points, impact areas and training ranges. Explosives in contaminated soils are possible sources for surface 

and ground water contamination, posing environmental and public health risks due to the compounds’ toxicity, 

carcinogenicity and mutagenicity. The analyses of explosive residues in soil are demanded by environmental monitor 

and protection agencies and the public authorities. The US EPA method 8330 is the current standard method for 

identification of explosive residues using HPLC separation and UV detection. It provides sensitive detection of 

14 nitro aromatic and nitro amine compounds. However, the lack of the selectivity of the UV detection employed 

in this method makes the identification process ambiguous. In this presentation, we show the development of 

ultra high performance liquid chromatography /mass spectrometry (UHPLC/MS) methods to separate, detect and 

quantitate explosive residues in soils (Figure 1). Explosive residues, extracted from soil samples with acetonitrile, 

were separated on a Hypersil GOLD PFP 1.9 µm, 2.1 x 100 mm column and detected by selected ion monitoring 

(SIM) on a single-quadrupole mass spectrometer. Employing this methodology we were able to identify and quantify 

nitro aromatics, nitro amines, nitrate esters and peroxide based explosives. This method offered a simple and 

efficient LC separation with a 10 fold increase in detection sensitivity with the possibility of employing additional MS 

spectrum confirmation. The identification of explosives was fast and easy by comparing the collected spectra to the 

comprehensive MS spectra library of the explosive residues. 


87


S2-04 FAST LC SEPARATIONS OF TRIAZINE HERBICIDES AT ELEVATED TEMPERATURE 

Guazzotti S., Jiang G. 



Temperature is a key variable in high performance liquid chromatography (HPLC), influencing solute diffusion rates, 

mobile phase viscosity, and solubility. As column temperature increases, analyte diffusion increases, generally 

leading to an increase in the optimum linear velocity of the separation. Furthermore, elevating the temperature 

reduces the operating backpressure. The net result is that separations can be performed faster without exceeding 

the pressure limitations of the instrument. This work will highlight the use of a porous graphitic carbon stationary 

phase thermostated in a high temperature column oven to separate triazine herbicides 5 to 10 times faster than with 

conventional HPLC. The performance of the high temperature liquid chromatographic method, including precision 

of retention time and peak area, resolution, and spike recovery from several environmental water matrices, will be 

discussed. Increasing the temperature of the separation has two important effects; first, the viscosity of the mobile 

phase decreases, reducing the backpressure of the separation system. Secondly, the water:acetonitrile mobile phase 

becomes more non-polar, so mobile phase elution strength increases. The result is that less solvent can be used, 

and temperature gradient programming is possible. High temperature helps by reducing the backpressure through 

the column. We were able to use five coupled columns at a flow rate of 500 µL/min without exceeding our column’s 

pressure limitation of about 7000 psi. The optimum linear velocity is shifted to higher flow rates as temperature 

increases, and maintains a relatively flat profile beyond the optimum. Chromatographic resolution follows a similar 

trend. For a given column, high temperature does not necessarily increase efficiency, because an increase in 

longitudinal diffusion offsets favorable improvements in mass transfer. High temperature does produce comparable 

efficiency in less time. A mixture of triazine herbicides, explosives and degradation products was analyzed by 

employing five columns coupled in series. More than twenty herbicides, explosives and their degradation products 

are separated in twenty minutes by using five 100 mm Hypercarb columns in series, a simple water:acetonitrile 

gradient and a column temperature of 160 °C. 

88 


ORAL 

SESSIONS 

S3 

LIQUID 

CHROMATOGRAPHY

ORAL SESSION 3 - LIQUID CHROMATOGRAPHY 

S3-01 SELECTIVE SAMPLE PRETREATMENT USING APTAMER-BASED EXTRACTION SORBENTS AND COMPARISON TO 

IMMUNOSORBENTS AND MOLECULARLY IMPRINTED POLYMERS 

Hennion M.C. - Madru B. - Thibert V. - Chapuis-Hugon F. - Pichon V. 

ESPCI, Paris 

The determination of compounds at a very low level of concentration is still a real analytical challenge in the 

various applications fields. In liquid chromatography, the evolution of the instrumentation in terms of separation 

and detection allowed a real improvement of the sensitivity and the analysis time. However, the analysis of ultra-traces 

from complex matrices still requires purification and preconcentration steps before the chromatographic analysis. 

Therefore, selective extraction sorbents based on a molecular recognition mechanism are still necessary for 

the selective extraction of a target molecule and its structural analogs for rendering their quantitative analysis 

more reliable and sensitive. The first selective sorbents that have been developed were immunoaffinity supports 

(immunosorbent, IS) based on the use of specific antibodies of the molecule of interest. The high selectivity and 

affinity of the antigen-antibodies interactions allows a selective clean-up with high enrichment factors. The same 

mechanism have been also exploited with the development of molecularly imprinted polymers (MIP) which synthesis 

leads to the formation of specific cavities mimicking the recognition site of the antibodies. Even if the potential of 

both selective materials is still largely demonstrated, the use of IS is expensive and the production of MIP can be 

limited by the availability of template in large amounts (molecule used to design the cavities). New selective support 

called oligosorbent (OS) has been recently developed in our laboratory using aptamers immobilized onto a solid 

support. Aptamers are oligonucleotides having a specific sequence able to bind a specific molecule with the same 

affinity as antibodies. Once the sequence identified, this new support is less expensive to produce and presents a 

higher stability than IS. It appears also as a good alternative to MIPs especially for expensive analytes because their 

production needs lower amount of target molecule. We describe here the characteristics of these new materials and 

their optimized use for extraction. Their selectivity is demonstrated by the analysis of cocaine in blood samples or of 

ochratoxins in wine. Advantages and limitations of this new type of extraction will be discussed and compared with 

IS and MIP with application to real samples. 

90 



S3-02 SUPERCRITICAL CO2 PREPARATION AND CHARACTERIZATION OF PHENYL AND FLUORO-PHENYL STATIONARY PHASES 

FOR USE IN LIQUID CHROMATOGRAPHY 

Ashu-Arrah A.B. 1 , Omamogho O.J. 1 , Yeman H. 2 , Albert K. 2 , Glennon D.J. 1 

1 


2 


Corresponding author e-mail: jdglennon@ucc.ie 

Phenyl and fluoro-phenyl silica stationary phases offer alternative selectivity compared to alkyl bonded C18 and C8 

stationary phases, through additional molecular interactions such as π–π interactions. [1] In contrast to alkyl and 

phenyl phases, the C-F bonds in fluorophases offer greater dipole interaction which increases the phase’s interaction 

with analytes particularly polar and halogenated analytes. A combination of additional solutes interactions with 

the C-F bond, π–π interactions couple with enhanced shape selectivity provided by the extra rigidity of the 

phases may provide the chromatographer with unique selectivity not obtain with alkyl phases.[2] Using a “green” 

chemistry approach which exploits the properties of supercritical carbon dioxide (sc-CO2), such as lower toxicity, 

programmable solvating power and enhanced diffusivity, organosilanes can be reacted with surface silanol groups 

for a clean, organic solvent-free synthesis of highly efficient silica bonded phases for liquid chromatography. [3] 

Phenyl and fluoro-phenyl silica bonded stationary phases were efficiently prepared in sc-CO2 at lower temperature 

and shorter reaction times, with extensive washing and drying of the product eliminated. The bonded phases were 

characterized by elemental analysis, thermogravimetric analysis, BET, and by solid state 13C and 29Si CP-MAS 

NMR spectroscopy. Chromatographic performance testing including the Neue test, is also reported. Literature: [1] 

P. G. Stevenson, S. Keyillow, G. R. Dennise and R. A. Shalliker, J. Liq. Chromatogr. & Rel. Technol. 2008, 324 [2] C. 

A. Rimmer, K. A. Lippa, M. E. Kern, L. C. Sander, M. Kuhnle and K. Albert, HPLC 2009 Dresden, Germany. [3] N.M. 

Scully, L.O. Healy, V.P. Owens, T. O’Mahony, J.D. Glennon, B. Dietrich, K. Albert, J. Chromatography A. 2008, 1191, 

99. 


91


S3-03 THE APPLICATION OF DYNAMIC TEMPERATURE PROFILES AND GRADIENTS FROM A PELTIER ARRAY BASED PLATFORM TO 

THE FABRICATION AND APPLICATION OF CAPILLARY HPLC COLUMNS 

Collins D. 1 , Nesterenko E. 1 , Connolly D. 1 , Macka M. 2 , Brabazon D. 1 , Paull B. 1 

1 


2 


It has been well documented that column temperature has a significant effect on separation selectivity and efficiency 

in various LC modes. However, longitudinal temperature gradients, which can potentially be used for focusing of 

strongly retained peaks or improve the resolution of early eluting peaks, through either heating or cooling particular 

zones of the column, have yet to be fully explored. To facilitate such complex thermal profiles the development 

of well defined and regulated temperature control with fast response is of primary importance. The aim of this 

work was to create the ability to apply complex gradients and profiles to LC capillary columns through the use of 

thermoelectric (Peltier) modules, which can provide very broad temperature ranges while also having a fast response 

time. Capillary columns were chosen as the subject of this study as they have low thermal mass and high thermal 

conductivity due to their size and material of construction, making them especially useful for the study of both 

stationary and dynamic temperature gradients and profiles along the column. The array of thermoelectric modules 

allowed rapid heating and cooling along segments of the column length and also permitted the thermal profile to 

be changed over time. This provided the capability to use dynamic gradients that can move along the column with 

specific individual peaks. It was shown that one of the main advantages of the developed array is the ability to 

rapidly heat or cool any section of the column at any time thus, allowing optimisation of the separation process. By 

using closed loop control of each thermoelectric element, very high accuracy (


S3-04 ESTIMATION OF THE FLUOROPHILIC-LIPOPHILIC-HYDROPHILIC BALANCE OF FLUORINE-CONTAINING SOLVENTS BY 

MEANS OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 

Nakajima Y., Fukuhara K., Kakiuchi T., Okamoto H., Yamamoto K. 

Asahi Glass Co., Ltd. 

Corresponding author e-mail: youji-nakajima@agc.co.jp 

Introduction of fluorine leads to large and unpredictable changes in the lipophilic and hydrophilic properties of 

organic compounds. Extensive fluorination finally generates a phase of liquid matter known as the fluorous phase 

which never mix with both lipophilic and hydrophilic phases. Therefore, quantitative estimation of the fluorophiliclipophilic-hydrophilic 

balance is important to characterize fluorine-containing compounds, and would be useful for 

screening the newly synthesized fluorine-containing compound to develop novel fluorine-containing solvents and 

surfactants. We have developed the following method to estimate the fluorophilic-lipophilic-hydrophilic balance of 

fluorine-containing compounds by means of a two-step high performance liquid chromatography (HPLC). At the first 

step, the fluorophilicity is estimated using a fluorine-fluorine interaction HPLC. Several kinds of stationary and mobile 

phases have been examined. Then, a fluorinated organic bonded silica-gel column and methanol/water are selected 

for the stationary and mobile phases, respectively. The fluorophilicity is expressed by logarithm of the retention 

factor. At the second step, the lipophilicity/hydrophilicity is estimated using an ODS bonded silica-gel column for the 

stationary phase and methanol/water for the mobile phase. The lipophilicity/hydrophilicity is expressed by logarithm 

of the retention factor. The fluorophilicity and lipophilicity/hydrophilicity values are cross-plotted to express the 

fluorophilic-lipophilic-hydrophilic balance of the compounds. Using the method developed, we have estimated the 

fluorophilic-lipophilic-hydrophilic balance of various fluorine-containing compounds. From a chromatographic point 

of view, selection of the appropriate fluorine-containing solvents for the mobile phase of liquid chromatography 

is important to analyze the molecular weight and chemical composition distributions of fluorinated polymers. The 

fluorophilic-lipophilic-hydrophilic balance of fluorine-containing solvents, including several novel solvents (HFC- 

52-13p, HFC-76-13sf and HFE-347pc-f) developed as alternatives for chlorofluorocarbons, are strongly related 

to the solubility of fluorinated polymers. The applications of those novel solvents for the mobile phase of liquid 

chromatography will be also presented. 


93

S4 HYPHENATED 

TECHNIQUES 

ORAL 

SESSIONS

ORAL SESSION 4 - HYPHENATED TECHNIQUES 

S4-01 ON LINE DERIVATISATION IN ON-LINE COUPLED NPLC-GC USING THE THROUGH OVEN TRANSFER ADSORPTION 

DESORPTION (TOTAD) INTERFACE: APPLICATION TO THE ANALYSIS OF TOTAL STEROLS IN EDIBLE OILS 

Toledano R.M., Cortés J.M., Aragón A., Vázquez A., Villén J. 


Corresponding author e-mail: jesus.villen@uclm.es 

On line derivatisation in on-line coupled NPLC-GC using the through oven transfer adsorption desorption (TOTAD) 

interface: application to the analysis of total sterols in edible oils. Toledano, R.M.2, Cortés, J.M.1, Aragón, A.2, 

Vázquez, A.2, Villén, J.1* 1Escuela Técnica Superior de Ingenieros Agrónomos, Universidad de Castilla-La Mancha, 

Campus Universitario s/n. 02071 Albacete. Spain 2Facultad de Educación de Albacete, Departamento de Química- 

Física, Universidad de Castilla-La Mancha, Plaza de la Universidad, 3. 02071 Albacete. Spain *corresponding author: 

email: jesus.villen@uclm.es; fax: 34-967-599229 Tel: 34-967-599200 (2839) Many analytes need to be derivatised 

before GC analysis. In on-line LC-GC the analytes may be derivatised off-line prior the sample is introduced into 

the HPLC, but the introduction of the derivatisation on-line presents numerous advantages (1). In the present work 

TOTAD interface (2, 3) has been modified to carry out on-line derivatisation combined with on-line coupled liquid 

chromatography-gas chromatography (LC-GC). A six port valve, situated after the HPLC system and prior the six 

port valve used to send the eluent coming from the HPLC to the waste or to the body of the TOTAD interface, is used 

to deliver the derivatised reagent after the transfer. The derivatisation process takes places in the adsorbent situated 

inside the liner of the TOTAD interface body. The system has been used for the analysis of total sterols of edible 

oils. The Official Method to determine total sterols in fats and oils (4) involves saponification, extraction of the nonsaponificable 

matter, separation by thin layer chromatography (TLC) and derivatization of the sterols prior to gaschromatography 

analysis. This procedure is tedious, time-consuming and subject to error due to the manipulation 

of the sample. TOTAD interface has been previously used to analyse free sterols in edible oils by RPLC-GC (5). The 

present method uses the TOTAD interface not only for on-line coupling normal phase liquid chromatography and gas 

chromatography (NPLC-GC) as well as to carry out the derivatisation reaction. In this method, the oil is injected into 

the HPLC system after a saponification and an extraction. LC replaces the thin-layer chromatography (TLC) step and 

the derivatization reaction is carried out inside the TOTAD interface. The method was applied to the determination 

of total sterols in certified samples and the results obtained were compared with those obtained with the Official 

European Method. Good agreements were found between both methods. 1. Hyötyläinen, T. and Riekkola, M.L. J. 

Chromatogr. B 817 (2005) 13-21 2. Pérez, M.; Alario, J.; Vázquez, A.; Villén, J. J. Microcol Sep. 1999, 11, 582-589 3. 

Pérez, M.; Alario, J.; Vázquez, A.; Villén, J. Anal. Chem. 2000, 72, 846-852 4. AOCS Official methods, 1991, Chap 

6-91; European Union Commission, 1991; International Standard Office, 1999 5. Cortés, J.M.; Sánchez, R.; Villén, J.; 

Vázquez, A. J. Agric. Food Chem. 2006, 54, 6963-6968 


95


S4-02 ANALYSIS OF MELAMINE-FORMALDEHYDE CONDENSATES AND METHYLATED MELAMINES IN REACTION MIXTURES BY CZE-MS 

Himmelsbach M., Vo T.D.T., Schwarzinger C., Buchberger W., Klampfl C.W. 

Johannes Kepler University Linz 

Corresponding author e-mail: Markus.Himmelsbach@jku.at 

Melamine is long known as a raw material for thermoset polymers. Classical melamine resins are made by the 

reaction of melamine with formaldehyde in an aqueous solvent. If melamine reacts with formaldehyde, it forms 

methylol melamines which are readily soluble in water. The reaction mixture consists typically of unreacted melamine 

and methylol melamines with a varying degree of methylolation. Additionally, methylolation is in concurrence with 

oligomerization, resulting from the condensation of methylol groups with free amino groups or other methylol 

groups. By varying the amount of formaldehyde and the pH value of the reaction the average degree of methylolation 

and condensation can be adjusted and thereby the processing properties and final product characteristics are 

predetermined. An improved method based on capillary zone electrophoresis (CZE) coupled to quadrupole-timeof-flight 

mass spectrometry (Q-TOF MS) for the analysis of melamine–formaldehyde condensates is presented. 

Employing a formic acid-based electrolyte containing 50% acetonitrile and MS detection, up to 13 compounds could 

be determined in lab-made resins, synthesized by mixing formaldehyde and melamine in different ratios ranging 

from 1:1.5 to 1:4. The use of a Q-TOF MS for detection allowed the assignment of molecular formulas for all 13 

separated substances with high accuracy. With migration times well below 20 min even for low mobility species the 

method can be regarded as fast compared to chromatographic methods. CZE–MS allows to determine the degree of 

polymerization (number of melamine molecules per unit) as well as functionalization (number of methylol groups per 

unit). Additionally structural isomers could be separated. Recent studies have used methyl melamines as building 

blocks for the synthesis of functional monomers with further application in polymer and coating materials or as 

fine chemicals. A new route for synthesis is the catalytic hydrogenation of melamine-formaldehyde resins, but with 

this method a very complex mixture of educts and products is obtained, comprising melamine, up to nine possible 

monomers of methylol melamines, a high number of oligomers, the desired methyl melamines, but also methylol 

methylmelamines which arise from incomplete hydrogenation. CZE coupled to Q-TOF MS allows the fast and reliable 

analysis of methylated melamines. Experiments with UV detection were performed using a background electrolyte 

containing formic acid and TFA to optimize electrophoretic conditions and to achieve baseline separation. Employing 

a TFA-based electrolyte containing 80% ACN and MS detection, nine different methyl melamines were determined in 

real samples, which were produced by catalytic hydrogenation of melamine/formaldehyde reaction mixtures. The use 

of a Q-TOF MS resulted in LODs better than 0.01 mg/L for all compounds and high mass accuracy with mass errors 

lower than 2.3 ppm. The short analysis time of 15 min is very useful if reaction control is required during synthesis. 

96 



S4-03 SCIENCE BASED CALIBRATION FOR THE EXTRACTION OF ‘ANALYTE-SPECIFIC’ CHROMATOGRAMS IN LC 

Kuligowski J., Quintás G., de la Guardia M. 


A multivariate approach named ‘Science Based Calibration’ [1,2] has been applied for the extraction of ‘analytespecific’ 

chromatograms in liquid chromatography in the presence of high spectral and chromatographic overlapping 

between the analytes of interest, co-eluting sample matrix constituents or mobile phase components. This 

multivariate method combines the main features of ‘classical’ and ‘inverse’ calibrations. By estimating the spectral 

signal in a physical way and the spectral noise in a statistical way, the SBC method combines the prediction 

accuracy of the ‘inverse’ approach with the low cost and ease of interpretability of the ‘classical’ models. Results 

obtained by SBC are based on user-estimates of both the analyte signal g (i.e. spectrum) and the spectral ‘noise’ 

to calculate an optimum regression vector bopt. Whereas its usefulness has been confirmed even when the analyte 

was injected in the presence of unknown interfering compounds, the benefits of the proposed approach are fully 

exploited when interfering compounds are considered for the calculation of the covariance matrix of the spectral 

‘noise’ matrix. This poster covers recent applications of SBC in LC systems, including LC-FTIR [3], GPC-FTIR [4] 

and LC-UV [5], and highlights the main advantages and drawbacks of this strategy. 1: Marbach, R., J. Biomed. 

Optics, 2002, 7, 130. 2: Marbach, R., J. Near Infrared Spectrosc., 2005, 13, 241. 3: J. Kuligowski, G. Quintás, S. 

Garrigues, M. de la Guardia, J. Sep. Sci., 2009, 32, 1. 4: J. Kuligowski, G. Quintás, S. Garrigues, M. de la Guardia, 

Chromatographia 2010, 71(3/4), 201. 5: J. Kuligowski, M. Martínez Galera, M.D. Gil García, M.J. Culzoni, H.C. 

Goicoechea, S. Garrigues, G. Quintás, M. de la Guardia, Talanta, 2010, submitted for publication. 


97


S4-04 POTENTIAL OF COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY COUPLED TO A VERY FAST QUADRUPOLE 

MASS SPECTROMETER (20000 AMU/SEC) 

Purcaro G., Quinto Tranchida P., Dugo P., Dugo G., Mondello L. 

University of Messina 

Corresponding author e-mail: lmondello@unime.it 

Comprehensive two-dimensional gas chromatography (GC x GC) is a very powerful technique, especially if coupled 

to the mass spectrometry detector (MS). The main requirement for a GC x GC detector is the acquisition speed to 

assure an optimal peak reconstruction (at least 10 points per peak), since narrow peak-widths are obtained after the 

second dimension. Over the past decade, it has been generally accepted that the most suitable MS instrument for 

GC x GC analysis is the ToF mass spectrometer; a negative aspect, also widely considered, is the high cost of such 

instrumentation. Quadrupole MS systems are less expensive but are characterized by much lower spectra production 

rates, because the mass analyzer scans individual ion groups on a m/z basis. Until present, the quadrupole MS has 

been considered suitable for identification but not for quantification purposes. 

The present research is focused on the employment of a recently-developed fast-scanning quadrupole mass 

spectrometer, in GC x GC experiments. The performance of the novel instrument has been evaluated in terms of 

number of data points per peak, peak skewing, mass spectrum quality, and application realm. The capabilities of 

simultaneous high speed scan/SIM acquisition has also been evaluated. 

98 



ORAL 

SESSIONS


S5-01 APPLICATION OF STIR BAR SORPTIVE EXTRACTION FOLLOWED BY COMPREHENSIVE TWO-DIMENSIONAL GAS 

CHROMATOGRAPHY-TIME-OF-FLIGHT MASS SPECTROMETRY FOR THE DETERMINATION OF TARGET AND NON-TARGET 

ORGANIC MICRO-CONTAMINANTS IN RIVER WATER 

Gómez Ramos M.J. 1 , Herrera S. 1 , Solé D. 1 , Fernández-Alba A.R. 2 

1 


2 

Almería University 

Chemical pollution of natural waters has already become a major public concern in almost all parts of the world, 

since it has largely unknown long-term effects on aquatic life and on human health. The list of priority chemicals 

included in various regulations such as the European Water Framework Directive, as well as the list of hazardous 

contaminants identified in the aquatic environment, are increasing at an accelerated pace. At the same time, 

many other organic contaminants from anthropogenic origin, many times called “emerging” contaminants, are 

being subject of high concern among the scientific community because of the frequent detection in wastewater 

effluents and to the potential contamination of surface water if the effluents are discharged directly into the water 

bodies. Therefore, there is a need for broad spectrum methods capable of simultaneously determining hundreds, 

if not thousands, of contaminants at low levels in a single analysis. For the analysis of non-polar or semi-polar 

contaminants in environmental matrices, comprehensive two-dimensional gas chromatography (GCxGC) coupled 

with a time-of-flight (TOF) detector is a powerful technique that allows the separation of many constituents of 

previously unresolved complex mixtures of contaminants in enviromental samples. In addition to the powerful 

chromatographic resolution, automated mass spectral deconvolution and identification system software enables 

a spectral deconvolution of closely eluting peaks. In this study a novel analytical approach to determine trace 

levels of 28 pesticides, 15 PAHs and 17 personal care products (PCP) in a total of thirty river water samples from 

the Henares River (Madrid, Spain) is proposed, based on stir bar sorptive extraction (SBSE) followed by GCXGC- 

TOF-MS. Excellent results have been obtained in terms of separation efficiency and, also, compound identification. 

The method detection limits (LODs) and quantification (LOQs) ranged from 0.1 to 13 ng/L and from 0.3 to 44 ng/L, 

respectively. Recoveries values were higher to 83% for all the target compounds. Good linearity (r2 > 0.98) and 

convenient precisions (RSD < 28%) were found for almost all cases. Results obtained during the monitoring program 

show that the contaminants more frequently detected, and at higher concentrations, were de PCPs at concentration 

in the range of 5-17350 ng/L, the musk fragrances galaxolide and tonalid were the most concentrated compounds 

in the samples. In most of the river waters were detected at least one pesticide and/or one PAH, the most common 

were diazinon, terbutylazine, naphthalene and phenanthrene. The result of this study presents SBSE-GCXGC-TOF- 

MS as a powerful tool for identifying and quantifying organic contaminants in waters. In addition to obtain analytical 

information such as identification and quantification of target analytes, with this technique it is possible to screening 

for unknowns, group type analysis and fingerprinting studies. The method has been applied to the screening of a 

large number of organic contaminants- not only to a subset of targets, in river waters. 

100 



S5-02 EVALUATION OF GLYPHOSATE IN SUPERFICIAL AND DRINKING WATERS OF AN AGRICULTURAL AREA OF RIO DE JANEIRO 

STATE, BRAZIL, BY HPLC-FLUORESCENCE 

Olivier B.C. 1 , Pereira Netto A.D. 2 

1 

National Institute of Technology, Brazil 

2 

Federal Fluminense University, Brazil 

Corresponding author e-mail: annibal@vm.uff.br 

Glyphosate, N-(phosphonomethyl) glycine is a systemic post-emergence non-selective herbicide of widespread use 

for controlling weeds. It is absorved through the plant tissue and inhibits the enzyme envolved in the synthesis of 

aromatic amino acids that are essential to vegetable growth. Its action is rapid, causing vegetation to dye in about 

twenty days that is observed by yellowish leaves. The Brazilian legislation established maximum concentrations of 

glyphosate in surface and drinking water of respectively 65 μg/L and 500 μg/L and therefore there is a clear need of 

sensitive methods for its determination in waters of different characteristics and uses, because in some agricultural 

areas and periods of the year, glyphosate is of widespread use. This work describes partial results of the application 

of a method for glyphosate determination in superficial and drinking water samples collected in the basin of the river 

Corrego Sujo – RJ, that is an agricultural area located around 150 km away of Rio de Janeiro City. High performance 

liquid chromatography with fluorescence detection (HPLC-Fluo) following the off-line derivatization of glyphosate 

with 9-fluorenylmethyl chloroformate (Fmoc-Cl) was employed. Excitation and emission at 270 and 315 nm, were 

respectively used. The results obtained by this technique were also compared with those obtained by HPLC-UV-Vis, 

because this is a comparatively more widespread detector with comparative low cost than the fluorescence one. 

Sample pre-treatment involved the filtration of samples in PTFE filters (0.45 μm) followed by derivatization reactions 

that were conducted by the addition of 1.5 mL of sample, 0.200 mL of sodium tetraborate at pH = 9.0 and 1.0 mL of 

Fmoc-Cl in acetonitrile (ACN) for 30 min at room temperature. Both HPLC systems consisted of Agilent 1100 series. A 

column Zorbax Eclipse XDB C18 (250 x 4.6 mm, 5 mm) and a mobile phase composed of ammonium acetate (45mM) 

and acetonitrile at final pH = 5.5 were used in both cases. Calibration standards with concentrations of 50.0, 100, 

250, 500, 750 and 1000 μg/L were used. The analytical curve was linear within this range yielding a R2 = 0.9998. 

The limit of detection (LOD) and a limit of quantification of (LOQ) of respectively 40 μg/L and 130 μg/L were found 

with the fluorescence detector. The results found with the UV detector showed a LOD of 400 μg/L thus indicating a 

lack of sensitivity to the determination of gliphosate in waters suspected to be contaminated with this herbicide, in 

the legislation limits. Recoveries between 84 and 96%, with coefficients of variation ≤ 2.6%, were found in different 

levels, by application of the HPLC-Fluo method. The evaluated samples showed no contamination with glyphosate, 

independent of sampling point localization. CNPq, CAPES, PROPP-UFF 


101


S5-03 HIGH PERFORMANCE LIGAND EXCHANGE CHROMATOGRAPHY AND FOURIER TRANSFORM MASS SPECTROMETRY — A 

COMBINATION TOWARD THE CHARACTERIZATION OF SULFUR AROMATICS IN ARABIAN HEAVY CRUDE OIL AND ITS 

DISTILLED FRACTIONS 

Panda S.K., Hajji A.A., Mueller H., Koseoglu O.R. 

Saudi Aramco 

Corresponding author e-mail: saroj.panda@aramco.com 

Refiners are processing heavier and sour crude oils. To handle such challenging feedstocks cost-effectively, an in-depth 

compositional characterization — of the heteroatom containing compounds (SNO) therein — is mandatory for 

feedstock management and process optimization. Many approaches have been attempted for the characterization 

of SNO in crude oils in the last decades. No single analytical technique is capable enough to provide an absolute 

compositional answer to the complex crude oil mixture. Gas chromatography, for example, is the most commonly 

used technique in the oil industry; however, its application is limited to fractions with boiling points less than 400 ºC. 

Although mass spectrometry (MS) can be used to analyze crude oil fractions in wide boiling ranges, the blindness of 

MS towards isomers — in conjunction with discrimination in the representation of SNO — limits its applicability for 

a comprehensive compositional characterization. A combination of liquid chromatography, selective derivatization, 

and mass spectrometry was employed in this study for a thorough characterization of sulfur compounds in Arabian 

crude oil and its distilled fractions (naphtha, gas oil and vacuum gas oil). The sulfur compounds were selectively 

separated from the aromatic fractions using ligand exchange liquid chromatography. This additional dimension 

in separation offers a class-based isolation of condensed and non-condensed thiophenes. In a subsequent 

derivatization reaction, such nonpolar sulfur heterocyclic species were converted to their polar analogs, so that they 

could be efficiently analyzed by positive electrospray Fourier transform mass spectrometery (FT-MS). This approach 

has produced comprehensive distribution patterns of the sulfur species in terms of their aromaticity and provides 

additional structural information in the different distilled fractions and whole crude oil. 

102 


ORAL 

SESSIONS 

S6 

FOOD QUALITY 

AND SAFETY

ORAL SESSION 6 - FOOD QUALITY AND SAFETY 

S6-01 INFANT EXPOSURE OF PERFLUORINATED COMPOUNDS: LEVELS IN BREAST MILK AND COMMERCIAL BABY FOOD 

Farré M. 1 , Llorca M. 1 , Picó Y. 2 , López-Teijón M. 3 , Alvarez J. 3 , Barceló D. 1 

1 

IDAEA-CSIC 

2 


3 

Instituto Marqués 

Infant exposure of Perfluorinated Compounds: Levels in breast milk and commercial baby food. Marinella Farré1, 

Marta Llorca1, Yolanda Picó2, Marisa Lopez Teijón3,4, Juan G Álvarez3,4, Damià Barceló1,5. 1 Department of 

Environmental Chemistry, IDAEA-CSIC, Barcelona, Spain 2 Nutrition and Food Chemistry Laboratory, University of 

Valencia, Valencia, Spain. 3 Servicio de Reproducción, Instituto Marqués, Barcelona, Spain. 4 Fundació Leonardo 

Marqués, Barcelona, Spain. 5King Saud University, Riyadh, Saudi Arabia. In this study, an analytical method to 

determine six perfluorinated compounds (PFCs) based on alkaline digestion and solid phase extraction (SPE) 

followed by liquid chromatography-quadrupole-linear ion trap mass spectrometry (LC-QqLIT-MS) was validated for 

the analysis of human breast milk, milk infant formulas and cereals baby food. The average recoveries of the different 

matrices were in general higher than 70% with a relative standard deviation (RSD) lower than 21% and method 

limits of detection (MLOD) ranging from 1.2 to 362 ng/L for the different compounds and matrices. The method 

was applied to investigate the occurrence of PFCs in 20 samples of human breast milk, and 5 samples of infant 

formulas and cereal baby food (3 brands of commercial milk infant formulas and 2 brands of cereals baby food). 

Breast milk samples were collected in 2008 from donors living in Barcelona city (Spain) on the 40 days postpartum. 

Perfluorooctanesulfonate (PFOS) and perfluoro-7-methyloctanoic acid (i,p-PFNA) were predominant being present 

in the 95% of breast milk samples. Perfluorooctanoic acid (PFOA) was quantified in 8 of the 20 breast milk samples 

at concentrations in the range of 21 - 907 ng/L. Commercial formulas and food were purchased also in 2009 from 

a retail store. The six PFCs were detected in all brands of milk infant formulas and cereals baby food analyzed, 

being perfluorodecanoic acid (PFDA), PFOS, PFOA and i,p-PFNA the compounds detected in higher concentrations 

(up to 1289 ng/kg). PFCs presence can be associated to possible migration from packaging and containers during 

production processes. Finally, based on estimated body weight and newborn intake, PFOS and PFOA daily intakes 

and risk indexes (RI) were estimated for the firsts 6 month of life. We found that ingestion rates of PFOS and PFOA, 

with exception of one breast milk sample did not exceed the tolerable daily intake (TDI) recommended by the EFSA. 

However, more research is needed in order to assess possible risk associated to PFCs contamination during early 

stages of life. 

104 



S6-02 DEVELOPMENT AND APPLICATION OF A HPLC-UV-ESI-MS METHOD FOR THE SIMULTANEOUS DETERMINATION OF UREA 

BY-PRODUCTS IN COMMERCIAL UREA 

Ferreira C.G. 1 , Albuquerque F.C. 1 , Pereira Netto A.D. 2 

1 

PETROBRAS-CENPES 

2 



The use of large concentrations of urea is essential for agriculture and livestock practices because this compound 

represents a source of nitrogen for amino- and nucleic acids synthesis. Official methods of quality control of 

urea in Brazil are established by the Ministry of Agriculture, Livestock and Supply. Two parameters (total nitrogen 

content and biuret concentration) are considered to represent the chemical composition of urea and are of great 

relevance in the quality control of the final product. Biuret is a dimer of urea formed during the solidification step 

of the industrial production of urea, which formation is increased above 132ºC. It is a toxic by-product because its 

uptake from soil leads to complexes formation with certain micronutrients, such as iron and manganese, in plant 

sap. As a consequence, the velocities of enzymatic processes catalyzed by the free metal ions are reduced. This 

effect is known as chlorosis that is evidenced by yellowish leaves and low quality harvests. Besides biuret, a trimeric 

structure (triuret) can be formed but its effects in crops are not well-known. Melamine may also result from the 

condensation of biuret molecules. This compound is used as a raw material in the petrochemical industry, especially 

to produce polymers, adhesives, pigments and flame retardant additives. Nevertheless melamine is not used as an 

ingredient in feed applications due to the nucleation of melamine-cyanuric acid hexamers into the renal tubules with 

subsequent crystal growth and kidney injury. Real examples of food contamination by melamine occurred in 2008 in 

USA, where cats were contaminated by pet food, and in China, where a widespread poisoning of babies was caused 

by melamine contaminated dairy products. This work describes the development of a HPLC method with UV or MS 

detection to identify and quantify urea contaminants, namelly melamine, ameline, amelide and cyanuric acid, which 

have aromatic s-triazine structures, and biuret and triuret, which have aliphatic structures, in one chromatographic 

run. An ion-chromatograph with UV-Vis detection (Metrohm, model 844) interfaced to a single quadrupole (Agilent) 

by an electrospray (ESI) interface was used. The separation was evaluated and optimized in a column Lichrospher 

RP-C8 (4.6 x 250 mm x 5 µm; 300 Ǻ). Isocratic conditions at 0.8 mL/min with a mobile phase composed of aqueous 

NH4Cl 2 mM and an aqueous buffer of pH = 5.0 composed of CH3COONH4 2mM and CH3COOH were used, 

respectively for UV and MS detection. Posteriorly it was observed that the addition of low concentrations of 

tetrabutylammoniun hydroxide to the mobile phase improved the separations. The detection of biuret at 215 nm 

led to linear responses in concentrations between 10 and 120 mg of biuret per kg of urea. The evaluation of the 

reference material “Urea fertilizer - BCR179” led to an estimated value (10.43±0.21) in good agreement with the 

certified value (10.37±0.11). The minoritary compounds were identified by MS in most samples but melamine was not 

found in any of them. The quantitative evaluation of these compounds is being studied. 


105


S6-03 NANO-LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY TO STUDY THE IN VITRO ABSORPTION AND METABOLIC 

CONVERSION OF OLIVE OIL POLYPHENOLS IN HUMAN BREAST CANCER CELLS 

García-Villalba R. 1 , Carrasco-Pancorbo A. 1 , Menéndez J.A. 2 , Segura- Carretero A. 1 , Fernández-Gutiérrez A. 1 

1 


2 

Catalan Institute of Oncology 

Corresponding author e-mail: rociogv@ugr.es 

Polyphenols from extra-virgin olive oil (EVOO), a main component of the Mediterranean diet, have demonstrated 

repeatedly anti-tumoral activity in several in vitro and in vivo studies. However, little is known about the efficiency 

of the absorption process and metabolic conversion of these compounds at the cellular level. In this study, a nano 

liquid chromatography-electrospray ionization-time of flight mass spectrometry (nanoLC-ESI-TOF MS) method was 

developed to study the cellular uptake and metabolism of olive oil phenols in JIMT-1 human breast cancer cells. After 

incubation for different time periods with EVOO- phenolic extracts, culture media, cytoplasm and solid parts of 

JIMT-1 breast cancer cells were separated, subjected to different extraction procedures and analyzed. A short 

capillary trapping column (100 μm ID, effective length 20 mm, 5 μm particle size) and a fused silica capillary column 

(75 μm ID, effective length 10 cm, 3 μm particle size), both packed with C18 stationary phase, were used. The mobile 

phase was a mixture of water + 0.5% acetic acid and acetonitrile eluting at 300 nL/min in a gradient mode. The 

nanoLC column was interfaced to the mass spectrometry using a commercial sheathless nano-spray interface with a 

tapered fused silica sprayer tip. Most of the free phenols, mainly hydroxytyrosol, its secoiridoids derivatives, and the 

flavonoid luteolin, disappeared in the culture media in different extent and at different times and the appearance of 

metabolites could indicate intracellular metabolism followed by rapid cellular export. Low intracellular accumulation 

was observed with only traces of some compounds detected in the cytoplasm and solid parts. Methylated 

conjugates were the major metabolites detected, suggesting a catalytic action of catechol-O-methyl transferase 

(COMT) in these cancer cells. NanoLC coupled to ESI-TOF has demonstrated to be a suitable technique with enough 

sensitivity for the determination of low concentrations of polyphenols in biological samples. 

106 


ORAL 

SESSIONS 

S7 

FUNDAMENTALS OF 

CHROMATOGRAPHY

ORAL SESSION 7 - FUNDAMENTALS OF CHROMATOGRAPHY 

S7-01 METHOD DEVELOPMENT IN LIQUID CHROMATOGRAPHY USING TEMPERATURE GRADIENTS 

Wiese S. 1 , Teutenberg T. 1 , Schmidt T.C. 2 

1 


2 


Corresponding author e-mail: teutenberg@iuta.de 

Increasing the temperature results in a change of the physico-chemical properties of the mobile phases commonly 

used in liquid chromatography: the static permittivity and concurrently the polarity of water-organic mixtures 

decreases with increasing temperature. Therefore, temperature gradients can be employed instead of solvent 

gradients to govern retardation in LC. Hence, it is possible to elute even hydrophobic compounds on a reversed 

phase column without changing the composition of the mobile phase. Moreover, pure water can be used as the 

sole eluent. This enables the use of new hyphenation techniques such as, e. g., LC-irMS or LC taste®. In these 

techniques, the optimization of separations cannot be performed by a classical solvent gradient. In LC-irMS, any 

organic carbon in the mobile phase will falsify the measured isotopic composition of the analytes. A similar problem 

exists in LC taste®, where only ethanol in a low concentration can be used as organic modifier. In other words, only 

the parameter temperature can be used to optimize the chromatographic separation. Furthermore, a requirement 

to use these methods in a routine environment is that a systematically method development can be performed. 

Therefore, in this presentation an approach adapted from temperature-programmed gas chromatography is 

introduced that allows to perform method development using temperature gradients in liquid chromatography based 

on a few initial experimental runs. 

108 



S7-02 SIMULTANEOUS EFFECT OF PH, TEMPERATURE AND MOBILE PHASE COMPOSITION IN THE CHROMATOGRAPHIC 

RETENTION OF IONIZABLE COMPOUNDS 

Agrafiotou P., Ráfols C., Castells C., Bosch E., Rosés M. 


Corresponding author e-mail: marti.roses@ub.edu 

pH, temperature and mobile phase composition are key factors in the optimization of chromatographic separations 

of ionizable compounds. In this work, we study several models to relate retention of acid-base compounds to 

these parameters which can be further used to predict retention in isocratic and gradient elution modes. The 

retention of 23 acid-base compounds in three mobile phase compositions (20/80, 40/60 and 60/40 % acetonitrile/ 

water) at three temperatures (25, 40 and 55 ºC) in 12 pH buffers of different type has been determined. The pH of 

the buffers has been also carefully measured at each mobile phase and temperature, and relationships between 

retention and pH, T, and mobile phase composition or polarity-related properties have been established. The 

classical sigmoidal relationships between k and pH (measured in the working mobile phase and temperature), on one 

hand, and linear Van’t Hoff plots (log k vs. 1/T), on the other hand, for each mobile phase composition have been 

obtained. Both equations can be combined in a general equation relating retention to pH and temperature. Several 

models relating the obtained fitting parameters to either mobile phase composition or polarity properties have 

been also investigated. From the final equations obtained general models are proposed, which can be implemented 

in procedures and software for the optimization of chromatographic separations of acid-base compounds. 

References M. Rosés, X. Subirats, E. Bosch. Retention models for ionizable compounds in reversed-phase liquid 

chromatography. Effect of variation of mobile phase composition and temperature. Journal of Chromatography A, 

1216 (2009) 1756–1775. P. Nikitas, A. Pappa-Louisi. Retention models for isocratic and gradient elution in reversedphase 

liquid chromatography. Journal of Chromatography A, 1216 (2009) 1737–1755. 


109


S7-03 DETERMINATION OF PERSISTENT ORGANIC POLLUTANTS IN FISH BY GAS CHROMATOGRAPHY–MS/MS 

Muñoz G., Pineda L., Serrahima E., Centrich F. 

Laboratori Agència Salut Pública de Barcelona 

Corresponding author e-mail: gmunoz@aspb.cat 

The polybrominated diphenyls ethers (PBDEs), polychlorinated biphenyls (PCBs) and polychlorinated naphtalenes 

(PCNs) are considered as a persistent organic pollutants (POPs). These compounds are used as flame retardants 

(BFRs), fungicides and in other industrial applications. All compounds have similar structural and chemical 

properties. 

As a routine, the Laboratory of Public Health Agency in Barcelona analyse organochlorine pesticides and PCBs in all 

kind of commodities by gas chromatography coupled with mass spectrometry single quadrupole (GC/MS SIM mode). 

After the European Union recommendation of the analysis of BFRs, the laboratory has validated the determination 

of PBDEs and PCNs in fish by gas chromatography coupled with mass spectrometry triple quadrupole (GC-MS/MS). 

The method for extracting pesticides is based on an accelerated solvent extraction (ASE) with ethyl acetate. After 

extraction, the matrix is purified by (with) GPC using ciclohexane / ethyl acetate as mobile phase. The same extract 

obtained is used for the determination of PBDEs, PCNs, organochlorine pesticides and PCBs and it is analysed by 

gas chromatography coupled with mass spectrometry. 

Surrogate matrix matched calibration curves are used for quantification. Blank extracts, recovery spiked samples, 

linearity and the use of internal standard are checked as quality control. 

The PBDEs congeners validated are: PBDE-28, PBDE-47, PBDE-99, PBDE-100, PBDE-153, PBDE-154, PBDE-183. 

Furthermore a polybrominated biphenyl (PBB-153 ) has been validated. 

The PCNs congeners validated are: 2,3,6,7-TetraCN; 1,2,3,6,7-PentaCN; 1,2,3,5,6,7-HexaCN; 1,2,3,4,5,6,7-HeptaCN 

and OctaCN. 

The limit of quantification (LOQ) has been established in 0.0050 mg/kg for each compound. 

The procedure is accredited by the Spanish body of accreditation ENAC for ISO /17025 according to NT-18. 

110 


S8 EXTRACTION 

ORAL 

SESSIONS

ORAL SESSION 8 - EXTRACTION 

S8-01 IONIC LIQUIDS IN MICROEXTRACTION TECHNIQUES COMBINED WITH CHROMATOGRAPHIC SEPARATIONS 

Cárdenas S., Lucena R., Aguilera-Herrador E., Cruz-Vera M., Valcárcel M. 


Ionic liquids are being consolidated as new, powerful extractant media in the analytical context. In addition to 

Chemistry, the multidisciplinary impact of these compounds affects also to important research areas such as 

nanotechnology and physics, among others. It can be ascribed to their peculiar characteristics, which includes 

a wide electrochemical window, negligible vapor pressure, great chemical and thermal stability and high 

extractant capacity for metals and organic compounds. The last property has been used in the chromatographic 

context to develop new and more efficient stationary phases as an injection media. When they are used in nonchromatographic 

separation techniques, the resulting procedures are highly selective and efficient [1]. The use and 

applicability of miniaturized extraction techniques in the analytical measurement process permits the reduction 

of the dimensions of the whole process. However, in order to maintain or even improve the basic analytical 

properties (sensitivity, selectivity and precision), the use of highly efficient tools should be employed. This 

communication summarized the analytical potential of ionic liquids in the development of new extraction modes 

that can be combined with chromatographic separations to improve the performance of the resulting analytical 

process. In sample preparation prior to gas chromatography, the ionic liquids can be used as extractant in single 

drop microextraction, being this step directly coupled to the instrument by means of a removable, reusable 

interface which permits the selective desorption of the analytes while prevents the extractant from reaching the 

chromatographic column. It has been successfully applied to the determination of volatile pollutants in waters. As 

far as sample preparation for liquid chromatography is concerned, ionic liquids have been used in dispersive and 

dynamic microextraction techniques. The analytes extraction is implemented using very simple devices and once 

isolates, the ionic liquid, enriched with the analytes, can be directly injected in the chromatograph including a simple 

dilution with the mobile phase. [1] E. Aguilera-Herrador, R. Lucena, S. Cárdenas, M. Valcárcel, Trends Anal. Chem., 

2010 DOI: 10.1016/j.trac.2009.11.009 

112 



S8-02 NOVEL UNBREAKABLE SOLID PHASE MICROEXTRACTION FIBER BY CHEMICALLY BONDED CARBON NANOTUBES ON 

MODIFIED GOLD SUBSTRATE 

Bagheri H., Ayazi Z., Sistani H., Aghakhani A. 

Sharif University of Technology, Chemistry 

Following our research interests regarding the development of new extracting media [1,2], a new technique for 

preparation of an unbreakable fiber for solid phase microextraction (SPME) was developed. In this technique 

primarily an intermediate film consisting of an ultrathin two-dimensional polymer was prepared by hydrolysis of 

a (3-mercaptopropyl) trimethoxysilane (ethanol, 10-3 M) self-assembled monolayer grafted onto gold [3]. Then a 

layer of trimethoxysililpropylamine (TMSPA) was introduced to the surface of modified gold substrate. At the final 

stage, a stationary phase of oxidized multiwall carbon nanotube (COOH-MWCNTs) was chemically bonded to the 

surface of substrate [4]. After preparing the fiber, scanning electron microscopy (SEM) technique was employed to 

study the surface characteristics of the coated fiber. The SEM of the coated surface revealed that the synthesized 

coatings possess porous structures, which should significantly increase the surface area availability on the fiber. 

The applicability of the prepared fiber coating was examined by SPME of diazinon and fenthion from aquatic media. 

Important parameters influencing the extraction process were optimized and an extraction time of 30 min at 60°C 

gave maximum peak area, when NaCl (30% w/v) was added to the aqueous sample. Limits of detection were at level 

of 0.2 ng mL-1 for diazinon and 0.3 ng mL-1 for fenthion using time scheduled selected ion monitoring (SIM) mode 

and relative standard deviation (RSD%) values were below 4.6% at concentration level of 1 ng mL-1. The developed 

method was successfully applied to real water samples while the relative recovery percentage (RR %) obtained for 

the spiked real water samples were 104% for diazinon and 97% for fenthion, respectively. References: [1] H. Bagheri, 

Z. Ayazi, E. Babanezhad, Microchem. J., 94 (2010)1-6. [2] H. Bagheri, E. Babanezhad, F. Khalilian, Anal. Chim. Acta, 

634 (2009) 209-214. [3] I. Piwonskia, J. Grobelnya, M. Cichomskia, G. Celichowskia, J. Rogowski, Appl. Surf. Sci., 

242 (2005) 147–153. [4] H. Liua, J. Li, X. Liua, S. Jianga, Talanta, 78 (2009) 929–935. 


113


S8-03 STIR MEMBRANE IN MICROSOLID PHASE EXTRACTION AND LIQUID PHASE MICROEXTRACTION APPROACHES COMBINED 

WITH GAS CHROMATOGRAPHY 

Lucena R., Alcudia-león M.C., Cárdenas S., Valcárcel M. 


The development of new extraction techniques which permits the efficient isolation and preconcentration of the 

target analytes before their chromatographic analysis remains as a key trend in Analytic Chemistry. Microextraction 

techniques (particularly solid and liquid phase microextraction) have gained in importance in recent years due 

to its efficiency, simplicity and versatility. In this context, polymeric membranes have been extensively used 

taking into account their high surface to length ratio, their availability, their wide chemical composition (covering 

different polarities) and their formats (flat sheet and follow fiber). Stir membrane extraction combines the extraction 

capabilities of polymeric membranes and the advantageous of continuous stirring in the same extraction device 

[1]. The analytes are extracted by adsorption in the membrane and later on are determined using an appropriate 

instrumental technique. This determination can be performed in the membrane surface by solid phase spectroscopy 

or it can be carried out after a chemical or thermal desorption. Stir membrane extraction allows the processing of 

high sample volume achieving good preconcentration factors. The extraction unit designed permits its use in both, 

microsolid phase extraction and liquid phase microextraction approaches by including a simple, non-permanet 

modification of its geometry. The use of these devices in liquid phase microextraction procedures is highlighted 

and discussed. The new extraction mode was characterized studying the main variables involved in the process. 

Once optimized, it was employed for the resolution of three model analytical problems, such as the determination 

of five polycyclic aromatic hydrocarbons (naphthalene, pyrene, fluoranthene, fluorene and benzo-anthracene); the 

hydrocarbon index and the chlorophenols content in water samples The novel approach resulted to be sensitive 

enough for the resolution of both problems with relative standard deviations lower than 8 %. [1] M.C. Alcudia-León, 

R. Lucena, S. Cárdenas, M. Valcárcel, Anal. Chem. 81, 2009, 8957-8961. 

114 


S9 SPECIATION 

ANALYSIS 

ORAL 

SESSIONS

ORAL SESSION 9 - SPECIATION ANALYSIS 

S9-01 INTERACTION OF THIOPHILIC ELEMENT SPECIES WITH PROTEINS 

Karst U. 


Many heavy metal species with low oxidation states of the metal are highly thiophilic and tend to form adducts with 

any available free thiol group in small molecules or proteins. This mechanism may be associated with both toxic 

effects of the species and their detoxification. We have investigated several thiophilic element species and their 

reaction products with biologically relevant thiols by liquid chromatography (LC) coupled to electrospray ionization 

mass spectrometry (ESI-MS) for identification purposes and to inductively coupled plasma mass spectrometry 

(ICP-MS) for quantification. This is presented for the example of thimerosal, a mercury-containing compound 

used as preservative in vaccines and other drugs in concentrations up to 100 mg/L. In multi-dose ampullae, its 

antibacterial and antifungal properties prevent microbial contamination during repeated removal of single doses. It 

was introduced in 1931 and has been banned in the European Union in 2001 due to suspected neurotoxic effects 

especially in children. A connection between THI-containing vaccines and an increased occurrence of autism could 

neither be proven nor excluded until today. As model proteins, β-lactoglobulin A from bovine milk and human serum 

albumin have been used. Physiological conditions upon an intravenous injection of thimerosal-containing drugs 

were mimicked. The formation of ethylmercury-protein adducts was proved and the identification of the binding 

site of ethylmercury, a free thiol residue in the peptide T13 was achieved after tryptic digestion of β-lactoglobulin A. 

Other examples for adduct formation of thiophilic species are silver ions generated from antibacterial surfaces and 

gold-based pharmaceuticals. In the respective experiments, it is observed that the products formed with gold and 

mercury species are much more stable during LC separation than those formed with silver species, which rapidly 

dissociate under the LC conditions. For the gold-based pharmaceuticals, even cluster ions with two and four gold 

atoms are formed, separated by reversed phase LC and detected by ESI-MS. 

116 



S9-02 ANALYSIS OF LABILE METAL COMPLEXES IN PLANTS BY HILIC-ESI/MS 

Köster J., von Wiren N., Weber G. 

Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Metabolomics 

Corresponding author e-mail: guenther.weber@isas.de 

Micronutrients such as iron, copper and zinc are limiting factors for plant nutrition especially on neutral or alkaline 

soils. In plants there are two different, genetically fixed strategies for metal uptake. The first one is based on 

enhancing the solubility by lowering of the pH and reduction (in the case of iron) with subsequent uptake of metal 

ions. The other mechanism is based on the release of low molecular weight chelators (phytosiderophores) in the 

rhizosphere and uptake of the intact metal complexes through a transport system in the root membrane. While 

the metal uptake in both cases is well understood very little is known about further steps of translocation and 

redistribution of metal species inside the plants. In order to elucidate these phenomena on a molecular level, it is 

necessary to identify the respective metal species and their transformation products. There are several candidate 

ligands, which have been proposed already for being involved in metal transport in plants, including organic acids, 

amino acids, small peptides, and others. Citrate is one of most prominent candidates for metal binding, esp. for 

iron, but a real proof of the presence and identity (incl. stoichiometry) of respective iron-citrate complexes in 

plants is very difficult, because the stability of such species is relatively low. Similarly, there is some evidence of 

copper binding to histidine but the analytical identification of the intact species in plant samples is hindered by 

the limited chromatographic stability. Generally, there is always a risk of unwanted species-redistributions during 

chromatographic separation. Comparative results are presented for the chromatographic separation of labile metal 

complexes, such as iron-citrates and copper-histidines, on several HILIC materials (incl. sulfobetain-, diol-, amino-, 

amido-, and pentafluorophenyl-based polar or charged stationary phases). Significant differences exist between 

columns with respect to species stability and chromatographic separation. In particular for iron citrate, which may 

(co-)exist in several stoichiometries, it is difficult to guarantee species integrity during chromatography. Examples 

are shown for the separation and identification of iron(III)-citrates of different stoichiometry. Moreover, it is shown 

that species present in standard solutions are also found in a series of real plant samples. The results are discussed 

with respect to species stability, separation mechanism, and remaining problems for application to different 

samples. 


117


S9-03 SPECIES ANALYSIS OF PLATINUM BASED CYTOSTATIC DRUGS AND BIOLOGICALLY RELEVANT THIOLS 

Brauckmann C., Karst U. 



Corresponding author e-mail: uk@uni-muenster.de 

In every second cancer chemotherapy platinum-based cytostatic drugs are administered. Cisplatin is the most 

common and also the most effective anticancer drug for germ cell cancer, osteosarcoma and neuroblastoma. It has 

already been applied for more than 30 years, although the reasons for serious side effects including nephrotoxicity 

and ototoxicity are not fully understood yet. Reactions of cisplatin with DNA cannot explain these side effects. 

Actually, it is known that many different reactions of Cisplatin may occur in the human body. In particular, reactions 

with thiols are assumed. In this presentation, we investigate the reaction of cisplatin towards biologically relevant 

thiols such as glutathione, cysteine or even plasma proteins. A new method for the analysis of platinum complexes is 

presented. A separation of strongly polar complexes requires the use of a dedicated stationary phase. In this case, 

a separation with a zwitterionic HILIC column has been used for separation of Cisplatin, its hydrolysis products 

and its adducts with amino acids and peptides. In case of experiments with proteins, a reversed phase column 

was used because proteins are less polar. The complementary use of electrospray ionisation (ESI) and inductively 

coupled plasma (ICP) mass spectrometry (MS) as detection techniques allows both, the identification of the platinum 

complexes and the quantification with low limits of detection in complex matrices on the other hand. In these 

experiments, the physiological conditions of human blood and cells were simulated, which differ mainly in their 

chloride concentration. Blood has a high chloride concentration whereas a low chloride concentration can be found 

in cells. The chloride concentration affects the reaction behavior of Cisplatin, because a high chloride concentration 

improves the stability of the platinum complex and the formation of highly reactive hydrolysis products is blocked. 

Regarding these affects, it was possible to investigate reaction of Cisplatin and cysteine, glutathione, methionine, 

Mesna and even proteins under physiological conditions in blood and in cells. In this presentation, we present a 

HPLC separation of cisplatin and the respective adducts, which were formed with biologically relevant thiols. We 

identified these adducts with the help of HPLC/ESI-ToF-MS. The complementary analysis with the help of HPLC/ICP-MS 

allowed quantifying the platinum concentration independently of the particular species. 

118 



S9-04 SPECIATION OF GADOLINIUM-BASED MRI CONTRAST MEDIA IN WASTEWATER TREATMENT PLANTS BY HILIC/ICP-MS 

Telgmann L., Künnemeyer J., Karst U. 



Since their introduction in the 1980s, the application of Gd-based contrast agents increased rapidly. Today, almost 

every second MRI procedure is enhanced by contrast agents, resulting in about 20,000,000 applications per 

year worldwide. For the application as contrast agent, Gadolinium is complexed by chelating agents. In general, 

contrast agent formulations are highly concentrated. This leads to a very high input of anthropogenic Gd into the 

environment. Therefore, Gd chelates are among the most important emerging environmental contaminants. Although 

this has already caused a strong anomaly for Gd concentrations in surface waters, only the sum concentration of 

Gd is typically determined by ICP-MS. However, there is little knowledge about the concentration of various Gd 

species in wastewaters and their behaviour in sewage treatment plants. This work describes method development 

and speciation analysis of gadolinium-based MRI contrast agents in wastewaters. This approach comprises the 

hyphenation of hydrophilic interaction chromatography (HILIC) with inductively coupled plasma mass spectrometry 

(ICP-MS). HILIC/ICP-MS exhibits high separation efficiency for the simultaneous separation of the five predominantly 

applied MRI contrast agents and the required selectivity and sensitivity for trace determination in wastewater 

samples. A zwitterionic HILIC stationary phase allowed the separation of the five most important contrast agents. 

ICP-MS provided quantification independent on the Gd species. Several pre-treatment-procedures have been 

examined: since silanised glass ware showed a decrease of the analyte concentration caused by adsorption, 

polypropylene bottles have been used for storing of the wastewater samples. Additional pre-treatment steps 

included filtration and in some cases pre-concentration of the samples. Sampling of wastewater has been carried 

out every ten minutes in the primary treatment tank and in the final clarification tank of the sewage treatment plant. 

By knowing the quantities of water that entered the sewage treatment plant during the period of the sampling, it was 

possible to state if Gd-based MRI contrast agents were removed during the sewage treatment. All water samples 

were not only analyzed for the particular contrast agents by triplicate HILIC/ICP-MS measurements, but also total Gd 

was determined by ICP-MS for validation purposes. The concentrations of the individual Gd species, the standard 

deviation of the mean values of the triplicate measurements, the sum of all Gd species determined by HILIC/ICP-MS 

as well as the total concentration of Gd determined by ICP-MS. It was demonstrated that the developed method is 

suitable for the speciation analysis of the five most important MRI contrast agents in real waste water samples. 


119


ORAL 

SESSIONS


S10-01 CHROMATOGRAPHIC METHODS FOR THE ANALYSIS OF POLYCYCLIC AROMATIC SULFUR HETEROCYCLES 

Nocun M., Andersson J.T. 



Modern fuels are desulfurized in the refineries to lower the sulfur content to below 10 parts per million as demanded 

by legislation. A major class of sulfur compounds remaining after the process are the so-called polycyclic aromatic 

sulfur heterocycles (PASH). Clarifying the relationship between their structure and recalcitrance to desulfurization is 

an important goal. 

Petroleum is the most complex mixture known and every speciation method relies on a simplification of this 

complexity. In our analysis we first separate the PASHs from all other groups of compounds through liquid 

chromatography on a phase containing Pd(II) ions. By using a silica gel with ligand-bonded Ag(I) ions we can achieve 

an efficient group separation of PASHs based on the number of aromatic C atoms. 

After conversion of the dibenzothiophene fraction to the corresponding sulfoxides, these can be separated on a 

diphenyl stationary phase according to their number of methyl groups. These subfractions are analysed with gas 

chromatography after a simple reduction step. Due to the characteristic chemical shifts for dibenzothiophene 

sulfoxides, they can also be analysed by 1H-NMR e.g. to determine the percentage and position of carbon side 

chains for these compounds. The goal is to establish the proportion of dibenzothiophenes with substituted 

4-position since these are most recalcitrant to hydrodesulfurization. 

These phases may have use outside fossil fuel characterization, e.g. in environmental analysis. 


121


S10-02 POLYCHLORINATED DIBENZO-P-DIOXINS, DIBENZOFURANS AND BIPHENYLS IN GENERAL POPULATION LIVING NEAR AN 

URBAN WASTE TREATMENT PLANT IN MATARÓ 

Parera J. 1 , Serra-Prat M. 2 , Moreno V. 2 , Martrat M.G. 1 , Palomera E. 2 , Abalos M. 1 , Rivera J. 1 , Abad E. 1 

1 

IDÆA-CSIC, Dioxins Laboratory, Mass Spectrometry Laboratory, Barcelona 

2 

Hospital of Mataró, Consorci Sanitari del Maresme, Research Unit, Barcelona 

Corresponding author e-mail: eaheco@iiqab.csic.es 

Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) constitute three classes 

of structurally related chlorinated aromatic compounds of concern since they represent potential risks for human 

health. These compounds possess a strong lipophilic character. Therefore, they may develop bioaccumulation 

phenomena in the different tissues of the living organisms and resulting of a biomagnification process trough the 

food chain1. In addition, it is reported their presence at ppt concentrations in biological tissues. In general, PCDD/ 

Fs occur as unwanted by-products in various industrial processes, where combustion, metallurgical and chlorinecompounds 

play a substantial role in producing these compounds. On the other hand, PCBs have been widely used 

as technical products in industry for many different applications2. Common practices for the analysis of PCDD/Fs 

and PCBs in blood samples involve several extraction methods3,4,5. From those, liquid-liquid extraction method 

ideally results in a complete extraction of lipids. However, the formation of emulsions and/or the consumption 

of large amounts of hazardous solvents are real drawbacks. The alternative to these methods is sorbent-assited 

method, which use a chromatographic glass columns packed with several layers of Chem-Elute6. Since this method 

is easier and with higher reproducibility it was selected for the determination of PCDD/Fs and PCBs in human whole 

blood samples. The aim of this study was to determine the concentration of PCDD/Fs and marker-PCBs in whole 

blood samples of people living near a solid waste incineration plant evaluated after 10 years of regular operations 

in the facility (1995-2006). In the present study, existing relationship between exposure biomarkers and the main 

variables: sex and age is also attended. In general, levels for PCDD/Fs varied between 13.2 and 20 pg WHO-TEQ/g 

fat and for marker-PCBs from 1.19 to 2.18 µg/L. In all cases, PCDD/Fs and marker-PCBs levels were higher in women 

than in men, although the differences were not significant. As expected, levels of PCDD/Fs and marker-PCBs were 

significantly higher in the oldest age-group, which is consistent with the fact that these are liposoluble substances 

that accumulate in the adipose tissue7. In general, our findings are consistent to those found for general population 

living in the surroundings of a municipal waste incineration plants8. References 1. P.Voogt, D.E.Wells, L.Reutergardh, 

U.A.Th.Brinkman, Int. J. Environ. Anal. Chemosphere. 40 (1990) 1-46. 2. D.Mukerjee, Org. Mass Spectrom. 26 (1998) 

157. 3. D.G.Pattersson, P.Fürst, L.R.Alexander, S.G.Isaacs, W.E.Turner, L.L.Needham. Chemosphere 19 (1989) 89- 

96. 4. R.R.Chang, W.M.Jarman, J.A.Hennings. Ana. Chem. 65 (1993) 2420-2427. 5. A.Covaci, P.Schepens. 2001. 

Chemosphere 43 (2001) 439-447. 6. O. Päpke, M.Ball, Z.A.Lis, K.Scheunert. Chemosphere 19 (1989) 941-948. 

7. E. Felip, A.Abballe, F.Casalino, A.Domenico, P.Domenici, N.Iacovella, A.M.Ingerido, E.Pretolani, M.Spagnesi. 

Chemosphere 72 (2008) 25-33. 8. M.F.Reis, J.P.Miguel, C.Sampaio, P.Aguiar, J.M.Melim, O.Päpke. Chemosphere 67 

(2007) 224-230. 

122 



S10-03 LC-MS/MS STUDY OF DISTRIBUTION OF PHARMACEUTICALS IN WATER, SUSPENDED SOLIDS AND SEDIMENTS OF THE 

EBRO RIVER 

Silva B.F. 1 , Jelic A. 1 , Lopez R. 1 , Mozeto A.A. 2 , Petrovic M. 1 , Barcelo D. 1 

1 

IDÆA-CSIC, Department of Envinromental Chemistry, Barcelona 

2 

UFSCar, Departamento de Química - Lab. Biogequímica, University of Sao Carlos 

Corresponding author e-mail: mpeqam@idaea.csic.es 

LC-MS/MS study of distribution of pharmaceuticals in water, suspended solids and sediments of the Ebro river 

Bianca Ferreira da Silva1,2, Aleksandra Jelic1, Rebeca Lopez1, Antonio A. Mozeto2, Mira Petrovic1,3, Damia 

Barcelo1,4 1. Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona, 18-26, 08034 Barcelona, Spain 

2. Departamento de Química, Lab de Biogeoquímica Ambiental, UFSCar, Rod Washington Luís Km 235, 13565-905, 

C.P. 676, São Carlos, São Paulo, Brasil 3. Catalan Institution for Research and Advance Studies (ICREA), Passeig 

Lluis Companys 23, 08010 Barcelona, Spain 4. CatalanInstitute for Water Research (ICRA), Parc Cientific i Tecnologic 

de la Universitat de Girona, Edifici Jaume Casademont, 17003 Girona, Spain Abstract High performance liquid 

chromatography (HPLC) coupled to a quadrupole linear ion trap mass spectrometer (QqLIT-MS) has been applied to 

study occurrence and distribution of pharmaceuticals in the aquatic environment. Concerning the pharmaceuticals 

market, Spain has raised its position over the recent years. Such high consumption may lead to the conclusion that 

the problematic associated with the aquatic contamination by pharmaceuticals may be an important issue that needs 

to be assessed and, since data regarding contamination of Spanish aquatic systems is still sparse, it is necessary to 

set up surveys at national or basin scale. In the light of these concerns, the aim of the study was to identify the loads 

of pharmaceuticals discharged into the aquatic environment through municipal wastewater effluents and to study 

their distribution between different phases, i.e. water, suspended solids and sediment. The list of target compounds 

included 73 pharmaceuticals of major consumption in Spain. The sampling of the sediment and the river water 

samples was done at 25 points along the river Ebro basin (North-East of Spain), and the effluent water was collected 

from 6 wastewater treatment plants that discharge treated water to the Ebro. To extract target compounds from 

solid samples pressurized liquid extraction followed by SPE clean up is used, while separation and detection is done 

using LC-QqLIT- MS. The obtained results showed presence of 32 compounds in effluent waters in concentrations 

ranging g/L (e.g. anti-inflammatory drugs), while in theμfrom low ng/L to a few river water samples concentrations 

were typically lower than 100 ng/L due to significant dilution factors. In sediment samples 25 pharmaceuticals were 

detected in ng/g concentrations. Acknowledgments The work was financially supported by the Spanish Ministry of 

Education and Science, project CEMAGUA (CGL2007-64551/HID) and Consolider-Ingenio 2010 project SCARCE 

[CSD2009-00065]. B. F. da Silva acknowledges the CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível 

Superior). 


123


S10-04 DERIVATIZATION AND FRAGMENTATION PROPERTIES OF THE TRIMETHYLSILYL (OXIME) ETHER/ESTER DERIVATIVES OF 

STEROIDS, BY GAS CHROMATOGRAPHY MASS-SPECTROMETRY: STEROID POLLUTANTS IN WASTEWATERS 

Andrási N., Helenkár A., Vasanits-Zsigrai A., Záray Gy., Molnár-Perl I. 

Eötvös Loránd University, Institute of Chemistry 

Corresponding author e-mail: perlne@chem.elte.hu 

The analysis of steroids by gas chromatography-mass spectrometry (GC-MS) is still a challenge to analytical 

chemists. Steroid profiling is of primary importance in the diagnosis of clinical disorders, in the recognition of drug 

abuses in sports doping control, in food analysis and most importantly in the pollutant analysis of environmental 

water samples. Recent reviews[1-3] and case studies [4-7] confirm the unambiguous fate of steroids, impairing 

wildlife. The authors’ goal was to extend our multiresidue analysis method already suitable for analyzing sixty-three 

compounds in waste water samples in a single injection [8], by increasing the number of analytes with numerous 

natural any synthetic steroids. These compounds can be sorted in five groups: natural androgens (androsterone, 

testosterone), natural estrogens (beta-estradiol, estriol), synthetic estrogens (mestranol, ethinylestradiol), synthetic 

progestogens (norethisterone, gestodene, levonorgestrel, etonorgestrel) and phytosterols (stigmasterol, betasitosterol). 

The first step of our basic research studies was the investigation of the effect of oximation on the 

response of the investigated ketosteroids (androsterone, testosterone, norethisterone, gestodene, levonorgestrel, 

etonorgestrel) in comparison to the only silylated derivatives. As the next step, the optimization of our earlier 

reported two-step derivatization process was done [8]. Experiments included varying reaction time and temperature 

in case of oximation (with hydroxylamine-hydrochloride in pyridine) and silylation; applying four different silylating 

agents: hexamethyldisilazane + trifluoroacetic acid (HMDS/TFA), N-methyl-N-(trimethylsilyl)-trifluoroacetamide 

(MSTFA), bis-(trimethylsiyl)trifluoroacetmide (BSTFA) and N-methyl-N-tert-butildimethylsilyl-trifluoroacetamide 

(MTBSTFA). After defining the optimum conditions of the derivatization process , the mass fragmentation 

pattern analysis of the investigated compounds was performed. Up to this point, androsterone, beta-estradiol, 

ethinylestradiol and estriol were identified and quantified in wastewater samples subsequently to their enrichment 

by solid phase extraction (pH 4, Oasis HLB cartridges). Investigation of optimal conditions of the extraction process 

is in progress. [1] V. Gabet, C. Miége, P. Bados, M. Coquery, Trends Anal. Chem. 26 (2007) 1113-1131 [2] G. Streck, 

Trends Anal. Chem. 28 (2009) 635-651 [3] H.S. Chang, K. H. Choo, B. Lee, S. J. Choi, J. Haz. Mat. 172 (2009) 1-12 

[4] N. G. Reyero, J. O. Grimalt, I. Vives, P. Fernandez, B. Pina, Environ. Poll. 145 (2007) 745-752 [5] A. V. López, E. R. 

Gallegos, M. G. Martínez, F. A. J. Orozco, E. G. Latorre, M. L. D. López, Comp. Biochem. Phys. C 145 (2007) 394-403 

[6] D. Schlenk, Mar. Poll. Bull. 57 (2008) 250-254 [7] J. R. Colman, D. Baldwin, L. L. Johnson, N. L. Scholz, Aquatic 

Tox. 91 (2009) 346-354 [8] Á. Sebök, A. Vasanits-Zsigrai, A. Helenkár, Gy. Záray, I. Molnár-Perl, J. Chromatogr. A, 

1216 (2009) 2288-2301 

124 


S11 

CLINIC AND 

PHARMACEUTICAL 

APPLICATIONS 

ORAL 

SESSIONS


S11-01 ALKYLATED POROUS GRAPHITIC CARBON FOR LIQUID CHROMATOGRAPHY 

Jensen D., Wiest L.A., Dadson A., Vail M.A., Linford M.R. 

Brigham Young University, Chemistry and Biochemistry 

Corresponding author e-mail: mrlinford@chem.byu.edu 

The covalent attachment of alkyl ligands to porous graphitic carbon (PGC) (HypercarbTM) imparts new properties to 

the support material. The attachment of alkyl ligands was achieved by using azo-tert-butane as the modifying agent 

in a gas phase reaction. At elevated temperatures azo-tert-butane thermally degrades to produce two tert-butyl 

radicals along with nitrogen as a byproduct. The tert-butyl radicals covalently bond to the PGC surface to produce 

an alkylated surface. The attachment of alkyl ligands to the PGC produces a more hydrophobic material. This new 

material has shown its use in reversed phase liquid chromatography (LC) and shows an increase in retention factor 

along with different selectivities compared to PGC for various alkyl benzenes. The resulting bonded PGC phase is 

stable under extreme pH conditions (pH < 0, pH > 14) and more chemically and thermally stable than commercially 

available silica-based stationary phases for LC. The modified PGC was characterized by Time of Flight-Secondary 

Ion Mass Spectrometry (ToF-SIMS) along with chemometrics to elucidate the modification of the PGC. Preliminary 

LC studies have shown an increase in the retention factor for the following analytes: benzene, toluene, ethyl benzene, 

propyl benzene, hexyl benzene, isopropyl benzene, 1,3,5-trimethyl benzene; with the mobile phase composition 

being 95:5 methanol:water. With the alkyl modification of this material, an increase in the retention factor of up to 

20% has been achieved. This new LC material can be used to separate biomolecules in their native state due to the 

pH stability of the chromatographic support and phase. 

126 



S11-02 CAPILLARY ELECTROPHORESIS PROCEDURE FOR THE SIMULTANEOUS ANALYSIS AND STOICHIOMETRY DETERMINATION 

OF A VINCA ALKALOID AND ITS COUNTER-ION BY USING DUAL-OPPOSITE END INJECTION AND CONTACTLESS 

CONDUCTIVITY DETECTION: APPLICATION TO CATHARANTHINE SULFAT 

Lopez C. 1 , Nehme R. 1 , Claude B. 1 , Morin P. 1 , Penna R. 2 , Max J.P. 2 , Filaquier J.P. 2 , Ribet J.P. 2 

1 

ICOA - University of Orléans 

2 

Institut de Recherche Pierre Fabre, Analytical chemistry, Boulonge Billancoutn 

Corresponding author e-mail: philippe.morin@univ-orleans.fr 

Catharanthine and vindoline are indole alkaloids present in Catharanthus roseus plant extract. The natural coupling 

of these two molecules results in formation of dimers (binary alkaloids), vinblastine, and vincristine, with proved 

anti-cancer therapeutic power. The very small quantities of dimer alkaloids (around 0.005%) in Catharanthus roseus 

was an hindrance overcome by non natural syntheses of vinblastine and vinorelbine; this last one was obtained from 

coupling of catharanthine and vindoline and is recommended in the treatment of advanced human non-small-cell 

lung cancer and breast cancer. Then, vinflunine, a fluorinated derivative of vinorelbine, was synthesized and is now 

in phase III trials, for the treatment of bladder cancer. A capillary electrophoresis (CE) procedure was developed 

for the simultaneous determination of alkaloids and its counter-ions. For this purpose, an uncoated fused-silica 

capillary, a low conductivity background electrolyte and a capacitively coupled contactless conductivity detector 

(C4D) were employed. This detection system is highly sensitive and enables detection of inorganic as well as organic 

ions. Moreover, to be able to simultaneously analyze the cationic drug (catharanthine+) and its anionic counter-ion 

(SO42−) in the same electrophoretic run, a dual-opposite end injection was performed. In this technique, the sample 

is hydrodynamically injected into both ends of the capillary. This CE-C4D procedure with dual-opposite end injection 

has been successfully validated and applied for the analysis of sulfate content in catharanthine sulfate. Thus, the 

developed method has been shown to be fast (analysis time < 12 min) and precise (repeatability of migration times 

< 0.4% and of corrected-peak areas < 0.8%; n = 6 for sulfate). The counter-ion tartrate in a bisindole alkaloid 

(vinorelbine ditartrate) was also quantified to determine the stoichiometry. The method was further applied to several 

counter-ions such as chlorure, maleate and citrate in pharmaceutical drugs. Keywords: Capillary electrophoresis ; 

Contactless conductivity detection ; Counter-ion ; Vinca alkaloids. 


127


S11-03 OPTIMIZATION OF SIMPLE IN SITU DERIVATIZATION REACTIONS FOR IBUPROFEN GAS CHROMATOGRAPHY ANALYSIS 

USING HEADSPACE GENERATION 

Casas Ferreira A.M., Fernández Laespada M.E., Pérez Pavón J.L., Moreno Cordero B. 


Corresponding author e-mail: anacasas@usal.es 

The use of headspace sampling (HS) solves many analytical problems by minimizing sample treatment. However, 

in many cases the limits of detection achieved are insufficient for the detection of the analytes of interest. The 

formation of more volatile derivatives is a widely used practice in gas chromatography and can also be used as a 

step prior to headspace sampling, allowing the use of HS for compounds to which, owing to their low volatility, in 

principle it would not be applicable. Non-steroid anti-inflammatory drugs (NSAIDs) form a group of drugs widely 

used as analgesic, antipyretic and anti-inflammatory agents. In particular, ibuprofen is among the most widely used 

pharmaceuticals in the world. The derivatization reactions most frequently used prior to their gas chromatography 

analysis involve silylation with MTBSTFA (N-methyl-N-(tert-butyldimethysilyl)trifluoroacetamide) or MSTFA 

(N-methyl-N-(timethylsilyl)trifluoroacetamide) [1], and alkylation, using diazomethane [2]. Other derivatization 

alternatives have also been proposed [3-5]. In all cases, a previous extraction of the analytes of interest must be 

performed from the aqueous matrix. A reported alternative in which the reaction is carried out in situ in the aqueous 

sample is derivatization with a carbodiimide and 2,2,2-trifluoroethylamine to produce an amide derivative [6], with 

subsequent liquid-liquid extraction using methyl tert-butyl ether (MTBE). The limits of detection obtained are high: in 

the order of mg/L. The aim of the present work is to optimize simple in situ derivatization reactions using headspace 

generation for the determination of ibuprofen in water samples. The proposed derivatization reagents used are: 

a) methanol in acidic medium, thus generating the methyl derivative, and b) 3-ethyl-1-[3-(dimethylamino)propyl] 

carbodiimide and 2,2,2-trifluoroethylamine, that generate the amide derivative. These reactions are quite simple. 

The instrumental configuration used in this study has the advantage that the derivatization and the instrumental 

measurement of the analytes (HS-PTV-GC-MS) are carried out on-line, with no need for intermediate steps. The 

limits of detection obtained (0.23 µg/L and 0.0096 µg/L, respectively), bearing in mind that no preconcentration or 

later clean-up steps are required, make these good methods for the analysis of ibuprofen in aqueous samples. [1] 

A. Sebók, A. Vasanits-Zsigrai, Gy. Palkó, Gy. Záray, I. Molnár-Perl, Talanta 76 (2008) 642. [2] S. Öllers, H.P. Singer, 

P. Fässler, S.R. Müller, J. Chromatogr. A 911 (2001) 225. [3] V. Koutsouba, Th.Heberer, B. Fuhrmann, K. Schmidt- 

Baumler,D. Tsipi, A.Hiskia, Chemosphere 51 (2003) 69. [4] S.S. Verenitch, C.J. Lowe, A. Mazumder, J. Chromatogr. A 

1116 (2006) 193. [5] W.C. Lin, H.C. Chen,W.H. Ding, J. Chromatogr. A 1065 (2005) 279. [6] Q.L. Ford, J.M. Burns, J.L. 

Ferry, J. Chromatogr. A 1145 (2007) 241. 

128 


S12 LIQUID 


ORAL 

SESSIONS


S12-01 NEW LC PUMP TECHNOLOGY TO IMPROVE PERFORMANCE UNDER ALL OPERATING CONDITIONS 

Preiswerk T. 1 , Guazzotti S. 2 , Boehm G. 1 

1 

Thermo Fisher Scientific, SID-Switzerland 

2 



Techniques for qualitative and quantitative analysis that offer improvements in productivity are currently in high 

demand. Ultra High Performance Liquid Chromatography (UHPLC) offers several advantages when compared 

to HPLC including shorter analysis times, excellent separation efficiencies, and increased sensitivity. In order to 

improve flexibility and performance, systems that can operate both under HPLC and UHPLC conditions have been 

designed. The main drawback of these systems is the loss of performance when operating outside of a specific set 

of conditions for which they have been optimized. Another limitation was the lack of quaternary capabilities since 

this requires low pressure mixing and in the past low pressure mixing demanded larger delay (dwell) volumes for 

optimum performance; this is no longer the case due to newly developed pump technology to be discussed in this 

presentation. This technology employees unique force sensors to modulate pumping efficiency based on assessed 

mobile phase compressibility, thereby enabling the delivery of accurate and reproducible gradients with extremely 

low delay volumes. The force sensors employed in these pumps measure the actual force that is needed to move 

each piston. This force sensor signal can be used to measure compressibility of the displaced solvent in real-time, 

therefore variations in compressibility due to changes in mobile phase composition, temperature, etc. are accounted 

and compensated during each individual piston stroke resulting in a true volumetric, virtually pulsation free flow from 

the pump over its complete dynamic working range. This outstanding flow and pressure stability eliminates the need 

for a pulse dampening device which increases the ease of use. This technological breakthrough results in increased 

accuracy, precision, and reliability in pump performance under all operating conditions, permitting the development 

of flexible HPLC/ UHPLC systems with outstanding characteristics. Details on the development and performance of 

these systems will be presented. 

130 



S12-02 ADVANCES IN CAPILLARY ION CHROMATOGRAPHY SYSTEMS USING ON-LINE ELECTROLYTIC ELUENT GENERATION AND 

SUPPRESSED CONDUCTIVITY DETECTION 

Divan K., Liu Y., Divan K. 

Dionex Corporation 

Corresponding author e-mail: yan.liu@dionex.com 

The operation of ion chromatography system in capillary format offers a number of advantages. Since the eluent 

consumption is very small, capillary IC systems can be operated continuously and therefore are always on and 

always ready for analysis. Capillary IC systems offer improved compatibility with applications where amount of 

sample is limited. The operation of capillary IC at low flow rates improves the system compatibility with mass 

spectrometer. The use of capillary separation columns can improve the separation efficiency and/or speed. Capillary 

column format opens the door for the possibilities of offering new selectivity for difficult applications using new 

columns packed with difficult-to-make stationary phases. Over the past several years, we have made consistent 

efforts to develop capillary reagent-free IC (RFICTM) systems with on-line electrolytic eluent generation and 

suppression. In this presentation, we will report the progress of our on-going efforts. We will discuss the preparation 

and optimization of key components used in the capillary RFICTM systems including capillary electrolytic eluent 

generators, continuously regenerated trap columns, separation columns, carbonate removal devices, and electrolytic 

suppressors. We will highlight the analytical capabilities of our prototype capillary RFICTM systems in the 

determination of target inorganic and organic analytes. 


131


S12-03 QUALITY BY DESIGN LC METHODS DEVELOPMENT USING ORTHOGONAL STATIONARY PHASES 


Waters Corporation 

Many tools are employed by method developers to achieve optimum separations for their compounds, including 

different columns, mobile phases, and software packages. Ideally, the such method would be obtained using the 

minimum number of screening runs possible. The most common and efficient methods development approach 

is to screen multiple columns and mobile phases to cover as much of the selectivity space as possible prior to 

optimization, which can be performed manually or with software. In this talk, we investigate the influence of the 

stationary phase on selectivity, peak capacity, and retention time drift after pH modification, especially with low 

ionic strength mobile phases (i.e., formic acid and ammonium hydroxide) for challenging basic, acidic, and neutral 

analytes. The wide range of selectivity provided by the innovative stationary phases benefits the automated LC 

methods development approach for the separation of very diverse compounds. By integrating Fusion Methods 

DevelopmentTM software with EmpowerTM 2 Chromatography software, the automated process can be designed 

according to Quality by Design (QbD) guidelines to ensure reliable and robust methods prior to validation. A new 

quaternary-based UPLC system was employed, which is ideal for methods development and due to the system’s 

ability to blend multiple solvents automatically it substantially improves chromatographic flexibility and throughput. 

In addition, the flow-through needle design of the instrument enables sequential development of methods using both 

reversed-phase and HILIC approaches. The system also allows seamless methods transfer from HPLC to UPLC and 

vice versa, and methods are shown to be easily transferred between HPLC and UPLC particle sizes. 

132 


ORAL 

SESSIONS 

S13 

FOOD QUALITY 

AND SAFETY


S13-01 FAST CHROMATOGRAPHY WITH SUB-2 µM PARTICLE-SIZE COLUMNS IN THE ANALYSIS OF HIGH-COMPLEX MATRICES: 

REALITY OR UTOPIA? VALIDATION OF A RAPID RESOLUTION LC-MS/MS METHOD FOR THE ANALYSIS OF MARINE TOXINS 

de la Iglesia P., Barber E., Giménez G., Diogène J. 

Institut de Recerca i Tecnologia Agroalimentàries (IRTA). Sant Carls de la Rapita 

Corresponding author e-mail: pablo.delaiglesia@irta.cat 

The application of rapid separation methods aimed at reducing the analysis time is highly demanded by analytical 

laboratories. In research, a short analysis time allows a rapid method development and validation. When routinely 

applied, rapid resolution methods not only improve the productivity of the laboratory but also may provide a more 

efficient response against food safety alerts. 

For chromatographic methods, the recent developments on instrumentation and column packing materials have 

promoted a rapid growth of rapid resolution applications using sub-2µm particle-size columns in the last few years. 

Nevertheless, methods for the analysis of samples with high-complex matrices are still limited. 

We present the application of rapid resolution liquid chromatography on a 1.8 µm particle-size column coupled 

with tandem mass spectrometry (RRLC-MS/MS) for the analysis of amnesic shellfish poisoning (ASP) toxins in 

shellfish, mainly domoic acid (DA) and epidomoic acid (EA). Complete resolution among DA and the isomers A, D 

and EA acid was achieved in less than 3 min. The method has been intra-laboratory validated for direct analysis of 

methanolic crude extracts (50:50) without further clean-up. With this aim, 6 different types of shellfish matrices with 

relevancy for the shellfish industry were selected, namely: blue mussel, pacific oyster, clam, smooth clam, wedge 

shell and a muricid gastropod. The method showed high sensitivity with limits of detection ranging from 0.01 to 

0.03 µg DA/mL (which corresponded to 0.05-0.09 mg/kg). Working range was selected bearing in mind the current 

maximum permitted level for DA of 20 mg/kg (4 µg/mL) and the level recently proposed by the European Food Safety 

Authority of 4.5 mg/kg (0.9 µg/mL). Linearity was demonstrated over the 0.1-4.0 µg/mL range (y = 17,899x – 1,067.8, 

R² = 0.9968) being adequate for quantitation. Matrix effects evaluation revealed slight drifts or drops on slopes 

compared to that obtained with external calibration, though they could be corrected. Additionally, confirmatory 

capabilities of MS/MS detection were demonstrated according to the Commission Decision 2002/657/EC criteria, 

with relative ion intensities higher than 20% for confirmation ions 248 and 193 m/z and variability from 2 to 7% below 

the maximum permitted tolerance of ± 25%. Robustness study revealed the percentage of formic acid as the most 

affecting parameter on ionization yields and therefore on quantitation. The comparison between RRLC-MS/MS and 

the reference LC-UV for the analysis of blind samples (n= 22) resulted in a lineal regression with a slope of 1.1721, 

intercept of 0.2061 and R2 = 0.9751. These results, together with those obtained in the inter-laboratory exercise 

organized by the Community Reference Laboratory on Marine Biotoxins during 2009 for ASP toxins analysis (z-score 

= -0.962 and 0.177 for a low and high contaminated sample, respectively), allow us to conclude that RRLC-MS/MS 

provided additional advantages for the analysis of ASP toxins while the performance and reliability of the results, 

even in very complex matrices, were conserved. 

134 



S13-02 DEVELOPMENT AND APPLICATION OF AN IMMUNOAFFINITY COLUMN FOR THE SOLID-PHASE EXTRACTION OF 

PYRACLOSTROBIN RESIDUES FROM FRUIT JUICES 

Esteve Turrillas F.A. 1 , Mercader J.V. 1 , Agulló C. 2 , Abad-Somovilla A. 2 Abad-Fuentes A. 1 

1 

Instituto de Agroquímica y Tecnología de Alimentos - CSIC, Valencia 

2 

University of València, Department of Organic Chemstry 

Corresponding author e-mail: aabad@iata.csic.es 

Pyraclostrobin (PY) is a fungicide of the strobilurin group, with a new action mode, that is widely used against 

several diseases affecting grape, strawberry, lettuce, pepper or tomato crops. The 2007 Annual Report on Pesticide 

Residues published by the European Food Safety Authority (EFSA) shows that PY traces were detectable in 3.74% 

of the fruit and vegetable samples analyzed in a surveillance campaign in 29 european countries. Moreover, 

maximum residue levels for PY in foods have been established by the European authorities with values ranging 

from 0.02 to 10 mg/Kg depending on the commodity. PY is often analyzed by multiresidue methods using solidphase 

extraction (SPE) with standard solid supports such as octadecil-silica (C18), primary secondary amine (PSA) 

or graphitized carbon black (GCB) with high-performance liquid chromatography (HPLC) or gas chromatography 

(GC) determinations. However, the development of new sorbents such as molecular imprinted polymers, carbon 

nanotubes or immunoaffinity supports has been a subject of great interest in the last years. The aim of this study 

was the evaluation of different antibodies for the production of immunoaffinity columns (IAC) for the SPE of PY 

residues in food samples. Three monoclonal and three polyclonal antibodies with different affinities to PY (ELISA 

IC50 = 1-668 nM) were considered in this study. Different immunosorbents were produced and characterized by 

the establishment of antibody immobilization efficiency (from 83.1 to 99.9%), immunosorbent capacity (from 13.3 

to 86.7 µgPY/g), elution solvent (75% acetonitrile in water) and reusability (more than 10 times). The best global 

performance was achieved with polyclonal antibody PYo5#2 and the efficacy of the produced IAC was manifested 

by the development of a SPE procedure coupled to HPLC with UV detection for the determination of PY residues in 

fruit juices. A 50-fold concentration was achieved using the developed IACs with a limit of detection of 0.005 mg/L. 

Finally, quantitative recoveries were obtained on apple juice and red grape must samples spiked with PY from 0.01 

to 1 mg/L. 


135


S13-03 DETERMINATION OF ISOTHIOCYANATES AND INTACT GLUCOSINOLATES IN FOODSTUFF USING GC-MS AND HPLC-MS 

Kuebler K., Paetzold R., 

Institute of Nutritional Science, Professorship of Food Sciences-Germany 

Corresponding author e-mail: Ralf.Paetzold@ernaehrung.uni-giessen.de 

Glucosinolates (GSL) are sulfur containing secondary plant metabolites mainly found in Cruciferae with promising 

potential in cancer prevention. They are found in 16 plant families including Capparaceae, Moringaceae, 

orBrassicaceae. GSL are stable components but disruption of plant tissue activates cell own myrosinase which 

catalyses decomposition to thiocyanates, isothiocyanates, and nitriles. The amounts of each resulting product 

depend on e.g. pH-value, temperature, and ascorbic acid content. 

We describe two different HPLC-methods, one using a C18-column modified for polar analytes (Varian Polaris® 

C18-Ether), the other working with HILIC-technology (phenomenex Luna® HILIC), for separation of 14 commercial 

available intact glucosinolates. They proved capable for detection of a broad range of GSLs in one analysis by a 

single column without desulphation step for getting unchanged intact GSLs ready for further activity studies or 

purified for using them as standard compounds. We also show contents of intact GSLs in samples of 10 “cress”- and 

8 sprout samples, mainly containing to the plant family Brassicacea. We e.g. found ranges of between 130 and 250 

mg/100g FW of glucoraphanin in various broccoli related samples, 15.8 mg/100g FW of Sinigrin in Mustard cress 

and 716 to 4431 mg/100g FW of glucoraphenin in radish related samples, whereas samples not belonging to the 

family Brassicaceae -as expected- didn’t contain any intact GSLs. In additionally analyzed dietary supplements 

or nutraceuticals (n=14) derived from e.g. maca, broccoli and cabbage we found the intact glucosinolates 

glucotropaeolin (115-462 mg/100g FW), glucoerucin (53.4 mg/100g FW), glucoraphanin (956 mg/100g FW), sinigrin 

(45 mg/100g FW), sinalbin (19.7 mg/100g FW), and methoxy-glucotropaeolin (41.8-156 mg/100g FW). Four samples 

did not contain any detectable GSLs inspite of their declaration. 

Using HILIC-technology Moringa (horseradish tree) leave powder, a food supplement in Far East, was analyzed. 

It is a traditional foodstuff e.g. in Korea and is derived from 4 different plants (M. oleifera, M. stenopetala, M. 

drouhardii, and M. peregrina). Monoacetyl-4-(alpha-L-rhamnopyranosyloxy)-benzyl-glucosinolate was detected 

in all four samples. 4-(alpha-L-rhamnopyranosyloxy)-benzyl-glucosinolate was detected in M. oleifera for the first 

time. 1-Methylethyl-GLS (Glucoputranjivin) was found in M. peregrina, additional an unknown acetyl-isomer in M. 

drouhardii. 

For health appraisement of all samples it’s also necessary to detect their amount of isothiocyanates (ITCs) as well. 

ITC’s are volatile compounds and we were able to develop a GC-SIM-MS method (column: Varian Factor Four® 

VF-35 ms) resolving 11 commercial available isothyocyanate standard substances without derivatization. The 

method was capable for detection of ITCs in different food matrices. We detected suspected addition of European 

horseradish material to wasabi, a Japanese relative which is much more expensive and is used for hot pastes and 

sushi. Three of six resolved commercial wasabi samples did contain phenylethyl-ITC (3.1-10.4 mg/100g FW) which 

is not a typical ingredient for wasabi. It is normally found (10.3-54.1 mg/100g FW) additional to allyl-ITC in European 

horseradish. 

So all shown methods will have benefits for use in food control and it will be possible to isolate intact GSLs for use 

as reference materials. 

136 



S13-04 ASSESSMENT OF THE EFFECT OF NON-VOLATILE WINE MATRIX ON THE VOLATILITY OF TYPICAL WINE AROMA 

COMPOUNDS BY HS-SPME-GC-MS ANALYSIS 

Rodríguez-Bencomo J.J., Muñoz-González C., Andujar-Ortiz I., Martín-Alvarez P.J., Moreno-Arribas M.V., Del Pozo-Bayón M. A. 


Corresponding author e-mail: mdelpozo@ifi.csic.es 

Volatile compounds of wines are responsible for the aroma quality of wines. The physico-chemical interactions of 

these volatile compounds with the compounds that form part of the wine matrix could produce variations in their 

volatility and solubility, varying their levels in the headspace phase and therefore affecting their sensorial impact. 

The effects of single compounds from the wine matrix (polyphenols, mannoproteins, ethanol, etc) in the volatility 

of several aroma compounds have been previously studied (1-3). However, the effect of the whole non volatile 

macromolecules from real wine matrices on representative wine volatile compounds has not been study so far. 

Therefore, the aim of this study has been to evaluate the effect of five different types of wine matrices, obtained 

from a white, sparkling, young-red, aged-red and sweet wine, in the effective volatility of several wine aroma 

compounds (alcohols, esters, volatile phenols, terpenes, lactones, etc) by comparing the calibration lines obtained 

by HS-SPME-GC-MS analysis. The calibration lines were built with the 5 previously deodorized-lyophilized wines 

and reconstituted at the same level of ethanol (12%). The behaviour of each aroma compound in the reconstituted 

wine matrices was compared to that of the same compound in a simple wine matrix with no-matrix effect (formed 

by tartaric acid and 12% ethanol). The results of the comparison of the slopes (expressed as % respect to the 

simple matrix) showed differences that depended on the type of aroma compound and the composition of the wine 

matrix. In general, a retention effect of most of the volatile compounds by the matrix producing lower slopes than 

those in the simple matrices was observed. Esters showed, in general, lower slopes in wines with low non-volatile 

residue (white and sparkling-white wines) compared to the simple matrix. However, wines with high non-volatile 

residue (aged-red and sweet wines) presented both effects, higher and lower slopes, depending on the ester type. 

In addition, most of the alcohols were affected by the matrix in the case of the sparkling and white wines, while 

the sweet wine was the less affected. However, white wines did not show a retention effect for most of the studied 

terpenes and C13 nor-isoprenoids. In general, volatile phenols, furanic compounds and lactones showed a matrix 

effect varying the calculated slopes between 83% lower than the slope in the simple matrix for the 4-vinylphenol in 

the young-red wine and 138% higher for the cis-whiskey lactone in the sweet wine. References 1. Chalier et al. Food 

Chem. 2007. 100. 22. 2. Pozo-Bayón et al. J. Agric. Food Chem. 2009. 57. 10784. 3. Robinson et al. J. Agric. Food 

Chem. 2009. 57. 10313. 


137


ORAL 

SESSIONS


S14-01 DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS USING MICRO EXTRACTION BY PACKED SORBENT 

Lezsak G. 1 , Eke Z. 1 , Rikker T. 2 , Torkos K. 1 

1 

Eötvös Loránd University Institute of Chemistry 

2 

WESSLING International Research and Educational Centre, Hungary 

Corresponding author e-mail: eke.zsuzsanna@wirec.eu 

Polycyclic aromatic hydrocarbons (PAHs) are members of a large group of organic compounds characterized by 

fused aromatic rings and no heteroatom. Even though their toxicity varies in a wide range, they have a general 

bad reputation for their genotoxic effects. PAHs are formed and released during incomplete combustion. They 

are one of the most widespread organic pollutants. In the European Union Directive 2008/105/EC of the European 

Parliament lists seven and in the US Appendix A to 40 CFR Part 423 lists nine more of them as priority pollutants 

regarding water. As a result of their health effects, wide occurrence and the existence of a legislative background 

the determination of PAH in environmental samples is a prevalent task in environmental analytical laboratories. The 

analysis in water samples is usually carried out using standardized methods (for example EPA Method 550) applying 

both liquid–liquid extraction (LLE) and solid-phase extraction (SPE). These methods typically process 1 L of sample 

using relatively large amount of organic solvents. Besides, they are both time-consuming and labour intensive. Micro 

extraction by packed sorbent (MEPS) is an easily automatable technique for miniaturized solid phase extraction (SPE) 

that can be connected on-line to either gas or liquid chromatography. The principles and the functions of MEPS are 

very similar to that of SPE. The differences result from the different layout, i.e. for MEPS the sorbent is packed in the 

needle of the syringe used also for injection. Thus there is no need for a separate device for the automatic sample 

preparation and both sample size and solvent usage can significantly be reduced. The developed method processes 

1.5 ml water sample using C8 sorbent in MEPS needle attached to a 100 µl syringe. Sample preparation and injection 

is carried out by a CTC Analytics CombiPAL Autosampler. Large volume injection is enabled by a programmable 

temperature vaporizator (GERSTEL CIS4) mounted on an Agilent 6890 gas chromatograph. The sixteen PAHs listed 

by EPA as priority pollutants as well as 1- and 2-Methyl-Naphtalene and Benzo[e]pyrene are detected in single ion 

monitoring mode by a 5973 Agilent mass selective detector. Limits of quantification (LOQ) vary from 5 to 10 ng/L. 

Linearity is tested in the range of LOQ-200 ng/L. Relative standard deviation proved to be less than 10% throughout 

the whole range. Since sample preparation including sorbent regeneration takes 25 min, which is a few minutes 

shorter than GC run time, the whole analytical cycle time equals the time demand of the chromatographic separation. 


139





IDAEA-CSIC, Barcelona 

Corresponding author e-mail: slbqam@idaea.csic.es 

Perfluorochemicals (PFCs) are globally distributed pollutants, environmentally persistent, bioaccumulative and 

potentially harmful to organisms. This study provides the first evidence of the sources and loads of PFCs to the NW 

Mediterranean Sea (Catalan coast). The presence of five PFCs ‹Perfluorobutane sulfonate (PFBS), perfluorohexane 

sulfonate (PFHxS), perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA) and perfluorononanoate (PFNA)› 

were analyzed by SPE-HPLC-MS⁄MS in 45 seawater samples (29 from coastal waters and 16 from ports). The study 

area comprises 400 km of a highly industrialized and urbanized coastal area. The inputs of PFCs in seawaters were 

estimated according to the levels of PFCs detected in 6 main Catalonia rivers waters and 8 wastewater treatment 

plant (WWTP) effluents discharging to the sea (via submarine emissaries) and their specific fluxes. In WWTP 

effluents, all target compounds were detected, being PFOS (2.79 to 72.1 ng ⁄l) and PFOA (3.47 to 61.9 ng ⁄l) the two 

major PFCs. The highest ΣPFCs levels were found in Tarragona (132 ng ⁄l) followed by Prat de Llobregat in Barcelona 

(76.0 ng ⁄l). Effluents discharges to the marine environment coming from the eight WWTP studied represent 34.7 g/d 

of ΣPFCs All river samples contained at least one PFC. PFOA (0.79 to 9.63 ng ⁄l) and PFOS (1.09 to 9.56 ng ⁄l) were the 

two major PFCs detected. The highest ΣPFCs concentration was found in the Llobregat River (21.9 ng ⁄l) followed by 

the Besos River (16.6 ng ⁄l) and the Ter River (16.3 ng ⁄l). Rivers were identified as the major transport route of PFCs 

to the sea. The six Catalan rivers studied represent a ΣPFCs discharge of 154 g ⁄d. Overall, a total load of 190 g ⁄d of 

PFCs are discharged to the NW Mediterranean coast. Concerning to PFCs in seawaters, the two majors pollutants 

detected were PFOS (from 0.05 to 3.93 ng ⁄l in coastal waters and from 0.09 to 8.38 ng ⁄l in ports) and PFOA (from 

0.07 to 1.86 ng ⁄l in coastal waters and from 0.38 to 2.25 ng ⁄l in ports). PFCs levels depended of the proximity of 

river outflows or submarine emissaries. The highest ΣPFCs concentrations were found in Barcelona port (13.0 ng ⁄l) 

and in San Carles de la Rapita (4.99 ng ⁄l), the last one near de Ebro river mouth To determine the environmental 

impact, the concentration of PFOS and PFOA detected were compared with the available values to protect the most 

sensitive aquatic species. PFOS and PFOA concentrations were several times lower than the calculated criteria 

maximum concentrations. Nevertheless, still there are a lot of uncertainties about the toxicities at low concentration 

levels. Although PFCs are diluted in seawater, the continuous discharge of PFCs to the sea may represent an 

emerging risk which may limit the use of coastal waters for human activity with serious implications in the long term. 

Understanding on the sources and pathways of contaminants to the sea, which will allow a better management to 

avoid anthropogenic organic pollution end up in oceans. 

140 



S14-03 DEVELOPMENT OF A QSRR MODEL FOR IDENTIFICATION OF UNKNOWNS IN ENVIRONMENTAL SAMPLES 

Ulrich N., Schymanski E., Brack W. 

Helmholtz Centre for Environmental Research-UFZ, Leipzig 

The identification of unknown substances in complex environmental mixtures plays an important role in effectdirected 

analysis (EDA). Our approach for the identification of unknowns is based on the generation of possible 

structures followed by the progressive exclusion of structures that do not match experimental chromatographic 

and spectroscopic behaviour sufficiently. Retention Indices (e.g. Kovat’s or Lee) alone are generally insufficient 

to exclude candidates successfully, due to large errors in prediction methods. However, more detailed 

relationships between various physicochemical properties and the retention of a compound could be useful for the 

characterization of molecules. Models such as the linear solvation equation (Abraham Equation) have been applied 

successfully in the past to differentiate structures and retention behaviour with greater accuracy than retention 

indices in liquid chromatography (LC). The aim of this study is to develop methods to derive retention estimates from 

the proposed structures using quantitative structure retention relationships (QSRRs) in order to reduce the number 

of candidate structures in EDA. This approach focuses on the determination of the hydrophobicity index CHI using 

gradient elution in liquid chromatography on a capillary LC system. The parameter CHI is used in a correlation with 

the Abraham descriptor A (hydrogen bond acidity) for the determination of the logKow. This provides us with two 

additional criteria to select or reject possible candidates. When the descriptor A is zero, this can be determined from 

the structure and used to eliminate all structures with A greater than 0, or vice versa. Additionally, the logKow can 

be predicted from the measured CHI value, allowing a more accurate prediction of logKow than, for example, using 

EPISuite and hence a better rejection of candidate structures with partitioning properties deviating from the sample. 


141

ORAL 

SESSIONS 

S15 

NEW CHROMATOGRAPHIC 

APPLICATIONS 

FOR BIOANALYSIS

ORAL SESSION 15 - NEW CHROMATOGRAPHIC APPLICATIONS FOR BIOMARKERS 

S15-01 LC-MS METABOLIC FINGERPRINTING OF SECRETOME FROM HUMAN ATHEROTHROMBOTIC ANEURYSMS 

Ciborowski M. 1,2 ; Martin-Ventura J.L. 3,4 , Meilhac O. 5 , Michel J-B. 5 , Tuñon J. 3,4 , Egido J. 3,4 , Barbas C. 1 

1 

Medical University of Bialystok 

2 


3 

Fundación Jiménez Díaz, Madrid 

4 


5 

Inserm, U698. Paris 

Corresponding author e-mail: cbarbas@ceu.es 

Atherothrombosis, and related complications are major cause of death worldwide in developed countries, whether 

the localization is related to coronary/carotid/peripheral artery disease or aneurysms of the abdominal aorta (AAA). 

The complex biological processes associated with atherothrombosis include oxidative stress and proteolysis in 

relation to neovascularization and intraplaque hemorrhages, leading to immuno-inflammatory response, cell death, 

and extracellular matrix breakdown. Intraluminal thrombus (ILT) is involved in the evolution of AAA, potentially linked 

to the proteolytic activities. Finally, the clinical consequences of wall rupture are arterial thrombosis in occlusive 

atherosclerotic plaques and hemorrhage in dilating AAA. Efforts to limit the mortality rate from AAA rupture depend 

on early detection and elective AAA repair. However, most AAAs shows discontinuous growth patterns and alternate 

periods of stability and non-growth with periods of acute expansion and occasionally rupture. For that reason, 

biomarkers of growth and rupture could give us a more nuanced indication for surgery and could also afford novel 

pathogenic pathways and may thus open possibilities for pharmacological inhibition of growth. We have developed 

a strategy for differential analysis of metabolites released by normal and pathological arteries in culture. In this 

way we try to find those molecules that would have a higher probability of later being found in plasma and could 

start signaling processes or be useful as diagnostic/prognostic markers. We used LC-MS-QTOF (Agilent) based 

metabolomic approach to analyze culture medium samples that had been in contact with human AAA (ILT and 

wall). AAA were divided on ILT (luminal and abluminal) and wall (media), and put in contact with culture medium to 

obtain the rate of exchange of metabolites between tissue and external medium, and the appearance or clearance 

of existing and/or new metabolites. As a comparison human healthy arterial wall were treated in the same way. Data 

were analysed with Mass Hunter and Mass Profiler Professional (Agilent) programs. The comparison of luminal vs 

abluminal layer of ILT, as well as wall of AAA in relation to healthy wall have shown statistically significant (p

ORAL SESSION 15 - NEW CHROMATOGRAPHIC APPLICATIONS FOR BIOMARKERS 

S15-02 NOVEL DERIVATIZATION STRATEGIES FOR THE LC-ESI-MS MEASUREMENT OF RELEVANT BIOMARKERS 

Kretschmer A., Giera M., Eggink M., Wijtmans M., Lingeman H., Niessen W.M.A., Irth H. 


Corresponding author e-mail: mgiera@few.vu.nl 

Carboxylic acids and aldehydes form two large groups of biologically important molecules that can both originate as 

oxidation products from unsaturated fatty acids, especially arachidonic acid. In the past decade, selected species 

from these two groups like prostanoids and alkenals have gained major interest as biomarkers of oxidative stress. 

Highly sensitive analytical tools are mandatory to measure these substances from biological fluids. Up to today, 

gas chromatography mass spectrometry requiring elaborate sample clean up procedures is still the most widely 

used technique. In this lecture, we focus on new derivatization strategies enabling the sensitive measurement of 

carboxylic acids and aldehydes by means of liquid chromatography coupled to positive ion electrospray ionization 

mass spectrometry (LC-ESI(+)-MS). 

The described derivatization strategy is based on the coupling of the aldehyde and carboxylic acid groups with 

empirically designed labelling components possessing a reactive amine group. The coupling of a carboxylic acid 

function with a primary amine mediated by a carbodiimide like N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide 

(EDC) is well known and frequently employed in organic synthesis, peptide synthesis, polymerization, protein 

immobilization and derivatization. On the other hand aldehydes can be coupled with amines by reductive amination 

employing sodium cyanoborohydride (NaBH3CN) as a reducing agent. 

Here, the development and optimization of a protocol for the EDC mediated amide coupling of a test set of 

prostanoids with the amine group of (2-(4-AminoPhenoxy)Ethyl)(4-Bromophenethyl)dimethyl ammonium bromide 

hydrobromide (4-APEBA), a novel derivatization reagent, is described. The same label can be used in an alternative 

reaction mechanism to selectively label aldehydes via reductive amination. This concept is explained on the earlier 

developed 4-Aminophenoxy-Choline (4-APC) label. Both derivatization reactions can be performed under ambient 

conditions and in aqueous solution. 

Both labels 4-APEBA and 4-APC contain a quaternary ammonium group, which significantly enhances ionisation 

efficiency in ESI-MS. Furthermore, 4-APEBA possesses a bromine atom for the easy identification of derivatives by 

isotope filtering. Selective identification of derivatives is further improved by specific fragmentations of the labels in 

MS-MS experiments. 

144 


S16 GAS 


ORAL 

SESSIONS

ORAL SESSION 16 - GAS CHROMATOGRAPHY 

S16-01 THERMAL DESORPTION GAS CHROMATOGRAPHY–MASS SPECTROMETRY AS AN ENHANCED METHOD FOR THE 

QUANTIFICATION OF POLYCYCLIC AROMATIC HYDROCARBONS FROM AMBIENT AIR PARTICULATE MATTER 

Van Drooge L.B. 1 , Pérez-Ballesta P. 2 

1 

IDÆA-CSIC, Environmental Chemistry, Bareclona 

2 

JRC-Ispra, Air quality and transport 

Corresponding author e-mail: barend.vandrooge@idaea.csic.es 

Polycyclic aromatic hydrocarbons (PAH) from ambient air particulate matter (PM10) were analysed by a two-step 

thermal desorption (TD) injection system integrated to a gas chromatograph–mass spectrometer (GC/MS). The 

operational variables of the TD method were optimised and the analytical expanded uncertainties were calculated to 

vary from 8% to 16% over the operative concentration range (40–4000 pg). The performance of the TD method was 

validated by the analysis of a standard reference material and by comparison of PAH concentrations in PM samples 

to those obtained by a conventional liquid extraction (LE) method. The TD method reported lower uncertainties than 

the LE method for the analysis of similar concentrations in air. The TD method also showed advantages for shorter 

sampling times (3 h) in comparison to 24 h for source apportionment applications. PAH were analyzed from ambient 

air PM10 that was collected near Lago Maggiore (Italy) in Northern Italy in November 2007 and from August 2008 

to January 2009. Northern Italy is characterised by relatively high PAH and PM10 concentrations in relation to calm 

wind conditions and intensive temperature inversions at ground level, especially during cold periods. These stagnant 

conditions are occasionally interrupted by North-Föhn events, which mix local air with air from the free troposphere. 

Large fluctuations of ambient air PAH concentrations have been observed in 3 h and 24 h time-resolution samples, 

between day and nighttimes and between days. The fluctuations are related to strength of local emission sources as 

well as influence of seasonal and short-term meteorological events. references: van Drooge, B.L., Nikolova, I., Pérez- 

Ballesta, P., (2009), Thermal desorption gas chromatography–mass spectrometry as an enhanced method for the 

quantification of polycyclic aromatic hydrocarbons from ambient air particulate matter, Journal of Chromatography 

A. 1216, 4030–4039. van Drooge, B.L. and Pérez-Ballesta, P., (2009), Seasonal and daily source apportionment of 

polycyclic aromatic hydrocarbon concentrations in PM10 in a semirural European area, Environmental Science & 

Technology, 43, 7310-7316. 

146 



S16-02 NEEDLE MICROEXSTRATION TRAPS: A POWERFUL, ROBUST AND SIMPLE TOOL FOR VOC ANALYSIS 

Alonso M., Muñoz S., Godayol A., Anticó E., Sanchez J.M., 


Analysis of Volatile Organic Compounds (VOCs) in air samples is extensively carried out by sorption on solid 

sorbents followed by thermal desorption and GC-MS determination (e.g., US-EPA methods TO-14 and TO-15). It 

requires the use of complex and expensive thermal desorption units using two desorption steps, which increases 

the probability of artifact formations. Since McComb et al. [1] introduced the inside needle capillary adsorption 

trap, different needle trap devices have been developed for sampling and determination of VOCs in aerosols and air 

samples with very promising results. Up to now, the most limiting parameter in the use of needle trap (NT) devices 

has been the reduced sampling flows achieved and the poor sampling flow reproducibility obtained from needle to 

needle. In this study, we have evaluated different NT designs with 22 gauge needles to overcome reproducibility 

problems. Injection parameters to GC instruments associated to NT devices are equivalent to those applied for 

solid-phase microextraction (SPME). For this reason, the different parameters that can affect the injection band 

broadening obtained with NT have been evaluated (e.g., injector temperature, liner inner diameter, splitless time, and 

conditioning time). Detection limits obtained range from 0.002 to 0.009 ng for the VOCs evaluated (i.e., from 2 to 9 

ng/m3 for one liter samples). Sensibility and resolution are equivalent to those obtained with conventional thermal 

desorption units. The simplicity of the NT methodology, together with its low cost, results in a very promising tool 

for routine environmental analysis. Once, the most appropriate experimental conditions have been found the NT 

methodology have been successfully applied to the analysis of VOCs in environmental air and breath samples. [1] 

M.E. McComb, R.D. Olechuk, E. Giller, H.D. Gesser, Talanta 44 (1997), 2137-2143 Acknowledgements: This study was 

financed by the MICINN (Spanish Ministry of Education and Science), project CTM2008-06847-C02-02/TECNO. 


147


S16-03 IN-TUBE EXTRACTION OF VOLATILE ORGANIC COMPOUNDS FROM AQUEOUS SAMPLES: AN ECONOMICAL ALTERNATIVE 

TO PURGE AND TRAP ENRICHMENT 

Laaks J. 1 , Jochmann M.A. 1 , Schilling B. 2 , Schmidt T.C. 1 

1 


2 

BGB Analytik AG-Switzerland 

Corresponding author e-mail: maik.jochmann@uni-due.de 

ITEX 2 is a novel in-tube extraction device, featuring a sorbent-packed steel needle with an integrated heating unit 

for thermodesorption. The analytes are enriched on the sorbent bed by several aspirating and dispensing cycles 

from the sample headspace, with a gas tight syringe. The system was evaluated for 20 hydrocarbons of regulatory 

and drinking water quality importance. Five commercially available sorbent traps were evaluated for their compound 

specific extraction yield; based on the results a custom mixed bed trap was prepared and evaluated. Several 

extraction parameters governing the extraction process were optimized to yield maximum sensitivity within the time 

of a GC run, to avoid unnecessary downtime of the system and allow higher sample throughput. Method detection 

limits (MDLs) of 1 ng L-1 to 10 ng L-1 were achieved for volatile organic compounds (VOC), which is similar to the 

MDLs of purge & trap (P&T) systems and much lower than demands by regulatory limit values in the European Union 

or the United States of America. Average relative standard deviations below 10% were achieved for all analytes and 

the recoveries from spiked tap water samples were between 90% and 103%, mostly. Depletion experiments with 

consecutive analyses from the same sample vial showed that the extraction is non-exhaustive, removing a fraction 

of 7% to 55% of the target compounds, depending on the air-water partitioning coefficients. The method was also 

tested with non-synthetic samples, including tap-, pond- and reservoir water and different soft drinks. Compared 

to P&T systems ITEX 2 requires only small sample volumes, it is less prone to contamination, because of missing 

tubing, and it is much simpler, more flexible and affordable. 

148 


S17 FAST 

SEPARATIONS 

ORAL 

SESSIONS

ORAL SESSION 17 - FAST CHROMATOGRAPHY 

S17-01 DATA ACQUISITION AND DATA HANDLING IN FAST LIQUID CHROMATOGRAPHY 

Felinger A. 


With the current trend of liquid chromatography, core-shell and sub-2µm particles yield rather fast separations. 

Highly efficient separations are achieved within minutes, thus the individual peaks are rather narrow. In order to 

properly characterize the retention time or peak width a proper data acquisition frequency is required. 

We study the effect of the stationary phase (particle size distribution, porous layer, etc.) on peak width and on 

separation efficiency. 

We will show the need for high data acquisition rate and demonstrate the possibilities and requirements to efficiently 

process rather narrow chromatographic peaks. 

Peak asymmetry due to extra-column volumes will also be discussed. We analyze the peak shapes recorded under 

various experimental conditions. We show the effect of high flow rate on peak shapes and associate the problem of 

data density with peak shape 

We present results obtained on nonporous, fully porous, as well as on shell particles. 

Multicomponent separations call for statistical analysis of the retention pattern and sample dimensionality. Efficient 

statistical tools (based on Fourier-analysis or autocorrelation function) are available to characterize complex 

multicomponent separations. 

150 


ORAL SESSION 17 - FAST CHROMATOGRAPHY 

S17-02 SEPARATION OF PARTICLES AND CELLS BY USING A HYDRODYNAMIC–ACOUSTIC CONTINUOUS SORTER (HACS) 

Hoyos M. 1 , Rarier C. 2 

1 

CNRS, UMR7636, PMMH, Paris 

2 


The understanding of hydrodynamic and mechanical behavior of rigid and deformable particle flowing through thin 

channels is of general interest in biomedical, biotechnological and environmental applications. Applications to cell 

sorting are ubiquitous and setting up micromanipulation techniques for separation needs is becoming challenging. 

For instance, separation of blood compounds from whole blood is one of the major issues in biomedical engineering. 

Platelet isolation, sickle cell detection and sorting, isolation of macrophages infected with parasites, detection of 

malaria-infected cells, are only a few examples of biomedical engineering challenges. We present the very first 

modeling and experimental work on an acoustic-fluidic technique for sorting out particles and biological objects 

without using membranes or filters. The Hydrodynamic Acoustic Continuous Sorter (HACS) generates continuous 

separations of particles of different sizes or different acoustic properties by combining ultrasonic standing 

waves and an axial flow in a Hele-Shaw cell provided with two or more inlets and outlets based on Split-flow Thin 

Fractionation channel, SPLITT design. This patented device generates very fast and efficient separations of microsized 

polystyrene particles. Separations 5-10, 7-15 µm particles will be presented, and an example of separation 

macrophage infected by Leishmania amazonensis parasite using only the fluidic chamber will illustrate the efficiency 

of the method. Analytical and high throughput separations can be conducted in function of the chamber size and 

flow rate used. We performed 3D in situ observations of separations with a digital holographic microscope with 

partially coherent illumination. 


151

ORAL 

SESSIONS 

S18 

LIFE SCIENCES 

AND BIOANALYSIS

ORAL SESSION 18 - LIFE SCIENCES AND BIOANALISYS 

S18-01 OPEN TUBULAR LC - ANY NEWS? 

Greibrokk T. 


Corresponding author e-mail: tygeg@kjemi.uio.no 

Due to the slower diffusivity in liquids compared to gases, open tubular columns never got a foothold in liquid 

chromatography, although there are some clear advantages compared to packed columns. Separation impedances 

are only about 1/100 allowing reduced analysis time, sample dilution in the column is only a fraction and the ability 

to analyze small samples is improved. However, in order to obtain the similar efficiency as a packed column, the 

internal diameters need to be of the order of 5-10 µm. With a thin film of stationary phase on the walls, the loadabilty 

is then severely reduced and the ability to find a suitable detector is highly limited. This problem is well known in 

capillary electrophoresis, but is even more marked when the column i.d. is reduced from 50-75 µm to 10 µm or less. 

A combination of fluorescent derivatives and laser induced fluorescence detection will solve some problems, but 

today the detection which is most suitable with OTLC is electro spray ionisation (ESI) mass spectrometry, since the 

sensitivity of ESI increases with reduced flow. Recently a hybrid between a monolithic and an open tubular column 

was presented, but so far the i.d. of this column (50 µm) is higher than the optimal bore for an OTLC. During the last 

3 years the most notable innovations have come from the Karger group in Boston., with 10 µm i.d. polymeric layer 

open tubular (PLOT) columns for separation of peptides, after enzymatic cleavage of proteins in solution. We have 

made 1 µm poly(styrene-divinylbenzene) films on 10 µm i.d. columns, according to Karger, but for proteins, not 

peptides (1). Even if bottom-up procedures are the most common in proteomics, there is a need for better resolution 

of proteins, particularly the post translational modifications, prior to enzymatic cleavage. One reason for using open 

tubular columns has been to avoid the losses of adsorbed large proteins that can take place in packed columns 

and to some extent in monoliths too. Thus, it has been our intention to use long PLOT columns with fairly high peak 

capacity of proteins, coupled to an immobilized enzyme column directly in front of the ESI MS. In the ideal world 

each protein elutes at a time from the separation column and the resulting peptides belonging to each protein can 

then be analyzed and quantified without fear of ion suppression from other coeluting peptides. In theory this allows 

quantitation without tagging and separation of isoforms or PTMs of proteins prior to enzymatic digestion. Some 

results will be presented. 1) M. Rogeberg, S.R. Wilson, T. Greibrokk, E. Lundanes, J. Chromatogr. A , 1217 (2010) 

2782-6 


153


S18-02 ACTIVITY ASSAY OF METHYLTHIOADENOSINE PHOSPHORYLASE IN TISSUE SPECIMENS BY MEANS OF STABLE ISOTOPE 

LABELED METHYLTHIOADENOSINE AND LC-MS/MS 

Stevens A.P. 1 , Kirovski G. 2 , Czech B. 2 , Dettmer K. 1 , Hellerbrand C. 2 , Bosserhoff A.K. 2 , Oefner P.J. 1 

1 

University Regensburg, Institute of Functional Genomics 

2 

University Hospital Regensburg, Internal Medicine I 

Corresponding author e-mail: axel.stevens@klinik.uni-regensburg.de 

5’-Deoxy-5’-(methylthio)adenosine (MTA) is a byproduct of polyamine synthesis in mammalian cells. Unlike the 

polyamines, MTA does normally not accumulate in cells, but is degraded by MTA phosphorylase (MTAP) to retrieve 

the essential amino acid methionine and adenine. Many tumors, however, lack the enzyme MTAP.[1] Recently, we 

developed a stable isotope ratio LC-MS/MS method and, subsequently, confirmed the hypothesized accumulation 

of MTA in melanoma cells lacking MTAP.[2,3] In an effort to further elucidate the molecular consequences of a 

lack of MTAP and a concomitant increase in intracellular MTA, we expanded the method to quantitate all key 

intermediates of the methionine and polyamine cycles in a single LC-MS/MS experiment using a triple quadrupole 

mass spectrometer (AB SCIEX 4000 QTrap®) in multiple-reaction monitoring mode (MRM) without prior enrichment 

of the analytes by solid-phase extraction.[4] To correct for variation in extraction and ion suppression, stable-isotope 

labeled standards were added to the samples prior to analyte extraction. In the course of the application of the 

method to tissue specimens from the livers of mice with fatty liver disease as well as various tumors, it was observed 

that the mRNA level of MTAP did not always correlate with the MTA concentration measured in the tissue samples. In 

an effort to elucidate whether alterations in posttranscriptional regulation of protein synthesis, MTAP activity, or MTA 

transport accounted for the lack of correlation, we developed an LC-MS/MS based assay for the measurement of the 

enzymatic activity of MTAP. Briefly, following homogenization of typically 20 mg of tissue in potassium phosphate 

buffer by means of a Precellys® 24-homogenizer, stable isotope-labeled MTA was added as a substrate and its 

decrease monitored over time to calculate rate constants. Under optimized experimental conditions, the assay was 

found reliable for the kinetic analysis of MTAP in tissue biopsies. Preliminary results show an inverse correlation 

between MTA concentration and MTAP activity per mg tissue. At present, we are working on an assay to measure 

MTAP abundance so that MTAP enzyme activity in tissues can be ultimately normalized against MTAP protein. 

Literature: 

[1] N. Kamatani, Cancer Res 1980, 40, 4178. [2] A. P. Stevens, J. Chrom. B 2008, 876, 123. [3] A. P. Stevens, J. Cell. 

Biochem. 2009, 106, 210. [4] A. P. Stevens, J. Chrom. A 2010, 1217, 3282. 

154 



S18-03 SEPARATION OF PHOSPHO AND GLYCO PEPTIDES USING POROUS GRAPHITIC CARBON FOR THE PROTEOMIC STUDY OF 

ONCOLOGY PATIENTS 

Barattini V. 1 , Pereira L. 1 , Smith D. 2 , Griffiths J. 3 

1 


2 

Patterson Institute for Cancer Research, NA 

3 

The University of Manchester, NA 

The separation and identification of protein molecules used in the study of metabolomics is an area of substantial 

interest. Unfortunately these compounds tend to be too large to analyse quantitatively in their native states and so 

a formal process of denaturisation, and digestion of the protein has to be undertaken. This results in the generation 

of a series of peptides and for this particular investigation it is the glycopeptides and phosphopeptides that are of 

particular interest. These types of compounds are very polar, which presents a challenge due to the poor retention of 

these compounds with standard reverse-phase LC packings. Ion-pairing chromatography has been used with limited 

success to increase the retention of the digested protein fragments on typical reverse-phase packings. However, this 

technique comes with a number of draw-backs, including long equilibration times and also low compatibility with MS 

detection. Moderate success has also been achieved using HILIC chromatography; however, this favours isocratic 

conditions due to the very long equilibration times required to coat the stationary phase with a stable aqueous 

layer. These challenges, when analysing very polar peptides, can be frustrating particularly when it is the more polar 

molecules that are of greatest interest. A different approach is clearly required if quantitation of the polar peptides 

is required. Porous Graphitic Carbon (PGC) has been shown to have a specific mechanism of interaction with polar 

species, therefore providing an excellent alternative to standard hydrophobic phases. Preliminary investigations 

have shown that analysis of complex mixtures containing polar species was possible on PGC. In the work presented, 

capillary-PGC columns were utilised for the analysis of polar peptides, showing the excellent retentive properties 

towards these species. The use of capillary columns allowed for increased sensitivity whilst accommodating fast 

elution methods. Complex mixtures of polar peptides were successfully analysed, thus showing the ability of PGC to 

complement reverse-phase analyses. 


155


S18-04 LITHOGRAPHICALLY DEFINED METAL SURFACES FOR BIOANALYTICAL APPLICATIONS 

Juskova P. 1 , Ostatna V. 2 , Palecek E. 2 , Foret F. 1 

1 

Institute of Analytical Chemistry, ASCR, v.v.i., Brno, Czech Republic 

2 

Institute of Biophysics, ASCR, v.v.i, Brno, Czech Republic 

Characterization of biological samples requires specialized analytical methods with high sensitivity, selectivity and 

reproducibility. While separations coupled to mass spectrometry are irreplaceable especially in the discovery phases 

of the research there is also a need for smaller, selective and less expensive detection techniques for the screening 

and diagnostic purposes. High resolution pattern transfer makes lithographic microfabrication techniques essential 

tool for constructing modern analytical tools with potential to fulfill these demands. Electrochemistry represents 

one of the promising methods for biomolecule detection in miniaturized systems. Practically all proteins (down to 

subnanomolar concentrations) produce well developed chronopotentiometric peak H at hanging mercury drop and 

bare solid amalgam electrodes. Unlike liquid mercury electrodes, solid amalgam electrodes could be prepared in a 

microarray format allowing automation and integration with microfluidic devices. In our work we present fabrication 

and characterization of simple amalgam electrode array for protein detection. Shape and surface of individual 

electrodes is defined lithographically on vacuum deposited thin layer of selected metal. Surface of electrodes is 

further modified by galvanic mercury amalgam formation allowing detection of proteins. In another application we 

utilized lithographic technique to construct high aspect ratio, free-standing structures with potential bioanalytical 

use. Optimized fabrication process results in ultra flat well defined and uniform sized mono or multimetallic particles. 

According to composition, particles with different surface chemistries on both sites and/or magnetic properties 

can be prepared. Proper surface modification of particles enables molecule targeting, addressable positioning and 

self-assembling into directed topologies. This work was supported by the Czech Grants: GAAV-KAN400310651, 

M20004090, and GACR-202/07/P497. MEYS CR (LC06035 and ME 09038) and institutional research plans AV0Z 

40310501, AV0Z50040507, and AV0Z50040702 are also acknowledged. 

156 



ORAL 

SESSIONS


S19-01 DEVELOPMENT OF NEW STATIONARY PHASE FOR SHAPE SELECTIVE SEPARATION OF SELECTED CARBAMATE PESTICIDES 

Omamogho J.O. 1 , Crean C. 1 , Stack E.M. 1 , Zhou L. 1 , Yeman H. 2 , Albert K. 2 , Glennon J.D. 1 

1 


2 

Universität Tübingen 

Corresponding author e-mail: j.glennon@ucc.ie 

The use of C18 columns for the separation of carbamates often result to long extended separation analysis time 

when using indispensible very aqueous mobile phase often sometimes with poor selectivity. 

We recently reported for the first time[1] the preparation of novel phenyl phase containing amino polar functionality 

(PAP) and employed for the rapid separation of 6 major toxic carbamate (see Fig. 1). Much improved separation 

was observed on the new phase in comparison to conventional C8 or C18 phases with respect to selectivity and 

efficiency. 

Further research involving synthesis of PAP ligand with mono- and trifunctional reactive silane groups and 

subsequent bonding onto the surface of silica particles and assessing thier chromatographic properties based 

on retention and selectivity of carbamate will be the focus of this study. 29Si and 13C solid state NMR study gave 

insight of the nature of the molecular order of the PAP phase and show significant differences in the resonances. 

The trifunctional PAP phase displayed some shape selectivity properties for the carbamate and gave good resolution 

for two carbamate pesticides (propoxur and carbofuran) that are extremely difficult to separate using conventional 

C18 columns. The PAP phase is regarded as a new shape selective phase for the carbamate pesticides 

The differences in particle types such as, fully porous and core-shell silica particle on the separation performance 

of the PAP phase is discussed and reveal eben better efficiency and separation speed. The mass transfer kinetics of 

the carbamate was also measured on the PAP phases prepared on the fully porous and core-shell particles. 

Reference 

[1] J. O. Omamogho, E. M. Stack, A. Santalad, S. Srijaranai, J. D. Glennon, H. Yeman, K. Albert; Anal. Bioanal. 

Chem. DOI: 10.1007/s00216-010-3816-3 (2010) 

158 








Perfluorochemicals (PFCs) are globally distributed pollutants, environmentally persistent, bioaccumulative and 

potentially harmful to organisms. This study provides the first evidence of the sources and loads of PFCs to the NW 

Mediterranean Sea (Catalan coast). The presence of five PFCs ‹Perfluorobutane sulfonate (PFBS), perfluorohexane 

sulfonate (PFHxS), perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA) and perfluorononanoate (PFNA)› 

were analyzed by SPE-HPLC-MS⁄MS in 45 seawater samples (29 from coastal waters and 16 from ports). The study 

area comprises 400 km of a highly industrialized and urbanized coastal area. The inputs of PFCs in seawaters were 

estimated according to the levels of PFCs detected in 6 main Catalonia rivers waters and 8 wastewater treatment 

plant (WWTP) effluents discharging to the sea (via submarine emissaries) and their specific fluxes. In WWTP 

effluents, all target compounds were detected, being PFOS (2.79 to 72.1 ng ⁄l) and PFOA (3.47 to 61.9 ng ⁄l) the two 

major PFCs. The highest ΣPFCs levels were found in Tarragona (132 ng ⁄l) followed by Prat de Llobregat in Barcelona 

(76.0 ng ⁄l). Effluents discharges to the marine environment coming from the eight WWTP studied represent 34.7 g/d 

of ΣPFCs All river samples contained at least one PFC. PFOA (0.79 to 9.63 ng ⁄l) and PFOS (1.09 to 9.56 ng ⁄l) were the 

two major PFCs detected. The highest ΣPFCs concentration was found in the Llobregat River (21.9 ng ⁄l) followed by 

the Besos River (16.6 ng ⁄l) and the Ter River (16.3 ng ⁄l). Rivers were identified as the major transport route of PFCs 

to the sea. The six Catalan rivers studied represent a ΣPFCs discharge of 154 g ⁄d. Overall, a total load of 190 g ⁄d of 

PFCs are discharged to the NW Mediterranean coast. Concerning to PFCs in seawaters, the two majors pollutants 

detected were PFOS (from 0.05 to 3.93 ng ⁄l in coastal waters and from 0.09 to 8.38 ng ⁄l in ports) and PFOA (from 

0.07 to 1.86 ng ⁄l in coastal waters and from 0.38 to 2.25 ng ⁄l in ports). PFCs levels depended of the proximity of 

river outflows or submarine emissaries. The highest ΣPFCs concentrations were found in Barcelona port (13.0 ng ⁄l) 

and in San Carles de la Rapita (4.99 ng ⁄l), the last one near de Ebro river mouth To determine the environmental 

impact, the concentration of PFOS and PFOA detected were compared with the available values to protect the most 

sensitive aquatic species. PFOS and PFOA concentrations were several times lower than the calculated criteria 

maximum concentrations. Nevertheless, still there are a lot of uncertainties about the toxicities at low concentration 

levels. Although PFCs are diluted in seawater, the continuous discharge of PFCs to the sea may represent an 

emerging risk which may limit the use of coastal waters for human activity with serious implications in the long term. 

Understanding on the sources and pathways of contaminants to the sea, which will allow a better management to 

avoid anthropogenic organic pollution end up in oceans. 


159


S19-03 CHROMATOGRAPHIC ISOLATION OF ANTIFUNGAL STILBENES FROM SHOREA BELANGERAN, DIPTEROCARPACEAE 

Kusuma I.W. 1 , Tachibana S. 2 

1 

Mulawarman University, Faculty of Forestry/Wood Chemistry-Indonesia 

2 

Ehime University, Faculty of Agriculture/Applied Bioresources Sciences-Japan 

Corresponding author e-mail: kusuma_iw@yahoo.com 

Dipterocarpaceae is one of the most -known plant family in tropical rain forest, while Shorea belangeran is one 

580 species in this family. Plants in this family have been reported to be a rich source of stilbene and its oligomers. 

Various biological propeties including antifungal, antibacterial, antioxidant and tyrosinase inhibitory activities have 

been reported fom the plants belong to dipterocarpaceae. Isolation and identification of stilbene oligomers fom 

dipterocarpaceae still remain a challenge because of complicated stereochemistry of the compounds. In the course 

of our research into biologically active compounds from dipterocarpaceae, here we reported chromatographic 

isolation and identification of antifungal compounds fom Shorea belangeran, a plant in dipterocarpaceae. 

Combination of silica gel column chromatography, normal phase MPLC, nomal- and reversed-phase preparative 

HPLC were applied to determine a convenience way to isolate the stilbene oligomers-related compounds fom the 

plant. The chromatographic separation guided by antifungal assay resulted in isolation of a new stilbene tetramer 

along with a known stilbene fom the heartwood of Shorea belangeran. Identification of the compounds conducted by 

spectral analysis (FAB-MS, HR-FAB-MS, 1H, 13C and two-dimensional NMR) established the isolated compounds 

as a new stilbene tetrame having tetrahydofuran ring system (1), C56H44O13, and a known stilbene, resveratrol (2). 

Absolute configuration of 1 was detemined by an x-ray chrystallography analysis. 

160 



S19-04 DISTRIBUTION OF PERFLUORINATED COMPOUNDS IN SEAGULL EGGS (LARUS MICHAHELLIS) FROM THE IBERIC PENINSULA 

Vicente-Bobes J., Meyer J.I., Bertolero A., Viana P., Lacorte S. 

IDÆA -CSIC, Barcelona 


Perfluorinated Compounds (PFCs) are emerging Persistent Organic Pollutants (POPs) widely present in the 

environment, wildlife and humans. PFCs have been used since 1950 in industry and in consumer goods, such as 

teflon (polytetrafluoroethylene) containing products. Once PFCs are released to the environment, they are adsorbed 

to sediments or accumulated in biota and may cause deleterious effects on the ecosystem. Birds have the capacity 

to bioaccumulate PFCs and are thereafter, PFCs are transferred from the mother bird to her eggs, therefore eggs 

have been proposed as a useful matrix for the biomonitoring of these environmental pollutants. Among bird species, 

seagulls have the capacity to bioaccumulate PFCs due to their specific biology: they live for more than 30 years and 

although they fly, they dwell in the same colony and lay eggs in the same nest throughout their life. The aim of the 

study was to evaluate the presence and distribution of PFCs in Yellow-legged gull eggs (Larus michahellis) collected 

from 8 National or Natural Parks from the Iberian Peninsula. After collection, pooled samples of 12 eggs were 

analyzed in triplicate using liquid-solid extraction and liquid chromatography coupled to tandem mass spectrometry. 

Perfluorooctanate sulfonate (PFOS) was the only compound detected in seagull eggs. PFOS was detected at 

higher concentrations in the north Mediterranean colonies than in southern Mediterranean or Atlantic colonies due 

to the more industrial and mass urbanization in this area. Accordingly, the Medes site, followed by the Ebro delta 

and Columbretes islands, all situated in the north Mediterranean coast, contained highest levels with 40.48-53.98 

ng/g-ww of PFOS. In all other colonies, PFOS were detected at levels of 10.10-18.59 ng/g-ww. Egg and eggshell 

parameters, such egg weight, length and breadth, dried eggshell weight and thickness, egg volume, eggshell 

thickness index (I) and desiccation index (Di), were studied to evaluate potential effect of PFOS in egg development. 


161

S20 INNOVATIVE 

CHROMATOGRAPHIC 

APPLICATIONS 

ORAL 

SESSIONS

ORAL SESSION 20 - INNOVATIVE CHROMATOGRAPHIC APPLICATIONS 

S20-01 MICRO-DISPERSE SINTERED NANO-DIAMONDS: A NEW VERSATILE STATIONARY PHASE FOR HPLC 

Nesterenko P. 


In the last decade an increased interest has been paid to the development of various carbonaceous adsorbents 

and their use as stationary phases in high-performance liquid chromatography. Carbonaceous stationary phases 

include various types of porous graphitic and porous glassy carbons, silica immobilised fullerenes and carbon 

nanotubes (CNT). However, significantly less information is known about using of other carbon allotrope – diamond. 

Clearly, diamond and diamond-like materials have so many advantageous properties including excellent mechanical, 

chemical and thermal stability together with excellent biocompatibility and extraordinary high thermal conductivity. 

Obviously the absence of diamond like materials with well developed surface area is one of limiting factors in the 

development of diamonds based stationary phases. However, this option is becoming more feasible with recent 

progress in the development of technology for the production of polycrystalline synthetic diamonds including micro 

disperse sintering detonation nanodiamonds (MSND). 

Detonation nanodiamonds is inexpensive material with particles of average size 5 nm. The structure of particles is 

not completely proved but it is accepted now that a single particle consists of central diamond core covered with 

graphite monolayer. The distinguishing feature of detonation nanodiamond is unusual adsorption properties, which 

are associates with the presence of big number of different functional groups connected with both sp2 (graphite) and 

sp3 (diamond) phases. The fine size of and strong aggregation of particles make difficult their use as adsorbent, so 

sintering, milling of sintered agglomerates and size fractionation is required to get particles suitable for the use as 

column packing. 

MSND wit average particle size 3-4 microns having specific surface area 150 - 250 m2/g and bimodal pore 

size distribution was used as a column packing after additional purification and fractionation on size. The 

chromatographic column was tested in reversed phase, normal phase, ion-exchange and hydrophilic interaction 

liquid chromatography (HILIC) modes. The preliminary results of investigation of the adsorption properties of bare 

MSND show the distinctive ability of this adsorbent to retain polar organic molecules with labile proton in functional 

groups (phenols, nucleotides, aromatic acids). The possibilities of using MSND in different chromatographic modes 

are discussed. The potential application for the use this adsorbents at elevated temperatures and high pressures is 

also demonstrated. 

1. P.N. Nesterenko, P.R. Haddad. Anal. Bioanal. Chem. 396(2010)205. 


163


S20-02 GCXGC WITH CAPILLARY FLOW MODULATION FOR THE SEPARATION OF SEVERAL FAME´S ISOMERS IN BROCCOLI LEAVES 

Manzano P. 1 , Bernal J.L. 1 , Diego J.C. 1 , Arnaiz E. 1 , Bernal J. 2 

1 


2 


Corresponding author e-mail: jlbernal@qa.uva.es 

Even though the principles and the first comprehensive two-dimensional gas chromatography (GCxGC) systems 

were developed more than 20 years ago, its use has greatly increased in recent years, as it can be deduced from 

the numerous research works and review papers published so far. In GCxGC, the whole first dimension effluent is 

transferred onto a second column, which must be operated at high speed. Therefore, a rapid sampling of the first 

column effluent must be achieved without affecting the second dimension analysis. In GCxGC, the interface between 

the two columns is a modulator whose main functions are to increase the amplitude of the signal and facilitate its 

transfer to the second dimension. This action can be done with flow switching or thermal modulators. In this work 

we explore the usefulness of a new modulator based on capillary flow technology (CFT). Recently, the study of 

fatty acids from vegetable oils and animal fats has drawn increasing attention in many fields of basic and applied 

research. This fact could be explained because of the great relevance of the fatty acids for the correct functioning of 

living organisms as well as the important demand of primary components for edible oil. The fatty acids are usually 

derivatized to methyl esters (FAME) prior to their GC analysis. The procedure presented in this work, involves the 

set-up of a method that allows the best separation of several FAME’s isomers in broccoli leaves samples by using an 

Agilent Tech. 7890A GCxGC equipped with a CFT modulator and a FID detector. Two different column combinations 

(orthogonal and non-orthogonal) were used for the experiments. The orthogonal set consisted on a first non-polar 

column DB5-MS (30m, 0.25mm, 0.25µm) and a highly polar column for the second dimension HP-INNOWax (10 and 

2m, 0.25mm, 0.25µm). Meanwhile, the non-orthogonal set is composed by a high polar column in the first dimension 

BPX-70 (30m, 0.25mm, 0.25µm) and a non-polar second dimension column ZB5-MS (10 and2m, 0.25mm, 0.25µm). 

Furthermore, it has been studied how affects the length of the second dimension column to the separation of 

FAME’s isomers. Acknowledgements The authors wish to thank the Junta de Castilla y León for the financial support 

(GIR127). J.B. would like to thank the Spanish Ministry for his “Juan de la Cierva” contract. P.M. thanks Junta de 

Castilla y León for her PhD grant. 

164 



S20-03 MULTIDIMENSIONAL HPLC+GC-MS: EXTENDED USE FOR NON-VOLATILE AND POLAR COMPOUNDS USING ON-LINE 

DERIVATIZATION 

Sarrión N., Gibert J.M., Alcón M.J. 

KONIK-Tech S.A., Sant Cugat del Vallès 

Corresponding author e-mail: nieves.sarrion@konik-group.com 

The innovative multidimensional on-line KONIK K2 AND K2Q12 HPLC+GC-MS system, based on the patented TOTAD 

interface (Patent Nº: US 6,402,947 B1 and others), marries in synergy the separation and fractionation potential of 

HPLC to the separation and selective detection and quantification capabilities of HRGC. Breakthroughs in analytical 

chemistry come from time to time. The TOTAD interface is one of them that has facilitated direct on-line coupling of 

HPLC to GC and GC-MS and hence the development of these “state-of-the-art” multi-compound analyzers resulting 

in simplified sample preparation, minimization of the use of solvents, lower detection limits, total automation and a 

substantial reduction of unit analysis cost. 

The HPLC stage, prior to the introduction into the GC column, contribute to simplify the sample preparation and 

its conditioning. The HPLC column and mobile phases used can contribute totally or partially to the sample clean 

up depending on the complexity of the matrix using both reverse and normal phase HPLC. Liquid samples can be 

directly introduced into the HPLC without any manipulation thus guaranteeing the sample integrity while improving 

quantification of analytes. The trap interface do the rest, selectively or universally, trapping the compounds of 

interest which are, after the complete solvent elimination, desorbed and transferred to the HRGC system, where the 

separation and detection take place. 

In previous works, the KONIK K2 HPLC+GC-MS system has been successfully applied to the analysis of compounds 

easily chromatographied by GC, for instance, analysis of pesticides in environmental, food and urine samples, 

analysis of PAHs and PCBs in industrial oils, determination of free fatty acids in plant extracts and separation and 

characterization of petroleum fractions among others. 

In the present work, an evaluation of the automated on-line derivatization KONIK HPLC+GC-MS multidimensional 

system coupled to ROBOKROM autosampler is presented for screening non-volatile or polar compounds. After 

fractionation of analytes using the HPLC system, the compounds are retained in the trap interface and derivatized 

there in few minutes by introducing the appropriate derivatization reagent using a second valve installed in the same 

autosampler. After complete solvent and reactive agent by-products elimination, the derivatized compounds are 

desorbed from the trap and transferred to the GC system for their analysis using MS or selective detectors. The 

system allows a selective derivatization of compound classes via esterification and acetylation or a more universal 

derivatization through a sylilation step. The on-line derivatizacion HPLC+GC-MS system can be used as a universal 

technique highly competitive with the expensive LC-MS-MS instrumentation as allows an easy identification of 

compounds based on GC-MS libraries and offers very good sensitivity and a cheaper cost in term of per sample 

analysis. 

In this work, an evaluation of the analytical power of the on-line derivatization system for several applications 

is performed: analysis of phenolic compounds in food and beverage samples (cocoa, wine and tea), analysis of 

impurities on solid-dose illicit drugs and determination of several drugs in biological samples. Quality parameters of 

the technique and analysis of real samples are presented for each application. 


165


S20-04 HYPERCROSSLINKED POLYMERS: ADVANCES AND PERSPECTIVES OF APPLICATION IN ADSORPTION TECHNOLOGIES AND 


Davankov V.A., Tsyurupa M.P. 

Institute of Organo-Element Compounds, Russian Academy of Sciences, Moscow 

Corresponding author e-mail: davank@ineos.ac.ru 

Hypercrosslinked polystyrene, introduced by present authors in the early 1970th, happened to be the first 

representative of a new class of polymeric networks and the first nanoporous polymer. While conventional 

porous materials present a plurality of pores confined between solid pore walls, hypercrosslinked materials 

are characterized by rigid open-network structure, where pores are nano-sized space within macrocycles and 

between individual polymeric chains that are held apart by numerous rigid spacers. Hypercrosslinked polymers are 

compatible with any type of mobile phases and display enhanced affinity to any organic adsorbate molecules. 

Commercialized in the 90th, new hypercrosslinked polystyrene-type adsorbents have found wide application in 

large-scale processes of water purification, recovery of organics from waste streams, sugar syrup decolorizing, 

purification of dry hydrogen chloride gas and many others. A promising process is the reagent-free preparative 

separation of concentrated solutions of acids, bases and salts by size-exclusion chromatography, where all 

separated components are isolated at enhanced concentrations, compared to the initial mixture. Excellent 

hemocompatibility of hypercrosslinked materials provides successful medical applications of the polymers. 

On the analytical scale, hypercrosslinked polymers are widely used as the most powerful materials for solid 

phase extraction of trace organics from liquid polar and non-polar media and air. Microbeaded hypercrosslinked 

polystyrene HPLC column packing materials are distinguished by unique selectivity due to the enhanced contribution 

from pi-pi-interactions with the solutes. An emerging area of research is preparation of monolithic separation phases 

for capillary HPLC and capillary electrochromatography. 

The paper presents an array of known and new application examples of various hypercrosslinked polymers [1]. 

[1] Davankov V.A. and Tsyurupa M.P., “Hypercrosslinked Polymeric Networks and Adsorbing Materials”, Elsevier, 

Sept. 2010. 

166 


S21 ELECTRODRIVEN 

SEPARATIONS 

ORAL 

SESSIONS

ORAL SESSION 21 - ELECTRODRIVEN SEPARATIONS 

S21-01 CAPILLARY ELECTROPHORESIS AS A NEW METHOD FOR THE SEPARATION OF POLYCYCLIC AROMATIC SULFUR 

HETEROCYCLES IN DIESEL FUELS 

Nolte T., Andersson J.T. 

University of Münster, Institute of Inorganic and Analytical Chemistry 

Corresponding author e-mail: anderss@uni-muenster.de 

Polycyclic Aromatic Sulfur Heterocycles (PASHs) are a class of compounds which are found in fossil fuels and refined 

petroleum based products. Because of the limited separation efficiency of HPLC and problems with volatility while 

separating high molecular PASHs with GC, capillary electrophoresis with UV detection is introduced as a new, highly 

efficient method to separate this class of compounds. The compounds are convertet to ions, needed for capillary 

zone electrophoresis, through S-methylation or S-phenylation. Standard PASHs with up to 32 carbon atoms that are 

expected to be present in industrially desulfurized fuels are separated by the CE method. RP-HPLC measurements of 

several of the analytes were performed for comparison. A linear relationship is found between migration time and the 

calculated volume of the alkylated polycyclic thiophene compounds. A separation of all monomethylbenzothiophenes 

was achieved which has never succeeded in capillary gas chromatography. The practicability of this method with 

respect to real world samples is demonstrated by separating the PASHs of desulfurized diesel fuels. The compounds 

are preconcentrated by ligand exchange chromatography on a Pd(II) containing phase and, after derivatization, 

separated using the CE method. The main components can hereby be identified. The electropherogram is compared 

with the chromatograms from GC and RP-HPLC measurements. 

168 



S21-02 ELECTROKINETIC ACCUMULATION FOR ON-LINE PRECONCENTRATION OF WEAK ACIDS IN CAPILLARY ELECTROPHORESIS 

Petr J., Maier V., Ranc V., Znaleziona J., Ginterova P., Sevcik J. 


Corresponding author e-mail: secjpetr@gmail.com 

Capillary electrophoresis (CE) used to suffer from a poor sensitivity with commercial UV detectors. However, the 

progress in CE led to development of on-line preconcentration approaches that allow detection and quantification 

of nano- and picomolar levels of many compounds with different structures. These approaches involve techniques 

as isotachophoresis, stacking, sweeping, dynamic pH junction and techniques with combined mechanism of 

preconcentration. In our lab, a new on-line preconcentration technique based on electrokinetic injection of weak 

acids in basic electrolyte to an acidic background electrolyte was developed. During this accumulation phase, 

analytes are concentrated to a sharp zone. Then the inlet electrolyte is replaced by mobilization acidic electrolyte 

containing SDS micelles. In the mobilization phase, analytes are separated thanks to the process of partitioning 

between free electrolyte and micelles. This set up could be easily used for separation of food preservatives 

as benzoic and sorbic acid or for separation of plant antioxidants like phenolic acids with approx. 5,000 fold 

preconcentration [1, 2]. Moreover, the accumulation/mobilization technique of on-line preconcentration allows 

easy and interesting methodological modifications. Here, two modifications will be mentioned. First, the addition 

of organic solvents to the injection electrolyte up to 80 % (v/v) has a great impact on the preconcentration in the 

accumulation phase. With 80 % (v/v) methanol, the preconcentration factor increased to 70,000 in comparison with 

classical MEKC. Second, change of the composition of the mobilization electrolyte from micellar solution to the 

electrolyte containing charged cyclodextrin, i.e. sulfated β-cyclodextrin, allows mobilization of preconcentrated 

compounds together with their separation. The separation mechanism is based on different interaction in 

comparison with using SDS micelles and also enables chiral recognition in nanomolar concentration level. From this 

point of view, the accumulation/mobilization technique represents a powerful tool for determination of weak acids 

by CE with on-line preconcentration. The financial support by the research project MSM 6198959216 is gratefully 

acknowledged. References: [1] J. Horakova, J. Petr, V. Maier, E. Tesarova, L. Veis, D. W. Armstrong, B. Gas, J. 

Sevcik, Electrophoresis 2007, 28, 1540-1547. [2] J. Petr, K. Vitkova, V. Ranc, J. Znaleziona, V. Maier, R. Knob, J. 

Sevcik, J. Agric. Food Chem. 2008, 56, 3940-3944. 


169


S21-03 AN INTEGRATED ON-CHIP SIRTUIN ASSAY 

Beyreiss R. 1 , Ohla S. 1 , Fan Y. 2 , Scriba G.K.E. 2 , Belder D. 1 

1 

University of Leipzig 

2 


Corresponding author e-mail: stefan.ohla@chemie.uni-leipzig.de 

Sirtuins, also called class III histone deacetylases, represent a family of nicotinamide adenine dinucleotide (NAD+)- 

dependent enzymes that catalyze the deacetylation of acetyl-lysine residues of histones, transcription factors 

and other proteins. The catalytic mechanism includes a transfer of the acetyl group to the cofactor NAD+ yielding 

nicotinamide, 2´-O-acetyl-ADP-ribose and deacetylated protein as products. Class III deacetylases have been 

associated with cellular functions that are involved in age-related diseases, e.g. obesity, type-II diabetes and 

neurodegenerative disorders like Alzheimer’s and Parkinson’s disease [1]. In order to investigate activities, substrate 

specifities or the effectiveness of potential activators and inhibitors it is important to develop powerful screening 

methods. In this context a microchip-based assay to monitor the conversion of peptide substrates by human 

recombinant sirtuin 1 (hSIRT1) is presented. For this purpose a fused-silica microchip consisting of a microfluidic 

separation structure with an integrated serpentine micromixer has been used. As substrate for the assay we used 

a 9 fluorenylmethoxycarbonyl (Fmoc)-labeled tetrapeptide derived from the amino acid sequence of p53, a known 

substrate of hSIRT1 [2]. The Fmoc group at the N terminus resulting from solid phase peptide synthesis enabled 

deep UV laser-induced fluorescence detection with excitation at 266 nm [3]. The enzymatic reaction of 0.1 U/µL 

hSIRT1 was carried out within the serpentine micromixer using a 400 µM solution of the peptide in buffer. In order to 

reduce protein adsorption, the reaction channel was dynamically coated with hydroxypropylmethyl cellulose (HPMC). 

The substrate and the deacetylated product were separated by microchip electrophoresis on the same chip. The 

approach was successfully utilized to screen various sirtuin inhibitors. [1] M. C. Haigis, L. P. Guarente, Gene Dev. 

2006, 20, 2913-2921. [2] Y. Fan, R. Ludewig, D. Imhof, G. K. E. Scriba, Electrophoresis 2008, 29, 3717-3723. [3] P. 

Schulze, M. Ludwig, F. Kohler, D. Belder, Anal. Chem. 2005, 77, 1325-1329. 

170 



ORAL 

SESSIONS


S22-01 COMPARATIVE STUDY OF TWO DIFFERENT LC-QLIT-MS/MS OPERATION MODES APPLIED TO THE ANALYSIS OF 

PSYCHOACTIVE STIMULATORY AND TRANQUILIZING DRUGS IN WASTEWATERS BY DIRECT INJECTION 

Martinez Bueno M.J. 1 , Uclés S .1 , Hernando M.D. 2 , Agüera A. 1 , Fernandez-Alba A.R. 1 

1 

Universidad de Almeria 

2 

Universidad de Alcalá 

Corresponding author e-mail: mjbueno@ual.es 

In order to evaluate the capability of a hybrid triple-quadrupole linear ion trap mass spectrometer (QqLIT) system, 

two methods based in the application of different operation modes have been compared. One of them uses selected 

reaction monitoring (SRM) mode, and the other is based on the use of an information dependent acquisition (IDA) 

experiment, which combines a SRM as survey scan and an enhanced product ion (EPI) scan, at two different 

collision energies, as dependent scan, in the same analysis. Both acquisition methods apply time scheduled 

SRM scan. Nowadays, the direct analysis of wastewater without prior treatment of the sample has not attained 

widespread use because of typical low sensitivity associated with the levels present in aquatic environment and 

the difficulties owed to the complexity of this kind of matrices. However, the direct injection methods offer the 

advantage of reduce sample preparation steps, and therefore improve reproducibility, decrease the use of solvents 

and sample pre-treatment time significantly. The criteria used for identification of target compounds in the samples 

for SRM mode was the presence of the two characteristic transitions at the correct retention time. For IDA mode 

were the presence of the characteristic transition (SRM1) at the correct retention time and good agreement between 

the reference spectrum in the library and the spectrum obtained from the samples. The two different operation 

modes (SRM and IDA) were compared and applied to wastewater samples by direct injection, for simultaneous 

determination of 17 psychoactive stimulatory and 7 tranquilizing drugs, including some of their major metabolites, in 

order to evaluate the efficiency and confirmation capability of both modes. As expected, quantification limits (LOQs) 

obtained by both operation modes were the same (lower to 0.7 µg/L for the most of compounds except paraxanthine, 

amphetamine, phenylephrine and methamphetamine), since in both modes the same transition was used to carry out 

the quantification of each compound. However, IDA mode provided detection limits (LODs) lower than SRM mode 

for 8 compounds for which the second qualitative transition was of low intensity (SRM2/SRM1≤0.3, paraxanthine, 

amphetamine, ephedrine, amphetamine, MDA, ethylamphetamine, phenylephrine and methamphetamine), providing 

additional product ion spectra at low concentration levels, allowing reliable identification. Figure 1 shows an example 

of the identification of morphine in an influent extract of a sewage treatment plants (STPs) of south-east of Spain, 

by IDA mode. ACKNOWLEDGEMENTS The authors acknowledge to the Spanish Ministry of Education and Science 

(Programa Consolider Ingenio 2010 CE-CSD2006-00044) and the Junta de Andalucía ( Proyect TEP232). M.J. 

Martínez Bueno acknowledges the research fellowship associated to project with Ref. TEP232. 

172 



S22-02 NEW STANDARDISED METHODS FOR THE BIOMONITORING OF PRIORITY PERSISTENT ORGANIC POLLUTANTS IN GULL EGGS 

Lacorte S .1 , Santos J. 2 , Abad E .1 , Olmos J. 2 , Meyer J. 1 , Abalos M. 1 , Morales L. 1 , Antunes P. 3 , Viana P. 3 , Bertolero A. 1 

1 


2 

University of Barcelona, Analytical Chemistry 

3 

Ministerio do Ambiente 

Corresponding author e-mail: slbqam@cid.csic.es 

The presence of Persistent Organic Pollutants (POPs) in birds has been documented in many countries. Birds 

have the ability to accumulate POPs through the diet and thereafter are transferred to the eggs. Recent studies in 

breeding-birds areas of special protection have reported unexpected high levels of contaminants, reinforcing the 

necessity of a better knowledge of the presence of halogenated compounds on sensitive areas which are refuges 

for numerous wildlife bird species. Eggs have become a suitable matrix for the biomonitoring of POPs, according 

to the Oslo-Paris Convention. In this study, we have developed a standardised biomonitoring method using gull 

eggs that includes from the sampling to the final analysis for all the POPs legislated according to the Stockholm 

Convention (REF). The chemical families included are: polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs), 

polybrominated diphenyl ethers (PBDEs) polychlorinated biphenyls (PCBs), organochlorinated compounds (OCs), 

chlorinated paraffins (CPs) and perfluorinated compounds (PFCs). Dioxins, furans and planar PCBs were Soxhlet 

extracted with toluene from freeze dried samples and the extracts were purified by means of an automated cleanup 

system (PowerPrep, FMS). These compounds were analyzed by gas chromatography coupled to high resolution 

mass spectrometry. Chlorinated paraffins, non-planar PCBs, PBDEs and OCs were liquid-solid extracted with 

hexane:dichloromethane (1:1) also from freeze-dried samples and purified using Florisil solid-phase extraction 

cartridges. These compounds were analyzed by gas chromatography coupled to mass spectrometry in electron 

ionization or negative chemical ionization, depending on the nature of compounds. Finally, perfluorinated chemicals 

(PFCs) were wet extracted with solid-liquid extraction using acetonitrile. The extracts were cleaned with ENVI-carb 

and then measured with ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC- 

MS/MS). The recovery rates for the PFCs were between 95 to 122 % for samples spiked with 5 ng/g ww of each of 

the PFCs and 84 to 120% for those spiked with 100 ng/g ww. OC pesticides, PBDEs and PCBs could be resolved 

in a single GC-MS method while for CPs, a short-GC column was used for achieving maximum sensitivity. Overall 

recovery rates for all these compounds were between 70 and 115%. Recoveries of dioxins and furans were evaluated 

using the 13C12-isotopically recovery standards and were between 60 and 120%. Method sensitivity, overall 

robustness of the developed methods and the various problems and solutions encountered will be discussed. Finally, 

to test and validate the protocol, Yellow-legged gull (Larus michahellis) eggs from the Ebro delta Natural Parks have 

been analyzed to evaluate the occurrence and distribution of the target compounds. 

Acknowledgements. This study has been financed by the Ministry of Environment, ref 2009/038. 


173


S22-03 NOVEL PRECONCENTRATION TECHNIQUES FOR ENHANCING SENSITIVITY IN THE DETERMINATION OF NON-STEROIDAL 

ANTIINFLAMMATORY DRUGS IN ENVIRONMENTAL SAMPLES BY CAPILLARY ELECTROPHORESIS 

Maijó I., Botello I., Borrull F., Aguilar C., Calull M. 


Corresponding author e-mail: irene.maijo@urv.es 

Nowadays, there is a growing interest in the determination of different emerging compounds such as non-steroidal 

anti-inflammatory drugs (NSAIDs). These compounds are present in the aquatic environment, and, thus, one of the 

main aims of different researchers is to develop methodologies capable to determine them in water samples from 

different origins. 

Liquid chromatography has been the preferred technique to determine them but also gas chromatography has 

also been used in some cases. An alternative is the use of capillary electrophoresis (CE) [1]. However, in this 

case, the limited sensitivity that one can get is an important limitation since these compounds are present at 

low concentration levels in environmental matrices. To solve this important drawback, several strategies have 

been introduced such as the use of electrophoretic preconcentration techniques such as stacking, sweeping or 

isotacophoresis, and also the use of chromatographic preconcentration techniques, such as solid phase extraction 

(SPE) [2]. 

We here present different novel preconcentration strategies coupled to CE to be able to determine NSAIDs 

at low concentration levels. In particular we are going to focus in the use of electrophoretic preconcentration 

techniques such as electrokinetic supercharging (EKS) [3] or sweeping [4], and also in the use of chromatographic 

preconcentration techniques in particular in the use of the in-line combination between SPE and CE. The main 

advantages and drawbacks of each approach are going to be discussed and also a comparison between the results 

obtained for all of them is presented. 

[1] Macià, A., Borrull, F., Calull, M., Aguilar, C., Trends Anal. Chem. 2007, 26, 133-153. 

[2] Breadmore, M. C., Thabano, J. R. E., Kazarian, A. A., Quirino, J. P., Guijt, R. M., Electrophoresis 2009, 30, 230–248. 

[3] Botello, I., Borrull, F., Aguilar, C., Calull, M., (Electrophoresis, in press). 

[4] Maijó, I., Borrull, F., Calull, M., Aguilar, C., (Electrophoresis, submitted). 

174 


S23 HYPENATED 

TECHNIQUES 

ORAL 

SESSIONS


S23-01 ON-CHIP LC-MSANALYSIS OF COCAINE FROM HAIR AND URINE 


UMR PECSA7195 CNRS - UPMC - ESPCI Paris 

Corresponding author e-mail: valerie.pichon@espci.fr 

Traditional laboratory scale methods for ultra-trace analysis of compounds from complex matrices consist of 

a step of solid phase extraction to clean-up the matrix and possibly to concentrate the sample, followed by a 

chromatographic separation. Recently, miniaturization of the analytical separation system has been developed in 

order to reduce consumption of reagents, and also to increase the sensitivity of the analysis. Moreover, the use 

of chips as micro-total analysis systems (µTAS) can provide a fast and fully integrated analysis. A chip integrating 

an enrichment column, a separation column and a nanoelectrospray tip is now available [1], the handling of fluids 

being performed by external pumps. This automated and miniaturized device has been already successfully applied 

to oligosaccharides analysis in milk [2], to proteomic analysis [3], even to biomarker discovery [4]. We studied the 

potential of this miniaturized device for the analysis of small molecules in complex matrices. Therefore, Cocaine 

and its main metabolite benzoylecgonine were analyzed from urine and hair extract on chip. The design of the chip 

is well adapted to trace analysis in complex matrices since large volumes can be injected (1-2 µL). Indeed, the 

compounds are first trapped in an enrichment channel and then eluted by the mobile phase to the analytical channel 

for the chromatographic separation. The optimization of the enrichment step is crucial for the analysis. This step 

was improved by the investigation of the analyte breakthrough from the C18 stationary phase. Several parameters 

were thus studied such as the injected volume, the amount of organic solvent in the sample, and the composition 

and volume of the flushing solution. This solution is used to transfer the analytes from the injection loop to the 

enrichment column and allows a sufficient clean-up without loss of compounds. The performances of the method 

were first evaluated by applying it to pure aqueous samples and then to urine samples and hair extracts spiked with 

the target analyte. Limits of quantification on aqueous media were as low as 0.1 µg/L for cocaine and 0.03 µg/L for 

benzoylecgonine. Taking into account the high sensitivity of the method, high dilution factor could be used for the 

application in spiked urine samples as well as spiked hair extracts in order to limit the ion suppression effect. [1] Yin, 

H., et al., Analytical Chemistry, 2004, 77, 527-533 [2] Ninonuevo, M., et al., Agric. Food Chem. 2006, 54, 7471-7480 [3] 

Yin, H., et al., Anal. Chem. 2005, 77, 527-533 [4] Fortier, M., et al., Anal chem. 2005, 77, 1631-1641 

176 



S23-02 APPLICATION OF ULTRA-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY COMBINED WITH HIGH RESOLUTION MASS 

SPECTROMETRY ON SCREENING OF VETERINARY DRUG RESIDUES IN MILK AND POWDERED-BASED MILK SAMPLES 

Romero-González R., Aguilera-Luiz M.M., Plaza-Bolaños P., Garrido-Frenich A., Martinez Vidal J.L. 


Corresponding author e-mail: rromero@ual.es 

The presence of veterinary drug (VDs) residues in dairy products such as milk is an important issue in Food Safety. 

Consequently, the European Union and US government agencies have established maximum residue limits (MRLs) 

for some VDs in milk, which has provoked the development of analytical methods for the determination of these 

compounds at trace levels. Current multi-residue methods for the monitoring of VDs are based on the use of 

liquid chromatography (LC) or ultra high performance liquid chromatography (UHPLC) coupled to tandem mass 

spectrometry (MS/MS), mainly applying low-resolution MS (LRMS) techniques. However, most of milk samples 

submitted to analysis fulfill the limits established by authorities or they do not show any VD residue. For this reason, 

screening methods can be applied, reducing the number of samples to be quantified. For that purpose, LRMS 

analyzers such as triple quadrupole (QqQ) show some drawbacks such as the required optimization of acquisition 

parameters for every single compound, resulting in time-consuming method set-up. To overcome this problem, high 

resolution MS analyzers such as time of flight (TOF) and Orbitrap can be used for screening purposes, improving 

the resolving power (up to 100.000 FWHM) and mass accuracy. Furthermore, fast extraction and chromatographic 

methods should be used in order to reduce analysis time. In this work a rapid multi-residue screening method to 

determine VDs in milk and powdered-milk based infant food by full-scan UHPLC single stage Orbitrap MS has been 

developed, with a chromatographic analysis time lower than 3 min. VDs have been extracted from milk samples 

using a rapid extraction procedure known as QuEChERS method without applying any further clean-up step. The 

identification of the target compounds was based on the retention time and the measurement of the accurate mass 

for each compound. Confirmation was carried out applying a pseudo-MS/MS process based on the selection of 

fragments generated in the multipole collision cell providing high energy collisionally activated dissociation (HCD). To 

carry out the detection of the compounds, a compromise solution has been adopted in the establishment of generic 

MS parameters, such as resolving power, scan rate and source conditions. The performance characteristics of the 

screening method have been evaluated (i.e. lineaarity, recovery, precision) in order to obtain reliable information 

regarding the presence or absence of the compounds below an established value, including unreliability region and 

cut-off values. Finally the proposed method has been compared with other screening methods based on the use of 

LRMS, namely QqQ and HRMS analyzers, such as Q-TOF, in terms of cut-off values and quantification capabilities, 

observing that the proposed method can be used in routine analysis. Acknowledgments We gratefully acknowledge 

Spanish Ministry of Science and Innovation-FEDER (SMSI, AGL2007-63569) for financial support. RRG is also 

grateful for personal funding through the Ramón y Cajal Program (SMSI-EFS). MMAL acknowledges her grant (F.P.U.) 

from the SMSI (Ref. AP2008-02811). PPB is grateful for personal funding through the Juan de la Cierva Program 

(SMSI-European Social Fund, EFS). 


177


S23-03 LIQUID CHROMATOGRAPHY- ISOTOPE RATIO MASS SPECTROMETRY: OPPORTUNITIES AND CHALLENGES FOR A NEW 

HYPHENATION 

Schmidt T.C., Kujawinski D.M., Zhang L., Jochmann M.A. 


Corresponding author e-mail: torsten.schmidt@uni-due.de 

Compound-specific isotope analysis of non-volatile and/or thermolabile organic compounds is a topic of 

increasing interest in research and laboratories working in the area of authenticity control. Although in some 

cases derivatization can be used to convert analytes of interest to derivatives amenable to GC separation, this 

approach has many drawbacks. Thus, there have been many attempts to develop an interface to hyphenate liquid 

chromatography and isotope ratio mass spectrometry (LC-IRMS). Only a few years ago, though, the first interface 

has been commercialized. Due to the design of the interface organic modifiers cannot be used to obtain the required 

elution strength since any additional carbon source would result in a severe decrease of sensitivity and in wrong 

δ13C values. Hence, the development of fully new separation approaches based on purely aqueous eluents is 

necessary except for methods based on ion exchange or size exclusion chromatography that have already been 

successfully used in LC-IRMS, e.g., for the isotope analysis of sugars, amino acids, and volatile fatty acids. For 

reversed-phase separations of nonionic compounds, temperature is the only parameter that might be used to 

sufficiently adapt elution strength. At high temperatures, the polarity of water decreases to such an extent that 

it behaves similar to an organic solvent with regard to elution strength. Temperature gradients instead of solvent 

gradients can then be used to separate compounds. However, for high temperature-LC-IRMS the stability of 

stationary phases at high temperatures needs to be carefully validated since column bleed will affect precision 

and accuracy of δ13C measurements. Results of such a column evaluation will be presented. The improved column 

efficiency at elevated temperature was demonstrated by increased peak height of caffeine on two C18 columns 

without sacrificing isotope precision and peak area. Furthermore, a successful separation and isotope measurement 

of sulphonamide antibiotics mixtures will be shown as a first example of RP-LC-IRMS of non-ionic compounds. Such 

methods open a new range of applications for isotope analysis. 

178 


ORAL 

SESSIONS 

S24 

LIFE SCIENCES 

AND BIOANALYSIS


S24-01 DETERMINATION RESVERATROL AND RELATED POLYPHENOLS IN ARMENIAN WINES BY HPLC/DAD 

Avakyan A.P., Gabrielyan E.S., G.Yu. 

Khachvankyan Scientific Center of drug and medical technology expertise, Expert analytical laboratory-Armenia 

Corresponding author e-mail: analytic@pharm.am 

Resveratrol is a phytoalexin with numerous pharmacological activities, such as antioxidant, anticancer, antiviral, 

chemo protector and others. The objective of this research is to determine the concentration of resveratrol 

in different types of Armenian wines. Two isomers of resveratrol and related polyphenols concentration were 

performed in different Armenian wines from endemic and European kinds of grape from one of the best Armenian 

winemaking region (Vayots Dzor).We are determined four endemic sorts of grape: Areni, Tozot, Malahi, Cat’s Eye. 

In this research, for the first time, we have been found that the best conditions of the resveratrol cummulation 

are:sort - Malahi, part of the plant – leaves, zone – 1100 – 1300 meter above see level, season – 39 -40th weeks of 

the year. The richest source of resveratrol is the roots of Polygonum cuspidatum Ko-jo-kon. The skins of grapes 

contain about 50–100 mg/resveratrol, and believed to be responsible for the cardioprotective properties of red wine, 

which contains about 0.2–7 mg/l of wine.A large variety of fruits including mulberry, bilberry, lingonberry, sparkleberry, 

deerberry, partridgeberry, cranberry, blueberry, and jackfruit, peanut, and a wide variety of flowers and 

leaves including gnetum, white hellebore, corn lily, butterfly orchid tree, eucalyptus, spruce, poaceae, scots pine, 

and rheum also contain resveratrol. Resveratrol is synthesized in response to environmental stressors that include 

water deprivation, UV irradiation, and, especially, fungal infection. Thus, the production of resveratrol in plants can 

be considered part of the defense mechanism. Almost 4500 years ago, Ayurveda, the ancient medicinal book of 

Hindus, lauded the health effects of darakchasava, the fermented juice of red grapes. Somewhat later, the Bible 

described red wine as a “gift of God,” presumably used in the service of both body and soul. In 1940, resveratrol 

was first identified as the medicinal component of grapes. Resveratrol, a polyphenol phytoalexin, possesses diverse 

biochemical and physiological actions, including estrogenic, antiplatelet, and anti-inflammatory properties. Several 

recent studies determined the cardioprotective abilities of resveratrol. Several recent studies determined the 

cardioprotective abilities of resveratrol. Both in experiments (acute) and in chronic models, resveratrol attenuates 

myocardial ischemic reperfusion injury, atherosclerosis, and reduces ventricular arrhythmias. It appears that 

resveratrol-mediated cardioprotection is achieved through the preconditioning effect (the best yet devised method 

of cardioprotection), rather than direct protection. Thus, resveratrol likely fulfills the definition of a pharmacological 

preconditioning compound. The long historical record concerning the health effects of wine production, along 

with current interest in resveratrol, has been an important element in the ascent of complementary and alternative 

medicine. The viticulture was known in Armenia, a country of ancient civilization, for thousands of years. Today vine 

plantations occupy in our country about 16 thousands ha and the vines’ variety includes several hundred sorts. 

Wine-making is a well-developed branch of the national industry. The residues of wine-making are used poorly. It 

seems reasonably to develop the extraction of resveratrol and other active grape components from waste winemaking 

products in Armenia to offer them to the market as biologically active food additives. 

180 



S24-02 VERSATILE, EASY AND RAPID ATMOSPHERIC MONITOR (VERAM) FOR THE PASSIVE SAMPLING OF VOLATILE ORGANIC 

COMPOUNDS IN AIR 

Ly-Verdú S., Esteve-Turrillas F.A., Pastor A., De la Guardia M. 

University of Valencia, Analytical Chemistry Department 

Corresponding author e-mail: miguel.delaguardia@uv.es 

The monitoring of volatile organic compounds (VOCs) in air has a relevant interest in environmental, industrial 

hygiene and air quality fields; taking into account that certain VOCs are hazardous to human health and even some 

of them are known as carcinogens. Monitoring campaigns are widely made in different scenarios to know the 

concentration levels of VOCs in air. In this sense, the use of passive samplers is preferred because of the simplicity, 

low prize and ease of use when they are compared with active samplers. Versatile, Easy and Rapid Atmospheric 

Monitor (VERAM) is a recent analytical strategy for the diagnostic of air quality, which uses a passive sampler based 

on a low-density polyethylene layflat tube filled with a solid-phase or a combination of solid-phases that is able 

to adsorb compounds with different physico-chemical properties [1]. The amount of VOCs retained in the sampler 

was directly measured by head space-gas chromatography-mass spectrometry (HS-GC-MS) without the need of 

any previous treatment. Nineteen VOCs were selected to develop a VERAM sampler after the evaluation of different 

solid phases, such as: activated charcoal, graphitized carbon black, BondElut (CH, C8, C18, PH and NH2), Bondesil 

SAX, Supelclean (LC-Diol and LC-CN), Tenax, alumina and Florisil supports. A summary of the air uptake of VOCs 

using different VERAM samplers is shown in Figure. A VERAM sampler made with 5 mg activated charcoal and 50 

mg Florisil (ACFL) was selected taking into account the high VOCs uptake and low price of the raw materials. Then, 

adsorption isotherms were carried out with ACFL-VERAM samplers and uptake constants, as sampling rate (Rs) 

and the sampler-air partition coefficient (Ksa), were established. Time-weighted average concentration of VOCs in 

air was established by using the amount of VOCs in the sampler, the uptake constants and the employed sampling 

time. An adequate sensitivity was obtained by using VERAM samplers with limits of detection ranging from 0.007 to 

0.200 mg/m3 for a sampling time of 24 h. Empirical equations have been also proposed to predict Rs and Ksa for any 

unexpected compound found in a sampling site. Finally, a monitoring campaign has been made in several package 

industries with high emissions of VOCs to the air, using the developed ACFL-VERAM sampler and semipermeable 

membrane devices (SPMDs) as reference, and the obtained results with both samplers were statistically 

comparable. [1] A. Pastor, M. de la Guardia, F.A. Esteve-Turrillas, Patent application number P200900912/6. 

Acknowledgements:This work was financed by Ministerio de Ciencia e Innovación (CTQ2008-05719/BQU) and 

Ministerio de Medio Ambiente (REACH-PACK, PC 2008-132-3-14.2) 


181


S24-03 NEW TRENDS ON PORTABLE INSTRUMENTATION FOR MICROCHIP CAPILLARY ELECTROPHORESIS 

Pozo-Ayuso D.F., Fernández-la-Villa A., Castaño-Alvarez M. 

Micrux Technologies, Research and Development 

Corresponding author e-mail: mcastano@micruxfluidic.com 

Microchips capillary electrophoresis (MCE) can be considered the first stage to get a point of care system (POC) in 

which would be integrated all the steps of an analytical process. These devices offer high speed, great versatility, 

high throughput, low cost, performance of parallel assays and negligible consumption of reagents/sample and 

waste generation [1]. However, the use of these miniaturized devices requires an additional instrumentation that 

in the most of the cases is not in concordance with the features of MCEs, specially, relative to miniaturization and 

portability. Clearly, microscale analytical devices have demonstrated to be more attractive than their macroscale 

counterparts [2]. Therefore, a new instrument (Figure 1) and graphical user interface (GUI) have been specially 

designed for microchip capillary electrophoresis with electrochemical detection. The instrument combines, for the 

first time, a bipotentiostatic system for dual-mode amperometric detection and a reversible polarity high voltage 

power supply. Electrochemical detection has been selected as signal transduction method because it is relatively 

easily implemented, since non-optical elements are required. The system is completed with a reusable chip holder 

(Figure 2) as easy-handle interface between the micro- and macro-world . This holder incorporates the electrodes for 

applying the high voltages and electrical contacts for the detection and voltage electrodes. The system is controlled 

from a desktop or laptop PC by means of a user-friendly software developed for microchip electrophoresis. The new 

system has been evaluated using single channel SU-8/pyrex microchips with integrated platinum thin film electrodes 

for the separation of the neurotransmitters dopamine, epinephrine and DOPA. References [1] P.S. Dittrich, K. 

Tachikawa, A. Manz, Micro Total Analysis Systems. Latest advancements and trends, Anal. Chem. 2006, 78, 3887 – 

3908. [2] D. Janasek, J. Franzke, A. Manz, Scaling and the design of miniaturized chemical-analysis systems, Nature 

2006, 442, 374-380. Caption Figure 1. New instrument HVStat Caption Figure 2. Chip Holder for easy-handling of 

SU8/Pyrex MCE with integrated Pt electrodes. This work has been supported by the Spanish Ministry of Science and 

Innovation (MICINN - PTQ-09-01-00263 and PTQ-09-01-00264) and Principality of Asturias (FICYT – EIBT08-08). 

182 



ORAL 

SESSIONS


S25-01 ADVANTAGES OF MONOLITHIC OVER PARTICULATE COLUMNS FOR SCREENING ANALYSIS OF ORGANIC POLLUTANTS BY 

IN-TUBE SOLID-PHASE MICROEXTRACTION COUPLED TO CAPILLARY LIQUID CHROMATOGRAPHY 

Moliner-Martinez Y., Carracedo-Marzo R., Molins-Legua C., Verdú-Andrés J., Herráez-Hernández R., Campins-Falcó P. 


Corresponding author e-mail: pilar.campins@uv.es 

The increasing demand of analytical results for monitoring environmental pollution make necessary the development 

of rapid, simple and cost-effective analytical procedures. In this respect, the coupling of in-valve in-tube solid-phase 

microextraction (IT-SPME) to liquid chromatography (LC) has emerged as a valuable tool for monitoring water quality. 

In particular, the combination of IT-SPME and capillary LC enables the simultaneous identification and quantification 

of several organic pollutants in water samples at sub-ppb levels [1]. In the present study the employment of 

monolithic media for separation has been tested for further improvement of IT-SPME-capillary LC performance 

in monitoring water pollution. Several compounds of different chemical structure and hydrophobicity have been 

used as model compounds: simazine, atrazine and terbutilazine (triazines), chlorphenvinphos and chlorpirifos 

(organophosphorous), diuron and isoproturon (phenylureas), trifluoralin (dinitroaniline) and di(2-ethylhexyl)phthalate. 

A Onyx Monolithic C18 has been used for separation, and the results have been compared to those obtained with 

conventional particulate column such as that utilised in [1]. The results obtained revealed that a monolithic column 

is clearly advantageous in the context of multiresidue organic pollutants analysis for a number of reasons: (i) The 

selectivity was considerably improved, especially for the most polar compounds triazines and phenyl ureas, which 

could not be resolved in the particulate column under any of the conditions assayed. Base-line resolution was found 

for the target compounds with the monolithic column. (ii) The time of analysis was substantially shortened. As an 

example, under optimized conditions the times of retention of the latest eluted compound, trifluoralin, were 24.4 

min and 36.1 min in the monolithic and particulate columns, respectively. (iii) The reduction of the retention also 

resulted in significant increment of peaks heights, and thus, in enhanced sensitivity, especially for the most retained 

compounds (organophosphorous and trifluoralin). The limits of detection obtained when using the monolithic column 

were in the range 0.25-20 ppt, which are values 4-100 times lower than those achieved with the particulate column. 

(iv) The coupling of the extractive column used to effect IT-SPME and the separative monolithic column was easier 

due to the high permeability of monolithic compared to the particulate column. From these results it can be deduced 

that IT-SPME-capillary LC with monolithic separation media is an excellent option for controlling different classes 

of organic pollutants in a single chromatographic assay. The reliability of the proposed conditions has been tested 

through the analysis of water samples of different origin. References [1] P. Campíns-Falcó, J. Verdú-Andrés, A. 

Sevillano-Cabeza, R. Herráez-Hernández, C. Molins-Legua, Y. Moliner-Martínez, J. Chromatogr. A 1217 (2010) 2695- 

2702. 

184 



S25-02 CONTINUOUS FLOW INTEGRATIVE SAMPLER (CFIS). AN NEW AND FUTURE ALTERNATIVE TO COMPLETE CONTROL THE 

WATER POLLUTION 

Llorca J. 

LABAQUA, Chromatography, Murcia 

Continuous Flow Integrative Sampler (CFIS). An new and future alternative to complete control the water pollution. 

Julio Llorca*, Rafael Tortajada, Juan Manuel Juarez and Ignacio Valor. LABAQUA S.A. c/ del Dracma, Parcelas 16- 

18 03114-Alicante, Spain * Julio.llorca@labaqua.com The actual passive sampling devices have to overcome many 

limitations to be able to assure a suitable control of the pollution in watery samplers. The range of possibilities to 

resolve some of these limitations, as turbulence effect and biofilm, is relatively restricted by the theoretical principles 

governing passive sampling. In addition, the passive samplers are limited to the analysis of the soluble fraction of 

the pollutants since they depend on processes of adsorption / absorption. A solution of these problems can be new 

devices based on Continuous Integrative Systems. The CFIS is a fully immersible device with small in size (20 x 7x 7 

cm), and consists of a small peristaltic pump powered by a lithium battery, which produces a constant flow through 

a glass cell and a filter. The sorbent is located inside this cell and a glass fibre filter is located in a monitor cassette 

holder. The device cannot truly be defined as a passive sampler because it needs an energy supply of 100mA to 

work the mini-pump, and so can be classified as “integrative”. Figure 1. The constant flow rate through the cell and 

filter will allow the collection of high sample rate values, which will be independent of any turbulence in the sampled 

medium. In addition allows the analysis of the pollution in the total fraction, making possible the simultaneous 

analysis of the soluble and particulate fraction. This new device, will allow autonomous sampling during long periods 

(up to 15 days) of both polar and apolar compounds alike, as well as heavy metals, depending on the sorbent, or 

combination of sorbents used. The high sample rate values permits LOD near to 1 or 0,1 pg/L. In this study, the 

results obtained from the CFIS device are presented for the emergent priority micropollutants. Results are also 

presented for the use of the device for the measurement of the contaminant load of priority pollutants of effluents 

from residual water treatment plants. The results allowed the identification of sporadic spillages which had not been 

detected by the classical spot sampling methods. 


185


S25-03 HYPHENATED µLC-SERS SYSTEM USING A NOVEL SILVER-QUANTUM DOTS “SPONGE” NANOCOMPOSITE 

Carrillo-Carrión C. 1 , Simonet Suau, B.M. 1 , Valcárcel M. 1 , Lendl B. 2 

1 

University of Córdoba, Department of Analytical Chemistry 

2 

Vienna University of Technology, Institute of Chemical Technologies and Analytics 

There is a continued interest in the development of highly sensitive, molecular specific detection techniques 

in miniaturised separation systems such as capillary based liquid chromatography. Surface enhanced Raman 

scattering (SERS) is a promising detection technique but in order to be truly useful, a reliable analysis system must 

be achieved. Without such robustness SERS detection will not gain widespread acceptance in a combined system 

for analytical chemistry. 

Here, we report the usefulness of an at-line capillary-liquid chromatography—(microdispenser)—surface-enhanced 

Raman spectroscopy coupling (Figure 1). The novel advances proposed here were the microdispenser used as 

interface and the type of SERS substrate employed for recording SERS spectra. As SERS-active substrate, a novel 

colloidal synthesis method where hydroxylamine based silver colloids are formed in the presence of CdSe/ZnS 

Quantum Dots (QDs) was reported. The QDs act as a co-reducer and form a link separator between the colloid 

particles establishing more controlled enhancement. 

Different pesticides were separated by capillary-column liquid chromatography (μLC) and then were immobilized 

on a moving CaF2 plate using a flow-through microdispenser interface. This interface generated array of crystalline 

microdeposits with diameters of ca. 10 μm. Next, the colloid silver-quantum dots (Ag-QD) solution was applied 

on each spot on the plate and SERS spectra were recorded (Figure 2). The high SERS-activity of the synthesized 

Ag-QD nanocomposite is due to its sponge-shaped structure that has many “hot spots”. The identification limits 

were 0.2−0.5 ng on column, depending on the pesticide selected, which corresponded to injected concentrations of 

0.2−0.5 μg•mL-1. 

Consequently, the combination of an adequate interface between the μLC system and the Raman spectroscopic 

detection with a very good substrate for the SERS technique was verified in the present paper on the example of a 

separation of a mixture of four pesticides. 

186 



S25-04 MONITORING OF ABRUPT CLIMATE CHANGE WITH CHROMATOGRAPHYC METHODS FOR MEASUREMENT OF PAST SEA 

SURFACE TEMPERATURES AND DEEP WATER FLOWS 

Rama O., Martrat B., Grimalt J.O. 

IDÆA-CSIC, Environmental Chemistry 

The present contribution is addressed to describe the chromatographic methods presently available for the 

description of abrupt climate changes. These changes are those occurring at higher rate that the orbitally induced 

climate transitions whose effect is faster that the driving processes. This type of changes can be taken as models for 

the potential climate evolution due to human influence. 

The characterization of a substantial amount of the processes related with these abrupt climate changes depend on 

the analysis of organic molecules. Thus, C37 alken-2-ones that are solely synthesized by Prymnesiophyceae flora 

allows to recording sea surface temperatures (SST). C26-C32 n-alkan-1-ols and C27-C33 n-alkanes that originate 

mainly from vascular plants are useful for the characterization of eolian inputs. The ratio between n-hexacosan-1-ol 

and n-nonacosane (hexacosanol index) is a good marker of the intensity of bottom marine currents. These organic 

molecules therefore provide evidence of atmospheric and sea surface and bottom rapid changes powered by abrupt 

climate transitions. 

The procedures and equipment used for extracting, isolating and quantifying these fossil compounds in 

paleoceanography studies have been previously optimised [J. Villanueva & J. O. Grimalt, 1996, J. Chromatogr. A, 

723: 285; J. Villanueva & J.O. Grimalt, 1997, Analytical Chemistry 69, 3329; J. Villanueva et al., 1997, J. Chromatogr. 

A, 757: 145; Grimalt et al., 2001, Paleoceanography 16, 226; Chaler et al., 2003, Journal of Chromatography 1012, 

87]. Now, the study of abrupt changes poses a need for analyses involving large numbers of samples and low 

sample amounts. New methods for handling this analytical needs must be implemented. In the present study several 

of these methods are reported. They are basically intended to increase the number of samples processed per week 

and to reduce interferences in detection and quantification of the compounds. Whenever possible, the analytical 

procedures consist in collection of only two fraction extracts (acidic and non acidic compounds) to further reduce 

time and cost in their chromatographic analysis. Criteria for adequate stationary phase selection in the capillary 

columns used to study the resulting complex biomarker mixtures are also reported. 


187

ORAL 

SESSIONS 

S26 

FOOD QUALITY 

AND SAFETY


S26-01 METHOD DEVELOPMENT AND VALIDATION OF A LC-MS/MS METHOD FOR PESTICIDE RESIDUES ANALYSIS IN FRUIT JUICES 

BY DIRECT INJECTION 

Ferrer C., Martínez-Bueno M.J., Lozano A., Fernandez-Alba A.R. 

University of Almería, Analytical Chemistry 

Method development and validation of a LC-MS/MS method for pesticide residues analysis in fruit juices by direct 

injection. Carmen Ferrer1, M.J. Martínez-Bueno1, Ana Lozano1, and Amadeo Fernandez-Alba1 1 Pesticide Residue 

Research Group. European Reference Laboratory EURL. University of Almeria. 04120 (Spain); e-mail: cferrer@ual. 

es Fruit juices are consumed daily in the European Union countries, due to their high nutritious values and to its 

benefits on the human health. For this reason, monitorization of pesticide residue levels in this kind of commodity is 

essential, specially taking into account its high consumption by children. Pesticide residue levels found in fruit juices 

depend on various factors such as type of pesticide, commodity, treatment applied and degradation processes 

involved. However, the concentrations are often quite small, and therefore, appropriate methods of analysis are 

needed. Matrix effect can be an important problem as it can severely compromise quantitative analysis of the 

compounds at trace levels as well as method reproducibility. A possible approach in order to avoid it can be dilution 

of the extracts, obtaining this way the injection of less matrix load into the chromatographic system. The appearance 

of new generation analytical systems in the market make possible this attempt, as highly sensitive instruments are 

available for these purposes. In this way, matrix effect can be avoided for many fruit and vegetable matrices. For this 

work, three different fruit juices (orange, pineapple and peach juice) were selected to develop an analytical method 

for the analysis of 50 pesticides by direct injection in LC-MS/MS. The preparation of the samples was very simple: 

an aliquot of the juice was centrifuged and filtered, and then it was ten-times diluted prior to analysis. Validation 

of the method was carried out in accordance with EU guidelines [1]. Calibration curves covering two orders of 

magnitude were performed, and they were linear over the concentration range studied for all the matrices (from 0.1 

to 100 μg/Kg). Practical detection limits (LOD) were in the low μg/Kg range, far below the maximum residue levels 

(MRLs) of the EU regulations, which don’t set specific MRLs for juices, and in this cases of processed food, MRLs 

of the raw product are applied. Repeatability of the instrumental method was studied in all matrices, obtaining 

good intra- and inter-day relative standard deviations (RSDs). The proposed method was applied to 55 real fruit 

juice samples purchased in different local markets in order to validate the suitability for routine analysis. 56% of the 

analysed samples gave positive results (higher than the practical LOD). The authors acknowledge funding support 

from the European Commission, DG SANCO (Specific Agreement No. 2007/1 to Framework Partnership Agreement 

No. SANCO/2007/FOOD SAFETY/025-Pesticides in Fruit and Vegetable) and the Junta de Andalucía (Regional 

Government of Andalusia, Spain) (Project ref. AGR-4047). References [1] Document SANCO/10684/2009 Method 

validation and quality control procedures for pesticide residue analysis in food and feed. 


189


S26-02 DETERMINATION OF PAHS IN MEZCAL BY SOLID PHASE MICROEXTRACTION FOLLOWED BY GAS CHROMATOGRAPHY-MASS 

SPECTROMETRY 

Alonso-Villegas R. 


Mezcal is a mexican artisan beverage with a cultural and economic importance in some regions of Mexico. Its 

peculiar flavor and taste is actually a delicate mixture of organic compounds present in an alcohol-water solution. 

The production of mezcal is still a handmade and not standardized process due to this, there is a possibility that 

during the stage of cooking, the agave plant could be incorporated by products originated from the incomplete 

combustion of organic matter; among them polycyclic aromatic hydrocarbons (PAHs) might be placed in the final 

product by its hydrophobic character since they can be accumulated in the lipid tissue of the plant. Upon being the 

PAHs priority organic pollutants (POPs), some of them can be potentially carcinogenic. Therefore it is important to 

identify through analytical and sensitive procedures since the presence of these compounds might cause a problem 

of food safety. A Solid Phase Microextraction followed by Gas Chromatography-Mass Spectrometry 

(SPME-GC-MS) was used to develop a methodology for the extraction, concentration and quantification of polycyclic 

aromatic hydrocarbons (PAHs) in mezcal samples from different regions of Mexico produced in this semi-distilled 

beverage process. This group of compounds was identified in the mezcal samples: naphthalene, acenaphthylene, 

acenaphthene, fluorene, phenanthrene, anthracene and 2-ethyl naphthalene, six of them were quantified. Limits of 

detection and quantification of the optimized method were in low range μg/L (ppb) for all compounds analyzed in this 

study. The develop of this methodology was the most critical point for the PAHs extraction in the samples analyzed 

therefore, the extraction conditions were: sample amount (1 mL sample), desorption time (2 min), extraction time 

(40 min), extraction temperature (20 ºC), pH (7) and NaCl (0 g) added. The method was linear in the range 1-2.5 μg/L 

(ppb) for naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene and anthracene with a correlation 

coefficient up to 0.9981 for all six PAHs. The precision of the method was less of 5%. The methodology was applied 

to the analysis of ten samples of mezcal from different regions of Mexico. It was found a wide range of the six PAHs 

(63-440 μg/L) in all mezcal samples. 

190 



S26-03 COMPARISON OF UV-VIS AND TANDEM-MS DETECTION OF SUDAN DYES AFTER RRLC SEPARATION 

Ochs S.M., Santos A.M.S., Pereira Netto A.D. 



Sudan dyes are carcinogenic azo-dyes that are prohibited in foods but were found in some spices and spice or 

pepper containing foods in the last ten years. Therefore, there is interest in sensitive methods for their evaluation 

in foods. Almost all methods of Sudan dyes determination are based on high performance liquid chromatography 

(HPLC) and detection by UV-Vis, mass spectrometry (MS) or tandem mass spectrometry. Different MS systems (ion 

traps, single or triple quadupoles etc) and interfaces such as electrospray (ESI), atmospheric pressure chemical 

ionization (APCI) or atmospheric photoionization (APPI) have been used. Comparable limits of detection (LODs) of 

Sudan dyes are obtained by UV-Vis detection and single quadrupoles, but the detection of appropriate transitions 

using multiple reactions monitoring (MRM) in tandem-MS systems leads to better LODs. ESI interfaces are frequently 

employed in the determination of Sudan dyes, but APCI ones show comparable LODs. However, there are few 

data comparing the detection of Sudan dyes using a HPLC system interfaced to the same MS system through 

different interfaces and also UV-Vis detection. Moreover, there are few data of Sudan dyes determination using 

rapid resolution liquid chromatography (RRLC) that leads to sharper and faster peaks than conventional HPLC and 

presents several comparative advantages. This study is focused on the evaluation and comparison of different 

detection conditions of five Sudan dyes using RRLC and detection by UV-Vis and tandem-MS following ionization in 

ESI or APCI interfaces. The data were also compared with those obtained using conventional HPLC-UV-Vis. A RRLC 

system (Agilent, 1200 Series) interfaced to an Ion-trap (Agilent, 6300) and a HPLC (Agilent, 1100 Series) were used. A 

column ACE 3 C18 (150 x 2.1 mm; 3 μm) that can be used under both HPLC and RRLC conditions was employed. The 

mobile phase consisted of (A) aqueous formic acid (0.1%) and (B) methanol:formic acid (0.1%). Isocratic conditions 

(98% of B) were employed during evaluation of the ESI interface to avoid variation of ionization conditions along 

the chromatographic run. Two strategies were evaluated to avoid non-proportional ionization in the ESI interface: a) 

column flow splitting before ionization and b) flow rate reduction, but the former led to the expected signal reduction. 

Calibration lines were obtained with triplicate injections of eight level standards and LODs were estimated after 6 

injections of the two less concentrated standards. The LODs obtained by tandem MS varied between 2 and 12 pg 

and 2 and 4 pg, using respectively the APCI and ESI interfaces. Sudan I that is of special concern, because of its 

known carcinogencic characteristics, showed the worst LOD under the APCI interface. The LODs found by 

tandem-MS were up to 20 times better than those found by UV-Vis detection that led to comparable LODs (between 

23 and 48 pg) by HPLC-UV-Vis or RRLC-UV-Vis. The reduction of mobile phase flow rate represented a drawback 

when using the ESI interface because of method throughput reduction. CNPq, FAPERJ, CAPES, PROPP-UFF 


191

ORAL 

SESSIONS 

S27 

LIFE SCIENCES 

AND BIOANALYSIS


S27-01 PREPARATION OF MONOLITHS IN GLASS/SILICON MICROFLUIDIC CHIPS AS STATIONARY PHASES AND FRITS FOR COLUMN 

PACKING 

Vázquez M., Collins D., Nesterenko E., Brabazon D., Paull B. 


The last decade has witnessed an increasing interest in the development of micro-fluidic devices integrating 

monolithic materials for chemical and analytical applications. These materials are amazingly versatile as they can be 

prepared with different porosities, pore sizes, and a wide variety of functionalities using many different precursors 

and chemistries [1]. Furthermore, they can be prepared in situ in micro-fluidic channels previously filled with the 

appropriate mixture of liquid precursors. Preparation of monoliths in specific areas of the micro-channel can be 

easily achieved by photo-initiated (UV-initiated) polymerisation employing customised photo-masks [2]. Another 

attractive characteristic of micro-fluidic systems is the possibility of integrating different analytical operations in one 

single chip. Micro-fluidic systems integrating monolithic materials and related porous polymer gels functioning as 

separation columns, ion-permeable membranes, extractors, preconcentrators, enzymatic and catalytic 

micro-reactors, micro-valves, electrokinetic pumps, electrospray emitters and micro-mixers have been successfully 

demonstrated [3]. In this work, we present the fabrication of monoliths in glass/silicon microfluidic chips for (bio) 

analytical applications. Monoliths to serve as stationary phases and frits for packed columns were prepared using 

localised UV- and thermally-initiated polymerisation. Particles of different sizes and varying size distributions 

(including mono-disperse particles) were employed for the column packing. The resulting monolithic beds and 

packed columns were characterised using optical microscopy, scanning electron microscopy (SEM) and capacitively 

coupled contactless conductivity detection (C4D). The latter was used as a non-destructive characterisation 

method, as opposed to SEM, for evaluation of the structural homogeneity and density of both monolithic and 

packed stationary phases prepared in the micro-fluidic channels. A commercial TraceDec C4D system (Innovative 

Sensor Technologies GmbH, Austria) coupled to copper sensing electrodes fabricated in-house was used for 

measurements. The column profile along its entire length was obtained by simply sliding the chip on top of the 

sensing electrodes and recording the corresponding conductivity values at every 1-mm increment. References 

[1] F. Svec, T.B. Tennikova, Z. Deyl, Eds., Monolithic materials: preparation, properties and applications, 1st ed., 

Elsevier, Amsterdam, 2003. [2] C. Yu, F. Svec, J. M. J. Frechet, Towards stationary phases for chromatography on 

a microchip: Molded porous polymer monoliths prepared in capillaries by photoinitiated in situ polymerization as 

separation media for electrochromatography, Electrophoresis 2000, 21, 120-127. [3] M. Vázquez, B. Paull, Review on 

recent and advanced applications of monoliths and related porous polymer gels in micro-fluidic devices, Anal. Chim. 

Acta 2010, 668, 100–113. 


193


S27-02 A NEW FAST WAY OF SCREENING MULTIPLE SOLVENTS AS ELUENTS FOR CHIRAL HPLC: APPLICATION FOR RACEMATE 

SEPARATION ON IMMOBILIZED POLYSACCHARIDE PHASES 

Duchateau A.L.L. Boesten J.M.M., Thomas-Verweij A. 

DSM Pharma Chemicals 

Corresponding author e-mail: lucien.duchateau@dsm.com 

For Chiral Stationary Phases (CSP) based on polysaccharides, the mechanism of enantioselectivity is mainly based 

on differences in hydrogen-bonding properties of the chiral solutes. Because of this phenomenon, the type of 

modifier (organic solvent) in the alkane-based eluent plays an important role in the chiral separation obtained. The 

availability nowadays of several types of polysaccharide CSP in their immobilized form allows the use of a wide 

range of organic solvents that can applied as eluent modifier in order to improve separation of enantiomeric pairs. 

Especially for preparative purposes, establishing a large selectivity by selecting the most suitable organic solvent 

as eluent in combination with a particular immobilized polysaccharide CSP, has a big impact on productivity of 

the preparative system. For testing large numbers (e.g. 40) of organic solvents as eluents, classical set-ups using 

solvent switching valves have severe drawbacks: storage place of bottles, time needed for flushing and filling of 

solvent transfer lines. By a slight modification of the injector of a standard liquid chromatograph (world-wide brand), 

we managed to use the 2 ml vials of the autosampler as eluent reservoirs. In combination with a 50 x 2.1 mm ID 

column filled with 3 µm immobilized polysaccharide phase, the complete sequence of condition the column with a 

particular eluent, injecting of the racemate and elution could be performed by the injecting valve (without the use of 

the standard pump). For detection, both UV and mass spectrometry have been evaluated. Advantages of the new 

set-up are: a) very low consumption of eluent – 160 ml for 100 injections-; b) few minutes analysis time per eluent; c) 

automated screening of up to 100 different eluents. Several examples of racemic molecules will be shown for which 

enhanced selectivity could be obtained by using a broad range of organic solvents as eluent modifier. 

194 



S27-03 DETERMINATION OF PROLINE ANALOGUES IN PLANTS SUBMITTED TO DROUGHT STRESS BY LIQUID CHROMATOGRAPHY 

AND MASS SPECTROMETRY 

Guignard C., Lefèvre I., Legay S., Evers D., Hoffmann L. 

Public Research Center Gabriel Lippmann, Luxembourg 

Corresponding author e-mail: guignard@lippmann.lu 

Water availability is one of the main limitations to plant productivity, so that drought represents a major threat 

to global agriculture production and food security. Drought stress, as other abiotic stress forms, can directly or 

indirectly affect the physiological status of plants by altering metabolism, growth and development. Active or passive 

accumulation of osmoprotectants is an important adaptation mechanism for plants in response to osmotic stresses 

including water deficit and high salinity levels. Thus, in order to keep an osmotic balance with the environment, 

many organisms synthesize solutes that help in retaining water in cells or protect organelles from dehydration. The 

study of nitrogenous metabolites like proline and its analogues, which are known to act as osmoprotectants, is 

thus of prime importance to study the response of plant metabolism during drought stress events. However, due 

to their high polarity and low concentrations, the determination of proline analogues in plants is often challenging. 

In addition to spectrophotometric procedures, which lack of specificity, several chromatographic methods have 

been developed, mainly based on HPLC with optical detection like fluorescence. However, according to our 

knowledge, none of these methods is able to quantify simultaneously a larger number of compounds of interest, 

namely proline, hydroxyproline, methylproline, glycine betaine and trigonelline. Moreover, the analytical throughput 

is often restricted by time-consuming sample preparation protocols, involving mostly chemical derivatization and 

solid-phase extraction or cleanup steps. In this work, a new method based on mass spectrometry coupled to 

high-performance ligand-exchange liquid chromatography (HPLEC) was developed for the simultaneous analysis 

of stress-associated solutes in plants. After a straightforward sample preparation by solid-liquid extraction with a 

methanol/chloroform/water ternary mixture, proline analogues were separated using an Aminex stationary phase 

and a Ca,Na2-EDTA solution as eluent. The detection was achieved by positive electrospray ionisation and singlequadrupole 

mass spectrometry operated in single ion monitoring (SIM) mode. The obtained method offers very 

good specificity and sensitivity, with quantification limits ranging from 0.1 to 0.6 µmol/L. Standard calibration curves 

showed also good linearity with correlation coefficients greater than 0.9995 in the measured range. Above all, the 

main advantage of the developed method, compared to previously published procedures using optical detection, 

is the simultaneous and direct determination of 5 nitrogenous osmolites plus an internal standard in a single 

chromatographic run. In addition, only a minimal sample preparation is needed, even for zwitterionic compounds like 

trigonelline and glycine betaine. This method was used to investigate the impact of drought exposure on nitrogenous 

osmoprotectants, among other metabolites, in two potato cultivars exhibiting different levels of drought tolerance. 

An increase of some osmolites, including proline and trigonelline was observed in response to water deficit. 

Additionally, this accumulation was significantly higher for the drought tolerant cultivar. Finally, these results were 

found to be in accordance with those obtained on gene expression by molecular biology. 


195


S27-04 TITANIUM DIOXIDE (TIO2) MONOLITHIC SUPPORTS FOR LIQUID CHROMATOGRAPHY AND SAMPLE TREATMENT 

Abi Jaoudé M., Randon J. 

University of Lyon, CNRS-UCBL 

Corresponding author e-mail: randon@univ-lyon1.fr 

Background 

So far, silica-based monoliths have represented an outstanding achievement in materials sciences for their use as 

viable substitutes to conventional particulate supports in liquid chromatography [1]. The unique 3D-geometry of 

micrometer-sized skeletal network and interconnected flow-through channels ensures the high permeability of these 

monoliths making them (i) adaptable for high efficiency separations with increase of their column length and (ii) 

suitable for high throughput analysis in view of their reduced column backpressure [2,3]. Despite their great potential, 

silica-based monoliths still suffer to date from chemical and thermal instability of their silica skeleton at extreme pH 

or high temperature of the mobile phase. Alternative approaches are therefore focusing on other inorganic materials, 

such as metal oxides (TiO2, ZrO2…) that not only withstand aggressive chromatographic conditions but additionally 

provide combined interactions (ion- and ligand- exchange) for original selectivities in complex separation sciences 

[4]. 

Results 

Our study was focused on the elaboration of titanium dioxide (TiO2) monoliths that would gather features of unique 

surface chemistry and innovative material design for future applications in liquid chromatography, targeting both 

conventional and miniaturized techniques. By controlling the sol-gel hydrolytic polycondensation of titanium 

n-propoxide (Ti(OnC3H7)4) and through an experimental design that covers the influence of each of the synthesis 

parameters on the resulting morphology of the monolith, we established a repeatable step-by-step synthesis 

protocol leading to monoliths with optimized network strength and geometry (Fig.1) compared to previously reported 

protocols [5-7]. Moreover, monoliths with high length (i.e. 10 cm) could be easily prepared at macro-scale level 

without problems of network collapse or crack formation. The same synthesis was lately adapted and transferred 

in situ to fit the capillary format of 50 µm and 75 µm of inner diameter. These monolithic structures were used for 

the separation of phosphorylated nucleotides and xanthines as probe molecules to evaluate their chromatographic 

characteristics. Such tools will be also applied to the specific enrichment of phosphorylated molecules in samples 

such as proteins, peptides and nucleotides. 

[1] G. Guiochon, J. Chromatogr. A, 1168, 101 (2007). 

[2] O. Núñez, K. Nakanishi, N. Tanaka, J. Chromatogr. A, 1191, 231 (2008). 

[3] T. Hara, S. Makino, Y. Watanabe, T. Ikegami, K. Cabrera, B. Smarsly, Nobuo Tanaka, J. Chromatogr. A, 1217, 89 (2010). 

[4] J. Nawrocki, C. Dunlap, A. McCormick, P. W. Carr, J. Chromatogr. A, 1028, 1 (2004). 

[5] J. Konishi, K. Fujita, K. Nakanishi, K. Hirao, Chem. Mater., 18, 6069 (2006). 

[6] J. Randon, J.-F. Guerrin, J.-L. Rocca, J. Chromatogr. A, 1214, 183 (2008). 

[7] J. Konishi, K. Fujita, K. Nakanishi, K. Hirao, K. Morisato, S. Miyazaki, M. Ohira, J. Chromatogr. A, 1216, 7375 (2009). 

196 


S28 LIQUID 


ORAL 

SESSIONS


S28-01 NEW NANOSTRUCTURED MATERIALS FOR HPLC AND TLC 

Linford M.R. 1 , Wiest L. 1 , Jensen D. 1 , Kanyal S.S. 1 , Hung D. 1 , Dadson A. 2 , Vail M.A. 2 , Davis R.C. 1 , Vanfleet R. 1 

1 


2 

US Synthetic 


Herein we describe two new types of nanostructured materials for HPLC and TLC: novel pellicular (core-shell) 

particles for HPLC and carbon nanotube templated structures for TLC. The core-shell particles were prepared by the 

layer-by-layer deposition of nanodiamond and polyallylamine, a water-soluble, primary-amine containing polymer, 

onto ca. 2 micron graphite cores. As a final step, the particles were crosslinked and end-capped with C18 chains, 

where the purpose for making these particles is to create solid supports for liquid chromatography that possess 

extraordinary stability under extreme pH conditions. The pore size of these materials is ca. 20 nm. The particles 

showed good stability and multiple analytes could be separated with low back pressures, where Van Deemter 

curves show the expected trends. Our preliminary study in this direction, which reported the preparation of more 

irregular all-diamond particles, was just published in Analytical Chemistry. The second new class of materials we 

have developed is derived from patterned, carbon nanotube-templated materials for thin layer chromatography. 

This procedure consists of depositing alumina, followed by a few nanometers of patterned iron onto a substrate 

material. Upon heating and reduction, the iron forms nanoparticles that then lead to nanotube forest creation. These 

patterned nanotube forests, which may be 200 microns high, are then infiltrated by low pressure chemical vapor 

deposition (LPCVD) with silicon, followed by oxidation of the silicon. The resulting TLC plates show high numbers of 

plates, rivaling the best HPTLC plates on the market, in addition to fast separation times. 

198 



S28-02 AN EFFICIENT APPROACH FOR ACCURATE GRADIENT ELUTION IN NANO-LC 

Palma P. 1 , Cappiello A. 1 , Famiglini G. 1 , Bergna M. 2 , Siviero A. 2 , Mantegazza A. 2 

1 

University of Urbino 

2 

DANI Instruments SpA 

Corresponding author e-mail: pierangela.palma@uniurb.it 

High-sensitive liquid chromatography-mass spectrometry detection is normally based on reduced flow rate delivered 

by a nano-LC column. However, when compared with conventional LC, most nano-LC systems suffer of less accurate 

gradient slopes, gradient transfer delays and an unacceptable waste of costly and toxic organic solvents when 

flow-splitting is used. These drawbacks may limit the appeal of nano-LC-MS. Here we present a new approach to 

generate accurate and reproducible nano-scale gradients in a simple, compact and robust device. The new device 

consists of a 14-port multiposition valve (MP) that supports six loops of a given volume for mobile phase storage. 

The loops are loaded using a binary system at higher flow rate. Each loop is loaded with a mobile phase of different 

composition. Computer controlled rotation of the valve delivers different solvent compositions to the column for 

binary solvent gradient combinations. Valve operation is performed at nano-scale flow rate using water as mobile 

phase in reversed phase conditions. Particular attention has been devoted to reduce the diameter of the loops 

and connecting tubings for a fast gradient delivery and to minimize turbulence and solvent mixing. Any gradient 

shape can be generated using the MP valve, by independently playing with eluent composition and switching time. 

Several aspects of the performance have been evaluated including precision and accuracy in a wide range of 

practical situations. This system has been coupled to a mass spectrometer via a Direct-EI interface and to an UV-vis 

detector equipped with a nano-flow cell. Several critical applications with different compounds of environmental and 

biological interest have been tested. 


199


S28-03 CORE-SHELL STATIONARY PHASES USING GRAPHITE CORES WITH POROUS NANODIAMOND SHELLS FOR HIGH PH 

REVERSED-PHASE HPLC 

Wiest L.A. 1 , Jensen D.S. 1 , Hung D. 1 , Olsen R. 1 , Vail M.A. 2 , Dadson A. 2 , Linford M.R. 1 

1 


2 



A new pellicular liquid chromatography phase for HPLC was created using 3 um graphite cores with 0.5 um porous 

nanodiamond shells. Our core graphite material was coated with an amine containing polymer (poly(allylamine)). 

After this initial polymer deposition, the particles were placed in a nanodiamond slurry which allowed nanodiamond 

to adhere to the surface in a self limiting process. Layer-by-layer depositions of polymer and nanodiamond were 

placed on the core particles until the desired thickness of our porous diamond shell was obtained. Our initial 

experiment involved the creation of two columns (3 cm × 4.6 mm ID) with only a single difference in their preparation. 

One was functionalized with a crosslinker, 1,2,7,8-diepoxyoctane, and the other was functionalized without 

crosslinker. The column that was not crosslinked showed reversed phase character, but showed rapidly increasing 

back pressures over the course of its stability test. The crosslinked column also showed reversed phase character, 

but was stable over the course of a 24 hour test period. During this period, back pressure increased by only 3% 

and the retention factors decreased by about 5%. The stable, crosslinked column was analyzed with a test mixture 

containing various analytes: benzene, ethyl-, propyl-, butyl- and hexylbenzene. A 60:40 acetonitrile/water mobile 

phase, with 0.1% triethylamine added to the mobile phase (giving a pH=11.3), was used. The highest plate count for 

this column was 56000 plates per meter. A flow rate of 0.5 mL/min gave a back pressure of 1000 psi (69 bar). The 

purpose in preparing this new stationary phase was to create a material with high stability at both very low and very 

high pH. This should allow pharmaceuticals to be separated using standard reversed phase separation techniques. 

Despite the crosslinked column lacking a plate count that is expected for industrial use, it is apparent from Van 

Deemter curves created using this column, that once we decrease our A term by narrowing down our particle size 

distribution we will achieve lower plate heights. This crosslinked column represents the first time that a 

reversed-phase diamond-based stationary phase has had a respectable plate count and good stability over an 

extended period of time. 

200 



S28-04 NOVEL MICROFABRICATED BINDER FREE THIN LAYER CHROMATOGRAPHY PLATES ASSEMBLED USING CARBON 

NANOTUBES 

Jensen D.S. 1 , Singh S. 1 , Song J. 1 , Vanfleet R. 1 , Davis R. 1 , Vail M.A. 2 , Dadson A. 2 , Linford M.R. 1 

1 


2 



Novel silica based thin layer chromatography plates (TLC) were prepared through a microfabrication process which 

uses carbon nanotubes (CNTs) as the framework. The use of CNTs as the framework provides the precise collocation 

of the chromatographic absorbent. Photolithography is used to place the catalytic material to grow CNTs. After the 

CNT growth the CNTs are infiltrated with elemental silicon by means of a chemical vapor deposition process. The 

resulting silicon coated CNTs are annealed in air. This annealing step removes the CNTs and converts the elemental 

silicon to silica. The resulting material is extremely white indicative of silica. After this annealing step the TLC plates 

are hydrated with a 0.1 M solution of HCl. This process produces silica based TLC plates that are very porous 

(image 1) and robust. The SEM micrographs (image 2 and 3) of the resulting microfabricated TLC plates demonstrate 

the precise placement of the adsorbent material. Being that this is a microfabrication process it excludes the need 

of a polymeric binder to keep the silica adhered to the TLC backing. These robust binder free TLC plates circumvent 

the possible secondary interactions between the binder and the species that are to be separated. It also allows for 

different staining techniques for detection. Being that there is not any binder present this will allow for a broader 

choice of solvents. The resulting normal phase microfabricated TLC plates have shown to give at least base line 

separation of a CAMAG (Muttenz, Switzerland) dye test mixture (five components) using 3 mL of toluene as the 

mobile phase. The performance of the microfabricated TLC plates meet and/or exceed that of high-performance TLC 

along with fast developing times (


SEPARATIONS 

ORAL 

SESSIONS


S29-01 ASSESSMENT OF CHIRAL STATIONARY PHASES FOR SUITABILITY FOR COMBINED ENANTIOMERIC IMPURITY / RELATED 

SUBSTANCES ASSAYS 

Perera R.W.H., Lough W.J. 

University of Sunderland 

Corresponding author e-mail: john.lough@sunderland.ac.uk 

The separation of enantiomers by LC has been a major success story since it became routinely possible in the late 

1990’s. This is so much so the case that in the field of chiral separations of pharmaceuticals there is little left to do 

by way of fulfilling genuine unmet needs. However, when a single enantiomer drug is tested analytically, the trace 

enantiomeric impurity is generally determined by a chiral LC test which is separate from the related substances test. 

Clearly it would be more convenient if the enantiomeric impurity and other related substances could be determined 

simultaneously using one set of LC conditions. This would also give a check on the specificity of the enantiomeric 

impurity test. Using N-acetyl-L-tryptophan as a model drug it has been demonstrated that this can be achieved by 

exploiting a specially-tailored combination of achiral and chiral stationary phases (CSP). However when extending 

this approach to a real drug example, it was found that when using reversed-phase conditions with the CSP it was 

necessary to use low percentages of the polar organic solvent in order to achieve the optimum chiral separation. 

As a consequence, this placed a limitation on the achiral phase that could be used in the combination system with 

the same mobile phase. At this point it was felt that, despite reversed-phase chiral LC method development by 

screening CSP having already been carried out, further retentivity, enantioselectivity and compound selectivity 

characterisation of CSP under reversed-phase conditions would be useful in informing attempts at conducting 

enantiomeric impurity and related substances determinations with one set of conditions. Compound selectivity 

was an issue in that a CSP may exhibit orthogonal compound selectivity to an achiral phase simply because it 

has little or no compound selectivity. In some cases this may be an advantage but it will be a disadvantage in a 

combined chiral/achiral system if a contribution from the CSP is needed to bring about resolution of all the related 

substances from one another. Accordingly CSP, focussing on those which might be expected to give retention and 

enantioselectivity with high amounts of polar organic solvent in the mobile phase, were evaluated under reversedphase 

LC conditions in order to ascertain which of them give large enough enantioresolution and suitable retention 

to be used in a combination column with an achiral C-18 silica to be able to determine the trace enantiomer and all 

the other related substances in one test. It was possible to demonstrate that there were several CSP that had similar 

retentivity to C-18 silicas and that achieving chiral resolution under reversed-phase LC conditions when using a 

high proportion of polar organic solvent was more facile than might have been imagined. Some examples of good 

compound selectivity were observed and so it was possible to identify chiral drugs for which the use of a tailored 

combined chiral/achiral system would be a viable approach to the simultaneous determination of drug enantiomeric 

impurity and related substances rather than just a curiosity which will only work for model compounds. 


203


S29-02 POLYSACCHARIDE-TYPE CHIRAL SELECTORS: THE IMPACT OF CHLORINE-CONTAINING GROUPS ON ENANTIOSELECTIVITY 

IN POLAR ORGANIC SOLVENT CHROMATOGRAPHY 

Ates H., Mangelings D., Vander Heyden Y. 


Corresponding author e-mail: yvanvdh@vub.ac.be 

As polysaccharide selectors are often used in chiral analyses, a comparison is made between two types of 

polysaccharide-based chiral stationary phases. The enantioselectivity of polysaccharides with chlorine-substituted 

selectors is compared to the selectivity of similar polysaccharide selectors without chlorine substituents. The four 

chlorine-containing selectors examined in this work, are cellulose tris(3-chloro-4-methylphenylcarbamate), amylose 

tris(5-chloro-2-methylphenylcarbamate), cellulose tris(4-chloro-3-methylphenylcarbamate) and cellulose tris(3,5- 

dichlorophenylcarbamate). These four selectors are recently marketed. They are compared to four non-chlorine 

containing selectors that are already for a longer time on the market: amylose tris(3,5-dimethylphenylcarbamate), 

cellulose tris(3,5-dimethylphenylcarbamate), amylose tris([S]-α-methylbenzylcarbamate) and cellulose 

tris(4-methylbenzoate). 62 pharmacologically and molecularly different drug compounds were analysed with 

the analysis conditions derived from a polar organic solvent chromatography separation strategy that has been 

developed earlier in-house. All eight stationary phases were screened with eight different mobile phases that 

only consist of polar organic solvents with basic and acidic additives. To try to increase the selectivity of the 

mobile phases, short-chained alcohols are added in small concentrations. The results show a slightly increased 

enantioselectivity for the chlorine-containing selectors compared with the classical polysaccharide-based ones: 

a total of 52 out of 62 compounds have been resolved on the four new CSPs compared to 49 separations on the 

four classical. The best performing chlorine-containing chiral stationary phase was Sepapak®-2 with 41 separated 

compounds compared with 33 compounds for the best performing non-chlorine containing stationary phase, 

Chiralcel® OD-RH. Furthermore it is remarkable that the mobile phases without additional alcohols – methanol, 

ethanol, 2-propanol and butanol – perform better than those that contain 5 vol% of a short-chained alcohol. This 

is clearly illustrated by the mobile phase ACN/DEA/TFA (100/0.1/0.1) (vol %) that obtained a separation of 49 

compounds of the test set. The addition of alcohols decreased the polarity and also the enantioselectivity leading to 

fewer separations. 

204 



S29-03 ENANTIOSEPARATION IN CHROMATOGRAPHIC LIQUID-LIQUID SYSTEMS 

Pérez A.M., Rubio N., Pérez E., Minguillón C. 

Parc Cientific de Barcelona / University of Barcelona 

Corresponding author e-mail: cminguillon@pcb.ub.es 

Countercurrent chromatography (CCC) is a liquid-liquid technique in which the stationary and the mobile phases 

are formed by immiscible liquids. In CCC the high volume ratio of active stationary phase/mobile phase and the 

accessibility of the liquid stationary phase lead to a much higher loading capacity with lower solvent consumption 

for a given amount of product processed [1]. These characteristics make CCC especially suited for preparative 

purposes. Moreover, the liquid-liquid constitution of the chromatographic system makes CCC highly versatile. 

Uncountable combinations of readily accessible solvents can be used as solvent system and a variety of eluting 

modes are allowed thanks to the absence of a solid material in the stationary phase. Regarding the separation of 

enantiomers, the preparative application of CCC offers the possibility to produce enantiomerically pure compounds 

at a lower cost than conventional liquid chromatography. As in other enantioselective separation techniques, a 

chiral selector (CS) is added, preferably to the liquid stationary phase, to produce enantioseparation [2,3]. The 

extent of the applicability of CCC in this field is dependent on the availability of CSs, experimental conditions 

and technical devices, which make the separation of a broad range of enantiomeric compounds feasible at a 

competitive level. Our research group has developed several kinds of CS and solvent systems and has applied 

them to enantioseparation using diverse eluting modes [4]. The presentation of our recent results in the field 

gives an indication of the possibilities of this technique when applied to enantioseparation. [1] A. Berthod (Ed.), 

Countercurrent Chromatography The Support-Free Liquid Stationary Phase, Comprehensive Analytical Chemistry, 

Vol. 38. Elsevier, Amsterdam, 2002. [2] E. Pérez, C. Minguillón, Countercurrent chromatography in the separation of 

enantiomers in G. Subramanian (ed.) Chiral Separation Techniques (3rd Ed.) Wiley-VCH, Weinheim, 2007 (Chap. 11). 

[3] N. Rubio, C. Minguillón, Enantioselective Recognition in Solution: The Case of Countercurrent Chromatography, in 

A. Berthod (ed.) Chiral Recognition in Separation Methods, Springer-Verlag Berlin Heidelberg, 2010 (Chap. 9). [4] a) 

P Franco, J Blanc, W R Oberleitner, N M Maier, W Lindner, C Minguillón, Anal. Chem., 74 (2002) 4175. b) E Gavioli, N 

M Maier, C Minguillón, W Lindner, Anal. Chem., 76 (2004) 5837. c) B Delgado, E Pérez, M C Santano, C Minguillón, J. 

Chromatogr. A, 1092 (2005) 36. d) E Pérez, M J Santos, C Minguillón, J. Chromatogr. A, 1107 (2006) 165. e) E. Pérez, 

C. Minguillón, J. Sep. Sci., 29 (2006) 1379. f) N Rubio, S Ignatova, C Minguillón, I Sutherland, J. Chromatogr. A, 1216 

(2009) 8505. g) A M Pérez, C Minguillón, J. Chromatogr. A 1217 (2010) 1094. h) N Rubio, C Minguillón, J. Chromatogr. 

A 1217 (2010) 1183. 


205


S29-04 POTENTIAL OF CAPILLARY ELECTROPHORESIS FOR ESTIMATION OF HUMIC SUBSTANCES PHYSICO-CHEMICAL PROPERTIES 

d’Orlyé F., Reiller P.E. 

CEA, CE Saclay 

Corresponding author e-mail: fanny.dorlye@cea.fr 

It has long been recognized that humic substances (HS) influence the transport and binding of trace metal elements 

in the environment. Despite the number of publications devoted to HS, both their structure and reactivity remain 

matters of debate. Over the last two decades, there has been an effort to extend capillary electrophoresis (CE) 

applications to HS characterization [1] as it allows the separation and detection of natural organic material (NOM) 

under conditions that are relevant to environmental systems. In this context, CE is mainly known to give information 

on the electrophoretic behaviour of HS. This in turn can provide deeper insight into their size, charge and structure 

according to appropriate electrokinetic models [2]. Besides, CE has recently proved to be well suited for performing 

Taylor dispersion analysis (TDA) in order to measure the average diffusion coefficients, D, and the equivalent 

spherical hydrodynamic radii, RH, of polydiperse samples such as polyelectrolytes [3] or nanoparticles [4]. The aim 

of the present work is to give an overview of the use of CE in the characterization of HS, and to emphasize on the 

factors that may impact their structural properties and reactivity. Hydrodynamic sizes and electrophoretic mobilities 

of three standard HS (Suwannee River fulvic and humic, and Aldrich humic acids) were determined in alkaline 

carbonate buffers, pH 10, which are inert toward NOM, ensure the global negative charge of these analytes, and 

thus prevent interactions with the inner surface of the fused-silica capillary. No evidence of ionic strength induced 

aggregation was found using CE coupled with TDA in the range of 1 to 250 mM. Nevertheless, TDA measurements 

highlighted the coexistence of a minor population of larger analytes. Broad electrophoretic profiles, characteristic of 

analyte samples presenting intrinsic size/charge polydispersity were observed. Negative electrophoretic mobilities 

(µep) monotonically decreased in absolute value from 1 to 250 mM ionic strength. In this range, a slight effect of 

the alkaline counterion nature was observed with increasing µep in absolute value from Li+ to Cs+. No evidence 

of HS concentration effect was found but HS from different origins can be distinguished on the basis of their µep. 

Interpretation of µep determined by CE is complicated and neither impermeable hard sphere nor fully permeable 

polyelectrolyte models seem to be appropriate. Modelling the HS as soft and permeable colloids appears to be more 

relevant in this case [5] and gives access to structural parameters such as the hydrodynamic permeability, λ0. 1. 

Schmitt-Kopplin, P., Junkers, J.: Journal of Chromatography A 998, 1 (2003). 2. Duval, J.F.L., et al.: Environmental 

Science & Technology 39, 6435 (2005). 3. Cottet, H., et al.: Analytical Chemistry 79, 9066 (2007). 4. d’Orlyé, F., et al.: 

Journal of Chromatography A 1204, 226 (2008). 5. Duval, K.F.L., Ohshima, H.: Langmuir 22, 3533 (2006). 

206 



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SESSIONS


S30-01 DETERMINATION OF CARBONYL COMPOUNDS BY RRLC-UV IN THE ATMOSPHERE OF NITERÓI CITY, BRAZIL 

Ochs S.M. 1 , Albuquerque F.C. 2 , Pontes Massa M.C.G. 2 , Pereira Netto A.D. 1 

1 


2 

PETROBRAS-CENPES, Leopoldo Américo Miguez de Mello Research and Development Center 


The monitoring of carbonyl compounds (CCs) in the atmosphere is of concern due to the toxicity shown by 

many of them. For example, formaldehyde and acetaldehyde are respectively classified by IARC as carcinogenic 

(Group 1) and possible carcinogenic (Group 2B) compounds. High Performance Liquid Chromatography with 

Ultraviolet detection (HPLC-UV), octadecylsilica columns and mobile phases composed of acetonitrile and water 

are often used in the determination of CCs after derivatization to the corresponding hydrazones, by reaction with 

2,4-dinitrophenylhydrazine (DNPH). The recent development of Rapid Resolution Liquid Chromatography (RRLC) 

techniques, results in analysis time reduction with improvement of resolution and method sensitivity. The goal of 

this study was the evaluation of the variation of atmospheric concentrations of CCs in Niterói City, RJ, during a 

week of January, 2010 using an optimized method of RRLC-UV. Samples were collected in the Valonguinho Campus 

of Federal Fluminense University, which is located in a mixed commercial-residential neighborhood of Niterói City 

Center. Samples were collected 5 meters above ground in an open area located 200 m of Guanabara Bay shore, 

facing an eleven lane traffic system. Daily samples of CCs were collected in 7 sampling intervals of 2 hours (from 

06:00 to 20:00) between January 9th and 14th, 2010, following the US-EPA Method TO-11. With this purpose, 60 

L of air were collected in silica cartridges impregnated with DNPH (Waters, USA) in series with an ozone scrubber 

containing KI (Waters, USA). Hydrazones were eluted with acetonitrile (5 mL) and the extracts were analyzed by 

RRLC-UV (at 360 nm) under optimized conditions using a Zorbax Eclipse Plus C18 column (50 x 2.1 mm x 1.8 

μm) and a gradient of a mobile phase composed of methanol, water, THF and isopropanol. Up to 23 CCs were 

determined. The total concentration of CCs varied between 10.89 and 24.61 ppbv but the average profile of total CCs 

varied along the different day periods (Figure 1). The maximum concentration of total CCs was observed between 

10:00 and 12:00 h that does not correspond to the maximum vehicular traffic in this area of the city, thus indicating 

that besides the traffic there may be other sources or factors that control the concentrations of CCs in this area. The 

variation of total concentration of CCs in the different periods of the weekdays was comparable to that observed on 

Saturday, but larger than that found on Sunday (Figure 2). This fact is certainly related to the characteristics of use of 

the studied area. Finally, our results showed that RRLC can be applied in CCs determination in air without the need 

of any change in the EPA TO-11 sampling conditions. 

208 



S30-02 FAST LIQUID CHROMATOGRAPHY-QUADRUPOLE-LINEAR ION TRAP MASS SPECTROMETRY FOR THE ANALYSIS OF 

PHARMACEUTICALS IN WATER RESOURCES 

Huerta-Fontela M. 1 , Galceran M.T. 1 , Ventura F. 2 

1 


2 

AGBAR, Analytical-Organic Chemistry 

The presence of pharmaceuticals and their metabolites in the aquatic environments has been undoubtedly 

demonstrated. These compounds can enter the water system by means of direct disposal or excretion and, 

depending on the efficiency of wastewater treatments, they may be present in surface waters at low concentration 

levels. Despite these low concentrations, the ubiquity of pharmaceuticals in the aquatic environment together with 

their persistent biological activities explains the concern over this specific group of water contaminants. Although 

first reports dealing with the presence of pharmaceutical residues in the environment were performed by using 

gas chromatography (GC) , nowadays liquid chromatography coupled to mass spectrometry (MS; MS/MS) is the 

preferred technique for multi-analyte determinations since it reduces analysis time and avoids derivatization 

procedures. A fast multi-residue method for the determination of 49 pharmaceuticals and 6 metabolites from 

different therapeutic classes, such as antihistaminics, angiotensin agents, psychiatrics or β-blockers, in water 

resources by means of ultra performance liquid chromatography (UPLC) coupled to tandem mass spectrometry was 

developed. The aim of this work was to explore the possibilities of combining improved chromatographic resolution, 

increased peak capacity and rapid elution of a fast LC separation with the dual quantitation and confirmation 

power of the hybrid mass analyzer QqLIT. An Acquity ultra-performanceTM liquid chromatograph from Waters was 

used and parameters such as mobile phase composition, ionic strength, flow rate, temperature and non-linear 

gradient profile were optimized for both positive and negative ionization modes. Under these conditions, the 55 

compounds were separated in less than 9 minutes (6.3 min positive mode and 2.7 min negative mode) with improved 

resolution. Unequivocal identification and quantification of the target compounds was performed by using the dual 

acquisition modes of the QqLIT system (3200 Qtrap from Applied Biosystems). Triple quadrupole mode by means 

of selected reaction monitoring (SRM) was used for quantification, whilst a second SRM transition together with 

information dependent analysis (IDA) experiments, were used for confirmation. These experiments consisting on a 

SRM acquisition as survey scan and an enhanced product ion (EPI) scan at three different energies as dependent 

scans, allowed the confirmation of those compounds exhibiting not enough intense or robust secondary transitions. 

A preconcentration step was also developed by using a general, single solid phase extraction (SPE) method with 

Oasis HLB cartridges. Under these conditions, quality parameters of the method in wastewaters were established 

obtaining low limits of quantification (from 0.02 ng/L to 50 ng/L), over 10 folds lower than those obtained when using 

conventional LC systems with similar MS instruments. Additionally, matrix effects were reduced probably due to 

the thinner chromatographic peaks obtained which allowed increasing resolution between both target compound 

peaks and matrix components. Finally, the optimized SPE UPLC/QqLIT method was evaluated for the analysis 

of pharmaceuticals in influents and effluents from six WWTPs. Thirty-one out of fifty-five pharmaceuticals were 

identified in the samples collected. 


209


S30-03 PESTICIDE ANALYSIS IN ENVIRONMENTAL WATERS BY DIRECT INJECTION IN AN ADVANCED LC-MS/MS SYSTEM 

Pareja L. 1 , Martínez-Bueno M.J. 2 , Cesio V. 1 , Heinzen H. 1 , Fernandez-Alba A.R. 2 

1 

Universidad de la República, Cátedra de Farmacognosia y Productos Naturales 

2 


PESTICIDE ANALYSIS IN ENVIRONMENTAL WATERS BY DIRECT INJECTION IN AN ADVANCED LC-MS/MS system. 

LPareja1,2, M. J Martínez Bueno1, V. Cesio2, H. Heinzen2, A. R. Fernández-Alba1,3 1Pesticide Residues Research 

Group, University of Almeria, 04120 La Cañada de San Urbano, Almeria, Spain. 2Cátedra de Farmacognosia y 

Productos Naturales, Facultad de Química, UdelaR, General Flores 2124, Montevideo, Uruguay. 3Fundación 

IMDEA-Agua, C/ Punto Net 4, 2ª planta, Edificio ZYE, Parque Científico Tecnológico de la Universidad de Alcalá. 

28805, Alcalá de Henares. Madrid, Spain. lpareja@fq.edu.uy Nowadays there is a public concern that chemicals 

currently employed in agriculture may have an adverse influence on the different compartiments of ecosystems, 

with a major impact on surface waters leading to its contamination. To monitor this situation, it is necessary to 

develop high through put analytical methodologies with high sensibility and reproductibility. Currently the principal 

methodology for contaminant analysis in waters involves the pre-concentration by solid phase extraction using 

different sorbents followed by instrumental chromatographic determination, which generally are costly and time 

consuming, when high through put analysis is considered. Direct injection to the LC- MS/MS system without 

any sample treatment is a more convienient procedure, as errors and artifact production are minimized, but in 

most cases, the detection level of the instruments does not allow to perform it. In the present communication, 

a multianalyte method has been developed for the confirmation and quantification of 77 pesticide residues in 

environmental waters by direct injection method using a hybrid triple quadrupole-linear ion trap-mass spectrometer 

(QqLIT system). The list of target pesticides included organophosphates, phenylureas, strobilurins, imidazolinones, 

acidic herbicides, triazoles, carbamates and thiocarmamates which are widely used in rice crops. Different operation 

modes have been compared and their efficiency evaluated for the analysis of the target pesticides in environmental 

waters by direct injection in the LC MS/MS instrument. For identification an quantification purposes, the MS system 

was operated in standard and scheduled mode, both in selected reaction monitoring (SRM) mode. The response 

of the pesticides at different pH (pH 3, 5, 7, 8) was evaluated in order to select the best conditions of the analysis. 

The best response was obtained working at pH 3 thus validation studies were performed at this pH. The validation 

parameteres, linearity, reproducibility, repeatability, LOD and LOQ were performed in paddy field water. Matrix 

effect was also evaluated by calculating, the slopes ratios (matrix-solvent) obtained in the calibration with solventbased 

and matrix matched standards for each pesticide. Calibration curves were performed and a response linear 

over three orders of magnitude were obtained for all the pesticides over the concentration range studied. Detection 

and quantification limits were below 0.1 and 0.5 µg L-1 respectively except for cyhalofop butyl, 2,4-dichloroaniline, 

2,4 D, bendiocarb, quinclorac, molinate and iprodione. The developed methodology proved to be a powerful high 

through put analytical technique for the rapid determination of pesticides at trace levels in environmental water.Its 

main advantage is that no sample preparation is neededimproving reproducibility, simplifying laboratory work and 

reducing analysis costs. 

210 



S30-04 EVALUATION OF EMPORE® DISKS VS POWDER RESINS AS RECEIVING PHASE FOR MONITORING OF HYDROPHILIC 

WATERBORNE MICROPOLLUTANTS IN WATERS BY PASSIVE SAMPLING AND UPLC-MS/MS 

de la Cal A. 1 , Dávila T.J. 2 , Céspedes R. 3 , Ventura F. 1 

1 

SGAB S.A. Laboratory of Organic Chemistry, Barcelona 

2 

Cetaqua, Laboratory of Organic Chemistry, Barcelona 

3 

SGAB and Cetaqua, Environmental Health R & D, Barcelona 

Passive sampling techniques are based on preconcentration of pollutants in situ, which simplifies the analytical 

procedure and improves detection limits regarding traditional protocols. Moreover, when used in an integrative 

mode, these techniques offer a more meaningful interpretation of results in monitoring campaigns, as they reflect the 

average concentration along the exposure period. 

Most studies on organic pollutants have focussed in devices that concentrate non polar compounds1, whereas the 

technique is still in an earlier development step for the monitoring of polar pollutants. The Polar Organic Chemical 

Integrative Sampler (POCIS) was developed in two different configurations for determination of pesticides and 

pharmaceuticals by Alvarez et al.2. The device consists of a receiving phase with high affinity for organic chemicals 

which is separated from the environment by a diffusion membrane, both fastened together with a rigid body. In 

the present work, different materials were compared as receiving phase for a range of polar contaminants of 

current interest, comprising pesticides as well as drugs and their human metabolites. The selected compounds are 

representative of a wide range of physicochemical properties (0.16≤logKow≤5.7; 1.6≤pKa≤11.1) so that the optimized 

methodologies could be applied to other pollutants. 

The two commercially available POCIS configurations were tested for each group of compounds. They use 

powder resins as receiving phase: Oasis HLB in the so-called “pharmaceuticals POCIS” and an admixture of 

Isolute ENV+:Ambersorb1500 (80:20/wt:wt) dispersed on S-X3 BioBeads in the “pesticide POCIS”. Besides, other 

POCIS were constructed using disks as receiving phase. In Empore® disks the sorbent particles are entrapped 

into a matrix of polytetrafluroethylene, forming a membrane that is more easily manipulated in laboratory than 

powder resins, avoid losses of sorbent and ensures an uniform sorbent distribution in the sampler. Disks of a 

polystyrenedivinylbenzene copolymer (SDB-XC), SDB with sulfonic acid groups (SDB-RPS) and C18 bonded silica 

were compared with powder resins in laboratory exposition experiments, taking into account the sampling efficiency 

for the target compounds and the simplicity of the laboratory procedure. Later, the kinetic regimes of accumulation 

were studied in a flow-throw system in order to test the applicability of the linear model and to determine the specific 

uptake rates of the device. 

The selected devices were evaluated in the field for determination of polar pollutants in surface and waste waters, 

comparing the accumulation in POCIS with grab samples measures. 

The water samples were analyzed by automated Solid Phase Extraction. Both extracts from water and POCIS were 

analyzed by Ultra Performance Liquid Chromatography coupled to Tandem Mass Spectrometry (UPLC-MS/MS). 


211

S31 MULTIDIMENSIONAL 


ORAL 

SESSIONS

ORAL SESSION 31 - MULTIDIMENSIONAL CHROMATOGRAPHY 

S31-01 SAMPLE SOLVENT STRENGTH AND VISCOUS FINGERING EFFECTS IN TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY 

Martin M. 1 , Mishra M. 2 , De Wit A. 3 , Grivel C. 4 , Heinisch S. 4 

1 


2 

IIT Ropar, Indian Institute of Technolgy Ropar-India 

3 

Free University of Brussels 

4 


Corresponding author e-mail: martin@pmmh.espci.fr 

In comprehensive two-dimensional liquid chromatography (2D-LC), sample component fractions collected from 

the effluent of a first chromatographic column are injected in a second chromatographic column. The nature 

and/or composition of the mobile phase within which they start their elution on the second column is different 

from that of the mobile phase from which they were collected from the first column. This difference in solvent 

nature or composition is the source of two solvent effects which have a potentially detrimental influence on the 

whole efficiency of the 2D process. First, the difference in viscosity between the two solvents is the source of a 

hydrodynamic instability at that sample/carrier interface where the less viscous fluid displaces the more viscous 

one (i.e. the upstream interface if the sample is more viscous than the carrier, the downstream interface in the 

opposite case). This viscous fingering instability develops as fingers of the less viscous fluid in the more viscous 

one and complementary fingers of the more viscous fluid in the less viscous one, leading to peak shape distortion 

and additional band broadening. Second, when the solvent strength of the sample solvent is larger than that of the 

carrier liquid, a deformation of the analyte zone occurs because the front part of this zone moves at a relatively large 

velocity with a low retention factor in the sample solvent while the rear part of the zone, more retained in the carrier 

liquid, moves at a relatively low velocity. The magnitude of these two solvent effects - the viscous fingering effect 

and the solvent strength effect - depends strongly on the sample volume. Understanding these effects is essential 

for the optimization of the 2D-LC process since the fraction collection volume is a key parameter to be optimized. 

In this study, the influences of these effects, individually as well as in combination, on the chromatographic 

performances are investigated by numerical simulations. Experimental data obtained in isocratic as well as gradient 

elution conditions are examined at the light of the results and of a simple model for the solvent strength effect. 


213


S31-02 A COMPREHENSIVE STUDY ON THE OPTIMIZATION OF TWO-DIMENSIONAL CHROMATOGRAPHIC SYSTEMS CONSIDERING 

LOSSES IN THEORETICAL PEAK CAPACITY IN FIRST- AND SECOND-DIMENSIONS 

Vivó-Truyols G. 1 , van der Waal S. 2 , Schoenmakers P.J. 1 

1 


2 

DSM Resolve 

Corresponding author e-mail: g.vivotruyols@uva.nl 

A method to optimize different objectives (total analysis time, total peak capacity and total dilution) has been applied 

to comprehensive two-dimensional liquid chromatography. The approach is based on Pareto-optimality and it yields 

optimal parameters (column particle sizes, column diameters and modulation times). Losses in the peak capacities 

in the first dimension (due to low detection frequency) and in the second dimension (due to high injection volumes) 

have been taken into account. Analytical expressions to calculate these effects under gradient and isocratic 

conditions are derived. Of particular interest is the study of the optimal modulation times, which corresponded to 

between 1.5 and 2 two-dimensional runs per one-dimensional peak. The effects of using gradient or isocratic elution, 

conventional (40 MPa) or “ultra-high” (100 MPa) pressures, and focusing between the first and second dimensions 

were also studied. A trade-off between total peak capacity, total analysis time, and total dilution can be established. 

214 



S31-03 COMPREHENSIVE TWO-DIMENSIONAL GC–FID AND GC–TOF MS WITH MULTIPLE COLUMNS IN THE SECOND DIMENSION: 

MATCHED FLOW CONDITIONS 

de Koning S. 1 , Janssen H-G. 2,3 , Peroni D. 3 ,Cochran J. 4 , van Egmond W. 5 

1 

LECO European Technical Centre 

2 

Unilever Research and Development, Advanced Measurement and Data Modeling 

3 


4 

Restek, New Business and Technology 

5 

NLISIS 

One of the most important recent developments in gas chromatography is comprehensive two-dimensional gas 

chromatography, or GC×GC. In this technique narrow time fractions eluting from the first dimension column are 

refocused and re-injected onto a second dimension (2D) column for a second, fast separation on a column with a 

different polarity. In this way a significant increase in separation power and detectability can be obtained. Because 

the 2D separations are recorded ‘on-the-fly’ as the first dimension (1D) separation progresses, the separation on the 

2D column has to be very fast. To obtain this high speed, short narrow-bore columns are generally used in the 2D, 

as opposed to the long normal-bore columns used in the 1D. A drawback of using different column diameters in the 

two dimensions is that the two columns can not both be operated at their respective optimum velocity at the same 

time. If a low column flow rate is applied, the 2D column is operated at its optimum velocity, but the 1D column is 

below optimum. Alternatively, at a higher carrier gas flow the 1D column is at its optimum, but the 2D column now is 

far above optimum. In practice this means that either a compromise has to be selected, or one of the columns has 

to operated (far) out of its optimum. This problem has been recognised by several authors and software to aid in 

selecting column diameters and flow rates has been presented [1]. One solution proposed is the use of a split point 

between the two dimensions [2]. Although this approach elegantly decouples linear velocities in the two dimensions, 

it can result in quantification problems due to changes in split ratio during the run. Moreover, it will also adversely 

affect the detection limits. Here we propose a novel strategy for operating both dimensions of a GC×GC set-up in 

the optimum at the same time: the use of multiple, parallel narrow columns in the second dimension. In this way the 

carrier gas flow eluting from the 1D column is divided over more flow channels which are combined again just before 

the detector. The use of optimum velocities in the wider bore 1D column now no longer results in above-optimum 

velocities in the narrower 2D column. As a result of this, both dimensions can be operated at their optimum velocities 

at the same time. Theoretical calculations are performed to determine the optimum 1D × multiple 2D column set. 

Column connectors for use of three to six parallel 2D columns are developed and evaluated. Evidently, the parallel 

columns used in the 2D need to be rigorously identical in terms of column length, diameter, film thickness etc. 

Theoretical and practical results for comprehensive GC × multiple GC are presented. Also some preliminary results 

on Time-of-Flight mass spectrometric detection will be shown. 1. J. Beens, H.-G. Janssen, M. Adahchour, U.A.Th. 

Brinkman, J. Chromatogr. A, 1086 (2005) 141-150 2. P.Q. Tranchida, A. Casilli, P. Dugo, G. Dugo G, L. Mondello, Anal. 

Chem., 79 (2007) 2266-2275 


215


S31-04 ADEQUATE CRITERIA FOR THE DEVELOPMENT OF ROBUST COMPREHENSIVE TWO-DIMENSIONAL CHROMATOGRAPHY 

METHODS 

Vanbel P.F., Vivo-Truyols G., Schoenmakers P.J. 


Corresponding author e-mail: pascale.vanbel@bluewin.ch 

Computer-assisted optimization of chromatographic separations is still a fruitful activity. In fact, advances in 

computerized data-handling should make the application of systematic optimization strategies much easier. Many 

different criteria have been suggested in the literature to assess the quality of one-dimensional chromatographic 

separations [1-3]. However, in most contemporary applications little attention is paid to the selection of optimization 

criteria. Existing criteria for optimizing one-dimensional separations will need to be modified if they are to be used 

for optimizing comprehensive two-dimensional chromatography (CxC) separations. One paper described a resolution 

metric for CxC [4]. Optimization criteria dedicated to the separation of selected target analyte(s) from irrelevant 

solute(s) have also been discussed [5]. CxC is one of the emerging techniques to achieve the analysis of complex 

mixtures. In two-dimensional separations the situation is a bit different. Group-type separations constitute another 

type of separation problem. The solutes belonging to a specific group do not need to be separated from each other. 

Considering robustness as an objective from the beginning of method development significantly reduces the chance 

of failure during the validation process. Robustness of the separation can be included in optimization strategies 

by using robustness criteria combined with other quality criteria (e.g. resolution and/or analysis time). Multicriteria 

decision making techniques are required [6-7]. The relevance of a Pareto-optimality approach in LC-LC for 

optimization of total analysis time, total peak capacity and total dilution was already demonstrated [8]. However the 

combination of robustness and other optimization criteria is a new concept in CxC. We will present examples related 

to different classes of compounds, such as pharmaceutical samples, oil products, and triglycerides. Literature [1] 

Optimization criteria in high-performance liquid chromatography, Vanbel P.F., Ph.D. thesis, Université catholique de 

Louvain, Bruxelles (B), 1997. [2] Vivo-Truyols G., Torres-Lapasio J.R., Garcia-Alvarez-Coque M.C., J.Chromatogr. 

A 2003, 991, 47-59. [3] Vanbel P.F., Schoenmakers P.J., Anal. Bioanal. Chem. 2009, 394, 1283-1289. [4] Peters S., 

Vivo-Truyols G., Marriott P.J., Schoenmakers P.J., J.Chromatogr. A 2007, 1146, 232-241. [5] Vanbel P.F., Tilquin B.L., 

Schoenmakers P.J., Chemom. Intell. Lab. Syst. 1996, 35, 67-86. [6] Vanbel P.F., Tilquin B.L., Schoenmakers P.J., J. 

Chromatogr. A 1995, 697, 3-16. [7] de Aguiar P.F., Vander Heyden Y., Massart D.L., Anal. Chim. Acta 1997, 348, 223- 

235. [8] Vivo-Truyols G., van der Wal S., Schoenmakers P.J., submitted for publication. 

216 


ORAL 

SESSIONS 

S32 

FORENSICS AND 

ENVIRONMENTAL 

RESEARCH

ORAL SESSION 32 - FORENSICS AND ENVIRONMENT RESEARCH 

S32-01 SIMULTANEOUS SEPARATION AND DETECTION OF ILLICIT DRUGS AND THEIR SALT FORMS USING LC/MS 

Guazzotti S., Jiang G., Preston K., Elmashni D. 



Identification of illicit drugs together with their salt forms is crucial for the quantitation and production purpose in 

forensic laboratories. Currently, to identify seized illicit drugs and their counter ions, at least two different analytical 

methods have to be used, making the approach time consuming and limiting overall throughput. Illicit drugs are 

separated on a reverse phase column and detected by ultra-violet (UV) or mass spectrometry detector. Inorganic 

anions are separated by ion chromatography or ion pairing chromatography and detected by conductivity or UV 

detector. In this study, we show the development of high performance liquid chromatography /mass spectrometry 

(HPLC/MS) methods to separate, detect and quantitate illicit drug compounds and their counter ions simultaneously 

in one chromatographic separation. The illicit drugs and their anions were separated on a Hypercarb 3 µm, 2.1 

x 50 mm column at 300 L/min of flow rate, 60 C of oven temperature. An MSQ Plus single-quadrupole mass 

spectrometer with polarity switching (positive/negative) was used as a detector. Amphetamine, methamphetamine, 

3, 4-methylenedioxymethamphetamine (MDMA) and cocaine were separated and detected, as well as their potential 

counter ions chloride, bromide, iodide, sulfate and phosphate (Figure 1). Part per billion limits of detection levels for 

chloride, bromide, sulfate and phosphate were achieved. Four orders of magnitude linear range (1 ppb – 10 ppm) 

was obtained with selective ion monitor (SIM) mode at low mass ranges, from 30 to 130 amu. This method offers 

simple LC separation, high detection sensitivity, and additional MS confirmation over the current methods applied in 

forensic laboratories. 

218 



S32-02 OPTIMISATION OF SOLID PHASE EXTRACTION FOR THE ANALYSIS OF BENZODIAZAPINES FROM PLASMA 

Edge A. 1 , Hui Y. 2 , Liddicoat T. 1 , Pereira L. 1 

1 


2 

Kings Colleague London 

Ion suppression has been demonstrated to have a significant effect on the quantitation of a range of molecules, 

particularly in the presence of a complicated matrix. One area where this has been prevalent is in bioanalysis, 

where the matrix will typically contain a range of salts, lipids, and proteins as well as many other components. 

These complicated matrices have been shown to cause a considerable amount of ion suppression which can 

affect the validity of the assay. The use of sample preparation techniques can significantly reduce the effects of 

ion suppression; however optimisation of this process is not routinely performed. This presentation will look at the 

use of solid phase extraction and the optimisation of the load, wash, and elution steps to determine the effect that 

this has on the degree of ion suppression. A series of Benzodiazapenes has been investigated to demonstrate the 

effect of optimisation of the extraction procedure in the removal of endogenous material. Of particular interest is the 

effect of the elution conditions, which typically defaults to 100% organic, which is not always optimal for selective 

extraction which will be demonstrated in this presentation. Data analysis of the samples will not only concentrate on 

the assay performance but data will also be presented which demonstrates the effect that differing conditions have 

on the residual matrix, with data being presented from post column infusion experiments, qualitative phospholipid 

concentration data and also TIC data demonstrating how the fingerprint pattern changes for the remaining 

endogenous matrix with differing experimental conditions. 


219


S32-03 OBJECTIVE ODOUR MEASUREMENT OF RECYCLED PAPERBOARD INTENDED FOR CONFECTIONERY FOOD PACKAGING, USING 

HS-SPME-GC-MS AND MS-BASED ELECTRONIC NOSE TECHNOLOGY 


Catholic University College Ghent, K.U. Leuven Association 

The organoleptic quality of food is the most important criterion for consumers’ preference. Recently, taste and odour 

contamination originating from packaging materials has become an important issue for the food industry. Especially 

confectionery products (e.g. chocolates) are very sensitive to packaging taints. Even though confectionery food 

is packed in glued, printed and varnished paperboard materials, the source of these taints may unfortunately be 

the recycled cardboard itself. For manufacturers of paper and paperboard, intended for confectionery packaging, 

it is fundamental to avoid as much as possible the presence of undesirable odours which could cause taints in 

food. In this work a new approach for fast and objective odour measurement of recycled paperboard intended 

for secondary food packaging is applied. Four recycled paperboard samples, originating from two different 

paperboard mills, were selected. Three paperboard samples were produced in a mill with a closed process water 

circuit. Sampling was done during the regular production (sample 1), during a start-up of the mill after a one month 

maintenance production-stop without recycling of the process water (sample 2) and during a start-up after a two 

week maintenance period with constant recycling of the process water (sample 3). The fourth sample was taken in a 

mill with open process water equipment during the regular production (sample 4). This sampling set-up was selected 

in order to investigate the influence of bacterial growth in the process water and more specific its immediate 

influence on the odour of the freshly produced samples. All samples were analysed by means of Headspace-Solid 

Phase Microextraction (HS-SPME) followed by Gas Chromatography - Mass Spectrometry (GC-MS). The HS-SPME 

extraction parameters, used in this research, were optimised in a previous study (1). Furthermore, the time-consuming 

HS-SPME-GC-MS technique was compared to fast mass spectrometry-based electronic nose (MS-nose) technology. 

Data obtained by HS-SPME-GC-MS as well as by MS-nose were statistically treated using Principal Component 

Analysis (PCA). The obtained classifications of both analysis techniques were compared. As well odorous volatiles 

originating from residual printing inks/varnishes (e.g. unsaturated aldehydes), as volatiles originating from bacterial 

contamination in the process water (e.g. butanoic acid) and volatiles originating from processing glues (e.g. acetic 

acid) could be discriminated between the selected samples. Additionally, the odour impact of the four samples 

was evaluated using descriptive sensory analysis. Results obtained by chemical-analytical and sensory analysis 

were statistically correlated using Partial Least Squares regression (PLS) to gain insight into the responsible 

odour impact components. (1) Van Caelenberg T. and Dirinck P. Optimisation and application of Headspace-Solid 

Phase Microextraction (HS-SPME) for odour analysis of paperboard materials intended for food packaging. Poster 

communication at the 11th International Symposium on Hyphenated Techniques in Chromatography and Hyphenated 

Chromatographic Analyzers (HTC-11), Bruges, Belgium, 25-29 January 2010. 

220 



S32-04 THE SCARCE CONSOLIDER PROJECT ON IBERIAN RIVER BASINS 

Navarro-Ortega A. 1 , Sabater S. 2 , Muñoz I. 3 , Sanchez-Vila X. 4 , Conde C. 5 , Picó Y. 6 , La-Roca F. 7 , Blasco J. 8 , Elosegi A. 9 , Schuhmacher M. 10 , Batalla R. 11 

Francés F. 12 , Barceló D. 1 

1 


2 

ICRA, Ecology of fluvial ecosystems, Girona 

3 

University of Barcelona, Flumen, ecology of rivers and reservoirs 

4 

Technical University of Barcelona, Hydrogeology 

5 

Technical University of Madrid, Numerical Techniques in earth Sciences 

6 

University of Valencia, Food and Environmental Safety 

7 

University of Valencia, Economy 

8 

ICMAN-CSIC, Ecology and ecotoxicology of estuarine ecosystems, Cadiz 

9 

University of the Basque Country, River Ecohydrology 

10 

University of Rovira i Virgili, Tecnatox, Tarragona 

11 

University of Lleida, Fluvial Geomorphology 

12 

Technical University of Valencia, Hydraulic engineering and environment 

Water resources in Spain are subjected to rising pressures, related to the socioeconomic activities of an increasing 

human population, expressed by accelerated land use changes, and the specific climate characteristic of 

Mediterranean countries. The main panels on climate change predict a future scenario of increasing frequency of 

floods and extended droughts in the Iberian Peninsula, mostly in the Mediterranean basin. This will certainly add to 

the currently existing problems, and will probably affect the available water resources, their quality, the functioning 

of associated ecosystems, especially rivers and their aquifers, and the ecosystem services they provide. In such 

context, SCARCE is a project that aims to describe and predict the relevance of global change impacts on water 

availability, water quality and ecosystem services in Mediterranean river basins of the Iberian Peninsula, as well 

as their impacts on the human society and economy. Hence, the project has assembled a multidisciplinary team 

of leading scientists in the fields of hydrology, geomorphology, chemistry, ecology, ecotoxicology, economy, 

engineering and modelling, in an unknown effort in the CONSOLIDER framework. The project also considers 

the active involvement of Water Authorities and other relevant agents as stakeholders. SCARCE will apply a 

multidisciplinary cross-scale approximation, with data mining and field based research in four representative basins 

in Spain: Llobregat, Ebro, Júcar and Guadalquivir. The selected basins cover a substantial area of the Mediterranean 

Spain, as well as a rich set of socioecological conditions: forested mountainous areas, highly populated basins, 

agricultural areas, and industrial clusters based on groundwater resources. - The Llobregat receives extensive 

urban and industrial waste water discharges that can not be diluted by its natural flow; this river experiences 

periodic floods and droughts which lead to frequent morphological variations in the river bed; waters have high 

concentration of pesticides, surfactants, pharmaceuticals and estrogenic compounds, with important effects on 

the biological communities. - The Ebro is largely regulated by dams and canals, which have altered its hydrological 

and sedimentary regime. Abstraction of ground and surface water, irrigation and industrial activities have also 

deteriorated soil and water quality, where pollution is relevant. - The Júcar basin has regulated surface water 

resources, though groundwater use is very intense. It is affected by water scarcity, salinisation and agricultural and 

urban pollution. - The Guadalquivir river basin is impacted by reservoirs and dams and its regime is rather artificial. 

The lower part of the river (including the estuary) has a high ecological value, but has been subjected to relevant 

transformations, and suffers from metal inputs and other pollution issues. The basic research element will be the 

kilometre-scale river reach, including the river channel, the alluvial plain and associated groundwater, as well as the 

dams that disrupt river continuity. At this scale, we will evaluate the impacts of global change on several processes 

affecting freshwater ecosystem services (e.g. nutrient processing and contaminant retention, sediment transport, 

community assembling, and habitat integrity). Results will be upscaled from the targeted river basins to the whole 

Mediterranean region in Spain. 


221

S33 

FOOD QUALITY 

AND SAFETY 

ORAL 

SESSIONS


S33-01 RP-HPLC ANALYSIS OF FUROSINE AND ACID-SOLUBLE ß-LACTOGLOBULIN TO ASSESS THE HEAT LOAD OF EXTENDED 

SHELF LIFE (ESL) MILK IN AUSTRIA 

Mayer H.K., Raba B., Meier J., Schmid A. 

BOKU University of Natural Resources and Applied Life Sciences 

Corresponding author e-mail: helmut.mayer@boku.ac.at 

The recent trend towards a longer keeping ability of pasteurized milk, without the negative flavour change normally 

associated with ultra-high-temperature (UHT) treatment, has resulted in the development of extended shelf life (ESL) 

milk. The currently used methods to produce ESL milk are microfiltration, direct heat treatment such as injection or 

infusion, or in some cases also indirect heat treatment. However, heating causes a significant loss of organoleptic 

and nutritional quality (e.g., vitamin destruction, precipitation of calcium phosphate, denaturation of whey proteins, 

Maillard reaction). Therefore, different time temperature integrators have been used to evaluate the heat load of 

ESL milk products (e.g., the milk enzymes alkaline phosphatase and lactoperoxidase, the native whey protein 

ß-lactoglobulin, hydroxymethylfurfural, lactulose, and furosine). The objective of this study was to improve published 

RP-HPLC methods for the analysis of furosine and native ß-lactoglobulin soluble at pH 4.6 in liquid milk using a 

Symmetry 300 column (Waters). Native polyacrylamide gel electrophoresis (Native PAGE) and SDS-PAGE were also 

used to assess the impact of a thermal process on milk and to distinguish different categories of heat-treated liquid 

milk samples to control nutritional value of ESL milk. The established RP-HPLC method enabled the separation 

of whey proteins within 21 minutes and was used for quantitative determination of acid-soluble ß-lactoglobulin. 

Furosine was analyzed by ion-pair chromatography RP-HPLC within 8 minutes. Native PAGE and SDS-PAGE were 

well suited for screening purposes to evaluate different heat loads of heat-treated milk samples. Approximately 55% 

of the samples designated as ESL milk product (n=71) showed ß-lactoglobulin contents lower than 1.800 mg per litre 

of milk, which had been discussed as threshold level in Austria. Most of these ESL milk samples with excessive heatload 

had a surprisingly low amount of native, non-denatured ß-lactoglobulin (< 500 mg/L) and a high furosine content 

(> 40 mg/100g protein), which was almost comparable to that of UHT milk. As ESL milk has shown a dramatic 

increase in Austria recently, and has been widely accepted in many other European countries (e.g., Germany) in 

the meantime, the nutritional and organoleptic quality of this new category of liquid milk has to be controlled in the 

future. Thus, electrophoresis of whey proteins and HPLC of furosine and native, non-denatured ß-lactoglobulin offer 

fast and reliable tools to evaluate and control the heat load of milk samples to minimize the loss of nutritional quality 

of milk with extended shelf life. 


223


S33-02 COMPREHENSIVE GCXGC(QMS) PESTICIDE ANALYIS PREPARED WITH QUECHERS: QUALITATIVE AND QUANTITATIVE 

ANALYSIS WITH AN ULTRA FAST QUADRUPOL MASS SPECTROMETER 

Baier H.U. 

Shimadzu Europa GmbH 

The QuEChERS preparation method for pesticide analysis is well established as it reduces sample preparation effort 

drastically compared to the former used method with a final GPC clean up step. On the other hand when injecting 

the extracts prepared by QuEChERS many matrix signals can be observed in the GCMS chromatogram.Therefore full 

scan modes are necessary to prevent false positive or false negative determination of target pesticides which could 

easily happen when running the GCMS in the more sensitive selected ion monitoring (SIM). To reach a high sensitivity 

for routine work in full scan here comprehensive GCxGC(q MS) was combined with Rapid large volume injection of 

30 microliter into the Optic 3 PTV. This was achieved by using a sintered glas liner i.e. a liner which has a rough inner 

wall surface and which was desactivated by a SILTEK desactivation. No additional packing material was necessary 

for the large volume injection due to the capacity of the large inner liner surface. A syringe was used with a side 

hole needle in order to spray the 30 microliter onto the liner wall. The solvent (ACN) was vented through the split line 

and this was monitored with a TCD in the split flow (solvent monitor). Best results were achieved with 55 °C initial 

PTV temperature followd by a 15 °C/sec ramp to 280 °C and a resulting venting time of 38 seconds. Comprehensive 

GCxGC with thermal modulation has a high separation power for complex matrices. In this technique analytes are 

continuously refocused between 2 columns and released in part to a second column. Here an RTX-1 30m, 025mm, 

0.25 micrometer was coupled to a BPX-50 1m, 0.15 mm, 0.15 with a loop of 1.6 m. The ZOEX ZX2 Modulator was 

used (ZOEX corporation) which allows thermal modulation without the use of liquid nitrogen. As MS detector, the 

high speed GCMS-QP2010 Ultra was used (max 100 spectra/sec,20000 amu/sec).The GC program started at 100 

°C with a ramp of 2.5 °C/min to 280 °C for 20 minutes. The modulation frequency was set to 8 sec which results in 3 

peaks per compound. The mass spectrometric detector was run in full scan mode with a mass range from 80 – 390 

amu and a sampling frequency of 50 scans/sec. This resulted in more than 15 data points across each modulated 

peak which ensured quantitative analysis with sufficient precision. For method validation a clean pesticide standard 

diluted in acetonitrile was measured and the image was compared to the data recorded with an apple matrix spiked 

with 54 seleted pesticides. The concentrations range was 0.01 to 0.2 mg/kg. The whole method covers more than 

500 pesticides. The limit of detection was below 1 micro g/kg. The GCMS EI classical spectra allow a precise 

identification of the target compounds using a search algorithm including linear retention index filtering. 

224 



S33-03 COMPARISON OF LIQUID-LIQUID EXTRACTION AND SOLID PHASE EXTRACTION FOR THE GC-MS ANALYSIS OF PHENOLIC 

ACIDS RESULTING FROM THE DEGRADATION OF WINE POLYPHENOLS BY FAECAL MICROBIOTA 

Muñoz-González C., Del Pozo-Bayón M.A., Rodríguez-Bencomo J.J., Martín-Alvarez P.J., Cueva C., Bartolomé B., Moreno-Arribas M.V. 


Corresponding author e-mail: mvmoreno@ifi.csic.es 

In recent years, it has been shown that some phenolic compounds naturally occurring in wine can suffer 

modifications in the gut, such as their transformation into different types of phenolic metabolites by colonic 

microbiota (1). These phenolic metabolites seem to modulate the oral and gut microbiota, and the development 

of beneficial or pathogenic bacteria (2). Therefore, the identification and quantification of phenolic metabolites 

in physiological samples is becoming an outstanding task in different fields, such as food science, nutrition 

and clinic. The availability of sensitive and roughness analytical tools is an aspect of great importance for their 

characterization. Although there are many studies based on the use of HPLC for the analysis of these compounds, 

GC-MS is an alternative analytical technique that can be adequate for the analysis of these compounds because 

of their sensitivity, roughness, the possibility of using mass spectra libraries for the identification and because 

of the fact that many of these metabolites exhibit very low molecular weighs. This, together with an adequate 

isolation technique may greatly improve the quality of the analysis. In this work, we have compared the analytical 

performance of two extraction techniques: liquid-liquid extraction (LLE) using ethyl acetate and solid phase 

extraction (SPE) using Evolute ABN cartridges in order to isolate the phenolic metabolites present in faecal samples. 

Forty three phenolic metabolites coming for the degradation of wine phenolic compounds were essayed and 

outstanding quality parameters of the method, such as detection limits, precision and recoveries were calculated. 

The selected method was applied for the characterization of the phenolic metabolites resulting from the incubation 

of a commercial grape seed extract and their corresponding flavan-3-ol monomeric and polymeric fractions, with 

faecal water from three healthy volunteers in an anaerobic fermentation system, at two different times (0 and 10 

hours). Results showed the suitability of both methods for the isolation of most of the assayed compounds, although 

the LLE procedure showed lower LDs, better repeatability, and very good recoveries ranging between 80 and 120 

% for most compounds. The application of this method to faecal samples remarks inter-individual differences and 

allows identification of a higher number of metabolites in the faecal water after 10-h incubation with the grape seed 

flavan-3-ol monomeric fraction . (1) Selma, M.V., Espín, J.C., Tomás-Barberán, F.A. J. Agric. Food Chem. 2009, 57. 

6485. (2) Cueva, C., Moreno-Arribas, MV., Martín-Álvarez, P.J., Bills, G., Vicente. M.F., Basilio, A., López Rivas.C.,Re 

quena,T.,Rodríguez.J.M., Bartolomé, B., Res.Microb.Doi:10.1016/j.resmic.2010.04.006 


225


SEPARATIONS 

ORAL 

SESSIONS


S34-01 VARIOUS STRATEGIES INVOLVING ON-LINE ELECTROKINETIC SAMPLE PRECONCENTRATION STEPS FOR CAPILLARY 

ELECTROPHORESIS ANALYSIS OF INORGANIC IONS AT TRACE-LEVEL IN REAL MATRICES 

Delaunay N. 1 , Sarazin C. 2 , Ghasemi M. 1 , Varenne A. 1 , Costanza C. 2 , Eudes V. 2 , Gareil P. 1 

1 

Chimie Paristech (PECSA), Paris 

2 

Central Laboratory of the Prefecture de Police, Paris 

Corresponding author e-mail: nathalie-delaunay@chimie-paristech.fr 

Capillary electrophoresis (CE) has several advantages over other separation techniques: high efficiency separation, 

minimum requirements of sample and chemical amounts, almost no need for organic solvents. One of the 

disadvantages of CE is generally thought to be poor concentration sensitivity of most popular photometric detectors. 

Apart from using high sensitivity detection modes, such as laser-induced fluorescence, one approach to solve this 

problem consists in on-line sample preconcentration, where a large amount of analytes compared to a conventional 

run is introduced into the capillary by pressurized or electrokinetic methods. The analytes are next focused into a 

narrow zone before separation. The focusing principle may be based on the velocity change of the analytes between 

the sample zone and the separation zone (stacking), caused by the change in conductivity and/or viscosity, or the 

change in effective charge of analytes (e. g. via pH). It may also involve transient isotachophoresis. These strategies, 

either alone or hyphenated, have been involved in our group for trace-level CE analysis of inorganic ions in various 

real matrices, such as pre- and post-blast residues and in fluids of nuclear power plants. Indeed, sensitivity 

improvement was achieved by on-line preconcentration employing either field amplified sample stacking (FASS) or 

field-enhanced sample injection (FESI) with or without an intermediary water plug for fast, selective, and sensitive 

analysis of inorganic anions and cations for the identification of explosives in detonator, post-blast or environmental 

samples. As real sample matrices, even of similar nature, can have very different electric conductivities, which can 

create stacking or destacking phenomena and lead to non reproducible analysis, adapted protocols were developed 

for the sample pre-treatment. As an example, for anion analysis, the addition of 10 % of background electrolyte (v/v) 

in the extract before its injection allowed to match the conductivity values, which in that case gives reproducible 

electrokinetic injection and preconcentration steps, precluding significant matrix effect (as demonstrated by a 

statistical approach). High sensitivity improvement has next been reached for analysis of Fe(II), Co(II), and Ni(II) 

in heat exchanger fluid matrices of nuclear power plants, containing 1000 ppm bore under borate form and 2 

ppm lithium ions, with a method involving both electrokinetic supercharging and transient-isotachophoresis 

preconcentration steps, CZE separation, and in situ derivatization with phenanthroline for direct UV detection. LODs 

in the low hundreds of ppt range were reached, taking advantage of the presence of lithium ion in the matrix to use it 

as a leading ion for the isotachophoretic step. 


227


S34-02 CE-TOF OPTIMIZATION FOR A METABOLOMIC APPROACH TO STUDY THE NUTRACEUTICAL EFFECT OF CYSTOSEIRA 

EXTRACTS ON DIABETIC RATS 

Moraes E.P., Rupérez F.J., Barbas C. 

University CEU San Pablo, Madrid 

Corresponding author e-mail: edgarmoraesusp@gmail.com 

Metabolite patterns of biological fluids can provide a comprehensive signature of the physiological state of an 

organism as well as insights into specific biochemical processes. Urine is a readily-collected, information-rich 

biofluid and, as a result, urine is often a focus in metabolomics investigations. Metabolites excreted in urine are 

commonly highly polar which render them hard to retain on reversed-phase stationary phases. This makes CE an 

attractive alternative technique, due to its ability to separate compounds in a purely aqueous medium, with high 

efficiency and speed. Other valuable features of CE are the small sample volumes and minimal sample preparation 

needed. Antioxidants supplementation of individuals with diabetes has been suggested because diabetes appears 

to increase oxidative stress and also diabetes complications are among the leading causes of death in diabetics. 

Studies have shown Cystoseira or its extracts possess antioxidant properties. Cystoseira is a brown alga in the 

order Fucales from Fucaceae family. It is known to be rich in beta-carotene, which is an antioxidant for lipids 

and other media. Cystoseira genus antioxidant activities are also the result of the presence in the extracts of 

tetraprenyltoluquinols, which are tocopherol-like compounds characteristic of these algae. In a previous paper 

[1], CE-UV urine fingerprints of control and diabetic rats have shown a clear effect of an antioxidant treatment on 

diabetic animals not seen in controls in a rapid, simple and cost-effective way. Hence, the present study investigated 

the potential nutraceutical antioxidative in vivo properties of a Cystoseira extract in STZ diabetic rats vs their 

controls by combining metabolic fingerprinting and target metabolite analysis and using univariate and multivariate 

statistical tools. The study comprised 18 urine samples of diabetic rats and 16 controls. Samples with and without 

treatment were available and were submitted to analysis by CE-MS. After optimization of several parameters, such 

as buffer 0.8% formic acid and 10% methanol, injection 50 mBar for 17 s, 30 kV, 20 oC, sheath-liquid composition 

comprised methanol/water (50% v/v) that contained 1 mmol.L-1 formic acid, 1.5 molL-1 purine and 0.75 molL-1 

HP-0921 and flow rate 4 L/min, nebulizing gas pressure 20 psig and the fragmentor conditions by Design-Expert 

software, the analysis were performed using a CE-ESI-TOFMS system (Agilent Technologies, Santa Clara, USA). 

Subsequent to alignment, normalization and filtering, Mass Profiler Professional was used to classify the samples. 

PCA (Principal Component Analysis) for this classification showed the principle of the separation. Using PLS-DA 

(Partial Least Squares Discriminant Analysis), we were able to predict with 97% precision and 100% using SVM 

(Support Vector Machines). Markers of the disease and evolution with the treatment are under study and will be 

presented. 1. M. Vallejo, S. Angulo, D. Garcia-Martinez, A. Garcia, C. Barbas, J. Chromatogr. A, 2008, 1187, 267. 

Acknowledgment: EADS_CASA 

228 



S34-03 SIMPLE AND RAPID CAPILLARY ELECTROPHORESIS METHOD FOR THE CHARACTERIZATION AND SEPARATION OF CDSE 

QUANTUM DOTS 

Carrillo-Carrión C. 1 , Moliner-MartÌnez Y. 2 , Simonet Suau, B.M. 1 , Valcárcel M. 1 

1 


2 


Corresponding author e-mail: qa2sisub@uco.es 

In this work we present a simple and rapid methodology to separate and characterize CdSe quantum dots (QDs) in 

aqueous medium by capillary electrophoresis. Firstly, we describe a controlled derivatization procedure to obtain 

water-soluble QDs. This derivatization methodology was based on the formation of a complex between the QDs 

and surfactants trioctylphosphine oxide/ trioctylphosphine (TOPO/TOP) and SDS. Different CdSe QDs core sizes 

were synthesised as function of the nanocrystals growing time. The fluorescence maximum values for the different 

synthetised QDs-TOPO/TOP-SDS were 522, 546, 572 and 591 nm for growing times. TEM images for the different 

QDs clearly showed the increase of the average size of nanocrystals as the growth time increased. The average sizes 

were 3.1, 3.6, 4.3 and 4.9 nm for growing times of 1, 3, 5 and 15 min, respectively. After derivatization, the different 

QDs-TOPO/TOP-SDS complexes were efficiently separated with capillary zone electrophoresis using 50 mM SDS 

in the electrophoretic buffer in order to avoid the breaking of the QDs-TOPO/TOP-SDS complexes. Satisfactory 

RSD values were obtained for the migration time (0.9 to 1.6 %) and for peak areas (4.6 to 5.4%). Finally, we describe 

the possibility of quantification the 3.1 nm QDs-TOPO/TOP-SDS complexes by using LIF detection (excitation 480 

nm and emission 520 nm). A log-linear regression was found between the peak area and the concentration of the 

QDs-TOPO/TOP-SDS complex. These results testified the utility fo CE for the characterization, identification and 

quantification of water solubles QDs with a simple procedures 


229


ORAL 

SESSIONS


S35-01 POROUS LAYER OPEN TUBULAR COLUMNS FOR SOLID PHASE MICRO-EXTRACTION 

Nesterenko E., Yavorsky A., Yavorska O., Paull B. 


Corresponding author e-mail: brett.paull@dcu.ie 

Porous layer open-tubular (PLOT) columns possess a porous layer of stationary phase on the inner surface of the 

capillary tubing, maintaining open-tubular structure after the completion of all column preparation steps. Modern 

PLOT columns are predominantly used in GC, and less in capillary electrochromatography, though PLOT columns 

with organic polymer monolithic stationary phases have potential for low pressure LC applications. Advantages of 

such an approach include, variation of surface chemistry through simple surface modification procedures, a high 

flow-through permeability and low column backpressure, and the simple column manufacture and low column costs. 

In addition, the column geometry permits its application as a micro-SPE platform for automated complex sample 

clean-up, or protein extraction and pre-concentration. In the work presented herein, glycidyl methacrylate - ethylene 

dimethacrylate (GMA-EDMA) PLOT columns (270 x 0.05 mm I.D., monolithic layer thickness - 5 um), fabricated 

using automated column scanning technique providing UV polymerisation at 365 nm, were chemically modified 

to obtain either diol or amino groups on the surface. The longitudinal homogeneity of the stationary phase was 

studied using capacitively-coupled contactless conductivity detector (C4D) before and after modification and it was 

shown that column-to-column production reproducibility was within 5%. The prepared columns were first tested to 

evaluate chromatographic stationary phase selectivity, efficiency and reproducibility. Columns were also applied 

to the extraction of proteins from test mixtures, and distribution coefficients and the extraction efficiencies were 

determined. The extracted samples were analysed by RP LC using an Acclaim C18 (100 x 1 mm I.D., 2 um) column 

with a simple acetonitrile gradient and UV detection at 210 nm. Finally the prepared PLOT columns were applied 

to the extraction of proteins from real samples, such as blood, saliva and synovial fluid, followed by analysis of the 

extracted sample by RP LC. 


231


S35-02 ELECTRODEPOSITION OF SILICA SOL-GEL ON METALLIC SUBSTRATE AS A NEW APPROACH TOWARDS UNBREAKABLE 

FIBERS IN SOLID PHASE MICROEXTRACTION 

Bagheri H., Sistani H., Ayazi Z. 


Commonly used fibers in solid-phase microextraction (SPME) possess a few disadvantages such as instability 

and swelling in organic solvents, relatively low recommended operating temperature, breakage of the fiber, and 

bending of the needle. Some of these problems could be obviated by covalent bonding of the polymer phase 

to the fused-silica substrate by sol-gel technology [1,2]. There have been some developments to replace the 

breakable fused silica [1,3] but more and extensive researches are needed to address these challenges. In this 

study a new methodology based on sol-gel technology for preparation of an unbreakable, thin and highly porous 

fiber coating for SPME [2,4] is presented. In this technique primarily an intermediate film consisting of an ultrathin 

two-dimensional polymer was prepared by hydrolysis of a (3-mercaptopropyl)trimethoxysilane (ethanol, 10-3 M) 

self-assembled monolayer grafted onto gold [5,6] then a stationary phase by electrodepositing of 3-trimethoxysilil 

propylmethacrylate as precursor, tetramethyl ortosilicat (TMOS) and polyethylene glycol (PEG) as coating polymer 

was produced. Scanning electron microscopy (SEM) analysis revealed that the synthesized fiber coating possesses 

porous structure, which should significantly increase the surface area availability on the fiber. The prepared fiber 

coating was used for SPME of polycyclic aromatic hydrocarbons (PAHs) as model analytes. Important parameters 

influencing the extraction process were optimized and an extraction time of 20 min at 50°C gave maximum peak 

area, when NaCl (15% w/v) was added to the aqueous sample. Limits of detection were at range of 10-20 pg mL-1 

using time scheduled selected ion monitoring (SIM) mode and relative standard deviation (RSD) values were all 

below 16.3% at 1 ng mL-1. The developed method was successfully applied to real water samples while the relative 

recovery percentage (RR %) obtained for the spiked real water samples were from 70 to 118%. References: [1] M. 

A. Azenha, P.J. Nogueira, A. F. Silva, Anal. Chem., 78 (2006) 2071–2074. [2] H. Bagheri, E. Babanezhad, F. Khalilian, 

Anal. Chim. Acta, 616 (2008) 49-55. [3] R. Jianga, F. Zhub, T. Luana, Y. Tongb, H. Liub, G. Ouyanga, J. Pawliszyn, 

J. Chromatogr. A, 1216 (2009) 4641-4647. [4] H. Bagheri, Z. Ayazi, E. Babanezhad, Microchem. J., 94 (2010) 1-6. [5] 

C.A. Goss, D.H. Charych, M. Majda, Anal. Chem., 63 (1991) 85-88. [6] I. Piwonskia, J. Grobelnya, M. Cichomskia, G. 

Celichowskia, J. Rogowski. Appl. Sur. Sci., 242 (2005) 147–153 

232 



S35-03 POLYPYRROLE-POLYAMIDE ELECTROSPUN-BASED SORBENT FOR MICRO-SOLID PHASE EXTRACTION 

Bagheri H., Aghakhani A., Akbari, Ayazi Z. 


Corresponding author e-mail: bagheri@sharif.edu 

Recently miniaturized solid phase extraction (SPE) have been developed which obviates some of its problems, 

using little amounts of solvents and the isolation process, like stir-bar sorptive extraction, could be performed on 

the sorbent surface [1, 2]. Electrospinning is a rather new method for producing nano-fibers using high voltages 

[3]. Previously electrospun nano-fibers were prepared and used for as packing in SPE cartridge [4]. In the present 

work, for the first time, polypyrrole-polyamide-based nanofibers were prepared by electrospinning at 13 KV in a 

way that a rather thin sheet of copolymer could be formed. The prepared sheet was used as a new micro solid 

phase extraction (µ-SPE) sorbent. This methodology needs no cartridge or any physical container and the sorbent 

is being used as a porous and nano-oriented structure sheet. Some important parameters affecting the nanosorbent 

structure were optimized. The influential parameters that optimized were included polyamide and pyrrole 

percentage, electrospinning voltage and time. The copolymer solution was loaded in an appropriate syringe and a 

syringe pump was used to produce a flow of 0.7 µL h-1. After electrospinnig, a nano-fiber sheet was produced on 

the Al foil collector. A piece of 1×1 cm of nano-fibe sheet was cut from the collector. Scanning electron microscopy 

(SEM) technique was employed to study the surface characteristics of the prepared nanofiber mat. The SEM of 

the fiber mat revealed that the electrospun copolymer possess very porous structures, which should significantly 

increase the surface area availability on the fiber. Comparing the polyamide nano-fibers with polyamide-polypyrrole 

nano-fibers revealed that the latter is more suitable for extraction of malathion. This is due to the presence of 

polypyrrole in the matrix. Important parameters influencing the extraction and desorption processes were optimized 

and an extraction time of 40 min gave maximum peak area, when NaCl (30% w/v) was added to the aqueous sample. 

Desorption was performed using 50 μL dichloromethane for 10 min. Limits of detection was at level of 0.1 ng mL-1 for 

malathion using time scheduled selected ion monitoring mode and relative standard deviation (RSD%) value was 2% 

at concentration level of 1 ng mL-1. The linearity of method was in the range of 0.1-1ng mL-1. The developed method 

was successfully applied to tap and Zayande-rood river water samples while the relative recovery percentage 

obtained for the spiked real water samples were 98% for tap water and 96% for Zayande-rood water samples, 

respectively. References: [1] C. Basheer, A. A. Alnedhary, B. S. M. Rao, S. Valliyaveettil, H. K. Lee, Anal. Chem., 78 

(2006) 2853–2858. [2] H. Bagheri, F. Khalilian, M. Naderi, E. Babanezhad, J. Sep. Sci., 33 (2010) 1132-1138. [3] N. 

Bhardwaj, S. C. Kundu, Biotechnol. Adv., 28 (2010) 325–347. [4] X. Kanga, C. Pana, Q. Xua, Y. Yaoa, Y. Wangb, D. 

Qib, Z. Gua, Anal. Chim. Acta, 587 (2007) 75–81. 


233

ORAL 

SESSIONS 

S36 

LIFE SCIENCES 

AND BIOANALYSIS


S36-01 AUTOMATED IMMUNOAFFINITY CAPILLARY ELECTROPHORESIS BASED ON MAGNETIC BEADS FOR THE DETERMINATION OF 

ALPHA-1 ACID GLYCOPROTEIN ISOFORMS PROFILE TO FACILITATE ITS USE AS BIOMARKER OF VASCULAR DISEASES 

Morales-Cid G. 1 , Puerta A. 1 , Díez-Masa J.C. 1 , Martín-Alvarez P.J. 1 , Martín-Ventura J.L. 2 , Barbas C. 3 , Tuñón J. 2 , Egido J. 2 , de Frutos M. 1 

1 

CSIC, Institute of Organic Chemistry, Madrid 

2 

IIS-Fundación Jiménez, Autonomous University of Madrid 

3 


Corresponding author e-mail: mfrutos@iqog.csic.es 

Changes in the isoforms (molecules of one glycoprotein differing in its peptidic and/or glycosidic moieties) of 

alpha-1 acid glycoprotein (AGP) have been related to some cancers and to liver or cardiovascular diseases (CVD). 

As differences in AGP glycosylation or amino acid composition can lead to differences in the total charge and/or 

size, CZE is a suitable technique for the analysis of AGP isoforms [1]. In order to perform CE analysis, AGP has to be 

isolated and concentrated from the biological fluid. 

In our group we have initiated studies aimed to allow investigating the role of the CE profile of AGP isoforms 

as biomarker of vascular diseases (abdominal aortic aneurysm and carotid atherosclerosis). In a first approach, 

AGP has been isolated from plasma samples using a home made immunoaffinity chromatography (IAC) column 

and ultracentrifuge filter devices [2]. Statistical methods applied to the CE profiles of AGP isolated in this way 

from plasma samples from healthy donors and patients with two vascular diseases has allowed 100% correct 

classification of samples. The whole analytical procedure, including sample preparation and electrophoretic 

separation took only 4.5 hours, which compares favorably to the conventional sample preparation process which 

took about 4 days with high reagents, sample and materials consumption. Nevertheless, these good experimental 

features could be improved even more by integrating the sample preparation inside of the electrophoretic capillary. 

This on-line procedure has been the second approach followed in our study. 

To perform this approach an immunoaffinity capillary electrophoresis (IA-CE) method for the analysis of AGP 

isoforms based in the use of tosyl-activated magnetic beads has been developed. Different parameters such as 

binding conditions of antibody to magnetic particles, effect of shape and positioning of magnets inside the capillary 

cartridge, pressure for trapping and releasing magnetic particles, conditions for capturing and desorbing AGP, and 

resolution and sensitivity enhancement have been optimized. 

Based on these preliminary results, it can be said that: i) neutral pH favors the binding of active antibody to magnetic 

particles, ii) using two high power disc-shaped magnets in an attraction configuration magnetic particles were 

trapped if introduced at low pressure and released when high pressure was applied, and iii) applying voltage (25 kV) 

using the low pH separation buffer allowed the desorption of AGP captured by the antibody bound to the magnetic 

particles. 

These preliminary results indicate that sample preparation to analyze AGP isoforms in plasma can be performed 

inside the capillary in a CE commercial equipment, allowing automation of the whole analytical process, and thus 

facilitating a broader of the potential of the CE profile of AGP as vascular diseases biomarker demonstrated by the 

off-line sample preparation approach. 

Acknowledgments 

Financial support: Comunidad de Madrid (Project S-GEN/0247/2006) and the Spanish Ministry of Science and 

Innovation (projects PCI2006-A7-0646, CTQ2009-09399, and PSE-010000-2008-6). Puerta, A. acknowledges the 

CSIC for a JAEdoc program contract. 

[1] Lacunza, I., Sanz, J., Diez-Masa, J. C., de Frutos, M., Electrophoresis 2006, 27, 4205-4214. 

[2] Ongay, S., Neususs, C., Vaas, S., Díez-Masa, J. C., de Frutos, M., Electrophoresis 2010, 34, 1796-1804. 


235


S36-02 RAPID ANALYSIS OF WHEY PROTEINS USING MICROCHIP CAPILLARY ELECTROPHORESIS 

González Crevillén A., Barrios Romero M., de la Puerta A., de Frutos M., Diez-Masa J.C. 

Institute of Organic Chemistry, Madrid 

Corresponding author e-mail: agusting.crevillen@iqog.csic.es 

Microchip Capillary Electrophoresis (MCE) allows separations in shorter time than CE in fused-silica capillaries with 

minimal consumption of sample and reagents. Also, microchips offer the possibility of integrating several analytical 

functions (i.e., sample clean-up, derivatization, preconcentration, separation, etc.) in the same platform, allowing the 

development of fully automatic analytical methods [1]. Microchips can be made of different materials, nevertheless 

plastics are becoming of particular interest because they can be fabricated by mass-production techniques. These 

techniques decrease the price of microchips making them disposable, which is a feature of interest in clinical 

and food analysis. However, the use of polymeric microchips is still a difficult task due to, from one side, the 

scarce knowledge about electrophoretical phenomena in polymeric channels and, from the other side, the lack of 

commercial instrumentation that permits one to run automatic analysis in microchips. Protein analysis is a relevant 

application for MCE, due to the interest of these biopolymers in clinical, pharmaceutical, and food analysis. In fact, 

there are some works in the literature dealing with the determination of proteins using polymeric microchips; however 

the protein adsorption on the micro-channel surface is still a serious limitation for the use of plastic chips [2]. In this 

communication we present the development of a microdevice, which integrates a commercial PMMA microchip and 

a home-made chip holder, with electrical and fluidic connections. Using this microdevice, whey proteins have been 

studied, as a model system, using size separation mode and fluorescence detection. A commercial polyacrilamide 

derivative, EOTrol (Target Discovery, Palo Alto, CA), was used as dynamic coating to avoid the proteins adsorption 

on the channel surface and to reduce the electroosmotic flow. The separation was performed using an aqueous 

borate buffer (5 mM borate pH 8, containing 0.1% (w/v) SDS) and two types of hydrophilic polymer -several cellulose 

derivatives [3] and polyethylene oxide (PEO) of different molecular weights [4]- for a comparative study. Aggregation 

and precipitation inside of the chips have been observed in separation buffers containing cellulose derivates and 

EOTrol when the electric field is applied. However, this effect has not been observed when PEO is combined with 

EOTrol. Finally, a separation method for whey proteins using PEO as sieving matrix was optimized studying several 

analytical parameters. Separations of the main whey proteins in less than 250 seconds are achieved. References 

[1] Benhabib M., Chiesl T.N., Stockton A.M., Sherer J.R., Mathies R.A., Anal Chem. 2010, 82, 2370-2379. [2] Tran 

N.T., Ayed I., Pollandre A., Taverna M., Electrophoresis 2010, 31, 147-173. [3] Okada H., Kaji N., Tokeshi M., Baba 

Y., J. Chromatogr. A 2008, 1192, 289-293. [4] Guttman A., Horváth J., Cook N., Anal. Chem. 1993, 65, 199-203. The 

authors acknowledge Spanish Ministry of Science and Innovation (Project PATSENS, ref. PSE-010000-2008-6 and 

CTQ2009-0939) and Comunidad de Madrid (Project S-GEN-0247-2006) for financial support. M.M. Barrios-Romero 

thanks the CSIC for a Ph. D. fellowship. 

236 



S36-03 FLUORESCENT DERIVATIZATION THROUGH THE AMINO GROUPS ALLOWS CE-LIF ANALYSIS OF PROSTATE-SPECIFIC 

ANTIGEN (PSA) GLYCOFORMS 

Garrido-Medina R., de Frutos M., Diez-Masa J.C. 

Institute of Organic Chemistry (CSIC), Madrid 

Corresponding author e-mail: mfrutos@iqog.csic.es 

PSA is a 28 kDa glycoprotein which can be found at very low concentration in serum. In the last twenty years, 

the PSA test in serum has been employed for the early diagnose of prostate cancer. However, the PSA test is 

not capable in too many cases to distinguish between benign prostate hyperplasia (BPH) and prostate cancer. 

Therefore, it would be of interest to find a more unequivocal biomarker for prostate cancer. In this sense, it is known 

that glycosylation of some proteins is changed under tumor pathologies. So, the quantification and comparison 

of the different glycoforms of PSA under benign or malignant prostate diseases, could potentially be a way for 

discriminating cancer and BPH. To analyze glycoforms of PSA in serum, CE-LIF seems the technique of choice 

because it combines the high resolution obtained by CE as separation technique and the excellent LOD achieved by 

LIF detection. High resolution permits the separation of structurally very close glycoforms while elevated sensitivity 

allows detecting the low concentration of the glycoprotein in serum. Unfortunately it is necessary, in most of the 

cases, to resort to a fluorescent derivatization and the methods commonly employed for it modify significantly the 

electrophoretical properties of the protein. In these cases, the fluorescent derivatization causes band broadening 

and lack of glycoforms separation. In this work, a fluorescent derivatization method which provides for the tagged 

PSA a separation resolution similar to the one obtained for the peaks of glycoforms of the untagged glycoprotein is 

presented. To do so, a commercially available pyrylium derivative fluorogenic dye (Chromeo® P503), which reacts 

through the positively charged amino groups of the protein was used. In a first approach off-column labeling was 

performed. Several temperatures and reaction times were assayed, showing that for 0.33 nmol of PSA derivatization 

was completed in 4 h at 50 ºC. Afterwards, derivatization speed, automation, and sensitivity were enhanced by 

performing on-capillary labeling. This operation mode allowed derivatizing 0.5 pmol of PSA in only 4 min at 35 ºC. 

The results indicate that the amino-derivatization of PSA with pyrylium dye can be achieved with a good LOD for 

the method and does not compromise the CE separation of peaks of PSA glycoforms, as it leds to a similar profile 

than the one obtained by CZE analysis of the non-labeled glycoprotein with UV detection. Moreover, comparing the 

two methods of reaction it is clear that the on-capillary method using a commercial CE instrument increases the 

labeling yield, enhances the analysis throughput, and allows automation, very important aspects for clinical analysis. 

Acknowledgments: The authors thank the Spanish Ministry of Science and Innovation (projects CTQ2009-09399 and 

HH2006-013) and the Comunidad de Madrid (project S-GEN-0247-2006) for financial support. Raul Garrido-Medina 

thanks the Spanish Ministry of Science and Innovation for a Ph. D. fellowship. 


237

POSTER 

SESSIONS 

TOPICS 

P01 FUNDAMENTALS OF CHROMATOGRAPHY 

P02 GAS CHROMATOGRAPHY 

P03 LIQUID CHROMATOGRAPHY 

P04 ELECTRODRIVEN SEPARATIONS 

P05 SUPERCRITICAL FLUID CHROMATOGRAPHY 

P06 MULTIDIMENSIONAL CHROMATOGRAPHY 

P07 HYPHENATED TECHNIQUES 

P08 COLUMN TECHNOLOGY 


P10 POLYMERS 

P11 ENANTHIOSEPARATIONS 

P12 FAST SEPARATIONS 

P13 NEW DETECTION METHODS 

P14 SPECIATION ANALYSIS 

P15 FOOD QUALITY AND SAFETY 

P16 CLINICS AND PHARMACEUTICS 

P17 LIFE SCIENCES AND BIOANALYSIS 

P18 FORENSICS 

P19 INDUSTRIAL PROCESS 


239 

251 

268 

325 

343 

345 

356 

370 

384 

388 

394 

411 

419 

422 

425 

528 

568 

601 

612 

616

P01 

FUNDAMENTALS OF 


POSTER 

SESSIONS

POSTER SESSION 01 - FUNDAMENTALS OF CHROMATOGRAPHY 

P01-001 PEAK HALF-WIDTH PLOTS TO STUDY PEAK PERFORMANCE OF BASIC DRUGS IN SURFACTANT-MEDIATED LIQUID 



1 


2 


Corresponding author e-mail: celia.garcia@uv.es 

A low concentration of an additive in a conventional mobile phase in reversed-phase liquid chromatography can 

alter the stationary phase surface and the partition characteristics of the analytes. The magnitude of the effect can 

be modulated by varying both the type and concentration of the additive. One interesting example is the addition 

of a surfactant above the critical micellar concentration (CMC). Owing to the existence of micelles, the technique 

has been called micellar liquid chromatography (MLC). However, the surfactant is also adsorbed on the stationary 

phase surface, giving rise to an open micelle-like structure, which is the main responsible of the observed behaviour. 

Concomitantly with the retention behaviour, the mass transfer kinetics on the modified stationary phase is affected. 

The MLC literature contains many comments on the reduced efficiency for compounds of different nature eluted 

with mobile phases containing exclusively a surfactant (either ionic or non-ionic). This has been explained by the 

thick SDS layer on the stationary phase, which gives rise to an increased carbon loading. The surfactant coverage 

produces also a significant decrease in the pore volume, dramatically reducing the active surface area. Alcohol 

addition and temperature raise have been given as solutions to decrease the amount of adsorbed surfactant, 

and consequently improve the observed efficiency. The significant enhancement of peak shape (i.e. increased 

efficiencies and symmetrical peaks) obtained by addition of the anionic surfactant sodium dodecyl sulphate (SDS) 

to hydro-organic mixtures of methanol, ethanol, propanol or acetonitrile with water, for a group of basic drugs 

(beta-blockers) chromatographed with a Kromasil C18 column, is here reported. The effect can be explained by the 

thin layer of surfactant associated to the hydrocarbon chain on the stationary phase in the presence of the organic 

solvents. The surfactant layer covers the free silanols on the siliceous support avoiding their interaction with the 

cationic basic drugs. These instead interact with the anionic head of the surfactant, increasing their retention and 

allowing a more facile mass transfer. The peak shape behaviour with the four organic solvents (methanol, ethanol, 

propanol and acetonitrile) was checked in the presence and absence of SDS. The changes in peak broadening 

rate and symmetry inside the chromatographic column were assessed through the construction of peak half-width 

plots (linear relationships between the left and right half-widths at 10% peak height versus the retention time). 

The examination of the behaviour for a wide range of compositions indicated that the effect of acetonitrile in the 

presence of SDS is different from ethanol and propanol, which behave similarly. Acetonitrile seems to be superior 

to the alcohols in terms of peak shape, which can be interpreted by the larger reduction in the adsorbed surfactant 

layer on the C18 column. However, the decreased efficiencies observed at increasing surfactant concentration in the 

mobile phase should be explained by the reduction in retention times, more than by a change in the stationary phase 

nature. 

240 



P01-002 ACETONITRILE AND ETHANOL: TWO BELITTLED MODIFIERS IN MICELLAR LIQUID CHROMATOGRAPHY 

Torres-Lapasió J.R. 1 , Ruiz-Ángel M.J., Carda-Broch S. 2 , García-Álvarez-Coque M.C. 1 

1 


2 


Corresponding author e-mail: Jose.R.Torres@uv.es 

The addition of a surfactant above the critical micellar concentration (CMC) in water to a reversed-phase liquid 

chromatographic (RPLC) system gives rise to significant modifications in the chromatographic performance (i.e. 

retention, selectivity and peak shape). This mode has been called micellar liquid chromatography (MLC). The 

presence of surfactant in the mobile phase allows the use of organic solvents which are non-miscible with water, 

reaching concentrations higher than in pure aqueous solution. In spite of the wide range of compatible solvents, only 

three are routinely used: 1-propanol, 1-butanol and 1-pentanol, being the former the most common. Surprisingly, 

there are only a few and recent reports on acetonitrile, which is the solvent of choice in hydro-organic RPLC, and 

there are no reported methods for ethanol, in spite that it is attracting much attention in recent time owing to its 

low toxicity (green chemistry). In the field of MLC, a particularly interesting case is the separation of basic drugs, 

such as beta-blockers, using an alkyl-bonded phase and mobile phases containing the anionic surfactant sodium 

dodecyl sulphate (SDS). In such systems, the surfactant monomers cover the stationary phase, the hydrophobic 

tail associated to the alkyl-chains bonded to the silica support, and the polar head group oriented away from the 

surface, to which the positively charged drugs are strongly attracted. This is revealed by the higher retention and 

enhanced peak profile (i.e. narrower and more symmetric peaks), with regard to conventional hydro-organic RPLC. 

The excess surfactant is dissolved in the mobile phase as monomers, associated in small clusters or forming 

micelles. These entities, together with the organic solvent molecules are responsible of the elution of the drugs. 

Different solutes experience the interactions with the components in the stationary phase and mobile phase in 

different degree, giving rise to diverse selectivity. In this communication, we report the performance of several 

organic solvents (methanol, ethanol, 1-propanol and acetonitrile) in MLC with SDS for the separation of eight betablockers 

(acebutolol, atenolol, celiprolol, esmolol, metoprolol, oxprenolol, pindolol, and timolol), using a Kromasil 

C18 column, which is compared with the conventional hydro-organic separation with each solvent. The exploration 

of the selectivity and resolution is based on a detailed description of the elution behaviour and peak properties in a 

two-factor space of concentrations of SDS and organic solvent. The results evidenced that acetonitrile and ethanol 

offer, by far, the best separations. 


241


P01-003 OPTIMAL EXPERIMENTAL DESIGNS IN RPLC AT VARIABLE PH BASED ON ERROR SURFACES AND GENETIC ALGORITHMS 

Torres-Lapasió J.R., Baeza-Baeza J.J., Pous-Torres S., García-Álvarez-Coque M.C. 


Corresponding author e-mail: Jose.R.Torres@uv.es 

Optimization in chromatography is usually carried out in two steps. In the first one, the most significant experimental 

variables are sought from fractional factorial designs or equivalent; in the second, these are finely tuned assuming 

second-order response surfaces. In the literature, a number of methodologies have been described for determining 

the optimal experimental design, in order to build second-order models. Usually, the construction of designs is 

based on geometrical considerations. This is the case of the designs proposed by Box and coworkers (G.E.P. Box, 

in The Design and Analysis of Industrial Experiments, edited by O.L. Davies, Oliver and Boyd, Edinburgh, 1954), 

Doehlert (D.H. Doehlert, Appl. Statist. 19 (1970) 231), or Hoke (A.T. Hoke, Technometrics 16 (1974) 375). A feature 

of these designs that some experimenters find appealing is that the number of different experimental conditions 

is the same as the number of terms in the polynomial models. An alternative is the maximization of the information 

contained in the design, which is measured through the inspection of several properties of the so-called “design 

matrix”, X. For example, a design is called D-optimal when the determinant of the product (X’X) is maximal. All these 

approaches are focused on general linear models (i.e. models involving linear relationships between parameters and 

responses). The pH is a key variable in the chromatographic separation of ionisable compounds, yielding sudden 

retention drops and multiple peak crossings. Owing to the difficulties involved in the optimisation of pH, analysts 

often fix it to a convenient value, or develop the optization in a narrow range, where the changes in retention can be 

approximated to linear or polynomial variations. An ideal optimization should be able to find out the best separation 

in a wide range of conditions. However, the non-linear (sigmoidal) nature of the changes of retention with pH makes 

the above designs less suitable. In this communication, we report a study to determine the best experimental 

designs able to model the retention of weak acidic or basic compounds (nine diuretics and two beta-blockers), 

eluted with mobile phases of acetonitrile-water (in the 25-45% v/v range) at varying pH (3-7), carried out at three 

temperature levels (20, 35 and 50 Celsius degrees). We developed two approaches, which generate and inspect the 

prediction error surface yielded from different designs, using the propagation error theory. In both approaches, a 

starting two-level design with five experiments (corners plus center), which will be called base design, is used. In 

the first approach, a sequential addition of new points is performed in the location of the design yielding the poorest 

predictions. The drawback of this approach is that the new points depend on the previous ones added to the design. 

The second approach solves this limitation by making a simultaneous addition of groups of two to six new points to 

the base design, using genetic algorithms. These prevent an exhaustive examination of all possible combinations, 

which is prohibitive in practice, reaching the solution in reasonable times. 

242 



P01-004 GLOBAL PARAMETERS ACCOUNTING FOR PEAK BROADENING AND SKEWNESS 

Baeza-Baeza J.J., Pous-Torres S., Torres-Lapasió J.R., García-Álvarez-Coque M.C. 


Corresponding author e-mail: Juan.Baeza@uv.es 

Peak broadening and skewness in chromatography results from several factors of intra- or extra-column origin. 

These are fundamental parameters, since they affect the resolution capability of closely eluting compounds. Hence 

the interest in developing descriptors that characterise column performance related to peak shape; the number of 

theoretical plates (plate count or efficiency), based on the plate theory described by Martin and Synge in 1941, being 

the most popular. A common practice to characterise chromatographic columns is to estimate the efficiency and 

asymmetry factor for the peaks of one or more solutes eluted under selected experimental conditions. This has the 

drawback that the extra-column contributions to the peak variance and skewness make the peak shape parameters 

depending on the retention time. In the literature, some attempts are found to estimate column performance based 

on global parameters (non-dependent on the retention time). The most common is the so-called “column efficiency”. 

Another widely used global parameter is the “peak capacity”, which is the maximal number of resolved peaks that 

fit in a chromatographic window. This parameter considers the peak broadening produced inside the column and 

the extra-column effects altogether. In this communication, we propose several alternatives to these parameters, 

according to three types of approaches, in order to characterise the chromatographic system as a whole, or 

distinguish the intra- and extra-column contributions to the peak broadening: (i) Estimation of column efficiency 

from the peak variance or the standard deviation. (ii) Estimation of column peak broadening and skewness from 

the half widths. (iii) Estimation of mean values of the observed efficiencies and asymmetry factors using simulated 

chromatograms. The proposed global parameters arise from different linear relationships that can be established 

between the peak variance, the standard deviation, or the half-widths with the retention time. The estimation of 

peak skewness was only possible for the approaches based on the half-widths. All global parameters distinguish 

between good and poor column (or system) performance. The use of one or another approach depends on the 

particular interest of the chromatographer: the description of the intrinsic behaviour of a chromatographic column, 

or the description of the chromatographic system as a whole (i.e. the observed peak behaviour taking into account 

the extra- and intra-column contributions). The proposed approaches should be useful for column development and 

selection, and were applied to the characterisation of different columns (Spherisorb, Zorbax SB, Zorbax Eclipse, 

Kromasil, Chromolith, X-Terra and Inertsil), using the chromatographic data obtained for several diuretics and basic 

drugs. 


243


P01-005 APPROACHES TO ESTIMATE THE RETENTION TIME IN CHROMATOGRAPHY 

Baeza-Baeza J.J., García-Álvarez-Coque M.C., Torres-Lapasió J.R. 


Corresponding author e-mail: Juan.Baeza@uv.es 

The accurate description of chromatographic peaks is an important step in the treatment of data oriented to 

the experimental design with prediction or optimization purposes, or to the qualitative/quantitative analysis of 

samples, either for isolated or overlapped peaks, which are far more demanding. The most important parameters 

that gather all peak information (position and shape) are the retention time, peak height or area, and the left- and 

right half-widths. The peak half-widths (instead of the peak width) are essential to make a reliable estimation of 

the efficiency for asymmetrical peaks. The accuracy in their measurement is decreased at increasing width and 

asymmetry, and depends on the correct measurement of the retention time value. This is one of the reasons of the 

scattering observed in the Van Deemter or half-width plots [1]. In this communication, we describe and compare 

three different approaches to measure the retention time, using the information of the experimental points in the 

nearby of the maximum of chromatographic peaks. The approaches are based on a reliable description of the peak 

profile using a parabolic-Lorentzian modified Gaussian (PLMG) model, developed in our laboratory [2]. The model 

is a Gaussian-based equation whose variance is a combined parabolic-Lorentzian function. The parabola accounts 

for the non-Gaussian shaped peak, whereas the Lorentzian function cancels the variance growth out of the elution 

region. The PLMG model adapts to a wide range of peak shapes, making a correct description of peaks showing 

a large asymmetry with positive and/or negative skewness, improved with respect to other models reported in the 

literature. The developments reported here were made based on the peaks of a set of probe compounds (alprenolol, 

bendroflumethiazide, benzothiazide, bumetanide and xipamide), showing different characteristics, which were 

eluted with acetonitrile-water mobile phases of diverse composition at different temperature levels, and using 

different columns. The proper evaluation of the accuracy in the estimation of retention times and heights was carried 

out through the simulation of peaks, using the parameters for several peaks of the probe compounds (showing 

different widths and asymmetries), to which different noise level was added. The proposed approaches make use of 

polynomial equations or simple equations based on the variation of the first derivative around the peak maximum. In 

each case, the optimal number of processed points was assessed. The three approaches were highly satisfactory. 

References [1] J.J. Baeza-Baeza, S. Pous-Torres, J.R. Torres-Lapasió y M.C. García-Álvarez-Coque, “Approaches 

to characterise chromatographic column performance based on global parameters accounting for peak broadening 

and skewness”. J. Chromatogr. A, 1217 (2010) 2147–2157. [2] R.D. Caballero, M.C. García-Álvarez-Coque, J.J. Baeza- 

Baeza, “Parabolic-Lorentzian modified Gaussian model for describing and deconvolving chromatographic peaks”, J. 

Chromatogr. A, 954 (2002) 59–76. 

244 



P01-006 CHANGES IN A SURFACTANT-MEDIATED CHROMATOGRAPHIC SYSTEM UPON ADDITION OF SHORT CHAIN ALCOHOLS 

AND ACETONITRILE 

Ruiz-Ángel M.J. 1 , Carda-Broch S. 2 , García-Álvarez-Coque M.C. 1 

1 


2 


Corresponding author e-mail: mjruiz@uv.es 

In 1980, a reversed-phase liquid chromatographic (RPLC) mode was first reported, where the mobile phases 

consisted of aqueous solutions of a surfactant at concentration levels above the critical micellar concentration 

(CMC). The addition of an organic solvent to the mobile phase was, however, soon suggested in order to enhance 

the low efficiencies and weak elution strength associated to the mobile phases that contained only micelles. In MLC, 

solute separation is achieved on the basis of the differential partitioning between the bulk aqueous phase and the 

micellar aggregates in the mobile phase, and the bulk aqueous phase and the monomers of surfactant coating the 

stationary phase. Several authors have shown that organic solvents affect these partitioning equilibria, and strongly 

influence micelle formation. Such is the enhancement of the chromatographic performance (i.e. elution strength, 

efficiency, selectivity and resolution) that most analyses in MLC are performed with mobile phases containing both 

a surfactant and an organic solvent. The amount of solvent is usually limited to preserve the formation of micelles. 

Recently, however, it has been shown that a chromatographic system with mobile phases containing organic solvent 

and surfactant monomers (without forming micelles) at high concentration can yield interesting resolution. The 

changes in the nature of an RPLC chromatographic system with mobile phases containing a short chain alcohol 

(methanol, ethanol or 1-propanol) or acetonitrile, with and without the surfactant sodium dodecyl sulphate (SDS) are 

here interpreted based in the changes in retention, elution strength and peak shape of several basic drugs. When 

SDS is added to the mobile phase, the free surfactant monomers bind the C18 bonded chains on the stationary 

phase, forming an anionic layer, which attracts strongly the positively charged analytes at the mobile phase pH. 

Changes in retention are yielded as a consequence of the solving power of the organic solvent, micelles and 

surfactant monomers. The organic solvents bind the micelles, modify their shape, and can avoid their formation. 

They also bind the monomers of surfactant, desorbing them from the stationary phase, which affects the retention. 

The remaining thin surfactant layer covers the free silanols on the siliceous support, avoiding the interaction with 

the cationic solutes. This increases the efficiencies and yields nearly symmetrical peaks for the basic drugs. The 

retention of these compounds results from a combination of electrostatic and hydrophobic interactions, the latter 

being weaker compared to the hydro-organic system. This is the reason that the requirement in conventional RPLC 

of performing gradient elution to achieve practical analysis times in a single run is not so imperative in the SDSmediated 

systems. The elution patterns for SDS and 1-propanol were examined in detail. The elution strength of 

the organic solvent decreased gradually at increasing concentration of the surfactant. Since micelle disaggregation 

seems to occur in the range 15–25%, at 35% 1-propanol, ion-pair interactions with surfactant monomers in the 

bulk mobile phase should replace those with micelles. In these conditions, the elution strength gets closer to that 

observed without surfactant (i.e. in the hydro-organic mode). 


245


P01-007 PERFORMANCE OF IONIC LIQUIDS IN REVERSED-PHASE LIQUID CHROMATOGRAPHY OF BASIC DRUGS 

Ruiz-Ángel M.J., Fernández-Navarro J.J., García-Álvarez-Coque M.C. 



A comparative study of the performance of several room-temperature ionic liquids (RTILs) in reversed-phase liquid 

chromatography (RPLC) is shown for a group of basic drugs. The cationic nature of these compounds produces 

broad asymmetrical peaks with conventional C18 columns and aqueous-organic mixtures, due to the ionic 

interaction of the charged solutes with the free silanol groups on the alkyl-bonded RPLC packings. In recent years, 

RTILs have attracted some attention as silanol-blocking agents. It has been demonstrated that the addition of these 

additives to hydro-organic mixtures of acetonitrile or methanol enhance the peak shape, but solute interactions 

with the modified stationary phase and the mobile phase are complex, and difficult to interpret. RTILs should 

be considered as modifiers with a dual character (cationic and anionic): when added to aqueous-organic mobile 

phases both the cation and the anion modify the stationary phase by coating it totally or partially. The stationary 

phase coverage will depend on the nature of these two oppositely charged ions. In order to test this behaviour, four 

different RTILs: 1-ethyl-3-methylimidazolium and 1-butyl-3-methyl imidazolium hexafluorophosphates (EMIMPF6 

and BMIMPF6), and 1-butyl-3-methyl imidazolium and 1-hexyl-3-methyl imidazolium tetrafluoroborates (BMIMBF4 

and HMIMBF4), were added to aqueous-organic mixtures containing 15% acetonitrile at concentrations ranging 

from 0.01 to 0.06 M. The solutions were buffered at pH 3. A group of eight basic beta-blockers (acebutolol, atenolol, 

celiprolol, esmolol, metoprolol, oxprenolol, pindolol, and timolol) were used as probe compounds. The changes in 

the nature of the chromatographic system with mobile phase composition were discussed considering the changes 

in retention, elution strength and peak shape. Peak broadening rate and symmetry inside the chromatographic 

column were assessed through the construction of peak half-width plots (linear relationships between the left and 

right half-widths at 10% peak height versus the retention time). This study revealed that EMIMPF6 and BMIMPF6 are 

the best efficiency enhancers. The plots of the retention factor against the concentration of ionic liquid, exhibited 

a change in the slopes. This was attributed to different degrees in column covering and changes in the nature 

of solute-stationary phase interactions, which can be hydrophobic/electrostatic mixed interactions, or mainly 

electrostatic at column saturation. This behaviour was more evident with RTILs containing the PF6 anion, and 

seemed to be less affected by the nature of the cation. Finally, mobile phases containing non-dual modifiers, such 

as triethylamine (cationic modifier) and the surfactant sodium dodecyl sulphate (anionic modifier) were also checked, 

and the results compared in terms of relative retention, analysis time and peak shape. Conclusions about the 

performance of each type of modifier are addressed. 

246 



P01-008 DETERMINATION OF THE HYDRPHOBICITY (LOGPO/W) OF ORGANIC BASES THROUGH A NEW CHROMATOGRAPHIC 

METHOD 

Pallicer J.M., Sales J., Rosés M., Ràfols C., Bosch E. 


Corresponding author e-mail: ebosch@ub.edu 

In this work, we determine the log Po/w of organic bases (pKa > 8) with different hydrophobicity (log Po/w values 

from 0 to 6) working with high pH mobile phases and HPLC columns with a wide range of pH stability (1-12). The 

selected chromatographic method has been published very recently [1]. This method is based on the experimental 

measurement of the solute retention factor, log k, in a characterized chromatographic system. The retention factor is 

converted to the polarity parameter of the solute, p, by means of the polarity model [2] 

log k = (log k)0 + p (PmN - PsN) 

where the PmN and PsN account for the mobile phase and stationary phase polarity parameters, respectively, and 

(log k)0 is the intercept of the correlation. The solute p value can be easily related to the log Po/w value and four 

parameters calculated solely from the structure of the solute [3] by means of 

logPo/w = 1.22p + 1.89 (HDCA-2) - 0.17 (HOMO-LUMO) + 1.98 (pol/d2) - 1.27•10-3 (DPSA-1) - 0.99 

where (HDCA-2) is a hydrogen bond descriptor, (HOMO-LUMO) accounts for the molecular polarizability, (pol/d2) is 

related to the molecular polarity and (DPSA-1) is an electrostatic descriptor. 

The log Po/w values determined from the chromatographic p values and the mentioned molecular descriptors agree 

with those available in the literature. 

References 

[1] J. M. Pallicer, S. Pous-Torres, J. Sales, M. Rosés, C. Ràfols, E. Bosch. J. Chromatogr. A 1217 (2010) 3026 

[2] E. Bosch, P.Bou, M. Rosés, Anal. Chim. Acta 229 (1994) 219 

[3] R. Bosque, J. Sales, E. Bosch, M. Rosés, M.C. García-Álvarez-Coque, J.R. Torres-Lapasió, J. Chem. Inf. Comput. 

Sci. 43 (2003) 1240 


247


P01-009 A WIDELY-APPLICABLE SYSTEM FOR STRUCTURE-BASED CHROMATOGRAPHIC RETENTION TIME PREDICTION 

Cimpan G., Kolovanov E., Vazhentsev A., Japertas P., McBrien M. 

ACD/Labs, UK 

The tooth is the hardest part of the human body with specific construction and constitution. It consists of enamel, 

dental pulp, and dentin. Dentin is the main part of the tooth lining the inner parts of the roots and crown. It is 

constituted by anorganic material (about 70%), organic material (about 20%), and water. The aim of this study was 

to identify dentin proteom. Six healthy permanent human molars from six adults were cut, pulverized, denaturated 

(by guanidine) and demineralized (by EDTA). Denaturation and demineralization steps were repeated twice. 

Extracted proteins were cleaved (by trypsin), separated, and detected using liquid chromatography-tandem mass 

spectroscopy (LC-MS/MS). For protein separation the ZipTip pipette tips containing reverse phase media were used 

too. We identified 58 proteins with at least two identified peptides, and another 10 proteins on the basis of only 

one characteristic peptide. The main part of the dentin proteins represents collagens (type I, III, IV, V, VII, X, XI, and 

XXVII), albumin, biglycan, vimentin, coagulation factors, alpha-2-HS-glycoproteins, and dentin sialophosphoproteins. 

We found several proteins that have never been detected before in human dentin (titin, collagen type X, and collagen 

type XXVII). This study is one of the first featuring the list of proteins detected in human dentin and introduces a new 

preparation procedure for calcified human tissue by combining denaturation and demineralization processes. 

248 



P01-010: DENTIN - PROTEOMIC ANALYSIS OF HUMAN TOOTH BY HPLC-MS/MS 

Eckhardt A.¹, Pataridis S.¹, Sedlakova P.¹, Lacinova K.¹, Zmatlikova Z.², Miksik, I.¹ 

¹Institute of Physiology, Academy of Sciences of the Czech Republic, Dpt. Analysis of Biologically Important Compounds 

²Institute of Analytical Chemistry, University of Pardubice 

The tooth is the hardest part of the human body with specific construction and constitution. It consists of enamel, 

dental pulp, and dentin. Dentin is the main part of the tooth lining the inner parts of the roots and crown. It is 

constituted by anorganic material (about 70%), organic material (about 20%), and water. The aim of this study was 

to identify dentin proteom. Six healthy permanent human molars from six adults were cut, pulverized, denaturated 

(by guanidine) and demineralized (by EDTA). Denaturation and demineralization steps were repeated twice. 

Extracted proteins were cleaved (by trypsin), separated, and detected using liquid chromatography-tandem mass 

spectroscopy (LC-MS/MS). For protein separation the ZipTip pipette tips containing reverse phase media were used 

too. We identified 58 proteins with at least two identified peptides, and another 10 proteins on the basis of only 

one characteristic peptide. The main part of the dentin proteins represents collagens (type I, III, IV, V, VII, X, XI, and 

XXVII), albumin, biglycan, vimentin, coagulation factors, alpha-2-HS-glycoproteins, and dentin sialophosphoproteins. 

We found several proteins that have never been detected before in human dentin (titin, collagen type X, and collagen 

type XXVII). This study is one of the first featuring the list of proteins detected in human dentin and introduces a new 

preparation procedure for calcified human tissue by combining denaturation and demineralization processes. 


249


P01-011 IMMUNOGLOBULIN G PURIFICATION WITH PROTEIN A IMMOBILIZED BIOCOMPATIBLE MICROPOROUS MEMBRANES 

Uzun L., Türkmen D., Karakoç V., Yavuz H., Çelik H., Denizli A. 

Hacettepe University 

Corresponding author e-mail: gabriela.cimpan@acdlabs.com 

A number of models have been proposed for the prediction of various types of chromatographic retention times 

based on chemical structures. These models are typically limited in their applicability, either in terms of the 

molecules for which accurate retention times can be performed, in terms of the type of chromatographic method, or 

both. Recently, a system for structure-based prediction of retention time for generic chromatographic methods was 

devised. It uses the concept of a “federation of local models” to give accurate prediction for diverse structures, even 

for gradient methods. This work describes further accuracy improvements through the incorporation of Abraham’s 

parameter prediction. The technique involves automated detection of when these parameters are applicable, 

and incorporates them accordingly. A diverse group of structures were studied under reversed-phase and HILIC 

methods. The accuracy of prediction will be compared, both with and without the use of this technology. 

250 


POSTER 

SESSIONS 

P02 GAS CHROMATOGRAPHY

POSTER SESSION 02 - GAS CHROMATOGRAPHY 

P02-001 IONIC LIQUID BASED HEADSPACE SOLID-PHASE MICROEXTRACTION-GAS CHROMATOGRAPHY FOR POLAR ORGANIC 

COMPOUNDS 

Ruiz-Ángel M.J. 1 , Carda-Broch S. 2 , Armstrong D.W.3, Berthod A. 4 

1 


2 


3 

University of Texas-Arlington 

4 



Solid-phase microextraction (SPME) devices incorporate an extraction fiber coated with different materials, which 

is exposed to the sample using different protocols. After the equilibrium between the analyte(s) and the coating 

material is established, desorption from the fiber coating is performed. When gas chromatography (GC) is the 

analytical technique of selection, analyte thermal desorption is directly achieved by exposing the SPME fiber to the 

hot injection port of the chromatographic system. This gives rise to a coupled solvent-free method that integrates 

extraction, concentration and sample clean up in one step. Fiber selection is an essential step before applying 

SPME to assure optimal selectivity and sensitivity of the extraction. Different types of fibers are available in the 

market but some drawbacks have been addressed, such as limited life span, relatively low operating temperatures 

in GC, and variable performance of the fiber depending on the manufacturer. For this reason, the exploration of new 

coating materials, such as ionic liquids (ILs), has recently attracted some attention. Their low volatility and relatively 

high and adjustable polarity make them potential candidate sorbents in SPME fibers. In this communication, SPME 

using IL-based fibers was developed for headspace extraction of a group of low molecular weight alcohols (ethanol, 

1-propanol, 1-butanol, and isopropyl alcohol), acetone, ethyl acetate and acetonitrile. Two different fibers (simple 

coating or chemically bonded) were prepared using two ILs of different characteristics. The ILs or ILs-bonded silica 

particles were sent to Supelco (Sigma-Aldrich group, Bellefonte, PA, USA), proprietary of the procedure to coat 

porous silica SPME fibers, which manufactured the fibers. The analytical capabilities of the different fibers were 

compared to two commercially available materials (polydimethylsiloxane/divinylbenzene and polyacrilate coatings 

from Supelco). The SPME extraction of analytes was optimized for time, temperature and NaCl salting out content. 

The headspace extracted analytes were determined by simple temperature desorption into the hot injection port of a 

gas chromatograph. The simple coated-IL fibers did not have enough extracting material to be useful. 

The bonded-IL-silica particle fibers had much more extracting material, yielding more satisfactory results. However, 

the extraction of the bonded-IL-silica fiber was inferior to those of the two commercial fibers, which contained a 

significantly higher amount of extracting material (85–100 micrometers versus approximately 40–50 micrometers 

for the bonded-IL-silica particle fibers). The linear concentration range for the bonded-IL-silica fibers reached up to 

120 microgram/mL. The recoveries, accuracy and precision (RSD) were in the 97.4-109.5%, 0.1-9.5% and 0.7-16.5% 

ranges, respectively. A specific affinity for ethanol was found, which gave a peak height equal or greater than that of 

the two commercial fibers tested with the same sample. 

252 



P02-002 CHARACTERIZATION OF RHODIOLA EXTRACTS BY MS AND GC-MS 

Rodríguez-Sánchez S. 1 , Díaz P. 2 , Sanz M.L. 1 , Sanz J. 1 , Martínez-Castro I. 1 

1 

Instituto de Química Orgánica General, CSIC, Madrid 

2 

Technical University of Madrid 

Corresponding author e-mail: iqomc16@iqog.csic.es 

Rhodiola rosea is a plant belonging to Crassulaceae family, whose root has been used in the popular medicine 

in Eastern Europe and Asia. It is defined as an adaptogen which produce favourable influence on a diversity of 

physiological functions (1). These properties are related to the presence of several classes of compounds such 

as phenyl propanoids, phenylethanol derivatives, polyphenols, terpenes and phenolic acids; this bioactivity and 

the limited habitat of the plant, make the commercial extracts expensive. The content of bioactive substances in 

the extracts varies with the geographical origin, culture conditions and extraction process. The quality control of 

these extracts defines their genuinity and also their content of bioactive substances. Analytical methods have been 

generally based on HPLC (2, 3) and CE (4). Taking into account the multiplicity of bioactive substances in Rhodiola 

rosea, we have selected MS and GC-MS as the techniques of choice because their high specificity. Different 

extracts of the ground roots using water, ethanol and dichloromethane for 3 hours at room temperature were 

obtained. Extracts were filtered using Whatman nº 40 filter paper. MS analyses were carried out by direct injection 

at 30 ºC for 0.5 min, then at 70 ºC for 0.5 min and finally at 450 ºC for 10 min. GC-MS analyses of extracts previously 

converted into their trimethylsilyl oximes (5) were developed using a capillary column coated with methylsilicone 

as stationary phase. Temperature was held at 60 ºC for 0 min, then raised to 300 ºC at a rate of 5 ºC min-1. The 

GC-MS method afforded the separation of a high number of compounds, including many previously described as 

bioactive. Mono- and disaccharides, individual phenyl propanoids and their glycosylated forms were well resolved 

from other substances. Sedo-heptulose and octaverine were detected by the first time in this plant by GC-MS and 

direct MS, respectively. This work was financed by projects PRONAOS (CDTI, CENIT-2008 1004) and ANALISYC-II 

(S2010/AGR-1464). 1.- F Khanum, A S Bawa, and B Singh. Compr. Rev. Food. Sci. Food. Saf. 4 (2005) 55-62 2.- M 

Ganzera , Y Yayla , IA Khan . Chem. Pharm. Bull. 49 (2001) 465-467. 3.- CY Ma, J Tang, HX Wang, XH Gu, GJ Tao. 

Chromatographia. 67 (2008) 383-388. 4.- GB Zhou, YQ Guan, HZ Chen, J Ye. J. Chromatogr. A. 1142, 2 (2007) 236 

-239 5.- E de la Fuente, ML Sanz, I Martínez-Castro, J Sanz. J. Chromatgr A. 1135 (2006) 212-218 


253


P02-003 NEW ATMOSPHERIC PRESSURE CHEMICAL IONIZATION SOURCE FOR PESTICIDE RESIDUES SCREENING BY GC-QTOF MS 

Portolés, T. 1 , Sancho J.V. 1 , Hernández F .1 , Newton A. 2 , Hancock P. 2 

1 


2 

Water Corporation 

Corresponding author e-mail: hernandf@qfa.uji.es 

The potential applications of a new prototype atmospheric pressure source for GC-MS analysis were investigated. 

Favoring the formation of the analyte molecular ion in this source is one of the main advantages. This is feasible 

thanks to the soft ionization produced in APCI in comparison to the most-widely used Electron Ionization that 

leads to highly fragmented EI spectra. This fact opens interesting possibilities for wide-scope screening of GCamenable 

organic pollutants, because the presence of the molecular ion can be searched for many compounds. The 

addition of water as modifier was tested in this work as a way to promote the generation of protonated molecules 

during the ionization process. Around 100 GC-amenable pesticides, including organochlorine, organophosphorus 

and organonitrogen compounds, were tested and their ionization/fragmentation behavior in the new source was 

studied. The use of water as a modifier favored the formation of the protonated molecule for 90% of the pesticides 

investigated. In most cases, the protonated molecule was the base peak of the spectrum, and very low fragmentation 

was observed in APCI ionization. These results facilitated the rapid and sensitive screening using GC-(APCI)-TOF 

MS, searching for the protonated pesticide molecule, which is typically absent in the highly fragmented EI spectra. 

The ChromaLynx XS application manager allowed a list of compound names, molecular formulae, and exact masses 

to be built making it easier the detection of these compounds in samples. The developed procedure was applied 

to pesticide residue screening in several food matrices (nectarine, orange and spinach), and it allowed detection of 

some pesticides, such as chlorpyriphos, deltamethrin and endosulfan sulfate. The availability of a QTOF instrument 

made feasible performing additional MS/MS experiments to go further in the confirmation of the identity of the 

detected compounds. Relative ion abundances in the positive samples were compared with reference standards, and 

all deviations were within the tolerances established by general guidelines in residue analysis. Chemical structures 

for the most abundant product ions were suggested based on the elemental compositions proposed accordingly 

to the accurate mass measurements given by TOF MS. Mass errors were also evaluated in positive findings, and all 

were below 2.7 mDa. In addition, retention times for the reference standard and sample peak were also compared, 

obtaining a deviation lower than 0.5%. The promising results obtained for these pesticides, taken as a model 

compounds, allow expecting interesting data in other applied fields, and for other organic pollutants and residues. 

In addition to facilitating the sensitive detection of the compounds in rapid screenings, with the low fragmented 

spectrum given by APCI, the precursor ion selection no longer requires a compromise between selectivity and 

sensitivity, making easier and more effective the performing of tandem MS experiments when appropriate MS 

analyzers are available. 

254 



P02-004 ORGANIC CHARACTERIZATION OF MURAL PAINTINGS FROM SPANISH ARCHEOLOGICAL SITES 

Rosales-Conrado N., Leon-González M.E., Pérez-Arribas L.V., Navarro-Villoslada F., Vidal-Cabeza L., Báez-Aglio M.I. 


Corresponding author e-mail: noerosales@quim.ucm.es 

Encaustic is practically the only pictorial technique cited in Ancient writings. There are two current hypotheses 

about the nature of this type of painting. The most widely-accepted is that encaustic was a technique based on the 

application of molten coloured waxes. The second hypothesis posits that in addition, Greek and Roman artists used 

a paint-based on wax emulsified with an alkali. However, chemical and historical studies would indicate that neither 

hypothesis fully accounts for its true nature. Currently, a multi-disciplinary study is in progress to characterize the 

organic and inorganic materials in pictorial remains to establish more precisely the pictorial procedure followed by 

artists in their mural paintings. 

This communication reports the results of organic analysis of mural painting samples from different Spanish 

Archaeological Sites: “Casa del Mitreo” (Mérida, 2nd century a.C.), “Casa de los Amorcillos” (Cartagena, 1st century 

b.C.), and two civil structures from Ampurias (2nd century a.C.) and Baelo (3rd century a.C.). Previous studies 

attempted to characterize natural organic binders potentially used in encaustic mural paintings were carried out [1-3]. 

Analyzed materials were: raw beeswax, bleached and aged beeswax, pine resins (glass colophony and essence of 

turpentine), vegetable oils (olive, walnut and linseed oil), pure lard and soap. 

Analyses were performed by gas chromatography with flame ionization detection (GC-FID) and Fourier transform 

infrared spectroscopy (FTIR). Characteristic hydrocarbon and fatty acid profiles were obtained. Prior to GC 

determination, a hydrolysis and a derivatization step were carried out to release and to form volatile derivates of fatty 

acids. 

Spectral and chromatographic data were modelled by principal component analysis (PCA). The second derivative of 

the raw spectra and the peak-area ratio between each fatty acid and palmitic acid and between each hydrocarbon 

and n-heptacosane were considered as variables. 

PCA allowed identifying clusters of the organic binders used in the encaustic pictorial works, accounting the first 

two principal components (PC) for more than 98% of the data variability. When data from real samples were included 

in the model, the first two or three PCs were necessary to explain high percentages of variability. For example, three 

PCs accounted for the 91% of the total data variability from Cartagena samples. In this case, materials such as 

bleached and aged beeswax, colophony, walnut oil and soap were identified, as well as drying linseed oil, which was 

probably used for subsequent treatment. 

Therefore, chromatographic and FTIR analysis together with chemometric tools allow discriminate between different 

sample compositions and identify both the materials employed for painting and those used for their preservation 

and/or restoration. 

References 

[1] N. Rosales et al., RSQ–Sigma Aldrich, CO6 (Santiago de Compostela, 2008). 

[2] M.E. León-González et al., 12as Jornadas de Análisis Instrumental, PO-CTQ-4 (Barcelona, 2008). 

[3] M.E. León-González et al., Euroanalysis XV, P103-B2 (Insbruck, 2009). 

The authors thank the Santander/Complutense research program (ref. PR34/07-15858) for financial support. 


255


P02-005 DETERMINATION OF CURRENTLY USED PESTICIDES (CUPS) IN FINE AIRBORNE PARTICULATE MATTER USING 

MICROWAVE-ASSISTED EXTRACTION AND GAS CHROMATOGRAPHY COUPLED TO TRIPLE QUADRUPOLE MASS 

SPECTROMETRY 

Coscollà C. 1 , Yusà V. 1 , Castillo M. 1 , Pastor A. 2 

1 

Public Health Research Center (CSISP) 

2 


Corresponding author e-mail: yusa_vic@gva.es 

A large proportion of pesticides spread on to agricultural crops are released into the atmosphere during the spraying 

process. Emissions during application can range from a few percent to 20-30% [1]. Moreover, post-application 

losses of pesticides by volatilization from soil and plants, and also wind erosion of soil particles containing sorbed 

pesticides, represent further significant pesticides input into the troposphere for several days or weeks after 

pesticide application [2]. The dominant factors that affect volatilization of pesticides are physicochemical properties 

of compounds (vapour pressure, solubility, adsorption coefficient, …), meteorological conditions and agricultural 

practices. Semi-volatile compounds such as pesticides present in atmosphere are know to be simultaneously 

present in both the gas and particulate phase. The distribution among these phases depends on the physicochemical 

properties of the compound considered, such as vapour pressure and water solubility [3]. A multirresidue method to 

determinate 42 CUPs in fine airborne particulate matter has been developed. The studied pesticides are diclorvos, 

ethoprophos, diphenylamine, trifluralin, chlorpropham, diazinon, pyrimethanil, chlorothalonil, pirimicarb, methyl 

chlorpyrifos, vinclozolin, tolclofos methyl, metalaxyl, pirimiphos methyl, fenitrothion, malathion, ethyl chlorpyrifos, 

triadimefon, isophenphos methyl, fipronil, cyprodinil, penconazole, tolylfluanid, procymidone, folpet, mepanipyrim, 

fludioxonil, bupirimate, buprofezin, kresomim methyl, quinoxyfen, bifenthrin, iprodione, propargite, lambda 

cyhalotrin, pyrazophos, permethrin, cyfluthrin, cypermethrin, deltamethrin, bupirimate, captan, Pesticides were 

extracted using microwave assisted extraction (MAE) at different temperatures. Finally, the extraction conditions 

were: 50ºC for 20 min., using a power of 1200W and 30 ml of ethyl acetate [4]. Samples were analysed by gas 

chromatography coupled to triple quadrupole tandem mass spectrometry (GC-MS/MS). Collision energy for each 

selected SRM transition was optimized. Different injection modes (PTV splitless, CT splitless and CT splitless/ 

surge) has been tested and the main parameters affecting the performance of the injection such as splitless time 

(ST) and surge pressure has been optimized. Good results were obtained using ST = 2.5s. During the extraction step 

many interfering (mainly organic) components are co-extracted together with target analytes and may interfere in its 

identification and quantification. Therefore, a Gel Permeation Chromatography (GPC) clean-up step performed after 

concentration was applied. The matrix effect was also evaluated. All compounds presented enhanced matrix effect, 

but this matrix effect could be decreased with a clean-up step. The method was validated. The limit of quantification 

(LOQ) ranged from 1 to 10 pg m-3, for collected air volumes of 760 m3. Good recoveries efficiencies were obtained. 

The method was applied to samples of PM 10 filters collected from monitoring network of the Regional Valencia 

Government (Spain). References [1] F. Van der Berg, R. Kubiak, W.G.Benjey, M.S.Majewski, R.R.Yates, G.L.Reeves, 

J.H.Smelt, A.M. A Van der Linden, Water Air Soil Pollut. 115, 195 (1999). [2] C. Bedos, P. Cellier, R. Calvet, E. 

Barriuso, Agronomie 22, 35 (2002) [3] C. Bedos, P. Cellier, R. Calvet, E. Barriuso, B. Grabielle, Agronomie 22, 21 

(2002) [4] C.Coscollà, V. Yusà, M.I. Beser, A. Pastor, J. Chromatogr. A 1216, 8817 (2009). 

256 



P02-006 GAS CHROMATOGRAPHIC DETERMINATION OF PHYTOSTEROLS AND THEIR OXIDATION PRODUCTS IN ENRICHED FRUIT 

BEVERAGES 

Alemany-Costa L., González-Larena M., García-Llatas G., Alegría A., Barberá R., Lagarda M.J. 


Corresponding author e-mail: m.j.lagarda@uv.es 

Due to their cholesterol lowering effects, phytosterols (PS) are added to foods. During food processing, PS are 

susceptible to oxidation, leading to the formation of phytosterol oxidation products (POPs). POPs have been mostly 

analyzed in lipid matrixes, but the incorporation of PS to foods rich in fat is contrary to the dietary recommendations 

for a healthy lifestyle. Fruit-skimmed milk beverages (where PS enrichment is allowed (1)) are appropriate for meeting 

the recommendations and are good sources of bioactive compounds. 

AIM 

To evaluate the influence of two ingredients used for PS enrichment on the PS and POPs contents in fruit beverages, 

using gas chromatography (GC). 

MATERIAL AND METHODS 

Samples: Four fruit beverages (Fb) with milk (M) enriched with two different ingredients serving as a source of PS 

(A: free PS from pine tree, and B: esterified PS from soybean, rapeseed, sunflower and corn oil), with similar PS 

contents (1.7g PS/100g). 

The method applied comprised the extraction of lipids with chloroform:methanol (2). PS: hot saponification (65ºC, 

1h) (3), extraction of unsaponifiable fraction with ether and derivatization in trimethylsilylethers (TMSE). POPs 

(4): saponification (room temperature, 18h), purification and enrichment by SPE, and derivatization to TMSE. The 

samples are injected into GC-MS for identification and GC-FID for quantification. The GC conditions are: 

RESULTS 

The PS (g/100g of sample) determined in samples with ingredient A, FbA and FbMA, are: beta-sitosterol 

(0.919;0.972), beta-sitostanol (0.141;0.145), campesterol (0.049;0.063), campestanol (0.012;0.012) and stigmasterol 

(0.017;0.014). With ingredient B, FbB and FbMB, the PS are: beta-sitosterol (0.549;1,460), beta-sitostanol 

(0.109;0.102), campesterol (0.107;0.245), campestanol (0.058;0.055) and stigmasterol (0.015;0.034). 

The POPs (mg/100g of sample) determined in A, FbA and FbMA, are: 7 alfa-hydroxysitosterol (0.091;0.061), 

7beta-hydroxysitosterol (0.363;0.300), alfa-epoxysitosterol (0.054;0.038), beta-epoxysitosterol (0.032;0.190) and 

7-ketositosterol (0.116;0.162). With ingredient B, FbB and FbMB, the POPs are: 7alfa-hydroxysitosterol (0.051;0.053), 

7beta-hydroxysitosterol (0.278;0.472), alfa-epoxysitosterol (0.036;0.060), beta-epoxysitosterol (0.046;0.126) and 

7-ketositosterol (0.064;0.167). 

CONCLUSIONS 

The most abundant PS is beta-sitosterol, followed by beta-sitostanol, campesterol, campestanol and stigmasterol. 

No statistically significant differences in beta-sitosterol and stigmasterol content are observed among the 

ingredients used. 

All the detected POPs derived from beta-sitosterol, because the latter is the main PS. The formation of POPs in the 

samples is independent of the PS source. 

REFERENCES 

(1): European Union Scientific Committee on Food. DOCE L105/49-51, 14th April 2004. 

(2): Boselli et al. J. Chromatogr. A. 2001, 917, 239-244. 

(3): Piironen et al. J. Food Comp. Anal. 2000, 13, 619-624. 

(4): Conchillo et al. J. Agric. Food Chem. 2005, 53, 7844-7850. 

ACKNOWLEDGEMENTS 

Study financed by AGL2008-02591-C02-01 (CICYT-FEDER), and partially funded by CONSOLIDER INGENIO 2010. 

Program FUN-C-FOOD CSD2007-063. Marina González-Larena holds a Danone grant from the Instituto Danone 

(Spain), and Laia Alemany-Costa is the holder of a grant from the Generalitat Valenciana (Spain). 


257


P02-007 A SIMPLE PARALLEL GC COLUMN SCREENING SYSTEM 

Schafer W. 1 , Pirzada Z. 1 , Hamilton S., Welch C.J. 1 

1 

Merck, Early Development Analytical Research 

2 

MSD, Early Development Analytical Research 

The use of chiral GC is well established for analyzing the stereoisomers of small molecules, especially those 

compounds lacking chromophores that are otherwise difficult to analyze by chiral HPLC or SFC. As it is often difficult 

to predict which chiral GC stationary phases will afford the best enantiomeric selectivity, multiple stationary phases 

must often be evaluated in order to obtain the desired separation. Unfortunately, there is no convenient GC analog to 

the column selection valves that are routinely used in automated HPLC or SFC method development. Consequently 

GC column screening can be tedious and labor intensive, as it requires manually exchanging columns on a single 

instrument. In addition, the shallow thermal gradients typically employed for chiral GC result in long run times and 

further limit the number of columns that can be evaluated within a normal working day. In this study, we describe 

a simple means of screening 4 columns on a single GC instrument using inexpensive y-splitters and a second 

autosampler tower that allows for the simultaneous evaluation of two columns. 

258 



P02-008 ANALYSIS OF RESIDUAL SOLVENTS USING GC/FID WITH HEADSPACE AND A CYANOPROPYLPHENYL POLYSILOXANE 

PHASE 

Khan A., Pereira L., Faulkner W., Barattini V. 


This poster describes the analysis of residual solvents according to the US Pharmacopeia (USP) revised method in 

effect from July 2008. This method is used for analyzing trace levels of solvents that are used in the manufacturing 

of Active Pharmaceutical Ingredients (API) in pharmaceutical industries. The USP 467 method involves the analysis 

of 53 solvents grouped in three classes according to their genotoxic hazards. Class 1 solvents should not be 

used in the manufacturing stages of API, whereas class 2 solvents should only be used in limited amounts; for 

class 3 solvents there is limited evidence of their hazard to health. The procedure involves GC/FID analysis with 

headspace sampling. The column used is a cyanopropylphenyl polysiloxane phase for the analysis of volatile organic 

compounds. This type of column is used for screening purposes only, for quantitation purposes the USP method 

recommends the use of a wax column. The analysis of Class 1 and 2 solvents is demonstrated in this work. The 

method presented in this work meets the resolution and signal-to-noise ratio criteria set in the USP467 method. 


259


P02-009 DETERMINATION OF SUSPECTED ALLERGENS IN COSMETIC PRODUCTS BY HEADSPACE-PROGRAMMED TEMPERATURE 

VAPORIZATION-FAST GAS CHROMATOGRAPHY-QUADRUPOLE MASS SPECTROMETRY 

del Nogal Sánchez M., Pérez Pavón, J. L., Moreno Cordero B. 


Corresponding author e-mail: mns@usal.es 

A new method for the qualitative and quantitative analysis of 24 volatile compounds listed as suspected allergens in 

cosmetics by the European Union is reported. Research was carried out on a GC device equipped with a 

headspace-sampler (HS), a programmed temperature vaporizer (PTV) and a MS detector unit. The use of a HS-PTV 

combination to introduce the sample provided satisfactory results. The volatiles of the sample were analyzed without 

interference from the non-volatile matrix. An important advantage of the methodology used here is that no complex 

treatment of the sample is required, thus minimizing the creation of analytical artefacts and the errors associated 

with this step of the analytical process. Two different injection techniques were used: solvent-vent injection and hotsplit 

injection. The first was used to detect suspected allergens in the cosmetic products. After that, quantification 

was performed using hot-split injection due to the high signal intensity of the detected compounds. Use of the 

standard addition procedure as a quantification technique overcame the matrix effect. The proposed methodology 

has proved to be useful for the rapid detection and quantification of suspected allergens in different type of cosmetic 

products. The information contained in the low-resolution chromatogram is sufficient for the identification of the 

compounds present in the samples. Plotting the chromatograms by contour surfaces with time and m/z axes allows 

rapid visualization of the results and detection of the allergens present in a sample. The method is rapid, simple and 

-in view of the results- highly suitable for the determination of suspected allergens in different cosmetic products. 

260 



P02-010 DETERMINATION OF BENZENE AND 22 ALKYLBENZENES IN SOIL WITH THERMAL DESORPTION-GAS 

CHROMATOGRAPHY-MASS SPECTROMETRY 

Kramarics Á. 1 , Szekeres Z. 1 , Eke Zs. 1 , Rikker T. 2 , Torkos K. 1 

1 


2 

WESSLING International Research and Educational Center 


Environmental pollution is one of the biggest problems that threaten the future of humanity. Volatile organic 

compounds (VOCs) are frequent pollutants of groundwater and soil samples. For analysis of soil samples 

solid-liquid extraction is the most commonly used method. However, it needs expensive ultraclean organic solvents, 

and handling of produced extracts is also a big problem regarding both financial and environmental aspects. 

Nowadays, solventless techniques gain more and more attention. Instead of solid-liquid extraction direct thermal 

desorption is a possible solution for analysis of soil samples. In this case soil samples are placed in glass tubes 

and analytes are thermally desorbed in a Thermal Desorption Unit (TDU). In order to refocus the components, a 

programmable temperature vaporizer inlet (CIS4) with a Tenax TA filled inlet liner is used. For identification and 

quantification of analytes gas chromatography-mass spectrometry is used. Increased level of benzene and alkylbenzenes 

refers to environmental pollution e.g. leaking of underground storage tanks, so determination of these 

compounds is very important in soils. In Hungary a regulation gives a limit for benzene, toluene, ethylbenzene, 

xylenes and for the sum of 16 defined alkylbenzene in soil. To meet the requirements of this regulation benzene and 

22 alkylbenzenes were investigated. Styrene was also included in the group of analytes due to its frequent industrial 

use. Method development and optimization were carried out. Linearity, precision and limits of detection were also 

determined. 


261


P02-011 INNOVATIVE USE OF IONIC LIQUIDS FOR CAPILLARY GC 

Sidisky L.M. 1 , Buchanan M. 1 , Stenerson K. 1 , Ferrari R. 2 

1 


2 


Corresponding author e-mail: katherine.stenerson@sial.com 

It is known that room temperature ionic liquids (RTILs) have wide applicability in many areas such as ‘green’ 

solvents, sensitivity enhancers for MS and Maldi reagents. It has also recently been found that unique combinations 

of cations and anions in ionic liquids can be tailored to provide a variety of innovative capillary GC stationary phases 

with new and alternative polarities. For the first time, high polarity phases that can operate at higher temperatures 

are possible. 

The characterisation and application of these ionic liquids as new GC stationary phases will be discussed. 

Characterisation shows in this study that the polarity of these stationary phases can extend beyond what is possible 

with traditional GC stationary phases. These columns showed high separation power, distinctly different selectivity, 

high efficiency and high thermal stability, indicating their applicability as a new type of robust GC stationary phase. 

The potential for using ionic liquid GC phases with air as a carrier gas, making environmental field work a simpler 

task will also be explored. 

Used individually, or in comprehensive GCxGC applications for complex separations, these phases offer a new, 

stable, MS-friendly and highly novel range of GC phases. This presentation will review the current state-of–the-art, 

R&D and applications. 

262 



P02-012 DETERMINATION OF PESTICIDE RESIDUES IN FOODS OF PLANT ORIGIN USING FUSED CORE RP AMIDE HPLC, MS/MS 

AND DISPERSIVE SPE - QUECHERS-METHOD 

Belotti E. 1 , Meni L. 1 , Ruggeri M. 1 , Ferrari R. 2 

1 

Water&Life Entratico (BG)-Italy 

2 


Corresponding author e-mail: roberto.ferrari@sial.com 

Recently, LC/MS/MS methods are proving to be very effective for the analysis of pesticides in food; several methods 

currently exist for the extraction and analyses of multi-residue pesticides from a variety of food matrices. A new 

method, known as the “QuEChERS” (Quick, Easy, Cheap, Effective, Rugged, and Safe) method, has recently 

been introduced and subsequently improved. This method employs dispersive solid phase extraction (SPE) and 

chromatography with mass spectrometric detection (GC-MS or LC/MS/MS) techniques. This method recently 

became European Norm (EN 15662). 

A total number of 190 pesticides divided into 7 mixes have been tested to develop and optimise the separation. For 

the validation of the method, a different representative fruit and plant origin matrix spiked with a mix of 29 pesticides 

have been chosen and used to follow the SANCO/3131/2007 document, ‘Method Validation and quality control 

procedures for pesticides residues analysis in Food and Feeds’. 

Most pesticide separation methods utilise C18 reverse phase HPLC columns. In this poster we describe an interlab 

validation procedure and its results using a embedded polar group (EPG) stationary phase (Ascentis Express) 

RP Amide HPLC column. These columns were found to provide a host of useful benefits that comes from both the 

phase technology and the particle technology. The particles have a solid core and a porous outer layer bonded on 

the surface, resulting in a highly ordered packed column bed that has very significantly less diffusion, with twice the 

efficiency compared to 3µm columns. They also exhibit similar high speed and high efficiency of sub-2 µm particles 

but at a much lower backpressure, making this method useable for conventional HPLC instrumentation and UHPLC 

alike. 


263


P02-013 DETERMINATION OF TRICHLOROANISOLE IN CORK PLANKS BY HS-SPME-GC/MS 

Martínez-Cañas M.A., Yuste-Córdoba F.J., 

Instituto del Corcho, la Madera y el Carbón Vegetal. Junta de Extremadura, Tecnología y Calidad-Spain 

Corresponding author e-mail: mmartinez@iprocor.org 

Cork is produced by the cambium in the outer bark of Quercus suber L. To date, the cork industry and the general 

public have viewed cork mainly in terms of wine-bottling stoppers. 

In the last few decades, the use of cork material has been put in question because of its potential influence on the 

delicate flavour of wine. Cork material has been blamed for imparting off-flavours (cork taint) caused by compounds 

such as 2,4,6-trichloroanisole (TCA) and its halogenated homologues, 2-methylisoborneol, geosmin, 1-octen-3-ol, 

1-octen-3-one, guaiacol, and certain methoxy-alkylpyrazines (1-3). 

A breakthrough was achieved when industry was able to detect TCA in cork lots at parts per trillion (ppt) levels by 

combining three powerful analytical tools: solid-phase microextraction (SPME) (4), capillary chromatography, and 

mass spectrometry under single-ion monitoring (SIM) (5-6). However, raw material (cork planks) never has been 

analyzed in order to prevent detectable TCA levels in cork stoppers. In this way, the aim of our study is to develop a 

method for quantifying TCA in cork planks. 

A linear relationship has been established between TCA/TCA-d5 peak area ratio and TCA concentration, such as 

repeatability and detection limit (Clayton’s method) for TCA. Influence of concentration has been also studied. 

The proposed method has been applied to the determination of TCA in cork planks samples. 

References. 

(1) Amon, J. M.; Vandepeer, J. M.; Simpson, R. F. Wine Ind. J. 1989, 4, 62–69. 

(2) Simpson, R. F. Wine Ind. J. 1990, 5, 286–293. 

(3) Sefton, M. A.; Simpson, R. F. Aust. J. Grape Wine Res. 2005, 11, 226–240. 

(4) Arthur, C. L.; Pawliszyn, J. Anal. Chem. 1990, 62, 2145–2148. 

(5) Evans, T.; Butzke, C.; Ebeler, S. J. Chromatogr., A. 1997, 786, 293–298. 

(6) Herve, E.; Price, S.; Burns, G.; Weber, P. ASEV Annual Meeting, Reno, NV, 1999. 

264 



P02-014 COMPARISON OF STIR BAR SORPTIVE EXTRACTION AND SOLID PHASE EXTRACTION FOR THE DETERMINATION OF 

POLYCYCLIC AROMATIC HYDROCARBONS IN WASTEWATER BY GAS CHROMATOGRAPHY COUPLED TO TANDEM MASS 

SPECTROMETRY 

Barco Bonilla N. 1 , Romero-Gonzalez R. 1 , Plaza-Bolaños P. 1 , Fernández-Moreno J.L. 2 , Garrido-Frenich A. 1 , Martinez Vidal J.L 1 

1 

Almeria University 

2 

Laboratorio Analítico Bioclínico LAB, Almeria 

Corresponding author e-mail: bbm809@ual.es 

Polycyclic aromatic hydrocarbons (PAHs) have been identified in wastewaters (WWs) and 16 of them are included 

in the list of priority pollutants established by the American Environmental Protection Agency (US-EPA). PAHs 

can be associated with the suspended particulate matter (SPM) that can be found in WWs. In this kind of 

multiphase samples, the analysis of both the aqueous phase and the SPM must be considered in order to avoid 

underestimations in the total PAH concentration. Despite solid phase extraction (SPE) is still the most common 

technique for the extraction of pollutants from aqueous samples, the development of miniaturized sample 

preparation methods such as stir bar sorptive extraction (SBSE) has become a dominant trend in Analytical 

Chemistry. Besides, for the determination of PAHs in WWs, SBSE could be suitable for the simultaneous extraction 

of the analytes dissolved in the liquid phase of the sample and sorbed into the SPM. In relation to the determination 

of PAHs in WWs, gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) has been the selected 

approach for the separation, identification, and quantification of these compounds. One of the main drawbacks 

of GC-MS/MS is that mass spectra of isomeric PAHs are essentially the same, and therefore co-elution of these 

particular compounds provokes quantification problems. Thus, the development of a chromatographic method 

capable to resolve these coeluted compounds becomes a challenge in PAH determination. In this study, a 

GC method was developed for the separation of three critical groups of PAHs: benzo[b,k and j]fluoranthenes; 

indeno[1,2,3-cd]pyrene - dibenzo[a,h]anthracene; and benzo[a]anthracene - cyclopenta[cd]pyrene – chrysene. 

For this, a specific capillary column developed for PAHs determination was used. This column presents some 

characteristics such as shorter length and lower film thickness than the conventional columns utilized in PAH 

analysis (e.g. DB-5-ms). Moreover, for the extraction stage, two methodologies based on SPE and SBSE were 

compared for the analysis of 24 PAHs (included in EPA and EU lists) in WWs effluents.It is important to notice 

that SPE was used for the determination of PAHs only in the aqueous phase, whereas SBSE was employed 

for the simultaneous extraction of the aqueous phase and the particulate matter. Since both methods showed 

adequate results, the SBSE-based methodology was eventually selected due to the low solvent consumption and 

the simultaneous analysis of both phases. Validation data for the SBSE methodology were generated, obtaining 

adequate precision values (RSD < 20%). The method showed limits of detection at 0.002-0.100 µg/L and limits 

of quantification from 0.005 µg/L (phenanthrene) to 1.000 µg/L (dibenzopyrenes). Acknowledgments The authors 

gratefully acknowledge Andalusian Regional Government (Regional Ministry of Innovation, Science, and 

Enterprise-FEDER) for financial support (Project Ref. P08-RNM-03892). NBB is grateful for her pre-doctoral grant 

from the aforementioned project. PPB acknowledges for personal funding through Juan de la Cierva Program 

(Spanish Ministry of Science and Innovation-European Social Fund, SMSI-ESF). RRG is also grateful for personal 

funding through Ramón y Cajal Program (SMSI-ESF). 


265


P02-015 VOLATILE COMPOUNDS AS MARKERS OF LA MANCHA D.O. RED WINES ACCORDING TO THE AGEING PERIOD BY GC/MS 

Castro-Vázquez L., Calvo E., Alañón E., Diaz-Maroto M.C., Pérez-Coello M.S. 

University of Castilla la Mancha 

The official Spanish legislation establishes three categories for aged red wines depending on the contact period 

with the oak barrel. This period constitutes a quality factor that clasified the wines in “Crianza”, “Reserva” and “Gran 

Reserva”. So, 6 months of wood contact are necessary in order to consider a red wine as Crianza; 12 months for 

Reserva and at least 16 months for Gran Reserva. 

The structural characteristics of wood (grain, porosity and permeability), chemical composition, oak specie, 

geographical provenance, toasting level and age of the barrel, influence the processes that take place during the 

oxidative ageing of wine in barrels, affecting the composition of wine (Díaz-Plaza, Reyero, Pardo, Alonso, & Salinas, 

2002; Fernández de Simón, Cadahía, & Jalocha, 2003; Fernández de Simón, Hernández, Cadahía, Dueñas, & Estrella, 

2003; Pérez-Prieto, López-Roca, Martínez-Cutillas, Pardo-Mínguez, & Gómez-Plaza, 2002). The variability of factors 

above mentioned influences the aroma composition of red wine aged in oak barrels. 

The objective of this work is to evaluate the evolution of the volatile composition of red wines from La Mancha 

denomination of origin (D.O) in relation with the aging period. For this purpose one hundred and four samples of the 

most utilized grape variety in wine-making from Castilla la-Mancha, Tempranillo, were used. Red wines were aged in 

oak barrel during 0, 3, 6, 8, 10, 12, 15, 24 and 36 months. The great number of samples analyzed allows us verify if 

the real time of contact with the oak barrel, an important quality parameter in Spain, corresponds with the category 

of “Crianza”, “Reserva” and “Gran Reserva” indicated on the sticky label. 

The volatile fraction was separated by adsorption/desorption on preconditioned styrene–divinylbenzene cartridges 

(Lichrolut EN, Merck, 0.5 g of phase) according to the method proposed by Sanchez Palomo et al. 2007. One 

hundred millilitres of wine was passed through the Lichrolut column at a flow rate of 1 ml/min. The column was rinsed 

with 50 ml of pure water to eliminate sugars and other low-molecular-weight polar compounds. The free 

fraction was eluted with 10 ml of dichloromethane. The organic phase collected was concentrated under nitrogen 

stream and analysed by GC/MS. 

The relationship between the concentration of esters, higher alcohols, phenolic compounds, lactones, furanic 

compounds and volatile phenols and the number of months that a red wine had been aging in oak barrels can be 

deduced using Multivariate Analysis. Results obtained demonstrate the essential role played by the concentration 

of eugenol, syringol, β-methyl-γ-octalactone, methylvanillate, vanillin and vanillin derivatives such as acetovanillone, 

during wine aging process. The concentration of several compounds in Tempranillo red wines from D.O. Mancha 

can be used as markers of the wood-wine contact period. This procedure could be considered a very useful tool to 

confirm the number of months that the wine has been in an oak barrel. 

266 



P02-016 ROLE OF GC-APCI-MAXIS MS IN FOOD METABOLOMICS: DETERMINATION OF POLYPHENOLS FROM EXTRA-VIRGIN OLIVE OIL 

García-Villalba R. 1 , Pacchiarotta T. 2 , Carrasco-Pancorbo A. 1 , Segura-Carretero A. 1 , Fernández-Gutiérrez A. 1 , Deelder A.M. 2 , Mayboroda O.A. 2 

1 


2 

Leiden University Medical Center 

Corresponding author e-mail: rociogv@ugr.es 

We describe the first analytical application involving solid-phase extraction and gas chromatography coupled 

to atmospheric pressure chemical ionization-MaXis mass spectrometry (GC-APCI-MaXis MS) for achieving the 

chemical characterization of the phenolic fraction of extra-virgin olive oils. The fact is quite relevant since only EI 

and CI (both operating under vacuum condition) have been used as ionization sources when GC-MS was applied for 

the analysis of phenols so far. Both chromatographic and MS parameters were optimized in order to maximize the 

number of phenolic compounds detected and the sensitivity of their determination. The BSTFA derivatized-phenols 

where injected in the instrument and separated in a HP-5-MS column (30 m, 0.25 mm ID, 0.25 mm film thickness). 

The use of our previous knowledge, commercially available standards and isolated standards (by semi-preparative 

HPLC), together with the capabilities of the analyzer used (MaXis-new generation of TOF instrument), gave us the 

opportunity to achieve a comprehensive characterization of the olive oil phenolic profile. Moreover, a complete 

validation of the method was carried out considering the specificity, linearity, sensitivity, precision and accuracy. The 

detection limits were found ranging between 0.50 and 4.20 ppm, for pinoresinol and homovanillic acid, respectively. 

Acceptable levels of precision were obtained for the developed method in terms of repeatability since in all cases 

RSDs calculated were lower than 6.07%. The accuracy ranged from 95.4% to 101.5%. The optima method was used 

to carry out an exploratory analysis of 25 samples belonging to 5 different olive oil varieties (from Spain and Italy). 

The data obtained were processed using statistic tools (mainly PCA and PLS) in order to discriminate between the 

samples and look for some varietal markers. 

Figure caption. Base Peak Chromatogram (BPC) of the Diol-SPE extract obtained from an extra-VOO obtained from 

a mixture between Arbequina and Picual oils under the optima GC-APCI-MaXis MS conditions. Elution windows of 

different families belonging to the phenolic fraction of EVOO are shown. Individual peaks with name in blue were 

identified with commercial standards; those with name in red were identified by using the isolated phenolic fraction 

(with semi-preparative HPLC); and those in purple were identified keeping in mind our previous knowledge about the 

fraction. In grey we can observe peaks with relevant intensity which were not fully identified. 


267

P03 LIQUID 


POSTER 

SESSIONS

POSTER SESSION 03 - LIQUID CHROMATOGRAPHY 

P03-001 QUANTITATIVE DETECTION OF CARCINOID TUMOR MARKERS BY HPLC–MS 

Jambor E., Patko B., Bona A., Maasz G., Mark L, Ohmacht R, Chemistry-Hungary 

University of Pecs-Hungary 

Corresponding author e-mail: eva.jambor@yahoo.com 

Carcinoid tumours are APUD-omas (characterised by amine precursor uptake and decarboxylation) that arise from 

enterochromaffin cells. Foregut carcinoids arise from the respiratory tract, pancreas, stomach and duodenum; 

midgut carcinoids from ileum and appendix; hindgut carcinoids from left colon and rectum. The tumours appear 

most frequently in midgut (about 70% of cases). Serotonin plays an important role in the aetiology of some symptoms 

of the carcinoid syndrome. Increase in the urinary excretion of 5-hydroxytryptamine (5HT; serotonin) and of its major 

metabolite 5-hydroxyindole-3-acetic acid (5HIAA) is one of the biochemical features of the carcinoid syndrome. 

Profiling of the urine indoles serotonin, 5-hydroxyindoleacetic acid (5-HIAA), vanillylmandelic acid (VMA), and 

homovanillic acid (HVA) are useful in the diagnosis and follow-up of patients with carcinoid tumors. We successfully 

detected these indoles in urine of healthy controls and patients with carcinoid tumors by 

reversed-phase high-performance liquid-chromatography (RP HPLC) coupled to quadrupol mass spectrometry 

detection (MS). Chromatographic separation on a C18 column provides sufficient specificity, allows selective 

detection of VMA, HVA and 5-HIAA. Newly developed core-shell type silica based reversed phase columns are well 

suited for rapid, high sensitivity, high reproducibility gradient separation of the above mentioned indol compounds in 

acidic water-acetonitrile eluents. 


269


P03-002 SIMULTANEOUS DETERMINATION OF ENALAPRIL AND LERCANIDIPINE BY HPLC-DAD TECHNIQUE IN PHARMACEUTICAL 

DOSAGE FORMS 

Gumustas M. 1 , Sanli S. 2 , Sanli, N. 2 , Ozkan S.A. 1 

1 


2 

Hitit University-Turkey 

Enalapril (ENA) and Lercanidipine (LER), a combination medicine, is often prescribed for the treatment of high blood 

pressure. An HPLC-DAD method is presented for the simultaneous determination of ENA and LER. In this method; a 

reversed-phase column (X-Terra RP-18 (250 x 4.60 mm ID x 5m) with the mobile phase assayed were 

methanol–water (55:45 ;v/v), containing 15 mM phosphoric acid. The pH of the mobile phase was adjusted at 2.7 

by addition of sodium hydroxide. 1.2 ml/min flow rate was used to separate both compounds with a detection of 

215, 240 and 210 nm for ENA, LER and IS, respectively. The chromatographic separation was performed at 40 oC. 

Ramipril was chosen as the internal standard (IS) because it showed a shorter retention time with better peak shapes 

and better resolution, compared to other potential internal standards. Using these conditions, the retention times 

were obtained as 2.76 min for ENA, 3.91 min for IS and 11.15 min for LER. 

The proposed methods have been extensively validated. System suitability tests were also carried out. Linearity was 

obtained in the concentration range of 0.50-20 mg mL-1 for ENA and LER. In order to demonstrate the validity and 

applicability of the proposed HPLC method, recovery tests were carried. The results of recovery tests are 100.236 % 

for ENA and 100.086 % for LER. High percentage recovery shows that the method is free from the interferences of 

the commonly used excipients and additives in the formulations of drugs. 

The present HPLC study purposes a rapid, simple, sufficiently precise and accurate method for the simultaneous 

determination of ENA and LER, in raw material and pharmaceutical formulations. The proposed method is suitable 

for quality control laboratories, where economy and time are essential. 

270 



P03-003 DEVELOPMENT AND VALIDATION OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) METHOD TO DETERMINE 

VANCOMYCIN IN PLASMA AND PULMONARY TISSUE 

Guerrero L., Martínez-Olondris P., Rigol M., Esquinas C., Piñer R., Esperatti M., Luque N., Liapikou M., Li Bassi G., Torres A., Soy D. 

Hospital Clinic Barcelona 

Corresponding author e-mail: lr.guerrero1@gmail.com 

BACKGROUND AND OBJECTIVE: Vancomycin is a glicopeptide antibiotic used in the treatment of serious 

gram-positive infections, mainly due to penicillin-resistant, staphylococci and enterococci. Its distribution to 

deep tissues, such as pulmonary segments, cardiac vegetations or osteoarticular material is quite variable and 

thus, microbiological failure could be expected. The aim of this study is to develop a high-performance liquid 

chromatographic (HPLC) assay to quantify vancomycin in plasma and lung tissue, obtained from a model of 

pneumonia in mechanically ventilated piglets. DESIGN: Plasma vancomycin controls were spiked with ceftazidime 

as internal standard. Afterwards they were precipitated with HClO4 3% (1/1.v/v). Tissue samples (0.5 g) were 

conditioned with 350µL of TBE and spiked with the internal standard (50 µg/mL). These specimens were 

homogenized, extracted and precipitated with HClO4 3%. Later, plasma and tissue samples were centrifuged and 

100µL of the supernatant were injected into the chromatographic system. The stationary phase was a silica based 

column Symmetry300TM® C18 (150*4.6 mm) with pre-column. The mobile phase consisted of 20% ultrafiltered water 

and 80% of (A) 75 mM sodium acetate buffer (pH=3) with (B) acetonitrile (92% / 8%;v/v). Isocratic flow rate was set 

at 0.8 mL/min and 0.7 mL/min for plasma and tissue samples, respectively. UV absorbance detection was set at 230 

nm. SETTING: Pharmacy Service, Hospital Clínic of Barcelona. MAIN OUTCOME MEASURES: Validation criteria: 

Accuracy, intra and inter precision, recovery, linearity. RESULTS: Vancomycin in plasma show good linearity results 

within 1.56-100µg/mL (r2= 0.99). Accuracy was 93.40-105.50%. Intra and inter precision were 2.55-5.30% and 

3.69-6.33%, respectively. Recovery resulted to be around 93.40%. For vancomycin in pulmonary tissue, the assay 

was lineal within 3.13-100µg/mL (r2= 0.99). Accuracy showed values from 90.78 to 106.49%. Intra and inter precision 

were 4.12-7.02% and 5.13-8.51%, respectively. In this case, recovery was close to 91.30%. Limit of detection 

for plasma and tissue were 0.80 µg/mL and 1.56 µg/mL, respectively. Lower limit of quantitation for plasma and 

tissue were 1.56 µg/mL and 3.13 µg/mL. CONCLUSION: Both HPLC assays to quantify vancomycin in plasma and 

pulmonary tissue are fast, easy and cheap. These methods could be helpful to develop further pharmacokinetic 

studies of vancomycin penetration in pulmonary tissue. Conflict of interest: CIBER de Enfermedades Respiratorias 

06/06/0028 and Fondo de Investigaciones Sanitarias (FIS) Grant: FIS PI070419. 


271


P03-004 ADVANTAGES OF MCROEMULSIONS AS MOBILE PHASES IN HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 

Pashkova E.B., Pirogov A.V., Shpigun O.A., 


Corresponding author e-mail: pirogov@analyt.chem.msu.ru 

The technique of microemulsion liquid chromatography (MELC) was first reported in 1992 and has been successfully 

applied for the analysis of pharmaceuticals in dosage forms and biological liquids since then. Nevertheless, such 

important questions as stability of the described mobile phases, ways to improve selectivity of the separation and 

features of sample pretreatment have never been touched upon. In current work different ways of the synthesis of 

the microemulsions were compared and the technique, allowing to obtain microemulsion, stable for two weeks at 

room temperature was chosen. Influence of the microemulsion composition on the eluting power of the mobile phase 

was investigated. An addition of the second surfactant was suggested as an additional way to affect selectivity of 

the separation. A methylene selectivity in MELC mode was investigated. It was found that in contrast to 

reversed-phase mode the retention dependence fit linear for all microemulsions. A compatibility of microemulsions 

with different types of detectors was studied. It was found that in case of fluorescent detection sensitivity may be 

increased up to three orders in comparison with RP-HPLC. It was also demonstrated that using microemulsions as 

a reaction media for on-line derivatization allowed to accelerate the reaction greatly. Advantages of microemulsions 

as extragents for the sample pretreatment were shown. Different types of objects with complex matrices (ointments, 

cosmetic products, food, biological liquids) were analyzed using the suggested technique. In comparison with other 

techniques (liquid-liquid, solid-phase or Soxhlet extractions) simple dissolvation of samples with the microemulsion 

gave better recoveries and was much more rapid and simple. A number of different applications of MELC for the 

analysis of pharmaceuticals, cosmetic products and food were suggested depending on the investigated features. 

Microemulsions of the L2 type (water-in-oil) were successfully used for the determination of highly hydrophobic 

compounds in complex matrices. 

272 



P03-005 DEVELOPMENT OF A STABILITY INDICATING HPLC METHOD FOR MELOXICAM, ITS RELATED SUBSTANCE 2-AMINO-5-METHYL 

THIAZOLE AND FORMULATORY ADJUVANTS IN AN AMPOULE DOSAGE FORM USING RETENTION MODELING 

Mokhtar H., Elian M. 

Medical union Pharmaceuticals Co.-Egypt 

Corresponding author e-mail: hatemmokhtar76@hotmail.com 

Retention Models are equations that describe the relationship between retention of any compound and a selected 

Chromatographic condition(s) e.g. % organic solvent in Mobile phase, column temperature , pH ,….etc. The 

use of retention models combined with proper experimental design using few initial experiments enables rapid 

development of robust HPLC methods by prediction of optimum values of the optimized conditions. This approach 

is used to develop a Method for separation of Meloxicam, Nicotinamide, benzyl Alcohol and the Meloxicam related 

substance, 2-amino-5-methyl Thiazole as well as other possible degradation products in a commercial Ampoule 

dosage form, Mobitil ampoules. The Column used for this work was Inertsil ODS-4 150 x 4.6 mm , The method was 

developed first using 4 experimental systems to simultaneously optimize % Organic solvent in mobile phase and 

column temperature then the selected optimum is further optimized for addition of Ion-pairing agent with another 3 

Experimental Systems using suggested hyperbolic equation as a retention model for ion pairing concentration. and 

the final method conditions are then established. Meloxicam retention time Predictions made by the suggested ion 

pairing concentration retention model was found to highly correlate with actual retention times with a correlation 

coefficient of 0.9988. The final method has a mobile phase composed of Organic solvent : Buffer ( 25:75), where 

organic solvent was Methanol : iso-Propanol (65:10) and Buffer was a solution of 0.5 g/l di-Ammonium Hydrogen 

Phosphate , 0.9mM Octane Sodium Sulfonate and 1.8 mM di-Sodium Hydrogen Phosphate , pH of the buffer is 

adjusted to 7, Column temperature of 40°C,flow rate of 1.5ml/min. and UV detection wavelength of 254 nm . The 

resulting System is convenient for both assay and related substances purposes. The method is validated for assay 

of Meloxicam and formula adjuvants ( Nicotinamide and benzyl alcohol). Good validation results are obtained and 

presented. Linearity is obtained for the related substance, 2-Amino-5-Methyl Thiazole with a correlation coefficient 

of 0.9999097. Limits of Quantitation and Detection were calculated to be 0.0272µg/ml and 0.009µg/ml respectively. 


273


P03-006 HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY: RETENTION STUDIES ON SUGAR BASED POLAR STATIONARY 

PHASES 

Schuster G., Hüser T., Lindner W. 


Corresponding author e-mail: georg.schuster@univie.ac.at 

In the early 1990s, aqueous normal phase chromatography was given a new name by Alpert [1]: Hydrophilic 

Interaction Liquid Chromatography (HILIC). While in organic normal phase mode, retention of analytes is 

predominantly governed by surface adsorption phenomena, in HILIC mode retention was originally thought to be 

achieved by partitioning mechanism. A water rich layer is partially immobilized on the surface of polar stationary 

phases by using mostly nonprotic organic solvents with a small amount of water and buffer components. The 

analytes then partition between this water-rich “quasi stationary phase” and the organic bulk eluent [2]. However, 

due to the development of new polar stationary phases in combination with HILIC mode (diol phases, anion/ 

cation exchangers, zwitterionic phases), retention is rather achieved as a combination of partition phenomena and 

other binding increments thus leading to a kind of mixed-mode retention mechanism [3]. In 2009, Sun and Yuan 

[4] presented the first enantioseparations under normal phase mode using mono- and disaccharides as chiral 

selectors bonded to silica gel. Inspired by their contribution we developed and investigated several saccharide 

based polar stationary phases mostly in the HILIC mode as analogs to diol bonded phases. The retention behaviour 

of various groups of Test analytes (acids, bases, purines, xanthines, polyphenoles, hydroxyacids, etc.) as well 

as the pH dependency of the HILIC type mobile phase composition was studied and compared to the retention 

characteristics of the more common and commercially available HILIC phases. The results will be discussed 

by the validation of main chromatographic parameters (retention, efficiency, asymmetry factor, etc.) and will be 

compared to gain retention mechanism insights. It was noticeable that the amine backbone of our phases, which 

diol phases are lacking, exhibits quite an apparent influence on the separation of purines and xanthines. Thus, it 

can be an advantage compared to diol phases for various separation applications. In this context it became evident 

that for identical mobile phase conditions the selectivity patterns of the compared stationary phases changed 

to some extend, which is a clear indication of retention increments caused by additional adsorption phenomena. 

Mechanistically one clearly deals with mixed modal acting “stationary phases”, which becomes more and more 

common sense for the interpretation of retention and selectivity data generated by HILIC systems. [1] Alpert, A.J., 

J. Chromatogr. A 1990, 499, 177 - 196 [2] Hemström, K. I., J.Sep.Sci. 2006, 29, 1784 - 1821 [3] Lämmerhofer, M., J. 

Sep. Sci. 2010, 33, 679 – 680; [4] Sun, W. Z., Yuan, L. M., J. Liq. Chromatogr. 2009, 32, 553 - 559 

274 



P03-007 DETERMINATION OF NITROSAMINES IN WATER AT THE NG/L LEVELS BY LIQUID CHROMATOGRAPHY COUPLED TO TANDEM 


Ripollés C., Pitarch E., Sancho J.V., López F.J., Hernández F. 

Research Institute for Pesticides and Water, University Jaume I, Castelló de la Plana 

Corresponding author e-mail: cripolle@qfa.uji.es 

DETERMINATION OF NITROSAMINES IN WATER AT THE ng/L LEVELS BY LIQUID CHROMATOGRAPHY COUPLED 

TO TANDEM MASS SPECTROMETRY 

N-nitrosamines are considered as carcinogenic to humans. They can enter drinking water supplies mainly during 

disinfection processes. In particular, N-nitrosodimethylamine (NDMA) formation in drinking water or wastewater is 

associated with the reaction of some organic nitrogen precursors with chloramine, being a potential disinfection 

byproduct from chloramines or, under certain conditions, from chlorine disinfection. The use of ozone as disinfectant 

product can also result in the formation of NDMA at low concentration levels. 

Because of the presence of these N-nitroso compounds in drinking water involves a significant risk for human health, 

strict regulations on the presence of NDMA and other volatile nitrosamines in drinking water have been adopted. 

Many countries have established a regulatory level of 10 ng/L for NDMA in drinking water. Therefore, there is a 

need to determine nitrosamines at the low nanogram per liter range in water samples. This determination is difficult 

because enrichment of very polar and uncharged compounds from water and selective detection of small molecules 

are both problematic. In recent years, sensitive methods based on solid phase extraction (SPE) with carbonaceous 

adsorbents and gas chromatography-mass spectrometry (GC-MS) have been developed. However, these GC-based 

methods are not the most suitable to analyze thermally unstable nitrosamines. 

In this work, we have developed a sensitive method for detecting and quantifying eight N-nitrosamines (NDMA, 

NMor, NMEA, NPyr, NDEA, NPip, NDPA and NDBA) in drinking water based on liquid chromatography coupled to 

tandem mass spectrometry, using atmospheric pressure chemical ionization (APCI) in positive mode, with a triple 

quadrupole analyzer. The simultaneous acquisition of two MS/MS transitions in selected reaction monitoring mode 

(SRM) for each compound together with the evaluation of their relative intensity, led to the reliable quantification 

and identification in water at ppt levels. Accurate mass and molecular formula of the product ions selected were 

confirmed by UHPLC-(Q)TOF MS analysis of reference standards. 

Prior to the measurement by LC-MS/MS, a preconcentration step by off-line SPE using coconut charcoal EPA 

521 cartridges (6 mL, 2 g), passing 500 mL of water sample, was necessary to meet regulation requirements. For 

accurate quantification, two isotope labelled nitrosamines (NDMA-d6 and NDPA-d14) were added as surrogate 

standards to the samples. 

The optimized method has been validated at two concentration levels (10 and 100 ng/L) in drinking water samples, 

obtaining satisfactory accuracy (recoveries between 90 and 120%), precision (RSD < 20%) and linearity (from 1 to 

100 mg/L, r > 0.99). Limits of detection were found to be in the range of 1 to 5 ng/L. The developed methodology has 

been applied to water samples from a drinking water treatment plant (raw water, prechlorinated, activated carbon 

and sand filtered, after chlorination and ozonation processes). 


275


P03-008 RP-CHIRAL LC-METHOD DEVELOPMENT WITH THE QUALITY BY DESIGN PRINCIPLES 

Vogel-da Silva F. 1 , Galushko S. 2 

1 

Novartis Pharma AG-Switzerland 

2 

ChromSword, Method Development-Germany 

Corresponding author e-mail: galushko@chromsword.de 

HPLC method development is still considered to be one of the crucial bottlenecks that impede productivity 

in analytical laboratories. The Quality by Design (QbD) approach for method development requires additional 

collecting knowledge about a sample to provide robust methods (sample profiling). In this presentation we explore 

an integrated solution based on ChromSwordAuto method development software and HPLC instrumentation. In 

our approach the system was specified to start with a user defined number of initial screening experiments with 

the following rapid optimization steps exploring the entire design space through software intelligence to find the 

best analysis conditions. Screening of different columns, solvents and buffers, instrument parameters as well as 

rapid optimization, fine tuning, robustness studies, and documentation have been implemented in one platform. 

The system was used effectively for chiral LC method development in the reversed-phase mode with the Quality 

by Design (QbD) principles. Our experiments have shown that the use of automatic procedures implemented 

in ChromSwordAuto® software can significantly reduce method development time. Results of separation of 

enantiomers on different RP chiral columns and mobile phases will be shown. 

276 



P03-009 DEVELOPMENT AND VALIDATION OF MICELLAR LIQUID CHROMATOGRAPHIC METHODS FOR THE DETERMINATION OF 

ANTIBIOTICS IN DIFFERENT MATRICES 

Rambla-Alegre M., Esteve-Romero J., Carda-Broch S. 


Corresponding author e-mail: mrambla@qfa.uji.es 

Antibiotics are the most important bioactive and chemotherapeutic compounds to be produced by microbiological 

synthesis and they have proven their worth in a variety of fields, such as medicinal chemistry, agriculture and the 

food industry. This growing interest in antibiotics has, at the same time, given rise to a high degree of productivity 

in the field of analytical applications. Therefore it is necessary to develop chromatographic procedures which are 

capable of determining various drugs simultaneously in the shortest possible time. Micellar liquid chromatography 

(MLC) is a reversed-phase technique with attractive advantages over conventional HPLC as far as sample 

preparation, selectivity and versatility are concerned. Its main advantage is that samples can be injected directly into 

the chromatographic system without the need for any previous step. The present work draws on the recent results of 

the authors’ own research to report on the column and mobile phase conditions for the various types of antibiotics 

(penicillins, quinolones and sulfonamides) in different matrices (pharmaceuticals, biological fluids and food). The 

MLC procedures are also compared with classical HPLC methods that use aqueous-organic mobile phases. 


277


P03-010 CATECHOLAMINE DETERMINATION IN PHEOCHROMOCITOMA PATIENTS BY LIQUID CHROMATOGRAPHY 

Esteve-Romero J. 1 , Carda-Broch S. 1 , Rambla-Alegre M. 1 , Bose D. 2 , Durgbanshi A. 2 , Agrawal N. 2 , Raviolo M.A. 1 , Ochoa-Aranda E. 3 

1 


2 


3 

Hospital Provincial de Castellón 

Catecholamines are important natural molecules containing a catechol ring in their structure, which act as 

neurotransmitters or hormones. Plasma level determination of catecholamines and their metabolites is necessary 

in order to evaluate neuroendocrine disorders. Recently, the measurement of plasma free metanephrine and free 

normetanephrine has been advocated as been more clinically sensitive than urinary free catecholamines and 

metanephrines. A procedure was developed to determine epinephrine and its two naturally occurring derivatives 

(metanephrine and normetanephrine) in serum samples with direct injection. The separation was achieved in less 

than 15 min using a micellar mobile phase of 50 mM sodium dodecyl sulphate and 10% butanol buffered at pH 3 

using a C18 column. Ultraviolet and electrochemical detection were employed. The main advantage of the method 

is the direct injection of the serum sample without any other pretreatment than filtration. The procedure was linear, 

with detection limits (ng/mL) in the 0.25-1.7 range. Repeatability and intermediate precision values were below 1.8%. 

Recoveries agreed with the concentration added. 

278 



P03-011 DETERMINATION OF BIOGENIC AMINES IN WINE 

Esteve-Romero J. 1 , Carda-Broch S. 1 , Rambla-Alegre M. 1 , Peris-Vicente J. 1 , Chin-Chen M.L. 1 , Bose D. 2 , Raviolo M.A. 1 , Agrawal N. 2 

1 


2 


Biogenic amines (BAs) can be formed and degraded as a result of normal metabolic activity in animals, plants and 

micro-organisms. Lactic acid bacteria are the responsible to transform amino acids in biogenic amines, growing 

in acidic fruit juice and wine, being involved in the decarboxylation process. In this work ompounds such as BAs, 

tyramine and tryptamine formed from decarboxilation of tyrosine and tryptophan have been studied in several types 

of wines samples after direct injection using pulse amperometric detection. Hybrid micellar mobile phase of sodium 

dodecyl sulfate (SDS) and propanol at pH 3 in a C18 column were used in the analysis. Results demonstrated that 

the best mobile phase was 0.15 M SDS-5% propanol- pH 3 using a C18 column. Method was validated, taking 

into account selectivity, linearity (0.1-10 μg/mL), repeatability and intermediate precision (RSD


P03-012 MICELLAR LIQUID CHROMATOGRAPHY DETERMINATION OF QUINOLONES IN FOOD SAMPLES WITH FLUORESCENCE 

DETECTION 

Rambla-Alegre M., Collado-Sánchez M.A., Esteve-Romero J., Carda-Broch S. 



Four quinolones (flumequine, marbofloxacin, danofloxacin and difloxacin) were separated and quantified in different 

milk and egg samples by liquid chromatography using a micellar mobile phase. Quinolones and fluoroquinolones 

are synthetic antibiotics whose action is based on their anti-DNA activity. These antibiotics are normally used to 

prevent diseases in animals such as cows and chickens. Lately, this practice could imply drug residues persisting 

in animal tissues. Consequently, the European Union has established maximum permitted levels of these 

antibiotics. Determination and separation were achieved by a sensitive and precise chromatographic procedure 

with no pretreatment step in a C18 column using a micellar mobile phase with sodium dodecyl sulfate, propanol 

and triethylamine at pH 3, with retention times below 18 min. Adequate limits of detection and quantification were 

obtained using fluorescence detection. This method was validated in accordance with European Union Decision 

2002/657/EC in terms of linearity, intraday and interday precision and accuracy, and robustness. Good recovery 

percentages were obtained in the analyses of the milk and egg samples. The results show that the validated 

procedure is suitable for routine analyses to control food quality. 

280 



P03-013 VALIDATION OF A LIQUID CHROMATOGRAPHIC PROCEDURE FOR THE DETERMINATION OF FIVE QUINOLONES IN FISH 

ACCORDING WITH REGULATION 2002/657/EC 

Rambla-Alegre M. 1 , Peris-Vicente J. 2 , Esteve-Romero J. 1 , Carda-Broch S. 2 

1 


2 

Química Analítica, QFA, Universitat Jaume I, Castelló, Spain 


Quinolones are used mainly in the treatment of human and veterinary diseases, and have been proved very useful 

in preventing diseases in animals. However, these practices imply drug residues persisting in the edible tissue 

derived from treated animals and, therefore, maximum permitted levels have been set for most of them. A simple 

and sensitive method was optimized and validated for the analysis of five quinolones (oxolinic acid, flumequine, 

enrofloxacin, difloxacin and sarafloxacin) in seven different fish muscles (gilthead, salmon, trout, sea bass, mussel, 

prawn and turbot). Only homogenization, extraction, dilution and filtration were required before sample injection, and 

no organic solvent was used in the pretreatment step. Separation was performed in a C18 column using as mobile 

phase an aqueous solution of sodium dodecyl sulfate, propanol and triethylamine at pH 3. The method was fully 

validated in accordance with European Union Decision 2002/657/EC. Selectivity, linearity, decision limit, detection 

capability, limits of detection and quantification, recoveries and robustness were studied. Finally, the developed 

micellar method was successfully applied to quantitatively determine quinolones in the muscle of medicated fishes. 


281


P03-014 STABILITY STUDIES OF 3’-AZIDO-3’-DEOXY-5’-O-OXALYL-N-LEUCINETHYMIDINE IN AQUEOUS SOLUTIONS AND 

SIMULATED GASTRIC AND INTESTINAL FLUID 

Raviolo M.A. 1 , Esteve-Romero J. 2 , Briñón M.C. 1 , Carda-Broch S. 2 , Rambla-Alegre M. 2 , Bose D. 3 

1 

Universidad Nacional Córdoba 

2 


3 


Corresponding author e-mail: moni_raviolo@yahoo.com.ar 

Novel derivatives of zidovudine (AZT) have developed, by association with different amino acids in order to develop 

antiviral agents (anti-HIV) with better properties. AZT-Leu was selected to study its stability in different media with 

the purpose to test the drug in different compartments of the body, using the following matrices: simulated gastric 

fluid (pH 1.2), simulated intestinal fluid (pH 7.4) and pH buffer at 1.2 and 7.4. Conventional HPLC (simple matrix) and 

micellar liquid chromatography (MLC) for complex matrices were used as analytical methods. The disappearance of 

reagents and the appearance of the only degradation product (AZT), were analyzed for each was the only product of 

degradation were analyzed for each one study. Both chromatographic techniques were properly validated. For HPLC 

the retention time (tr) were: AZT-Leu 19.3 min and AZT tr: 5.3 min. In CLM the tr were: AZT-Leu 9.5 min and AZT tr: 

3.9 min. The half-times (t1/2) of AZT-Leu in the different media were: simulated gastric fluid (pH 1.2): 14.6 h; buffer 

pH 1.2: 25.5 h; simulated intestinal fluid (pH 7.4): 2.35 min and buffer pH 7.4: 76.2 min. The degradation of AZT-Leu 

in all cases follows a pseudo-first-order kinetics. The life time varied depending on the medium and clearly reflects 

the influence of pH, it being more stable in acid pH than pH 7.4. In turn, in this pH the complex matrices (containing 

pepsin and pancreatin), have greater influence on the stability of the compound. 

282 



P03-015 ANALYSIS OF HYDROXYTYROSOL IN OLIVE EXTRACT SAMPLES BY LIQUID CHROMATOGRAPHY USING A SURFACTANT- 

MEDIATED MOBILE PHASE 

M. Rambla-Alegre M. 1 , Marco-Peiró S. 1 , Peris-Vicente J. 1 , Beltran-Martinavarro B. 1 , Collado-Sanchez M.A. 1 , Carda-Broch S. 1 

Esteve-Romero J. 1 , Bose D. 2 

1 


2 



Hydroxytyrosol (3,4-dihidroxyphenil ethanol) has a potential interest for the natural food additives, pharmaceutics 

and cosmetics markets, all of which are currently highly receptive to products of a natural origin. The high added 

value of hydroxytyrosol is due to not only its important bioactive properties, but also its antioxidant action on the 

human organism. A micellar liquid chromatography method to determine hydroxytyrosol in olive extract samples 

is described. The resolution from the matrix was performed using a Kromasil C18 column and a micellar mobile 

phase of sodium dodecyl sulfate buffered at pH 7. Detection was performed in the UV region. Samples were directly 

injected into the chromatographic system after dilution with 0.05 M SDS. Method validation was performed following 

the U.S. Food and Drug Administration guideline. The main analytical parameters studied were: linearity 

(0.06 - 250 μg/mL; r2 = 0.999), LOQ and LOD, and intra-and inter-day precision (RSD (%)


P03-016 TERNARY ISOCRATIC MOBILE PHASE OPTIMIZATION USING A PEAK WIDTH PREDICTION MODEL 

Kawabe T. 1 , Tomitsuka T. 2 , Takemura A. 2 , Kishi N. 2 , Toyo’oka T. 1 

1 

School of Pharmaceutical Sciences, University of Shizuoka 

2 

Daiichi Sankyo Co., Ltd., Analytical & Quality Evaluation Research Laboratories 

Corresponding author e-mail: kawabe.takefumi.vr@daiichisankyo.co.jp 

Recently, the quality guidelines in the International Conference on Harmonization (ICH) show a way of thinking 

about Quality by Design (QbD) and Design Space which affect the method development for quality control of 

pharmaceutical compounds. As for the QbD approach on the liquid chromatography (LC) method development, we 

often use chemometrics for the systematic study instead of a try and error approach. Chemometrics, especially for 

the design of experiment (DOE), shows a suitable data acquisition parameter and the result of analysis provides the 

design space of separation. However, a regression model regarding an optimization factor is nessesary to estimate 

using the DOE because the DOE is normally generated based on multivariate analysis. 

Pharmaceutical impurities are usually a complex mixture of compounds which show widely different retention, 

including similar retention in a reversed-phase LC. From the viewpoint of quality control, it is desirable to separate 

peaks as much as possible. The solvent effect between acetonitrile and methanol sometimes provides a successful 

separation. Therefore, a prediction system of retention behavior against mixed solvents is necessary to find the 

appropriate LC condition. 

A resolution is one of the best indexes on separation. The equation of resolution is composed of the retention time 

(tR) and the peak width of half height (w0.5). Regarding the model of tR on ternary isocratic mode, which uses 

acetonitrile, methanol and pH-controlled buffer, it is well known that multiple regression gives a good correlation 

between the natural logarithm transformed tR and the quadratic equation of solvent strength. On the other hand, 

regarding the model of w0.5, it does not give good correlation like the model of tR. The establishment of an accurate 

prediction model for the w0.5 seems to be an important issue to obtain good prediction results of resolution, 

because calculation using the w0.5 value with errors generates a much larger error on the predicted resolution value. 

The model of w0.5 on ternary isocratic mode was verified by observed retention behavior of several pharmaceutical 

compounds, which indicate different structures. Statistical analysis in this study was performed by JMP® ver. 

8.0.1 (SAS Institute, Tokyo, Japan). The w0.5 of all pharmaceuticals showed correlation with the tR, but not with 

the ternary isocratic solvent strength. The results suggest that the solvent strength affects the tR, and the w0.5 

correlates to that tR. Therefore, the w0.5 was predictable by the tR on the ternary solvent strength change. By this 

study, the resolution could be predicted accurately based on both predicted results of tR and w0.5. A change in 

predicted resolution against the ternary solvent strength was indicated on a resolution map, which is the response 

surface generated by an artificial neural network. This resolution map could indicate the design space of separation. 

284 



P03-017 RP-HPLC DETERMINATION OF HYDROPHOBIC PROPERTIES OF NOVEL SUBSTITUTED BENZOTHIAZOLE DERIVATIVES 

Oktabec Z. 1 , Imramovsky A. 2 , Pejchal V. 2 , Jampilek J. 1 

1 

Zentiva, k. s./ Faculty of Pharmacy, Charles University, Development-Czech Republic 

2 

Faculty of Chemistry and Chemical Technology, University of Pardubice 

Corresponding author e-mail: zbynek.oktabec@zentiva.cz 

Various substituted benzoxazoles and benzothiazoles have shown a wide range of biological activities [1,2]. Drugs 

most frequently cross biological barriers through passive transport, which strongly depends on their lipophilicity. 

Therefore log k data characterizing the hydrophobicity of the studied compounds are presented, which is an 

important factor in membrane permeability [3]. Substituted (S)-1-[(R)-1-(6-fluoro-2,3-dihydrobenzo[d]thiazol-2-yl) 

ethylcarbamoyl]-2-methylpropylcarbamates were designed as potential antimicrobial agents. Eleven compounds 

substituted with an aliphatic chain moiety in the carbamate part of the molecule were analysed using the RP-HPLC 

method for the lipophilicity measurement. The procedure was performed under isocratic conditions with methanol 

as an organic modifier in the mobile phase using an end-capped non-polar C18 stationary RP column. In the present 

study the correlation between the RP-HPLC retention parameter log k and log P data calculated in various ways is 

shown, and the relationships between the lipophilicity and the chemical structure of the studied compounds are 

discussed. The distributive parameters of various substituents are listed for the mentioned studied compounds. 

The determined distributive parameters of substituents can be used for describing relationships between the 

physico-chemical properties and biological activity of prepared benzothiazole derivatives. [1] Vinsova J, Cermakova 

K, Tomeckova A, Ceckova M, Jampilek J, Cermak P, Kunes J, Dolezal M, Staud F. Synthesis and antimicrobial 

evaluation of new 2-substituted 5,7-di-tert-butylbenzoxazoles. Bioorg Med Chem 2006;14:5850-5865. [2] Rana A, 

Siddiqui N, Khan SA. Benzothiazoles: A new profile of biological activities. Indian J Pharm Sci 2007;69:10-17. [3] 

Kerns EH, Li D. Drug-like properties: Concept, structure design and methods. Elsevier: San Diego, CA, USA, 2008. 


285


P03-018 HPLC EVALUATION OF THE FUROSINE CONTENT IN INFANT FORMULAS DURING THEIR SHELF-LIFE 

Salcedo J. 1 , Lacomba R. 1 , Alegría A. 1 , Barberá R. 1 , Vasallo I. 2 , Lagarda M.J. 1 , 

1 


2 


Corresponding author e-mail: jaime.salcedo@uv.es 

INTRODUCTION 

Furosine is an indicator of the first stages of the Maillard reaction generated during the acid hydrolysis of Amadori 

compounds (lactulosyl-lysine)1. Its determination has been widely used to control protein quality in infant formulas 

(IFs) 2. 

AIM 

To evaluate the influence of storage Maillard reaction by furosine determination in starter and follow-up IFs stored at 

room temperature, using HPLC-UV. 


Two commercial powder IFs (IF-A starter, IF-B follow-up) were analyzed every 2 months during their shelf-life 

(IF-A 18 months, IF-B 24 months), stored at room temperature. The composition of IF-A (g/100g) was: protein 10.2, 

carbohydrates 56.9, fat 27.5, while that of IF-B was: protein 12, carbohydrates 53.2, fat 25. 

The method described by Resmini et al3 was employed. Briefly, furosine was generated mixing 0.34 g of sample with 

8 ml of HCl 8N and heated for 23 hours at 110ºC. The hydrolyzed sample was filtered through paper filter and diluted 

with HCl 3N (1:5, v/v). 500ml of diluted sample were purified with SepPak C18 cartridges previously activated (5 ml of 

methanol and 10ml of deionized water) and eluted with 3ml of HCl 3N. 

The HPLC conditions were: Alltech furosine dedicated C8 column (250 x 4.6mm i.d.) and C8 precolumn (7.5 x 4.6mm i.d.) 

at 34ºC. Mobile phase: A) 0.4% (v/v) acetic acid, B) 0.27% (w/v) potassium chloride in solvent A; linear gradient: 9 min 

– 0% B, 17 - 21.5 min – 50%, 23 - 32 min – 0% B. Injection volume: 20ml, l = 280nm. Quantification was made by an 

external calibration curve ranging from 0.360 to 2.016mg/ml. 

RESULTS 

Furosine content varied over time in IF A from 64.47mg/100g product (t=0 months) to 85.70mg/100g product (t=18 

months), while in IF B it ranged between 67.68 mg/100 g product (t=0 months) and 108.11mg/100g product (t=24 

months), implying an increase of 33% and 42%, respectively, in furosine content. This fact can be explained by 

the same quality of raw milk and thermal treatment, similar protein content and the equal whey protein:casein ratio 

(50:50) present in the two infant formulas. 

Both samples showed significant differences (p < 0.05) between the start (t=0 months) and the end of the study 

(t=18-24 months), while no significant differences (p < 0.05) where found between samples. Similar increases have 

been described by Guerra et al.3 in IFs stored during 3 months at 20ºC. 


This work has been funded by the Hero Institute for Infant Nutrition, and has been partially funded by the 

CONSOLIDER INGENIO 2010 program FUN-C-FOOD CSD2007-063. Jaime Salcedo is the holder of a grant from the 

Hero group. Ramon Lacomba is the holder of a V Segles–Hero group grant. 

REFERENCES 

1 Erbersdobler H., Somoza V. (2007) Mol. Nutr. Food Res. 51, 423-430. 

2 Guerra-Hernández E. et al. (2002) Int. J. Dairy Technol. 55, 171-176. 

3 Resmini P. et al. (1990) It. J. Food Sci. 3, 173-183. 

286 



P03-019 HPLC DETERMINATION OF SIALIC ACID (NEU5AC AND NEU5GC) CONTENTS IN INFANT FORMULAS 

Lacomba R. 1 , Salcedo J. 1 , Alegria A. 1 , Barberá R. 1 , Matencio E. 2 , Lagarda M.J. 1 

1 


2 


INTRODUCTION 

Sialic acid is an essential biocompound that can be found in animal tissues and fluids in several forms. The 

main representative forms are N-acetylneuraminic (Neu5Ac) acid and N-glycolylneuraminic (Neu5Gc) acid. 

Spectrophotometry has been used to determine the total sialic acid content in human milk and infant formulas (Ifs)1, 

but is unable to differentiate between Neu5Ac and Neu5Gc2. A HPLC method with fluorescent detection to determine 

both compounds in infant formulas has been proposed by Martín et al. (2007)3, who validated the determination of 

Neu5Ac. In a previous work we validated Neu5Gc determination by this same method4. 

AIM 

To analyze the Neu5Ac and Neu5Gc contents in infant formulas by HPLC with fluorescence detection. 


Powdered or lyophilized samples were hydrolized with H2SO4 0.05M (80ºC/60min). Hidrolizate was centrifuged (200 

x g/4ºC/10 min) and purified by ion exchange (2ml of Dowex 1x8). An aliquot of purified sample was ultrafiltered with 

Microcon Ultracel YM-10 (13000 x g/4ºC/10min). Fifty ml of the filtered sample were derivatized with 50 ml of DMB 

reagent (8 mM) (50ºC/2.5 hours). 

HPLC conditions: Hidrosorb RP-18 (250x4.6 mm, 5 mm) column and Hidrosorb RP-18 (5 mm) guard column, mobile 

phase water:methanol:acetonitrile (85:7:8 (v/v/v)), flow 0.9 ml/min, detection λexc=373 nm, λem=448 nm, gain: 1, 

attenuation: 64, response: 5 s. 

Seven starting formulas from different manufacturers and their respective counterpart follow-up formulas were 

analyzed. 

RESULTS AND CONCLUSIONS 

Contents of Neu5Ac in starter and follow-up formulas ranged from 171.1 to 498.5 mg/l (131.6 to 369.3 mg/100 g) 

and 208.9 to 476.4 mg/l (160.7 to 352.9 mg/100 g), respectively, while Neu5Gc ranged from 2.3 to 7.7 mg/l (1.8 to 5.7 

mg/100 g) and 3.6 to 7.3 mg/l (2.8 to 5.4 mg/100 g) for starting and follow-up formulas. The values obtained are in 

agreement with the literature sources3. 

For a common manufacturer, starting formulas contain less sialic acid than follow-up formulas. The range of sialic 

acid content in starting and follow-up infant formulas includes the sialic acid content in mature milk (340 mg/l)1. 

A significant linear correlation (p=0.0077; a=0.01), was found between the contents of Neu5Ac and Neu5Gc 

(Neu5Ac=53.44+47.72xNeu5Gc; R2=0.49). This is the first time that the contents of Neu5Gc are reported, along with 

their correlation to Neu5Ac, in infant formulas. 


This work has been funded by the Hero Institute for Infant Nutrition, and has been partially funded by the 

CONSOLIDER INGENIO 2010 program FUN-C-FOOD CSD2007-063. Jaime Salcedo is the holder of a grant from the 

Hero group. Ramon Lacomba is the holder of a V Segles – Hero group grant. 

REFERENCES 

1 Carlson S. (1985) Am. J. Clin. Nutr. 41, 720-726. 

2 Lacomba R. et al. (2010) J. Pharm. Biomed. Anal. 51, 346-357. 

3 Martín M. J. et al. (2007) Anal. Bioanal. Chem. 387, 2943-2949. 

4 Salcedo J. et al. (2009) Nutr. Hosp. 25, 163-164. 


287


P03-020 OPTIMIZATION OF LC METHODS FOR THE SIMULTANEOUS DETERMINATION OF BISOPROLOL AND 

HYDROCHLOROTHIAZIDE IN THEIR PHARMACEUTICAL DOSAGE FORMS 

Gumustas M., Bozal B., Dogan-Topal B., Ozkan S.A., Uslu B. 


Bisoprolol (BIS) and hydrochlorothiazide (HCT) have been recently suggested as a combination therapy for the 

treatment of hypertension and chronic heart failure. HCT, is one of the oldest thiazide diuretics, often described 

in combination with other drugs such as , ACE inhibitors, angiotensin II receptor blockers or B-blockers [1]. BIS, 

is a highly selective B1- receptor blocking agent used for the treatment of hypertension and angina pectoris 

[2]. Throughout this study, the mobile phase were assayed ACN - water containing 15 mM phosphoric acid. The 

results indicate that good chromatographic separation can be obtained for the compounds with 25 % (v/v) of 

acetonitrile when the pH of the mobile phase is 3.0.Due to the excellent separation efficiency about BIS, HCT 

and I.S. (moxifloxacine) the proposed method is suitable for mixture of these compounds. The method developed 

was successfully applied to the simultaneous determination of BIS and HCT in their commercial dosage forms. 

Pharmaceutical dosage forms were analyzed using this optimized method. The calibration curves and equations 

for BIS and HCT were calculated by plotting the peak area ratios of these compounds to I.S. (moxifloxacine) versus 

concentration of the compounds in the range of 0.5–12 ppm for BIS, , and 0.2-8.0 ppm for HCT. The retention 

times are 2.783 for HCT and 5.058 for BIS. When working on standard solutions and according to the obtained 

validation parameters, results encourage the use of the proposed method described for the assay of simultaneous 

determination in their pharmaceutical dosage forms. The quantities found were in conformity with the values claimed 

by the manufacturers. [1] N. Mougenot, O. Médiani, P. Lechat, Pharmacol. Res. 51, 395, 2005. [2] V. Papademetriou, 

L.M. Prisant, J.M. Neutel, M.R. Weir, Am. J. Cardiol. 81,1363, 1998. 

288 



P03-021 OPTIMISATION OF A LIQUID CHROMATOGRAPHIC METHOD FOR THE SEPARATION OF HYDROXYLATED 

POLYCHLORINATED BIPHENYLS ON A POLAR-EMBEDDED STATIONARY PHASE APPLYING EXPERIMENTAL DESIGN 

PROCEDURES 

Galindo-Iranzo P. 1 , Quintanilla-López J.E. 2 , Lebrón-Aguilar R. 1 , Gómara B. 2 

1 


2 


Corresponding author e-mail: bgomara@iqog.csic.es 

Polychlorinated biphenyls (PCBs) are a group of ubiquitous pollutants that are usually present in environmental and 

biological samples as complex mixtures. In living organisms, PCBs are metabolised leading to polar metabolites 

which are suppose to be more easily excreted, such as their corresponding hydroxylated metabolites (OH-PCBs). 

However, some studies have shown that OH-PCB metabolites can be bound to specific plasma proteins, being 

retained and accumulated in different species. So, nowadays, it is still not clear if PCBs’ toxicity is only due to PCB 

concentrations or is also due to the presence of PCB metabolites in the same individual. All of this demonstrates that 

PCB metabolites should be considered as a secondary class of contaminants of concern. 

OH-PCBs are usually determined by gas chromatography (GC) with electron capture detectors (ECD) or coupled to 

mass spectrometry (MS). However, a previous derivatisation of the OH-PCBs into their methoxylated derivates is 

mandatory for their GC separation. In order to avoid this extra step, the development of a liquid chromatographic 

method will became a useful analytical tool for the separation of these new contaminants. At the moment, their 

separation on octadecylsilane stationary phases is not always satisfactory. A coelution of isobaric compounds is 

frequently observed, being therefore impossible to differentiate them from their mass spectra either. A solution would 

be the use of chromatographic columns with stationary phases (SPs) showing a greater selectivity towards this type 

of compounds. It has been reported that an amide group embedded into an alkyl chain presents an especially high 

affinity towards substances capable to give hydrogen bonds, that it is the case of OH-PCBs. Consequently, this type 

of SPs was selected for the present study. 

However, once chosen the chromatographic column, it is not a simple task to find the optimal analytical conditions 

for the separation due to the large number of variables involved in the process. For this reason, it is a normal practice 

to resort to techniques of experimental design and especially to the so called Response Surface Methodology. 

Therefore, the objective of this work was to develop and optimise a liquid chromatographic method for the separation 

of OH-PCBs on a stationary phase with an amide group embedded, by means of the use of experimental design. 

The Response Surface Methodology was carried out applying a Box-Wilson Central Composite design, choosing 

the initial content of methanol in the mobile phase, the gradient time, and the concentration and the pH value of 

the buffer (ammonium formate/formic acid) as relevant parameters. A global optimum was obtained by using the 

Derringer’s function value and selecting the elution time, the sensitivity and the overall resolution as responses to 

optimise. 

Acknowledgements 

This study was supported by CSIC (project 200880I192) 


289


P03-022 APPLICABILITY OF SOLID PHASE EXTRACTION WITH MICELLAR DESORPTION (SPE-MD) COMBINED WITH HPLC TO THE 

DETERMINATION OF FLUOROQUINOLONES IN HOSPITAL AND MUNICIPAL WASTEWATERS 

Montesdeoca-Esponda S., Sosa-Ferrera Z., Santana-Rodríguez J.J. 

University of Las Palmas de Gran Canaria 

Corresponding author e-mail: jsantana@dqui.ulpgc.es 

A method based in Solid Phase Extraction (SPE) followed by elution with surfactant solution (SPE-MD) combined 

with high performance liquid chromatography (HPLC) is applied to the determination of Fluoroquinolones (FQs) 

residues in hospital and municipal wastewaters. These antibiotics are a new and synthetic generation of quinolones 

family, used in human and veterinary medicine [1] against several infections diseases. They are partly be excreted 

in an unmetabolized form and can even survive the passage through the sewage treatment plants [2]. The target 

FQs are in very low concentrations in sewage waters, for that, they need to be extracted and preconcentrated prior 

their analysis. We proposed a novel variant of solid phase extraction procedure replacing the organic solvents 

used conventionally for the elution, by a micellar solution. The parameters that affect the extraction process were 

optimized for achieve a simple, fast, safe and non-contaminant extraction method [3]. Finally, the results obtained 

by this process are compared using both fluorescence and mass spectrometry detection. References: [1] K. Kaur, 

A. Kumar, A. Kumar Malik, B. Singh, A.L.J. Rao, Crit Rev Anal Chem 2008, 38, 2-18. [2] W.W. Buchberger, Anal Chim 

Acta 2007, 593, 129-139. [3] S. Montesdeoca-Esponda, Z. Sosa-Ferrera, J.J. Santana-Rodríguez, unpublished data. 

290 



P03-023 SIMULTANEOUS DETERMINATION OF ENDOCRINE DISRUPTING CHEMICALS IN WASTEWATER TREATMENT PLANT 

SLUDGES BY MICROWAVE ASSISTED EXTRACTION AND LC-ESI-MS/MS 

Vega-Morales T., Sosa-Ferrera Z., Santana-Rodríguez J.J. 

Universidad de Las Palmas de Gran Canaria 

Corresponding author e-mail: jsantana@dqui.ulpgc.es 

Recently, many chemicals released into the environment have been shown to mimic endogenous hormones such 

as estradiol [1]. It has been demonstrated that these compounds cause several adverse effects on wildlife and 

humans, such as the feminisation of animal species, development of physical abnormalities and birth defects, and 

reproductive failure [2]. It is happens that the complete degradation of these endocrine disrupting chemicals (EDCs) 

in conventional wastewater treatment plants is not achieved, and therefore, a complex mixture of these xenobiotics 

could eventually enter the environment where aquatic organisms are exposed to them. Moreover, in many cases the 

biodegradation processes leads to the formation of sub-products more toxic, more lipophilic, more estrogenic and 

more persistent than the parent substances, as occurs with alkylphenol polyethoxylated [3]. The determination of 

these substances in the sewage sludges has a great importance due to they tend to bind tightly to the particulate 

matter and sediments. In addition, the use of sewage sludge from wastewater treatment plants as organic 

amendment has become usual in Europe during the last decade to mitigate the low productivity or profitability of 

several agriculture soils [4], which facilitates the “arrival” of these pollutants to humans through the food chain. 

Therefore, the objective of this work focuses on the development of a simple and fast analytical procedure for the 

simultaneous extraction and determination of bisphenol-A, 17α-Ethynylestradiol, 17β-estradiol and its two main 

metabolites (estriol and estrone), and nonylphenol, octylphenol and their corresponding ethoxylates (1-12) for the 

quantitative analysis of these EDCs in sewage sludge samples by liquid chromatography tandem mass spectrometry 

(LC-MS/MS). For the extraction of the analytes, we use microwave assisted extraction, which needs a small amount 

of sample, provides satisfactory recoveries, and requires low time consumption and low volumes of organic solvents. 

[1] R. P. Schwarzenbach, B. I. Escher, K. Fenner, T. B. Hofstetter, C. A. Johnson, U. von Gunten, B. Wehrli, Science, 

313 (2006) 1072. [2] C. Sonnenschein and A. M. Soto, J. Steroid Biochem. Molec. Biol., 65 (1998) 143. [3] T. Vega 

Morales, M. E. Torres Padrón, Z. Sosa Ferrera, J. J. Santana Rodríguez, Trends Anal. Chem., 28 (2009) 1186. [4] V. 

Andreu, E. Ferrer, J.L. Rubio, G. Font, Y. Picó, Sci. Total Environ., 378 (2007) 124. 


291


P03-024 CHARACTERISATION OF THE LIQUID CHROMATOGRAPHIC BEHAVIOUR OF HYDROXYLATED POLICHLORINATED 

BIPHENYLS ON AN AMIDE-TYPE STATIONARY PHASE 

Quintanilla-López J.E. 1 , Galindo-Iranzo P. 2 , Gómara B. 1 , Lebrón-Aguilar R. 2 

1 


2 


Corresponding author e-mail: rlebron@iqfr.csic.es 

It is well-known the toxic effects related to the presence of polychlorinated biphenyls (PCBs) in living organisms. 

PCBs are metabolised leading to polar metabolites which are suppose to be more easily excreted, such as their 

corresponding hydroxylated metabolites (OH-PCBs). However, several authors have established that they can 

present agonist or antagonist interactions with hormone receptors and/or induce hormone-receptor-mediated 

responses. Therefore, the PCB metabolites should be considered as a secondary class of contaminants of concern. 

At the present time, the liquid chromatographic separation obtained for OH-PCBs is not always satisfactory on the 

widespread octadecylsilane stationary phases. Since this kind of separation is driven by hydrophobic interactions, 

a coelution of isobaric compounds is frequently observed, being consequently impossible to differentiate among 

them by their mass spectra either. For this reason, the evaluation of other stationary phases (SPs) capable to provide 

selective interactions with these new contaminants would be very promising. Therefore, chromatographic columns 

with SPs able to promote hydrogen bonds with the hydroxyl group of the OH-PCBs, as those with an amide group 

embedded into an alkyl chain, were chosen for this study. 

A deeper knowledge of the retention mechanism involved in the separation of OH-PCBs in an amide-type column 

could be of the greatest interest and would simplify the optimisation of the chromatographic process. Hence, the 

objectives of this work were to study the chromatographic behaviour of the OH-PCBs on this type of columns and to 

characterise the retention mechanism that regulates their separation in an amide-type column. 

In order to carry out the retention study, an OH-PCBs mixture was analysed under isocratic conditions, using 

percentages of methanol in the mobile phase ranging between 65 and 98%, all of them with 0.1 mM of ammonium 

formate. The retention factors so obtained for the OH-PCBs studied were correlated with their pKa and log D values, 

chosen as the molecular descriptors more related to their chromatographic retention. Results showed that the 

prevalent interaction between OH-PCBs and the amide-type SP takes place by hydrogen bonds, although dispersion 

forces also play an important role for moderate content of methanol in the mobile phase. Furthermore, the Snyder 

solvent strength model showed that the mobile phase pH value is the most important parameter controlling the OH- 

PCBs separation, since it allows modulation of the hydrogen bonds strength. 


This study was supported by CSIC (project 200880I192) 

292 



P03-025 A NEW CHROMATOGRAPHIC MODEL TO PREDICT THE RETENTION OF IONIZABLE ANALYTES UNDER GRADIENT ELUTION 

RP-HPLC 

Andrés A., Téllez A., Rosés M., Bosch E. 

Factulty of Chemistry, University of Barcelona 

The retention of a ionizable solute in gradient RP-HPLC is a pretty difficult parameter to predict, because it strongly 

depends on its pKa and the pH of the mobile phase, and both parameter vary with the change of the mobile phase 

composition. Eq 1 has been proposed to estimate retention in gradient elution [1]: 

(1) where tD* is the dwell time and kφ accounts for the retention factor in a mobile phase with a given fraction of 

organic modifier φ. Several equations can be used to relate kφ to φ. One of them, explains the behavior of neutral 

compounds by means of the expression [2]: 

(2) where a, b and c are fitting parameters. The polarity parameter model (Eq 3) has also been successfully employed 

for the prediction of the retention of neutral solutes in isocratic [3] and gradient [4] RP-HPLC: 

(3) where p is a polarity parameter of the solute, (log k)0 and PSN are polarity parameters of the chromatographic 

system (column-organic modifier), and PmN is a polarity parameter of the mobile phase. If the system is properly 

characterized, Eq 3 becomes a one parameter model (p). A variation of the polarity parameter model, which 

involves two parameters, has also been proposed [3,5]: 

(4) Eq 5 has been used when ionizable compounds are chromatographed in isocratic RP-HPLC [6]: 

(5) where kHA is the retention factor of the neutral form of the solute, D is the ionization degree and f is the fraction 

between the retention factors of the solute in its ionic and neutral forms. In this work, Eq 5 has been combined 

with equations 2-4 in order to predict retention of ionizable analytes under gradient elution RP-HPLC. The 

agreement between the experimental and calculated retention times obtained is good using each model. The 

combination with Eq 2 is the one that gives better results. The combination with Eq 4 shows results almost as 

good as the others but it involves one parameter less, which is an advantage because it translates into less 

amount of previous experimental work to obtain the fitting parameters of the model. The combination with Eq 3 

gives also good results for most compounds, but not nearly as accurate as the ones obtained from the other two 

models. 

Literature: [1] P. Nikitas, A. Pappa-Louisi, J. Chromatogr. A, 2005, 1068, 279. [2] P. Nikitas, A. Pappa-Louisi, Anal. 

Chem., 2005, 77, 5670. [3] E. Bosch, P. Bou, M. Rosés, Anal. Chim. Acta, 1994, 299, 219. [4] A. Téllez, M. Rosés,. 

E. Bosch, Anal. Chem., 2009, 81, 9135. [5] J.R. Torres-Lapasió, M.J. Ruiz-Ángel, M.C. García-Álvarez-Coque, J. 

Chromatogr. A, 2007, 1166, 85. [6] M. Rosés, D. Bolliet, C. F. Poole, J. Chromatogr. A, 1998, 829, 29. 


293


P03-026 HOP’S (HUMULUS LUPULUS SP) PRENYLATED FLAVONOIDS EXTRACTED BY PLE AND ANALYZED BY LC-ESI-MS/MS 

Gil-Ramírez A., Mendiola J.A., Marín F.R., Ibáñez E. 

Instituto de Investigación Ciencias de la Alimentación CIAL-CSIC 

Corresponding author e-mail: j.mendiola@ifi.csic.es 

Hops (Humulus lupulus sp) contain a wide range of compounds with different properties and activities, among them 

are the prenylated flavonoids such as the Xanthohumol (XN) and Isoxanthohumol (IXN). In aqueous medium XN it’s 

isomerized to IXN, who produce 8-prenilnaringenin (8PN) by the action of gut microbiota. It’s raising the interest in 

8PN by its functional properties: potential estrogen only about 1000 times less than estradiol with ability to bind to 

both receptors, ERα and ERβ (Zanoli and Zavati, 2008). 

The first step to get 8PN is by isolation of its precursor: IXN (or XN in either case). 

Hop pellets (normally used for animal feed) were used for the extraction by two methods: 

• Solid-liquid extraction to determine the initial amount of both compounds: cryogenically crushed hops mixed with 

DMSO, and left in agitation and darkness 24 hours. It is filtered and the cake obtained again underwent the same 

process. This was repeated until no XN nor IXN were detected in the filtrate. 

• Pressurized Liquid Extraction (PLE) hop powder mixed with sea sand (1:2 ratio) were extracted using following 

conditions: water at 150 º C (6 cycles of 5 minutes), ethanol at 150 ° C (6 cycles of 5 minutes ), sequential extraction 

(hexane followed by ethanol and finally water; 20 minutes each fraction). All samples were evaporated and/or freeze 

dried. 

In order to have an idea of the amount of total phenols extract, Folin-Ciocalteu method was used, as described 

by Kosar et al (2004). Extracts obtained by both methods was analyzed by HPLC-ESI-MS/MS (triple quadrupole). 

Multiple reactions monitoring analysis was carried out for quantification. In this sense, direct infusion of 

standards (XN, IXN and 8PN) was used to optimize MS transitions and voltages. Moreover two full scan analysis 

simultaneously, with and without source fragmentation. 

The results indicated that there was a considerable amount of XN in hops (around 50 mg/g hops). The pressurized 

water extraction at 150 ºC showed a high selectivity towards IXN (10 times more than XN) that reflects the 

isomerization described previously (Stevens and Page, 2004), while ethanol was more selective to extract XN, 

because no isomerization took place. In hexane and water fractions of sequential extraction the amount of XN and 

IXN was not relevant (less than 0.40 mg/g extract) although it is in ethanol (that is where more of both substances 

extracted, however there is no selectivity). 

The next step would be to conduct trials of digestion and bioavailability of the extracts of interest, to determinate the 

synthesis yield of 8PN from our extracts. 

References: 

- Kosar M et al. 2004. Food Chemistry 91: 525-533 

- Stevens, JF and Page JE. 2004. Phytochemistry 65: 1317-1330 

- Zanoli, P and Zavatti, M. 2008. Journal of Ethnopharmacology 116: 383-396. 

294 



P03-027 HOW CLEAN ARE YOUR VIALS AND CLOSURES ? 

Pereira L., Edge A., Shick L., King B., 


Improvements in chromatographic techniques, instrumentation and sample handling continue to push the limits of 

detection in trace analysis. As such, the cleanliness of all apparatus used in the total workflow becomes even more 

important to reduce the potential for interferences and contamination that ultimately will reduce the sensitivity of the 

analysis. The selection of the right auto-sampler vial and closure becomes an important consideration. Vials that are 

not processed can introduce particulate matter that can cause blockages and accumulation of foreign material at the 

head of the GC or LC column and therefore deterioration of the chromatographic separation. Additionally, residual 

organic compounds that might survive the glass forming process or that might leach from the closure when exposed 

to the sample solvent can reduce the analysis sensitivity. The work present in this poster evaluates the performance 

of a new pre-cleaned vial and an ultra high pure bonded PTFE/silicone closure and compares them with other 

leading commercially available products. Vials and closures were exposed to acetonitrile at room temperature and 

50°C for 2 hours. Potential non-volatile organic compounds were determined using LC/UV and LC/MS with several 

ionization techniques: positive electrospray, negative electrospray and positive atmospheric pressure ionization 

(APCI). Potential semi-volatile compounds were determined by GC/MS with electron impact (EI) ionization. 


295


P03-028 CHARACTERIZATION OF INDUSTRIAL ENZYMES BY HPLC OF THE INTACT PROTEINS AND THEIR TRYPTIC DIGESTS USING A 

POLYMERIC MONOLITHIC COLUMN 

Beneito-Cambra M. 1 , Herrero-Martínez J.M. 1 , Ramis-Ramos G. 1 , Lindner W. 2 , Lämmerhofer M. 2 

1 


2 


Corresponding author e-mail: michael.laemmerhofer@univie.ac.at 

Enzymes for cleaning products constitute the largest segment of the world market for industrial enzymes. The role 

of enzymes in cleaning products has changed from one of a minor additive to becoming a key ingredient. Cleaning 

power enhancement by enzymes leads to a reduction of washing times and temperatures, with the subsequent 

savings of water and energy. Further environmental advantages arise from the lower consumption of aggressive 

chemicals, with the additional benefit of a better care of fabrics during washing. In this work, enzymes commonly 

used in the detergent industry were identified and characterized. For this purpose, enzyme industrial concentrates 

of the protease, lipase, amylase and cellulose classes were studied. Two approaches were tried: first, the HPLC 

separation of the intact proteins, and second, to digest the enzymes with trypsin in the presence of dithiothreitol, 

and to use HPLC to separate the resulting peptides. In both cases, a polymeric monolithic column (ProSwift 

RP-1S, Dionex) and UV detection were used. Separations were performed by gradient elution with water/acetonitrile 

containing 0.1% trifluoroacetic acid as mobile phase. Both approaches gave enough information for enzyme class 

prediction using chemometric tools (as LDA); however, HPLC of the tryptic digests provided superior reliability 

for enzyme identification. The retention times of the peaks and either normalized peak areas (divided by the sum 

of the peak areas of the chromatogram) or ratios of pairs of peak areas as predictor variables. Application to the 

classification and identification to enzymes in detergent bases and laundry cleaning products, including binary 

mixtures of enzymes of different classes, is in progress. 

Acknowledgements: Project CTQ2007-61445 (MEC and FEDER funds) and V-Segles-Empresa grant for PhD studies 

(M. B-C., University of Valencia and Químicas Oro). 

296 



P03-029 NOVEL POLYSTYRENE-DIVINYLBENZENE ANION EXCHANGERS FOR ION CHROMATOGRAPHY 

Zatirakha A.V., Smolenkov A.D., Shpigun O.A. 


One of the most important directions of research in the field of ion chromatography is development of new stationary 

phases, providing high efficiency and good selectivity of separation. Polystyrene-divinylbenzene (PS-DVB) anion 

exchange resins are the most popular stationary phases for ion chromatography due to their high stability, but there 

is a number of problems caused by strong specific interactions of polarizable ions, such as nitrate and bromide, with 

aromatic rings of PS-DVB. Negative influence of these interactions results in high retention times and low efficiency 

for ions of nitrate and bromide. Therefore search for new approaches to the synthesis of PS-DVB anion exchangers 

and methods of reducing the influence of matrix on the retention of polarizable anions is very important scientific 

problem. Novel method for preparation of anion exchangers based on PS-DVB was presented. It included 

Friedel-Crafts acetylation of PS-DVB, reductive amination of carbonyl groups and following alkylation with 

epichlorohydrin. PS-DVB with cross-linking degree of 25%, beads diameter of 3,3±0,2 microns and average pore 

diameter of 6 nm was used as a matrix for synthesis. Chromatographic properties of obtained stationary phase 

with functional group of 3-chloro-2-hydroxypropyl-N,N-dimethylammonium were studied by means of suppressed 

ion chromatography with conductometric detection. This anion exchanger provides good selectivity for separation 

of the mixture of seven polarizable and nonpolarizable anions, namely, fluoride, chloride, nitrite, nitrate, bromide, 

phosphate and sulfate in 30 minutes in isocratic mode. No abnormal retention of nitrate and bromide ions is 

observed, indicating a decrease in the influence of matrix on the retention of polarizable anions. The efficiencies 

of the column with obtained anion exchanger using carbonate buffer or sodium bicarbonate solution as eluents are 

50000 and 35000 theoretical plates per meter respectively (for phosphate-ion). 


297


P03-030 THE EFFECT OF HYDROTHERMAL TREATMENT ON COLUMN PERFORMANCE FOR MONOLITHIC SILICA CAPILLARY COLUMNS 

Hara T., Mascotto S., Weidmann C., Heuser S., Smarsly B. 

Justus Liebig University of Giessen-Germany 

Corresponding author e-mail: Bernd.Smarsly@phys.Chemie.uni-giessen.de 

In this study, monolithic silica capillary columns with i.d. 100 mm and monolithic silica rods were prepared 

by different hydrothermal treatments with urea at 80 °C or 120 °C. One type of monolithic silica columns was 

prepared only with tetrametoxysilane (TMOS) and the others were obtained using a mixture of TMOS and 

metyltrimethoxysilane (MTMS). The former is recognized as a “TMOS column” and the latter as a “Hybrid column”. 

Nitrogen physisorption was applied for the determination of pore size distribution and surface area for monolithic 

silica rods. The characterization of porosity of the monolithic silica capillary columns was carried out by Inverse size 

exclusion chromatography (ISEC). Using nitrogen physisorption, it was possible to observe the change of pore size 

distributions and surface area for monolithic silica rods corresponding to the change of the hydrothermal treatment 

and the precursors. The results from ISEC for monolithic silica capillary columns agreed well with the results 

from nitrogen physisorption regarding the pore size distribution. In addition, the HPLC properties of monolithic 

silica capillary columns modified by octadecyl-silylation were measured in MeOH/H2O (v/v) = 80/20 at 30 °C 

using alkylbenzenes (n = 1-6). The retention factors for hexylbenzene could also support the results from nitrogen 

physisorption. 

Furthermore, column efficiency for the monolithic silica capillary columns was evaluated with the alkylbenzenes 

and three kinds of peptides, Leucine-enkephalin (Mw = 555), Angiotensin II (Mw = 1046), and Insulin (Mw = 5770) at 

linear velocities from 0.08 mm/s to 6.0 mm/s. Column efficiency using the alkylbenzenes was quite similar between 

a capillary column with and without the hydrothermal treatment at 120 °C. Even for TMOS columns, there was no 

significant difference in column efficiency for the peptides despite the difference in hydrothermal treatment for 

the columns. In contrast, for hybrid columns, it was possible to observe the effect on hydrothermal treatment at 

120 °C due to the difference in column efficiency, especially for Insulin. This difference supports the results from 

both nitrogen physisorption and ISEC because of the presence of more small pores for a hybrid column without 

hydrothermal treatment at 120 °C. 

In conclusion, it can be suggested that the effect from the presence of small pores on column efficiency is negligible 

with small molecules like the alkylbenzenes even using a capillary column without hydrothermal treatment at 120 

°C. However, the results using peptidesrecommendthat hydrothermal treatment for a hybrid column with higher 

temperature or longer time is necessary compared to that for a TMOS column. 

298 



P03-031 GREEN CHROMATOGRAPHY DETERMINATION OF PARACETAMOL, PROPYPHENAZON, CAFFEINE, AND P-AMINOPHENOL IN 

PHARMACEUTICAL PREPARATIONS 

Brabcová I., Šatínsky D., Solich P. 

Charles University in Prague Faculty of Pharmacy in Hradec Králové 

The aim of the work was to develop chromatographic method for determination of paracetamol, propyphenazon, 

caffeine and p-aminophenol in pharmaceutical preparations. 

These analytes show different polarity and acidobasic properties. P-aminophenol - degradation product of 

paracetamol shows high hepatotoxic effect and level of this impurity must be controlled. The application of 

traditional octadecylsilane stationary phases for their simultaneous determination is problematical. 

The chromatographic separation of analytes under a isocratic elution was achieved at room temperature with 

a Supelco Disovery HS PEG (15 × 4 mm, 3 μm) column, mobile phase containing acetic buffer pH 4.5, 0.04% of 

triethylamine, flow rate 1 mL min-1. UV detection was at 254 nm. The analysis time was 7 min. Benzoic acid was used 

as internal standard. 

Optimal conditions for chromatographic separation of all substances were found in green chromatography mode. 

The mobile phase used was free of organic solvents (methanol, acetonitril). Polyethylenglycol (PEG) column in 

reverse phase mode was used for the efficient treatment of problems of common determination of substances 

different polarity. 

Validation parameters of the method (linearity, precision, accuracy, robustness and selectivity) were tested and they 

showed very good results. 

The method was found to be applicable for the routine analysis of determination of paracetamol, propyphenazon, 

caffeine and p-aminophenol in pharmaceutical preparations. 

The authors gratefully acknowledge the financial support of the Ministry of Education of the Czech Republic, MSM 

002162082 and Grant Agency of Charles University Project No. 34609/2009. 


299


P03-032 APPLICATION OF AN HPLC METHOD FOR A RAPID DETERMINATION OF -TOCOPHEROL, ERGOCALCIFEROL AND 

CHOLECALCIFEROL IN DIFFERENT LIQUID FOODS AND CEREALS 

Barba F.J., Esteve M.J., Frigola A., 


Corresponding author e-mail: maria.jose.esteve@uv.es 

Introduction 

There are differences in the spectral characteristics of a-, δ- and γ-tocopherol, ergocalciferol and cholecalciferol 

(265–452 nm), which presents a problem when it comes to determining these vitamins together, so that traditionally 

they have been determined separately, lengthening analysis time. 

The technique of choice for fat soluble vitamins (FSV) determination is liquid chromatography. The main problem is 

that nearly all foods contain other lipid components which may interfere in the determination, introducing the need 

for a semi-preparative stage which is generally laborious and prolongs analysis time. 

Two procedures can be used for preparing the sample: saponification and extraction of the vitamins from the 

non-saponifiable fraction or extraction of the fat, saponification and extraction. The factors to be optimized in the 

saponification process are: sample size, quantity and concentration of potassium hydroxide in the solution, and 

saponification temperature and time. After saponification, generally a liquid-liquid extraction is performed with nonpolar 

solvents. 

Aim 

The aim of the present study was to establish a method for the simultaneous determination of vitamins E 

(a-, d-, γ-tocopherol) and D (ergocalciferol, cholecalciferol) in liquid foods and in cereals. 

Samples 

Liquid foods: milk and by-products, beverages based on fruit juice and milk and vegetable beverages. 

Cereals: bread, corn, wheat, oats and malt. 

Material and Methods 

The LC system consisted of two isocratic pumps (Prostar 210, Varian Inc, USA) with degasser, column thermostat 

(Prostar 510, Varian) and UV-vis detector (Varian Inc, California, USA). The column of choice was the Kromasil 100 

C18 (5 µm, 150x4.6 mm) (Scharlab, Spain). Mobile phase: Acetonitrile:Methanol (90:10 v/v). 

Results and discussion 

Saponification 

Liquid Foods: 20–25 g of sample was taken, 25 mg of BHT was added as antioxidant, and it was saponified with 15 

mL of KOH in ethanol (50%, v/v) for 1 hour at ambient temperature in nitrogen atmosphere and in darkness. 

Cereals: It was obtained the fat portion of 15–20 g of sample using Soxhlet method with diethyl ether (6h, 60ºC), 

after 25 mg of BHT was added as antioxidant, and it was saponified with 15 mL of KOH in ethanol (50%, v/v) for 1 

hour at ambient temperature in nitrogen atmosphere and in darkness. 

Extraction 

The greatest yield in liquid foods and cereals was obtained when the extraction was performed twice with 50 mL of 

hexane, agitated for 2 minutes and hexane fractions were combined. 

In the figure are shown the chromatograms for a fruit juice-milk sample with added vitamins A, D and E (a) ); where: 

(1) retinil acetate, (2) δ-tocopherol, (3) ergocalciferol, (4) cholecalciferol, (5) γ-tocopherol, (6) a-tocoferol, and samples 

of vegetables beverage (b), and corn germ (c). 


This study was carried out with funds from the Spanish Ministry of Science and Technology and European Regional 

Development Funds (Project AGL-2006-13320-C03-03) and the Generalitat Valenciana’s Aid for Research Groups 

3/147 and GV/2007/048. F.J. Barba holds an award from the Generalitat Valenciana. 

300 



P03-033 ANALYSIS OF CHEMICAL MARKERS IN MEAT AND MEAT PRODUCTS BY HILIC 

Mora L. 1 , Hernández-Cázares A. 1 , Aristoy M-C. 1 , Toldrá F. 1 , Reig M. 2 

1 

Instituto de Agroquímica y Tecnología de Alimentos, Ciencia de la Carne, Valencia 

2 


Introduction There are some chemical compounds in meat products that are analysed because of its use as markers 

of the final quality (i.e.- specific peptides, creatinine/creatine, nucleotides) [1]. Recently, the hydrophilic interaction 

chromatography (HILIC) was reported to have good performance for the separation of peptides [2] creatine, 

creatinine, nucleotides and nucleosides [3, 4]. The objective of this work was the use of HILIC chromatography for 

the analysis of relevant chemical quality markers like nucleotides and nucleosides, creatine (Cr) and creatinine (Cn) 

and dipeptides carnosine (CAR), anserine (ANS) and balenine (BAL) in meat and meat products (cooked ham and 

dry-cured ham). Materials and methods Samples used for this work were pork meat (glycolytic muscle Longissimus 

dorsi and oxidative muscle Masseter), cooked ham and dry-cured ham ripened for 7 and 11 months. Compounds 

were extracted with 0.01 N HCl and deproteinized with 2.5 volumes of acetonitrile (peptides, Cr and Cn), or 

extracted-deproteinized with perchloric acid solution (nucleotides and nucleosides), centrifuged and filtered. 

Chromatography was performed in a HPLC Agilent 1100 series system. Two different methods were used in 

this work: a) Separation of Cr, Cn and dipeptides: An Atlantis Silica® (150mm x 4.6 mm, 3mm) from Waters was 

used. Mobile phases consisted of solvent A, containing 0.65 mM ammonium acetate, pH 5.5, in water/acetonitrile 

(25:75), and solvent B, containing 4.55 mM ammonium acetate, pH 5.5, in water/acetonitrile (70:30). The separation 

conditions were a linear gradient from 0% to 100% of solvent B in 13 minutes at a flow rate of 1.4 mL/min. The 

detection was UV at 214 nm for Cr, CAR and ANS and 236 nm for Cn. b) Separation of nucleosides and nucleotides: 

A ZIC® pHILIC (150mm x 4.6 mm, 5mm) from SeQuant was used. Chromatographic conditions are given in table 1. 

Flow rate was 0.5 mL/min and detection was fixed at 254 nm. Results and Discussion As it is shown in Figures 1 to 

4, HILIC methods developed using Atlantis Silica® and ZIC® pHILIC columns showed very good performance for the 

analysis of biochemical compounds like nucleotides and nucleosides, Cr and Cn and dipeptides CAR, ANS and BAL 

as quality markers in complex food matrices like meat products. Conclusion The developed analytical methodologies 

are simple when compared with other rather conventional methods as reversed-phase. Thus, described methods are 

simple, fast and reliable avoiding complex sample preparation procedures. Furthermore, HILIC methods shown in 

this study might be compatible with further mass spectrometry analysis. References [1] Scheffler,T.L. & Gerrard,D.E. 

2007. Meat Science 77, 7-16. [2] Yoshida,T. 2004. Journal of Biochemical and Biophysical Methods 60, 265-280. [3] 

Mora, L., Sentandreu, M.A. & Toldrá, F. 2008. Journal of Agriculture and Food Chemistry, 55, 4664-4669. [4] Mora, 

L., Hernández-Cazares, A., Aristoy, M.C. & Toldrá, F. 2010. Food Chemistry, doi:10.1016/j.foodchem.2010.05.072. 

Acknowledgments Grant AGL2007-65379-C02-01 from MICINN (Spain), FPU scholarship from MEC are 

acknowledged and CONACyT and Colegio de Post-graduados, México. Work under Unidad Asociada IIAD-IATA. 


301


302 



P03-034 HOW THE DISSOLUTION SOLVENT AFFECTS PEAK SHAPES IN HYDROPHILIC INTERACTION CHROMATOGRAPHY? 

Josephine R. 1 , Guillarme D. 1 , McCalley D. 2 , Rudaz S. 1 , Veuthey J-L. 1 

1 

University of Geneva 

2 

University of the West of England 

Corresponding author e-mail: Jean-Luc.Veuthey@unige.ch 

Reversed-phase liquid chromatography (RPLC) is a powerful and versatile technique for the separation of a wide 

range of compounds. However, the separation of polar analytes is often very challenging because of the weak 

retention of such compounds with RPLC conditions. An alternative approach to RPLC for the separation of polar 

compounds is hydrophilic interaction chromatography (HILIC) [1]. In HILIC, analytes interact with a hydrophilic 

stationary phase and are eluted with a relatively hydrophobic binary eluent in which water is the stronger eluting 

solvent. The separation mechanism of elution is most probably a combination of partitioning, electrostatic 

interactions and hydrogen bonding to the stationary phase [2]. Unfortunately, peak shapes are sometimes 

problematic in HILIC compared to RPLC because of problems related to overloading of the stationary phase, 

insufficient mobile phase ionic strength or unsuitable dissolution solvent. The latter is certainly one of the most 

important parameter to improve peak shapes and has been thoroughly evaluated in the present study, using low 

molecular weight pharmaceutical analytes (i.e. neutral, acidic and basic) and peptides (with molecular weight 

ranging between 1000 and 5000 Da) as model compounds. Various solvents were tested as dissolution solvents 

including water, acetonitrile, methanol, ethanol, propan-2-ol, DMSO and mixtures of them. Two different HILIC 

materials were investigated, namely bare silica and silica amide. It appears that the dissolution solvent has a strong 

impact on peak shape in HILIC mode. For small compounds, even if ACN remains the best choice in terms of peak 

shape, the solubility needs to be considered as it is particularly critical with polar analytes. On the other hand, for 

peptides analysis, a compromise should be made between peak shape, solubility and stability. Thus, to obtain 

good chromatographic performance, some alternative solutions are proposed and discussed. [1] A.J. Alpert, J. 

Chromatogr. 499 (1990) 177. [2] P. Hemström, K. Irgum, J. Sep. Sci. 29 (2006) 1784. 


303


P03-035 DEVELOPMENT OF A VALIDATED LC METHOD FOR THE SIMULTANEOUS DETERMINATION OF AMLODIPINE AND PERINDOPRIL 

IN THEIR COMMERCIAL DOSAGE FORMS 

Gumustas M., Ozkan S.A., 

Ankara University Faculty of Pharmacy 

Amlodipine (AML); R, S-2 [(2-aminoethoxy) methyl]-4-(2chlorophenyl)-ethoxycarbonyl-5-methoxycarbonyl-6methyl-1, 

4-dihydropyridine, is a potent calcium channel blocker used in treatment of hypertension and angina pectoris. 

Perindopril is the first member of a new chemical class of non-peptide angiotensin II receptor antagonists. The 

first approved indication for perindopril is for hypertension. The combination therapy with ACE inhibitors and 

calcium channel antagonists may exert more beneficial effects on cardiovascular diseases than monotherapy. The 

combination of amlodipine and perindopril are consistently reduces blood pressure. In this study, HPLC-AD method 

has been developed for the simultaneous determination of amlodipine and perindopril in their pharmaceutical 

dosage forms. The following chromatographic conditions were used for the analysis of these compounds. Mobile 

phase were assayed 70:30 v/v acetonitrile - water containing 15 mM phosphoric acid, the pH of the mobile phase 

is 5.0. The working temperature for column oven programmed 45 oC with the flow rate of 1.2 ml/min. Atenolol was 

chosen as the internal standard (IS) because it showed a shorter retention time with better resolution compared to 

the other potential compounds for using internal standard. Detection wavelength was chosen 215 nm. The developed 

method was successfully applied for simultaneous determination of AML and PER with IS within a shorter analysis 

time in their commercial dosage forms. Using these conditions, analysis is completed about 5 minutes. Also the 

proposed method has been fully validated. Linearity was obtained in the concentration range 1-20 ppm for AML 

with the detection limit 0.017 ppm, and 5-100 ppm for PER with the detection limit 0.336 ppm. The present study 

purposes precise, accurate, simple, enough sensitive and rapid method for the simultaneous determination of 

AML, PER. Also the aim of this study was to develop and validate HPLC method suitable for the purity and stability 

evaluation of AML and PER and subsequently to employ these methods in stress tests addressing their chemical 

stability. The accelerated degradation studies were performed to provide an indication of the stability of the 

compounds and specify of the method. 

304 



P03-036 INVESTIGATION OF THE SELECTIVE DEPLETION OF PHOSPHOLIPID INTERFERENCES UTILIZING HYBRID-SPE TECHNOLOGY 

FOR LC-MS 

Buckendahl K. 1 , Aurand C.R. 2 , Brandes H. 2 , Bell D.S. 3 , Gutierrez P. 4 , Ferrari R. 5 

1 

Sigma-Aldrich, Sales & Marketing 

2 

Supelco, Research & Development 

3 


4 


5 


Corresponding author e-mail: dave.bell@sial.com 

Sample preparation is critical in pharmaceutical bioanalysis due to the complexity of biological matrices. Common 

sample preparation techniques include 96-well protein precipitation, liquid-liquid extraction, and solid phase 

extraction. SPE provides superior selectivity relative to simpler techniques; however, it is the most time-consuming, 

requiring multiple steps. Protein precipitation is highly generic with few processing steps. As a result, it is widely 

adopted for analysing plasma samples, although it removes only proteins. Other critical endogenous interferences, 

such as phospholipids, will remain in the sample. It is well known that phospholipids cause ion suppression in MS 

analysis, leading to low recovery and high variation of analytical results. Additionally, elution of phospholipids from 

an HPLC column require longer run times or gradient methods because of the presence of phospholipids. 

In this work, we discuss the development of the technology behind a sample prep platform that combines protein 

precipitation with SPE enabling the selective removal of both proteins and phospholipids. The technology utilizes a 

zirconia-coated particle that exhibits selective binding towards phospholipids whilst remaining non-selective towards 

other compounds. Comparison data with standard protein precipitation and SPE will be shown. Principles and 

mechanisms will be discussed to enable analysts to successfully use this technique for their assays and expand it for 

other applications that can benefit from the interactions offered by this innovative material. 


305


P03-037 MOBILE PHASE CONSIDERATIONS FOR IMPROVED LC-MS AMENABLE PEPTIDE SEPARATIONS 

Brandes H.K. 1 , Claus J.E. 1 , Aurand C. 1 , Bell, David S. 1 , Gutierrez P. 2 , Ferrari R. 3 

1 


2 


3 


Corresponding author e-mail: craig.aurand@sial.com 

Elution of peptides on silica-based, reversed-phase alkyl phases under the common MS-compatible condition of 

dilute formic acid is generally not as efficient when compared with the traditional UV-based methods with dilute 

trifluoroacetic acid (TFA). This is a complex phenomenon that may involve charge-charge interactions between the 

peptide analytes themselves as well as between the peptide and silica surface. Therefore, such interactions may 

be mitigated by pH and/or mobile phase additives that may function as counter-ions in an ion-exchange process. 

This paper describes the chromatographic behavior of reversed-phase peptide separations as a function of pH and 

counter-ion concentration. The impetus to explore this is driven by rapid growth in peptide drug candidates and 

peptide-based active pharmaceutical ingredients. 

Utilising a new, wide pore Fused Core C18 phase developed for peptide analysis, peptides of various basicity were 

chromatographed, and peak efficiency and symmetry recorded as a function of acidic modifier, pH and/or counter-ion 

concentration. Data analysis deciphered mechanistic reasons for poorer peak shape of peptides with formic acid (as 

compared to traditional methods with TFA). These studies were performed with designed synthetic peptides as well 

as enzymatic protein digests. 

It was found that optimal mobile phase conditions for reversed-phase, LC-MS amenable peptide chromatography 

depends in part, on the nature of the peptides being analyzed. Not only is peak shape and selectivity varied by 

relatively minor changes in pH, but the results are also affected by the peptide pI. Implications are also raised for 

deactivation of the silica surface through control of the mobile phase composition. The results of this work provide 

insights leading to facile development of robust and rugged LC-MS methods for peptide analysis. 

306 



P03-038 PURIFICATION OF ANGIOTENSIN-CONVERTING-ENZYME FROM PIG LUNG 

Eisele T., Stressler T., Kranz B., Fischer L. 

University of Hohenheim, Biotechnology-Germany 

Corresponding author e-mail: lfischer@uni-hohenheim.de 

Angiotensin-I-converting-enzyme (ACE; EC: 3.4.15.1) is a membrane bound zinc-metallopeptidase which plays a 

major role in the renin-angiotensin system[1,2]. ACE hydrolyses the dipeptide histidylleucine from the decapeptide 

angiotensin I and generates the potent vasoconstrictor angiotensin II[3], an octapeptide. Furthermore, ACE 

inactivates bradykinin, a vasodilating peptide[4,5]. ACE was solubilised from pig lung with 50 mM Tris-buffer pH 8.5 

over 48 h. In cell free supernatant after centrifugation a volumetric activity of 2134 U/L, and a specific activity of 

0,24 U/mg protein was detected. ACE was purified in a five step purification method including CaCl2 precipitation, 

a strong anion exchange, hydrophobic interaction chromatography, Cibacron and glycly-proly-EAH-Sepharose 4B 

affinity chromatography. ACE activity was determined with Hippuric-His-Leu (HHL) as substrate using UHPLC. After 

purification the specific activity of ACE was 300 times increased to final specific activity of 72 U/mg with a yield 

of 26 %. The purified ACE was revised by SDS PAGE (silver stained) and showed a major band at ~ 175 kDa which 

is in accordance to the literature[6]. In order to investigate the purified ACE sample for other peptidase activities 

angiotensin I was tested as substrate and analysed by UHPLC-MS. Since only angiotensin II was detectable it can 

be concluded that the purified ACE (72U/mg protein) is free of other peptidases. Inhibitory studies showed that ACE 

was completely inibited by EDTA (1 mM) and Lisinopril (10 µM), a specific pharmaceutical inhibitor. Literature: [1] 

K.K.F., NG and Vane, J.R., (1967). Conversion of Angiotensin I to Angiotensin II. Nature. 216 (5117). 762-766. [2] D.W. 

Cushman and Cheung, H.S., (1971). SPECTROPHOTOMETRIC ASSAY AND PROPERTIES OF THE ANGIOTENSIN- 

CONVERTING ENZYME OF RABBIT LUNG. Biochemical Pharmacology. 20 (7). 1637-1648. [3] L.T. Skeggs, W.H. 

Marsh, J.R. Kahn and Shumway, N.P., (1954). THE EXISTENCE OF TWO FORMS OF HYPERTENSIN. The Journal of 

experimental medicine. 99. 275-282. [4] S.H. Ferreira and Vane, J.R., (1967). THE DETECTION AND ESTIMATION 

OF BRADYKININ IN THE CIRCULATING BLOOD. British Journal of Pharmacology. 29 (3). 367-377. [5] F. E. Dorer, 

J.W. Ryan and Stewart, J.M. (1974) Hydrolysis of Bradykinin and its Higher Homologues by Angiotensin-Converting 

Enzyme. Biochemical Journal. 141 (3), 915-917. [6] M. Andujar-Sánchez, A. Cámara-Artigas and JaraPérez, V. (2003). 

Purification of angiotensin I converting enzyme from pig lung using concanavalin-A sepharose chromatography. 

Journal of Chromatography B. 783 (1), 247-252. 


307


P03-039 PURIFICATION OF A PROLINE SPECIFIC EXOPEPTIDASE FROM LACTOBACILLUS HELVETICUS 

Stressler T., Eisele T., Kranz B., Fischer L. 

University of Hohenheim, Biotechnology-Germany 

Corresponding author e-mail: lfischer@uni-hohenheim.de 

The cyclic structure of proline is unique among the 20 proteogenic amino acids. Proline introduces a fixed bend 

into the polypeptide chain and serves as a structure breaker [1]. The generation of biologically active peptides in 

human body from its precursors involves the action of endopeptidases, which cleave the peptide chain at marked 

positions. The shortened chain is exposed to the proteolytic action of exopeptidases. In most cases the resulting 

peptides have proline residues at the ends of the polypeptide chain (Xaa-Pro bond) and limiting the susceptibility 

to proteolytic degradation [2]. This kind of protective effect is also important for the incomplete digestion of food 

proteins in industrial protein hydrolysis. Additionally the length of the resulting peptides and the position of the 

proline residues are major factors for the bitterness of protein hydrolysates [3]. The proteolytical systems of lactic 

acid bacteria are a rich source of proteolytic enzymes. The exopeptidases are classified by their specificity, like 

the general aminopeptidases and the proline-specific exopeptidases (e.g. PepX) [4]. With the use of proline specific 

exopeptidases, a further hydrolysis of proline containing peptides is possible [2, 3]. So the bitterness, as well as the 

allergenic potential could be reduced [3, 5]. Beside the debittering, the application for the proline specific peptidase 

PepX is to produce bioactive peptides (e.g YP and FP). It is known that small peptides, such as di- and tri-peptides, 

are easily resorbed in the intestine [6]. In our studies a X-prolyl-dipeptidyl aminopeptidase (EC 3.4.14.11, PepX) from 

Lactobacillus helveticus ATCC 12046 was produced by cultivation in MRS broth, the cells were disrupted with glass 

beads and the PepX was purified. The purification included a strong anion exchange chromatography step and two 

affinity chromatography steps (Zn2+ IMAC and glycyl-prolyl-EAH-sepharose 4B). The purified enzyme appeared as 

a single band on native polyacrylamide-gelelectrophoreses (PAGE) with silver staining and had a molecular weight 

of about 160 kDa. The SDS PAGE showed a strong band which was calculated with 96 kDa, so the enzyme is most 

likely a homodimere. The purification factor was 171 with a yield of 5.3%. The purified PepX had a specific activity 

of 369 nkat/mg with H-Ala-Pro-pNA as substrate (pH 6.5 / 37°C). Literature [1] Cunningham, D. F. and O’ Conner, 

B. 1997. Biochim. Biophys. Acta-Protein Struct. Molec. Enzym. 1343 (2): 160-186. [2] Yaron, A. 1987. Biopolymers. 

26: 215-222. [3] Fitzgerald, R. J. And O’ Cuinn, G. 2006. Biotechnol. Adv. 24 (2): 234-237. [4] Kunji, E.R.S., Mierau, 

I., Hagting, A. Poolman, B. and Konings, W.N. 1996. Antonie van Leeuwenhoek. 70 (2-4): 187-221. [5] Wang, W. and 

Gonzalez De Mejia, E. 2005. Compr. Rev. Food. Sci. Food Saf. 4 (4): 63-78. [6] Yamamoto, N., Ejiri, M. und Mizuno, S. 

2003. Curr. Pharm. Des. 9 (16): 1345-1355 

308 



P03-040 RP-HPLC OF MALONDIALDEHYDE IN BLOOD PLASMA OF PATIENTS WITH ACUTE MYOCARDIAL INFARCTION 

Tomandl J., Pes O., Parenica J. 


Corresponding author e-mail: tomandl@med.muni.cz 

Malondialdehyde (MDA), one of by-products of both enzymatic or oxygen radical-induced lipid peroxidations, is 

widely used as a lipid peroxidation marker. In vivo MDA exists as a free molecule or bound to proteins or nucleic 

acids. When heated with 2-thiobarbituric acid (TBA) at acidic pH a red fluorescent MDA(TBA)2 adduct is formed, 

which may be detected by spectrofluorometry. We present here a new reverse-phase HPLC of MDA in plasma 

samples from healthy subjects and from treated patients with acute myocardial infarction with cardiogennic shock. 

First, samples were derivatized with TBA and then formed adducts were isocratically separated on a reversedphase 

column and fluorimetrically detected at an excitation wavelength of 532 nm and an emission wavelength of 

551 nm. The method was linear over 2 orders of magnitude with limit of detection as low as 0.2 nmol/L. Intra- and 

inter-assay precisions were within 2.8 and 6.2 %, respectively, for plasma MDA concentration of 540 nmol/L. The 

presented method was compared with other one commonly used (1). Moreover, stability of formed adduct and 

also the effect of fat-soluble antioxidant BHT on the stability MDA during plasma sample processing was verified. 

The proposed method was used to evaluate a week course of plasma MDA level in patients with acute myocardial 

infarction and cardiogennic shock. The method has been sufficient for the precise and reliable determination of MDA 

in plasma sample, although, some drugs may interfere and are discussed here. Acknowledgements: This work was 

supported by Ministry of Education (LC06023 and MSM 0021622402) and by the Grant Agency of Czech Republic 

(P206/10/0057). Reference: (1) GA Khoschsorur et al.: Chromatographia 2000, 52, 181-184. 


309


P03-041 APPLYING QUALITY BY DESIGN CONCEPTS TO CHROMATOGRAPHIC METHOD DEVELOPMENT 

Ponzio T., Sinclair T., Cimpan G. 

ACD/Labs, D 

Corresponding author e-mail: gabriela.cimpan@acdlabs.com 

Quality by Design is a concept that garners much attention by the pharmaceutical industry in the quest for greater 

safety and speed in bringing novel compounds to market. To be successful, Quality by Design must be applied 

at every stage in the development and manufacturing process; however, one can consider processes within drug 

discovery on an individual basis to apply Quality by Design principles. One such process is the development of 

chromatographic methods for impurities and degradant studies. Ensuring both robustness and optimization from an 

efficiency standpoint is time-consuming and difficult. Generally method development is carried out using a 

trial-and-error approach, and requires a large amount of manual data interpretation. Quality is achieved when 

Quality by Design is applied to the stability study process by producing well developed, traceable, error free results, 

and finally into the drug product by bringing novel products to market faster, and more cost effectively. 

310 



P03-042 HPLC DETERMINATION OF CAFFEINE AND PARAXANTHINE IN SALIVA AS A METHOD FOR CYP1A2 METABOLIC ACTIVITY 

ASSESSMENT IN HUMANS 

Jurica J., Zendulka O., Tomandl J. 


Corresponding author e-mail: tomandl@med.muni.cz 

Introduction: Caffeine (1,3,7 trimethylxanthine) is widely used as a probe substrate for phenotypization of 

N-acetyltransferase and cytochrome P450 1A2 (CYP1A2) enzymes. For the latter reason, N-demethylation of 

caffeine is used as a marker reaction. Ratio of molar concentrations of caffeine and its N-demethylated metabolite 

paraxanthine (1,7 dimethylxathine) serves as a measure of CYP1A2 metabolic activity. Methods: Presented 

method allows assessing concentrations of caffeine and paraxanthine in human saliva after oral administration. 

Paracetamol (4-hydroxyacetanilide) was used as an internal standard (I.S.) to ensure precision and repeatability of 

the method. After an addition of I.S. and pH adjustment, the samples were extracted into the mixture of solvents 

(chloroform: isopropanol 85:15, v/v), and dried out under a gentle stream of nitrogen. The residues of the extracts 

were dissolved in 250 ul of mobile phase (methanol:acetic acid 0.05%; 25:75 v/v) and 50 ul was injected in the HPLC 

system (Shimadzu LC 10 A vp). The samples were separated on Kinetex PFP column (150x4.6 mm, 2.6 um) using 

isocratic elution (0.55 mL/min). The analytes were quantified at 268 nm using diode array detector. The usability of 

the method was proven with real samples of saliva from 3 healthy volunteers. The marker was administered as an 

oral capsule (250 mg) after at least 24 hour wash-out period (no caffeine in diet). Saliva samples were collected 3, 

4 and 6 hours after caffeine administration. Results: The retention times of all the analytes were up to 13 min. The 

limits of detection were 3.54 and 4.35 ng/mL for caffeine and paraxanthine (S/N ratio = 5). The extraction recoveries 

of the analytes were better than 69 %. Inter-day repeatability of the method was sufficient for routine usage; RSD 

does not exceed 10 %. The concentrations of the analytes in real samples were far above the limits of detection. 

Conclusion: Thanks to the simplicity, precision, sensitivity and repeatability, this method seems to be suitable for 

CYP1A2 phenotypization in human pharmacokinetic studies. Supported by the Ministry of Education projects (MSM 

0021622404 and LC 06023) and by the Grant Agency of Czech Republic (P206/10/0057). 


311


P03-043 INFLUENCE OF EXTRACTION VARIABLES ON FATTY ACID COMPOSITION AND TRIACYLGLYCEROL PROFILE OF DURIAN 

SEED GUM 

Hamed Mirhosseini 

Faculty of Food Science and Technology, Universiti Putra Malaysia 

Influence of Extraction variables on fatty acid composition and triacylglycerol profile of Durian Seed Gum Bahareh 

Tabatabaee Amid, Parviz Kavousi, Farhad Farivar and Hamed Mirhosseini* Department of Food Technology, 

Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 

*Corresponding author. Tel.: +603-89468390; fax: +603-89423552. E-mail address*: hamedmi@food.upm.edu. 

my Abstract The present study was conducted to evaluate the effect of type of solvent on extraction efficiency, 

fatty acid composition and triacylglycerol profile of jackfruit seed oil. For different solvent mixtures namely 

petroleum ether plus methanol (PEM), hexane plus methanol (HM), ethanol plus methanol (EM) and 60% hexane 

plus 40%isopropoanol and methanol (HIPM) were employed in this study. It should be noted that the same volume 

of methanol was considered for all the treatments in order to bind the moisture and subsequently improve the 

extraction efficiency. The results indicated the highest and least extraction efficiency was obtained by using solvent 

EM and HIPM, respectively. As shown in the results, the extraction using ethanol and methanol significantly 

(p < 0.05) resulted in the highest color intensity compared to the oils extracted by using the other solvents PEM, HM 

and HIPM. The major fatty acid compositions identified by using gas chromatography-mass spectrometry (GC-MS) 

were: palmitic (P, C16:0), stearic (S, C18:0), oleic (O, C18:1), linoleic (L, C18:2), linolenic (Ln, C18:3; α, γ unspecified), 

gadoleic (G, C20:1), arachidic (A, C20:0), behenic (B, C22:0) and lignoceric (Li, C24:0). In the present study, the liquid 

chromatography-mass spectrometry (LC-MS) was applied to identify the main triacylglycerol (TAG) profile of jackfruit 

seed oil extracted by using four different solvents. The results also indicated that the main triacylglycerol profile of 

jackfruit seed oil composed of LLL, POP, LLO, LnOO, OOO, POO, POS, PLP, OLO, PLS and PLL. 

312 



P03-044 MULTI-MYCOTOXIN ANALYSIS IN EGGS USING A BASED QUECHERS EXTRACTION PROCEDURE AND ULTRA-HIGH- 

PRESSURE LIQUID CHROMATOGRAPHY COUPLED TO TRIPLE QUADRUPOLE MASS SPECTROMETRY 

Gómez Perez M.L., Romero-Gonzalez R., Garrido Frenich A., Martinez Vidal J.L. 


Corresponding author e-mail: rromero@ual.es 

Mycotoxins are secondary metabolites generated by many species of filamentous fungi, and currently, more than 

400 mycotoxins have been identified in the world. They are toxic and pose a health hazard to humans and animals. 

This toxicity can range from the induction of carcinogenic, teratogenic or mutagenic activities to the production of 

several hormonal disorders or immunosuppression. The presence of these compounds and their metabolites in food 

of animal origin such eggs could be consequence of feed contamination. In this sense, there is not specific European 

legislation about maximum limits of mycotoxin concentration present in egg samples but it should be established for 

a better control of human safety. For this reason the development of analytical methods that allow an unambiguous 

identification, quantification and detection at very low concentration levels are necessary. For detection and 

quantification, liquid chromatography coupled to mass spectrometry (LC-MS) is a suitable technique for the analysis 

of polar substances, like mycotoxins. For instance, LC using several analysers such a single quadrupole, time 

of flight (TOF) or tandem mass spectrometry (MS/MS) have been the most applied methods. In recent years, the 

introduction of ultra high performance liquid chromatography (UHPLC) has provided a new potential for method 

development and analysis. The purpose of this work has been to develop a simple and efficient UHPLC- MS/MS 

multi-mycotoxin analytical method for the simultaneous determination of enniatins A, A1, B1, aflatoxins B1, B2, G1, 

G2, citrinin, ochratoxin and beauvericin in eggs at trace levels with a chromatographic analysis time lower than 7 

min. Mycotoxins have been extracted from egg samples using a based QuEChERS extraction procedure without 

applying any further clean-up step. Extraction, chromatographic and detection conditions were optimised in order 

to increase sample throughput and sensitivity. Full-scan mass spectra and product ion scan were acquired in order 

to obtain at least one precursor and two product ions for each compound for both identification and quantification 

purposes, selecting the most abundant product ion for quantification and the second one for confirmation. The 

performance characteristics of the developed method have been evaluated (i.e. linearity, recovery, precision) in order 

to obtain reliable information regarding the presence or absence of these compounds. Matrix-matched calibration 

was used for quantification and recoveries of the extraction process ranged from 70% to 110%, with relative 

standard deviations lower than 25% in all the cases, when samples were fortified at 10, 25, 50 and 100 μg/kg. Limits 

of detection ranged from 0.5 μg/kg (for aflatoxins B1, B2 and G1) to 5 μg/kg (for enniatin A, citrinin and ochratoxin) 

and limits of quantification ranged from 1 μg/kg (for aflatoxins B1, B2 and G1) to 10 μg/kg (for enniatin A, citrinin 

and ochratoxin). Acknowledgments We gratefully acknowledge Spanish Ministry of Science and Innovation-FEDER 

(SMSI-FEDER, CTQ2009-07686) for financial support. RRG is also grateful for personal funding through the Ramón y 

Cajal Program (SMSI-EFS). 


313


P03-045 EVALUATION OF POROUS AND CORE-SHELL PARTICLES FOR USE IN LC-MS BIOANALYSIS 

Butchart K. 1 , Woodruff M. 1, Saunders K. 2 

1 

Pfizer, Sandwich, Kent 

2 


Corresponding author e-mail: kenbutchart@fortis-technologies.com 

In recent years much has been made of speed in ultra high pressure liquid chromatography (UHPLC) which has 

become ever more popular, analysts can now utilise smaller particles to increase efficiency of the separation, leading 

to increased sensitivity, speed and/or resolution. Another option that has appeared is the use of “fused-core” or 

“core-shell” particles, which have the potential benefit of efficiency without the pressure of UHPLC particles. 

In this poster we discuss the various options available for high throughput bioanalysis and assess some of the 

implications of these technologies in comparison to traditional 3mm particle HPLC configurations. Spiked blood 

samples are analysed, at typical concentrations from pharmokinetic studies. 

We consider fundamental issues such as peak capacity, peak width, surface area and matrix effects and how they 

may compromise the resolution that can be achieved. 

314 



P03-046 RESOLUTION VERSUS EFFICIENCY OF COMPLEX MIXTURES IN UHPLC 




The current trend towards using high pressure in LC is well-documented, high efficiencies; good resolution and fast 

throughput can all be achieved. However this requirement for speed and its relation to generic gradient conditions 

has negated one of the most important needs in chromatography; selectivity. 

Whilst MS can also provide resolution, for critical applications LC resolution is still necessary in order to reduce 

matrix effects and maintain sensitivity. We discuss the use of LC across the entire, low, mid and high pH range. The 

ability to move to smaller particles and the implications that this has on analyte response, selectivity and loadability. 

We discuss the use of phase chemistry within UHPLC, and its use in order to regain the selectivity that has been lost 

by the use of C18 chemistry and generic gradient conditions alone. 

Applications highlighting resolution, efficiency and sensitivity gained by small particle efficiency and alternative 

phase chemistries are all highlighted. Resolution of complex mixtures can be achieved by the correct selection of 

phase chemistry, gradient length and flow rate for 2.1um particles, providing the analyst with a powerful analytical 

tool. 


315


P03-047 WHAT SHOULD WE RELY ON IN UHPLC? ARE WE REALLY BEING MORE PRODUCTIVE? 

Woodruff M., Butchart K. 



The Carr equation outlines the contribution of each variable in the resolution equation. Recent interest has revolved 

around the ability to move to smaller and smaller LC particles in order to increase efficiency and gain the advantages 

that this can provide as the inlet to MS. 

Whilst MS can provide resolution, for critical applications LC resolution is still necessary in order to reduce matrix 

effects and maintain sensitivity. We discuss the use of LC across the entire, low, mid and high pH range and the 

ability to move to smaller particles and the implications that this has on analyte response, sensitivity and loadability 

in the MS detector. 

We show the compatibility of the LC system and the MS detector with particular reference to the variables, flow rate, 

temperature and pH and the effect this has on sensitivity, resolution and peak capacity. 

How do these compatibility issues influence our ‘real world’ results and thought process of increasing speed. Does 

more speed automatically lead to more productivity? How does the robustness of the whole process affect our 

process? 

The Carr equation shows us that selectivity actually provides more resolution than efficiency alone, therefore should 

we rely on UHPLC at all as an interface for MS. 

We show how achieving large peak capacities in the separation process is still necessary for highly complex 

samples. How can we gain peak capacity? we look at the options! 

Overall we compare the compatibility of UHPLC with MS, are we becoming more or less productive with reliance on 

UHPLC? does speed gain us everything? 

316 



P03-048 APPLICATIONS OF A NEW HILIC STATIONARY PHASE 




Hydrophilic Interaction Chromatography (HILIC) is an ever popular and growing area of interest for the retention 

and separation of more polar analytes than are achievable on traditional reversed phase stationary phase systems. 

Moving towards the use of normal phase conditions HILIC chromatography offers the ability to retain very polar 

molecules without the need for complex mobile phase systems, ion-pair reagents or other buffers that weaken the 

ability to utilise the advantages of MS as a detection technology. 

We discuss the use of a new HILIC stationary phase for the separation and retention of polar analytes, we compare 

this to other LC techniques for the retention of polar analytes, such as high pH and ion-pair chromatography, and we 

highlight the relative strengths and weaknesses. 

Applications highlighting the unique selectivity that can be achieved with simple mobile phases and a HILIC 

stationary phase are shown. Examples of the molecular structures that can be successfully retained are shown in 

comparison with their reversed phase comparisons. The advantages of HILIC chromatography as a technique are 

discussed. 


317


P03-049 INVESTIGATION ON THE ANALYSIS AND QUANTIFICATION OF FULLERENES IN REAL AEROSOL SAMPLES 

Sanchis J., Berrojalbiz N., Caballero G., Dachs J., Farre M., Barceló D. 

IDÆA-CSIC, Departament de Química Ambiental, Barcelona 

Corresponding author e-mail: mfuqam@cid.csic.es 

Carbon-based nanopartícules are present in the environment due to different causes including natural events, 

incidental sources, as industrial and combustion processes, and during the recent years due to the use 

and production of carbon based nanomaterials for nanotechnological applications. Therefore, occurrence 

assessment of carbon-based nanopartícules is required for determining their environmental cycling and impacts. 

This work describes a new method to assess the presence of natural and synthetic fullerenes (C60, C70, 

N-methylfulleropyrrolidine, C60 pyrrolidine tris-acid ethyl ester, [6,6]-Phenyl-C61 butyric acid butyl ester and 

[6,6]-Thienyl C61 butyric acid methyl ester) in airborne particles based on ultrasonic extraction followed by 

LC-MS/MS, is presented. This method has sensitivities in the pg/m3 range, with repeatabilities between 3,7% and 

15,6% and it has been applied to the study of aerosol samples from the Mediterranean Sea atmosphere. While the 

presence of fullerenes in wastewater [1] and its occurrence in the atmospheric particulate (mainly associated with 

coal combustion processess [2] and domestic kitchen stoves burning natural gas/air and propane gas/air mixtures 

[3]) have already been reported, to our knowledge, this work is the first study to be about the occurrence and 

quantification of fullerenes in sea airborne. The obtained results can be reasonably related to incidental emission 

and posterior atmospheric transport and deposition, underpinning the need of studying the possible risks associated 

to the presence of carbon nanopartícules in the environment and the need of evaluating the possible consequences 

of its ubiquitous distribution, which is facilitated by long range atmospheric transport, and their increase during 

next coming years. Keywords: Fullerenes, C60, C70, N-methylfulleropyrrolidine, nanomaterial, nanoparticles, 

wastewater, aerosols, LC-MS/MS, LC-(ESI)-MS [1] M. Farre et al. “First determination of C60 and C70 fullerenes 

and N-methylfulleropyrrolidine C60 on the suspended material of wastewater effluents by liquid chromatography 

hybrid quadrupole linear ion trap tandem mass spectrometry”, Journal of Hydrology, 2010, 383 (1-2):44-51 [2] S. 

Utsunomiya et al. “Uraninite and Fullerene in Atmospheric Particulates”, Environmental of Science and Technology, 

2002, 36 (23): 4943-4947 [3] L. E. Murr et al. “Carbon nanotubes, nanocrystal forms, and complex nanoparticle in 

common fuel-gas combustion sources and the ambient air”, Journal of Nanoparticle Research, 2004, 6(2): 241-251 

318 



P03-050 IN VITRO TRANSPORT EVALUATION OF CHITOOLIGOSACCHARIDES MIXTURES THROUGH INTESTINAL EPITHELIA BY SEC-HPLC 

Fernandes J.C. 1 , Cardelle-Cobas A. 2 , Pintado M. 1 , Corzo N. 2 

1 

Escola de Biotecnologia e Quimica Fina. Universidade Católica Portuguesa 

2 


Corresponding author e-mail: acardelle@ifi.csic.es 

Chitooligosaccharides (COS) – depolymerized products of chitosan obtained by chemical or enzymatic 

hydrolysis, have recently attracted much attention as a potential therapeutic agent. These chitosan derivatives 

(generally, the molecular weight of COS is 20 kDa or less), also seem to possess several biological properties as 

immunostimulatory, anti-inflammatory, antioxidant or antibacterial among others. Furthermore, their ready uptake 

by cells and intestine makes theoretically possible for COS to be accessible to the entire human body, which 

enhances the range of possible applications. The aim of the present study was to evaluate the transport through 

the intestinal epithelia of two different COS mixtures (COS3 and COS5) at different concentrations (0.1, 1, 5 and 10 

mg/mL). Transport through the intestinal epithelia was evaluated in vitro using Caco-2 cells (as they form confluent 

monolayers of well-differentiated enterocyte-like cells, with the functional property of transporting epithelia), and 

using also a mixture (10:1) of Caco-2 and mucus cells. Transported COS were measured by sampling at 15, 30 

60 and 180 minutes. Solutions of COS, before and after to pass through cells, were analyzed by Size Exclusion 

Chromatography (SEC). Analyses were carried out using a TSKGel column (G2500PWXL ) (7.8 mm ID x 30.0 cm 

L) thermostated at 25ºC and refractive index detection (RID-10A Shimadzu). Separations were performed at a flow 

rate of 1.0 mL.min-1 and the elution was in isocratic using as mobile phase a solution of 0.5M AcOH-0.2M AcONa 

(pH 4.4-4.5). Sample injection was 50L. Pullulan of different molecular weights were used as standards, and 

quantification of COS transported through cells was performed by external calibration using chitobiose as standard. 

Acquisition and processing of data were achieved with Chromeleon software version 6.7. 

SEC-HPLC analysis of solutions obtained from cellular transport showed, in all performed assays, the presence of 

COS. This content increased with treatment time and concentration. When using the mixture of Caco-2 and mucus 

cells, COS concentrations after transport, independently of the sampling time or initial concentration, were higher 

than in the Caco-2 cells system. No significant differences between both COS mixtures were found. When 0.1, 1 

and 5 mg/mL of COS where added to cells, an increase of COS content was observed up to 60 min of transport. 

However, when 10 mg/mL were used, a significant decrease in the concentration after 30 min was observed, which is 

most probably related with reported cytotoxic effect by COS, upon human cells. 

Acknowledgements: This work was financed under the projects AGL 2008-00941/ALI, ALIBIRD-CM-P 2009/AGR 

1469and CONSOLIDER Ingenio 2010Fun-C. Food CSD 2007-00063. 


319


P03-051 TEMPERATURE ASSISTED HILIC SEPARATION OF NUCLEOSIDE TRIPHOSPHATES 

Wilson S., Johnsen E., Odsbu I., Lundanes E. 


Corresponding author e-mail: stevenw@kjemi.uio.no 

Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, 

dCTP, dGTP and dTTP, were separated with two 15 cm, 2.1. mm I.D. ZIC-pHILIC columns coupled in series, using 

simple LC-UV instrumentation. A polymer based ZIC-pHILIC column gave vastly better separations and peak shape 

than a silica based ZIC-HILIC column. Curiously, better separations and peak shapes were obtained with isocratic 

elution as compared to gradient elution. The temperature markedly affected the selectivity and could be used to 

fine-tune the separation. The analysis time was also affected by temperature, as lower temperatures surprisingly 

reduced the retention of the nucleotides. An explanatory model is presented in the poster. The model is based on the 

hypothesis of an existence of nucleotide/water clusters, which disassociate at elevated temperatures, shifting the 

equilibrium towards the aqueous layer HILIC stationary phase. The dNTP/NTPs were separated in 35 minutes with 

ACN/100mM ammonium carbonate (70/30, v/v) with a flow rate of 0.2 mL/minute. 

320 



P03-052 DEVELOPMENT OF A STABILITY INDICATING METHOD FOR SIMVASTATIN USING A QBD APPROACH WITH 

EXPERIMENTAL DESIGN 

Alden P.G., Potts W., Moore D., Yurach D. 

Waters Corporation, Pharmaceutical Market Development-USA 

Corresponding author e-mail: peter_alden@waters.com 

Throughout the drug development process, methods are developed at various stages, often consisting of samples 

that vary in complexity. Due to the inherent nature of this process, redundant efforts take place across an 

organization, resulting in a very costly and time consuming process. If we can streamline the process by which we 

develop methods, products can be brought to market faster and in a more cost effective manner. 

Many different approaches are typically used to develop chromatographic methods today including trial and error, 

method/column scouting, and software approaches such as first principles approaches and simplex optimization 

procedures. All these approaches are time consuming and suffer from the inability to determine complex 

interactions effects between method variables or measurably consider method robustness during the method 

development process. 

This paper describes a novel method development approach using Quality by Design (QbD) with Design of 

Experiments to develop HPLC and/or UPLC® methods resulting in optimally performing analytical methods while 

simultaneously applying robustness limits to ensure success in final method validation and ultimately in method 

transfer. 

Simvastatin, (marketed under the trade names Zocor, Simlup, Simcard, Simvacor, and others, as well as 

generically) is a hypolipidemic drug belonging to the class of pharmaceuticals called “statins”. It is used to control 

hypercholesterolemia (elevated cholesterol levels) and to prevent cardiovascular disease. Simvastatin is a synthetic 

derivate of a fermentation product of Aspergillus terreus. 

The development of a stability indicating method for the separation of impurities in forced degradation samples 

of Simvastatin using a QbD with Design of Experiments approach is shown. Forced degradation samples can 

be quite complex mixtures of impurities making method development extremely challenging. Many variables are 

studied including different column chemistries, buffer pH, organic mobile phase, flow rate, column temperature, and 

gradient conditions and the different types and degrees of variable interactions are discussed. In addition to method 

development, a measure of robustness of the analytical method is determined giving confidence that the method can 

be successfully validated without the need to re-optimize. 


321


P03-053 REFERENCE METHOD FOR THE ABSOLUTE QUANTIFICATION OF TROPONIN I IN SERUM USING HIGH PERFORMANCE 

LIQUID CHROMATOGRAPHY AND TRIPLE QUADRUPOLE MASS SPECTROMETRY 

Dullnig V. 1 , Kobold U. 2 , Hallermayer K. 2 , Huber C.G. 1 

1 

University of Salzburg- Austria 

2 

Roche Diagnostics, Penzberg-Germany 

Corresponding author e-mail: verena.dullnig@sbg.ac.at 

Introduction: Troponin (Tn) is a regulatory protein of the thin filament of striated muscle. It consists of three subunits 

(C, I and T) and is essential for the calcium-mediated regulation of skeletal and cardiac muscle contraction. The 

cardiac forms of Tn (cTnT and cTnI) are important markers for diagnosis of acute myocardial infarction, which are 

usually measured by immunoassays. Due to the lack of calibration standards of known absolute concentration, we 

aim in this study at developing a reference method capable of determining the absolute concentration of cTnI in 

serum with a lower limit of detection in the clinically relevant low femtomol per milliliter range. 

Methods: Software-supported method setup (Thermo Scientific Pinpoint) was utilized to create a starting method for 

multiple reaction monitoring (MRM) in a triple quadruple mass spectrometer (Thermo Scientific TSQ Vantage) of 68 

candidate peptides with 1667 MRM transitions and theoretically predicted collision energies. Prior to analysis, cTnI 

was denatured, reduced, alkylated, and tryptically digested. After trapping in a 10 x 0.2 mm i.d. monolithic poly 

(styrene-divinylbenzene) (PS-DVB) column with 0.1% heptafluorobutyric acid in water, the peptides were separated 

by capillary ion-pair reversed-phase HPLC (Dionex Ultimate3000) in a 300 x 0.20 mm i.d. monolithic PS-DVB column 

with an acetonitrile gradient in 0.050% trifluoroacetic acid. Upon experimental evaluation using the cTnI digest, the 

list of candidate peptides was narrowed down to 8 peptides with 53 transitions. The optimal collision energies for 

all transitions were determined experimentally. Results: Using the established MRM assay the limit of detection for 

a digested cTnI standard was determined at 800 fmol/mL. Further system miniaturization by using a smaller column 

inner diameter of 0.10 mm facilitated the detection 50 fmol/mL cTnI. In serum, substantial ion suppression was 

observed, which necessitated further sample preparation by chromatographic prefractionation and the application 

of longer gradients in order to increase the peak capacity for the peptide separation. For the measurement of clinical 

samples we therefore followed two different strategies, namely the depletion of high-abundant proteins by ion-pair 

reversed-phase chromatography or the direct analysis of digested serum using miniaturized capillary HPLC. Novel 

aspect: Traditionally, immunoassays are used for clinical validation of biomarkers because of their high sensitivity 

and specificity. At the moment however quantitative results of cTnI immunoassays from different vendors show 

considerable variance. Analysis using the proposed reference method will allow to assign an absolute concentration 

to the different immunoassays available, which will then facilitate to define an absolute scale for cTnI concentrations 

causing clinically relevant conditions. 

322 



P03-054 NUCLEOTIDE AND SUGAR NUCLEOTIDE ANALYSIS BY LC-ESI-MS ON PRETREATED POROUS GRAPHITIC CARBON 

Pabst M.., Grass J., Léonard R., Altmann F. 

University of Natural Resources and Applied Life Sciences, Department of Chemistry-Austria 

Corresponding author e-mail: friedrich.altmann@boku.ac.at - Muthgasse 18 - 1190 - Vienna – Austria 

Nucleotides and nucleotide sugars are pivotal components of metabolic and anabolic pathways of cells. Their polar 

nature and chemical lability as well as the occurrence of isobaric structures render their determination a difficult 

task. Here we examined the analysis of a wide range of nucleotides and nucleotide sugars by chromatography on a 

reduced porous graphitic carbon column in capillary format with mass spectrometric detection using a fully volatile 

buffer without ion pairing reagents. Column regeneration and reduction/oxidation steps ensured stable elution times 

and good peak shapes of all analytes. A rapid sample preparation procedure was developed that allowed handling 

and detection of the very short-lived bacterial CMP-2-keto-desoxy-octulosonic acid in E.- coli. The chromatography 

revealed unexpected isomers of cyclo-, mono-, di and triphosphates of various nucleotides. Rarely considered 

nucleotide sugars such as UDP-L-arabinopyranose, UDP-L-arabinofuranose, GDP-L-galactofuranose, 

UDP-L-rhamnose, GDP-sulfoquinovose, ADP-ribose as well as ADP-glucose were detected in plant extracts. 

Corresponding peaks for UDP-Xyl as well as UDP-Arap were found in CHO-cells. The labile CMP-Neu5Ac as well 

as CMP-Neu5Gc were found in liver extracts from mouse. The overall time consumption from harvesting the plants 

(1-10 mg of fresh weight) or cells (CHO-cells, 105) to the ready prepared samples for LC-ESI-MSMS analysis is 

approximately 1 hour. Cycle times for the LC-ESI-MSMS runs can be scaled down to 15 minutes. Peak detection and 

quantification was done employing the automatic data mining software MassMap. 


323


P03-055 Development of a low cost, analytical process to extract, separate and determine efavirenz and rifampicin plasma 

concentrations in HIV/TB co-infected patients 

Fox D. 1 , O’Connor R. 2 3 , Mallon P. 4 , McMahon G. 1 

1 

School of Chemical Sciences, Dublin City University 

2 

National Institute of Cellular Biotechnology, Dublin City University 

3 

School of Nursing, Dublin City University 

4 

Mater Misericordiae University Hospital, Dublin 

Tuberculosis (TB) complicating HIV-1 infection is a persistent significant clinical concern, particularly in resource 

limited settings. Treatment of HIV-infected patients with TB often comprises efavirenz-containing antiretroviral 

regimens particularly when on TB treatment containing rifampicin. Although rifampicin may reduce EFV 

concentrations, little is known of the effect of the HIV virus or efavirenz on the pharmacokinetics of rifampicin, 

particularly in resource limited settings where the burden of disease exists. 

Our focus has been the development of a rapid, simple and novel analytical protocol for extraction, separation and 

determination of both efavirenz and rifampicin (and the major metabolite of rifampicin) with a view to being able to 

monitor plasma levels of these drugs simultaneously over time. This work is part of an EU SPhEAR (Study on the 

Pharmacokinetics of Efavirenz And Rifampicin) project which will examine the effects of efavirenz medication on the 

pharmacokinetics of oral rifampicin in the treatment of TB in co-infected patients. The novel protocol will be used to 

analyse the blood samples collected from patients on the SPhEAR study. 

Despite the drugs very different physiochemical properties (affecting hydrophobicity, solubility and acidity) we 

developed and validated a low-cost, simple analytical method for the accurate determination of efavirenz, rifampicin 

and deacetylrifampicin in plasma using high-performance liquid chromatography (HPLC) employing UV detection. 

Recovery for plasma samples spiked with the drugs were >90% for rifampicin and deacetylrifampicin and >70% for 

efavirenz. Intra- and inter-assay precision relative standard deviation (RSD) values were

P04 ELECTRODRIVEN 

SEPARATIONS 

POSTER 

SESSIONS

POSTER SESSION 04 - ELECTRODRIVEN SEPARATIONS 

P04-001 IDENTIFICATION OF ENZYMES IN DETERGENTS BY CAPILLARY ELECTROPHORESIS 

Beneito-Cambra M., Herrero-Martínez J.M., Ramis-Ramos G. 


Today enzymes constitute important components of laundry, dishwashing and other cleaning products. Enzymes 

for cleaning products constitute the largest segment of the world market for industrial enzymes. Cleaning power 

enhancement by enzymes leads to a reduction of washing times and temperatures, with the subsequent savings of 

water and energy. Further environmental advantages arise from the lower consumption of surfactants, hypochlorite 

and alkalis, with the additional benefit of a better care of fabrics during washing. In this work, enzymes for the 

detergent industry, including proteases, lipases, amylases and cellulases, were classified and identified using 

capillary electrophoresis with UV detection at 214 nm. For this purpose, the proteins present in industrial enzyme 

concentrates were diluted with water and aliquots were injected in the capillary. First, an aqueous BGE containing 

50 mM iminodiacetic acid, 6 M urea and 0.5% hidroxyethylcellulose at pH = 3.1, aqueous proteins solutions 

containing 3 M urea was used. With this acidic BGE, almost half of the proteases gave electropherograms showing 

two intense peaks. All the others enzymes gave electropherograms with a single predominant peak. Second, an 

aqueous BGE containing 50 mM sodium dihydrogen phosphate, 25 mM borax, 0.1% polyvinylalcohol (PVA) and 

NaOH up to pH = 9.0, was also used. With this alkaline BGE, all samples gave a single predominant peak. The 

baselines were more stable in the basic medium, which made possible to recognize several small peaks which 

were also useful as additional features for the identification of the enzymes. Further information was obtained 

from the relative and absolute sensitivities, i.e. peak area ratios within each electropherogram, and peak area/ 

enzyme concentration ratios, respectively. In order to establish these later ratios, the enzyme concentrations in the 

industrial concentrates were measured by using the Bradford’s method. The proposed procedure was applied to the 

identification of individual enzymes in detergent bases of different composition and commercial cleaning products. 

For this purpose, the enzymes were previously precipitated with acetone and re-dissolved with water. 


(M. Beneito-Cambra, University of Valencia and Químicas Oro). 

326 



P04-002 DEVELOPMENT OF A CAPILLARY ELECTROPHORESIS METHOD FOR THE DETERMINATION OF POLYPHENOLS IN WINES 


1 


2 


Polyphenolic compounds are responsible of relevant sensory characteristics of wines such as color, flavour and 

astringency [1-2]. In this communication, a new capillary electrophoresis (CE) method is developed for the separation 

and quantification of the principal polyphenols occurring in wines. The proposed method has been thoroughly 

optimized to get the best separation of the most significant compounds. The influence of variables such as applied 

potential, injection mode, pH, organic solvent percentage and buffer concentration on the separation has been 

evaluated using experimental design. Multicriteria decision making has been utilized for achieving a reasonable 

compromise among number of separated compounds and analysis time. Selected electrophoretic conditions are as 

follows: fused-silica capillaries of effective length of 58.7 cm and 75 µm I.D. (375 µm O.D.) are used. The separation 

buffer consists of 20 mM aqueous sodium tetraborate solution (pH 9.2) + 2-propanol (20%, v/v). The capillary 

temperature is 25 ºC. The running voltage is 25 kV, which provides a current intensity of 40 µA, approx. The sample 

is injected hydrodynamically for 2 s at 50 mbar. Electropherograms are monitored at 230 nm. Figures of merit 

including accuracy, precision, and detection limits have been established under selected experimental conditions 

using red wine samples. This method will applied be to characterize red wines from different Spanish regions using 

their CE profile fingerprints as a rich source of information. References: [1] E. Boselli et al., Eur. Food Res. Technol., 

227 (2008) 709. [2] S. Kallithraka et al., J. Agric. Food Chem., 55 (2007) 3233. 


327


P04-003 CHARACTERIZATION OF WINES THROUGH THE ELECTROPHORETIC PROFILES OF POLYPHENOLS 


1 


2 


Corresponding author e-mail: xavi.saurina@ub.edu 

The characterization of wines based on compositional profiles of polyphenols as a source of information is here 

investigated. Contents of polypenolic species seem to be dependent on climatic, agricultural and winemaking issues. 

As a result, this family of natural wine components can be exploited as potential descriptors of wine features (e.g., 

color and taste properties) as well as quality [1-2]. In this study, capillary electrophoresis (CE) is utilized to generate 

quantitative data on polyphenol contents. Further analysis of the electrophoretic profiles relies on chemometrics as 

a way of facilitating the extraction of information dealing with the wine properties. For this purpose, cluster analysis, 

principal component analysis and related methods are used for discrimination, classification and correlation 

purposes. The CE method is applied to characterize wines from different Spanish regions. A set of 40 wines are 

analyzed. The resulting electropherograms consist of complex profiles containing peaks of polyphenolic compounds 

which sometimes overlap with other wine matrix components. Contents of major polyphenolic constituents, 

including gallic, coumaric, vanillic acids, catechine, trans-resveratrol, quercetin, etc. are preliminarily considered as 

descriptors of wine variety and ageing. References: [1] E. Boselli et al., Eur. Food Res. Technol., 227 (2008) 709. [2] I. 

Budic-Leto et al., J. Food Agric. Environ. 6 (2008) 138. [3] D.P. Makris et al., Talanta 70 (2006) 1143. 

328 



P04-004 CATIONIC COPOLYMERS AS PHYSICALLY ADSORBED COATINGS FOR CAPILLARY ELECTROPHORESIS: CONNECTIONS 

BETWEEN STRUCTURE AND PERFORMANCE 

Bernal J. 1 , Herrero M. 1 , Velasco D. 2 , Elvira C. 2 , Cifuentes A. 1 

1 


2 

Institute of Science and Technology of Polymers (CSIC), Madrid 

Corresponding author e-mail: jbernal@ifi.csic.es 

In capillary electrophoresis (CE), fused-silica capillary surface has long been known to cause adsorption problems 

during the analysis of high positively charged compounds (e.g., basic proteins) due to electrostatic interactions 

between the solute and the capillary wall. Moreover, the fused silica wall is also responsible of the irreproducibility 

frequently observed between injections and capillaries. The use of capillary coatings is a good strategy to avoid 

these harmful effects. In this work, the properties of four copolymers synthesized in our laboratory are studied as 

physically adsorbed coatings for capillary electrophoresis (CE). Namely, the four copolymers investigated were 

poly(N ethyl morpholine methacrylamide-co-N,N-dimethyl-acrylamide), poly(N ethyl pyrrolidine methacrylate-co- 

N,N-dimethylacrylamide), poly(N ethyl morpholine methacrylate-co-N,N-dimethylacrylamide) and poly(N ethyl 

pyrrolidine methacrylamide-co-N,N-dimethylacrylamide). The capillary coating is easily obtained by simply flushing 

into the tubing the dissolution containing the copolymer. The stability and reproducibility of each coating were tested 

for the same day, different days and different capillaries. It is demonstrated that the use of these coatings in CE can 

drastically reduce the analysis time and/or to improve the reproducibility of the separations. It is also shown that 

all the coated capillaries allow the separation of basic proteins by reducing their adsorption onto the capillary wall. 

Links between their molecular structure, physico-chemical properties and their performance as coatings in CE are 

discussed. 


329


P04-005 SEPARATION OF PHENOTYPICALLY INDISTINGUISHABLE CANDIDA SPECIES, ORTHOPSILOSIS, METAPSILOSIS AND 

PARAPSILOSIS, BY CAPILLARY ELECTROMIGRATION TECHNIQUES 

Horká M. 1 , Ružicka F. 2 , Kubesová A. 1,2 , Šlais K. 1 

1 

Institute of Analytical Chemistry of the ASCR, v.v.i., Brno, Czech Republic 

2 

Masaryk University Brno, Czech Republic 

Corresponding author e-mail: horka@iach.cz 

C. parapsilosis is the second most common yeast species isolated from bloodstream infections [1]. Simultaneously, 

the mortality rate of candidaemia due to C. parapsilosis is higher than that associated with C. albicans [2]. C. 

parapsilosis forms a complex composed of three genetically distinct groups (groups I, II, and III) from which 

genotypes II and III have been designed as the separate species Candida orthopsilosis and Candida metapsilopsis 

[3, 4]. Moreover microbial infection is preceded by adherence and biofilm formation. Rapid diagnosis especially 

for uncommon or newly identified fungal species [5] is crucial for prompt management of infection with tailored 

antifungal treatments. But the greater genetic variability of these newly described yeasts compared with C. 

parapsilosis has caused difficulties in the development of molecular techniques for the subtyping of these yeasts [4, 

6]. In this study, ways and means of the separation and possible identification of the selected strains of Candida, C. 

orthopsilosis (biofilm-negative) and C. metapsilosis and C. parapsilosis (biofilm-positive and biofilm-negative), are 

outlined. The capillary isoelectric focusing in the pH gradient pH range 3.6-4.0 and capillary electrophoresis with 

UV/Vis detection were successfully used for the on-line rapid separation and focusing of these yeasts. The pH 

gradients were traced by the low-molecular-weight pI markers. These techniques were verified on the group of thirty 

selected Candida strains and appear to be suitable for their use in the diagnostic microbiology. 

References: 

[1] Messer, S. A., Jones, R. N., Fritsche, T. R. J. Clin. Microbiol. 2006, 44, 1782–1787. 

[2] Miranda, L. N., van der Heijden, I. M., Costa, S. F., Sousa, A. P. I., Sienra, R. A., Gobara, S., Santos, C. R., Lobo, R. D., 

Pessoa, V. P., Jr., Levin, A. S., J. Hospi. Infect. 2009, 72, 9-16. 

[3] Kocsube, S., Toth, M., Vagvolgyi, C., Doczi, I., Pesti, M., Pocsi,I., Szabo, J., Varga, J. J. Med. Microbiol. 2007, 56, 190-195. 

[4] Tavanti, A., Davidson, A. D., Gow, N. A., Maiden, M. C., Odds, F. C. J. Clin. Microbiol. 2005, 43, 284-292. 

[5] Campa, D., Tavanti, A., Gemignani, F., Mogavero, C. S., Bellini, I., Bottari, F., Barale, R., Landi, S., Senesi, S. J. Clin. 

Microbiol. 2008, 46, 209-215. 

[6] Lasker, B. A., Butler, G., Lott, T. J. J. Clin. Microbiol. 2006, 44, 750–759. Acknowledgements This work was supported by 

the Grant Agency of the Academy of Sciences of the Czech Republic No. IAAX00310701 and by the Institutional research 

plan AVO Z40310501. 

330 



P04-006 APPLICATION OF AFFINITY CAPILLARY ELECTROPHORESIS AND DENSITY FUNCTIONAL THEORY TO INVESTIGATION OF 

HEXAARYLBENZENE-BASED RECEPTOR INTERACTIONS WITH ALKALI METAL AND AMMONIUM IONS IN METHANOL 

Ehala S. 1 , Toman P. 2 , Rathore R. 3 , Makrlik E. 4 , Kasicka V. 1 

1 

Institute of Organic Chemistry and Biochemistry AS CR, v.v.i. 

2 

Institute of Macromolecular Chemistry AS CR, v.v.i. 

3 

Marquette University 

4 

University of West Bohemia 

Corresponding author e-mail: ehala@uochb.cas.cz 

Novel hexaarylbenzene derivatives could possibly be employed in various applications in the continuously growing 

fields of molecular electronics and nanotechnology. Recently, polyaromatic hexaarylbenzene-based receptor 

(R) was synthesized for potential usage in sensing devices for metal cations [1]. Our interest was to describe 

quantitatively interactions of receptor R with several alkali metal ions, such as Li+, Na+, K+, Rb+ and Cs+, and 

ammonium ion. Capillary electrophoresis (CE), especially its affinity mode (ACE), is acknowledged as an efficient 

method for studying non-covalent interactions of (bio)molecules and for determining binding constants of their 

complexes in aqueous, mixed hydro-organic or nonaqueous media [2, 3]. The advantages of employing ACE to 

study molecular interactions comprise low sample size requirement, high separation efficiency, high resolving power 

and mostly short analysis times. Because of above merits, herein, receptor R interactions with several alkali metal 

and ammonium ions were quantitatively evaluated by ACE. The apparent binding (stability) constants (Kb) of the 

M-R+ complexes in methanol were obtained from the dependence of the effective electrophoretic mobilities of the 

receptor R (corrected to reference temperature, 25ºC, and constant ionic strength, 25 mM) on the concentration 

of alkali metal or ammonium ions in the background electrolyte using non-linear regression analysis. Additionally, 

by means of quantum mechanical density functional theory (DFT) calculations, the structural details of the studied 

complexes, such as the position of the central M+ ion in the cavity of the receptor R and the interatomic distances 

within the complexes were determined. In conclusion, the employed ACE method proved to be an appropriate and 

efficient tool for the quantitative evaluation of weak as well as strong interactions between receptor R and alkali 

metal and ammonium ions in methanol. The selectivity of R expressed by binding constants Kb towards alkali metal 

and ammonium ions was found to increase in the order of Na+ (log Kb ≈ −0.7)


P04-007 PARTIAL FILLING ELECTROKINETIC CAPILLARY CHROMATOGRAPHY – A USEFUL TOOL FOR INTERACTION STUDIES BETWEEN 

LIPID BILAYER DISKS AND PHARMACEUTICALS 

Vainikka K. 1 , Reijmar K. 2 , Edwards K. 2 , Riekkola M-L. 1 

1 

Univeristy of Helsinki 

2 

Uppsala University 

Corresponding author e-mail: marja-liisa.riekkola@helsinki.fi 

Artificial membranes, mimicking biological membranes, have been extensively used for studying interactions 

between compounds and membranes. The commonly used phosphatidylcholine-based membranes suffers 

from limited stability, hence these are often stabilized by polyelectrolytes or polymers. Polyethylene glycol 

(PEG)-lipid stabilized bilayer disks, that are planar and circular in shape and exhibit good long-term stability, 

have been employed as model membranes in partition and interaction studies [1]. In this work studies on the 

interactions between phosphatidylcholine-based bilayer disks and selected positively charged pharmaceuticals 

were carried out. Capillaries for electrokinetic chromatographic studies were coated either non-covalently with 

a poly(1-vinylpyrrolidone)-based copolymer [2] or covalently with polyacrylamide [3]. Both coatings mask the 

negative charges of the fused-silica capillary wall and, hence, minimize interactions between positively charged 

pharmaceuticals and the capillary wall. The developed non-covalent copolymer coating method is very fast; a twominute 

flush with the polymer dispersion is enough to strongly adsorb the polymer onto the fused-silica surface 

via electrostatic interactions. However, the covalent polyacrylamide coating was shown to be more stable at 

physiological pH (7.4) values. Distearoylphosphatidylcholine (DSPC)/cholesterol/distearoylphosphatidylethanolamine 

(DSPE) -PEG5000 lipid disks dispersed in phosphate buffer at pH 7.4 were employed as pseudostationary phase in 

electrokinetic capillary chromatography, using the partial filling mode. The migration times of the pharmaceuticals 

were proportional to the amount of lipids in the pseudostationary phase. The partitioning constant was successfully 

determined for the pharmaceuticals selected for the study. The results demonstrate that lipid bilayer disks can 

successfully be used for studying analyte-membrane interactions in electrophoresis. References: [1] Boija, E., 

Lundquist, A., Nilsson, M., Edwards, K., Isaksson, R., Johansson, G., Electrophoresis, 2002, 29, 3377-3383. [2] 

Wang, A-J., Witos, J., D’Ulivo, L., Vainikka, K., Riekkola, M.-L., Electrophoresis, 2009, 30, 1-8. [3] Molina, M., 

Wiedmer, S. K., Jussila, M., Silva, M., Riekkola, M.-L., J. Chromatogr. A, 2001, 927,191-202. 

332 



P04-008 COMPARISON OF ALKYL ACRYLATE MONOLITHIC COLUMNS FOR CAPILLARY ELECTROCHROMATOGRAPHY USING CHEMICAL 

INITIATION 

Ten-Doménech I., Bernabé-Zafón V., Simó-Alfonso E.F., Herrero-Martínez J.M. 


Corresponding author e-mail: jmherrer@uv.es 

Several alkyl acrylate-based (butyl (BA), lauryl (LA) and octadecyl acrylate (ODA)) monolithic stationary phases 

for capillary electrochromatography were synthesized, using a chemical system as initiator of polymerization. BA 

monoliths were initiated with ammonium peroxodisulfate, whereas LA and ODA columns were obtained with lauroyl 

peroxide as initiator. For each alkyl acrylate, the influence of porogenic solvent composition on both morphological 

and electrochromatographic properties of resulting monolith was investigated and comparatively discussed. Under 

their respective optimum conditions, satisfactory separations of a test mixture of polyaromatic hydrocarbons with 

similar efficiencies (minimum plate heights of 2.6-13.1 um obtained from Van Deemter plots) were achieved for 

investigated alkyl acrylate monoliths. In all cases, satisfactory run-to-run (RSD < 6.6%) and column-to-column 

reproducibilities (RSD < 10%) were achieved. The capability of separation of these monolithic beds was compared 

by the analysis of complex mixtures of neutral compounds. LA and ODA columns showed similar separation 

performance followed by the BA monoliths. 

Acknowledgements: Project CTQ2007-61445 (MEC of Spain and FEDER funds). 


333


P04-009 DETERMINATION OF MUSCLE RELAXANTS PANCURONIUM AND VECURONIUM BY CAPILLARY ELECTROPHORESIS WITH 

CAPACITIVELY COUPLED CONTACTLESS CONDUCTIVITY DETECTION 

Petru K., Siroka J., Luzova V., Jac P., Polasek M. 

Faculty of Pharmacy, Charles University-Czech Republic 

Corresponding author e-mail: klara.petru@faf.cuni.cz 

A new capillary elecrophoretic method for the separation of pancuronium bromide (PMB) and vecuronium bromide 

(VMB) in pharmaceuticals utilizing capacitively coupled contactless conductivity detection was devised and 

validated. The separation was carried out in 75 cm x 50 µm i.d. fused-silica capillary (45 cm to the detector) at 25°C. 

A 50 mM borate buffer of pH 9.5 (adjusted with NaOH) containing 12.5 mg/ml of hydroxypropyl-gamma-cyclodextrine 

was selected as the optimal background electrolyte. The voltage +30 kV was applied. The samples were injected 

hydrodynamically at 1000 mbar for 3 s. The separation of PMB, VMB and phenyltrimethylammonium iodide (internal 

standard) was achieved in less than 4.5 min. The calibration curves were linear for both PMB and VMB in the range 

25 to 200 µg/ml (r² > 0.9977). The limits of detection were 7 µg/ml for PMB and 6 µg/ml for VMB. The method was 

applied to the assay of PMB (2 mg/ml) in Pavulon inj. and VMB (4 mg/ml) in Norcuron inj.sicc. The content of PMB 

and VMB found (5 independent measurements) was 98.72 % (RSD = 1.34 %) and 99.22 % (RSD = 3.85 %) of the 

nominal value, respectively. The inter-day precision (n = 18) of peak area ratios for real samples of PMB and VMB are 

characterized by RSD = 2.79 % and RSD = 2.49 %, respectively. The accuracy was tested by recovery experiments 

at three concentration levels; the recoveries ranged between 96.72 % and 103.47 % (n = 3) with RSD < 2.2 %. 

The work was supported by the grants GAUK 123709, MSM 0021620822 and SVV-2010-261-001 

334 



P04-010 PREPARATION AND CHARACTERIZATION OF OCTADECYL ACRYLATE MONOLITHS FOR CAPILLARY 

ELECTROCHROMATOGRAPHY BY THERMAL, CHEMICAL AND PHOTOCHEMICAL INITIATION 

Ten-Doménech I., Escrig-Doménech A., Bernabé-Zafón V., Simó-Alfonso E.F., Herrero-Martínez J.M. 



Octadecyl acrylate-based (ODA) monolithic columns for capillary electrochromatography (CEC) were prepared 

using either thermal, chemical initiation or by UV-irradiation in the presence of lauroyl peroxide as initiator. Thermal 

polymerization was carried out at 70ºC for 20 h, whereas chemical (using N,N,N’,N’-tetramethylethylenedediamine as 

activator) and photopolymerization were carried out at room temperature for 24 h and 10 min, respectively. For each 

initiation process, and using a 1,4-butanediol/1-propanol mixture as porogenic solvent, the influence of composition 

of polymerization mixture (ratios of monomers/porogenics solvents, 1,4-butanediol/1-propanol and ODA/crosslinker) 

on the morphological and CEC properties of beds was evaluated. After optimization of polymerization mixture for 

each initiating system, photochemically and chemical initiated ODA stationary phases showed higher permeabilities 

and better efficiencies than those prepared by thermal initiation. The potential of separation of the different initiating 

ways was also evaluated by the analysis of complex mixtures of neutral compounds. 

Acknowledgements: Project CTQ2007-61445 (MEC of Spain and FEDER funds). 


335


P04-011 PREPARATION AND CHARACTERIZATION OF POLY(LAURYLMETHACRYLATE) MONOLITHS WITH SILVER NANOPARTICLES BY 

PHOTOPOLYMERIZATION FOR CAPILLARY ELECTROCHROMATOGRAPHY 

Navarro-Pascual-Ahuir M., Lerma-García M.J., Simó-Alfonso E.F., Ramis-Ramos G., Herrero-Martínez J.M. 



Organic-polymer based monoliths are one of the major categories of monolithic materials. Some of their advantages 

are simple preparation, no need of retaining frits, high permeability, and easy tuning of pore size, surface area and 

column functionality. They are often made by a one-step polymerization of a mixture consisting of one or more 

functional monomers, including a cross-linker, porogenic solvents and an initiator. 

On the other hand, nanoparticles and nanostructured materials are currently having a significant impact in 

many scientific fields, including chemistry, material sciences, physics, medicine and electronics. Among these 

nanomaterials, nano-sized metal particles display novel properties, such as high catalytic activities, interesting 

optical properties, and large surface-to-volume ratios, which can be useful to increase mass transfer rates. Then, 

the combination of monolithic column technology and the specific features of metal nanoparticles could be an 

attractive way to obtain novel stationary phases for separation techniques, with an increased efficiency and different 

selectivities. 

In this work, silver nanoparticles were photogenerated in situ in poly(lauryl)methacrylate monoliths by UV irradiation 

of the polymerization mixtures. Both polymerization and silver ion reduction was simultaneously produced during 

irradiation in the UV-transparent capillaries. Using a 1,4-butanediol/1-propanol mixture as porogenic solvent, 

the influence of the composition of this mixture and jointly with the silver nanoparticles concentration on the 

morphological and chromatographic properties of polymer matrix (monolith) was investigated. The morphology 

of the resulting columns (nanocomposites) was characterized by scanning electron and transmission electron 

microscopies, whereas the CEC performance of different monoliths was evaluated by the separation of test mixtures 

of non-charged solutes. 

Acknowledgements: Project CTQ2007-61445 (MEC and FEDER funds). M.J. L-G. thanks the Generalitat Valenciana 

for an FPI grant for PhD studies. 

336 



P04-012 ANALYSIS, PURIFICATION AND CHIRAL SEPARATION OF BETA-ALANYL-D,L-TYROSINE AND ITS DERIVATIVES BY CAPILLARY 

AND FREE-FLOW ZONE ELECTROPHORESIS 

Sazelova P., Solinova V., Schimperkova T., Masova A., Jiracek J., Kasicka V., 

Institute of Organic Chemistry and Biochemistry AS CR, v.v.i.-Czech Republic 

Corresponding author e-mail: sazelova@uochb.cas.cz 

The problem of growing resistance of microorganisms to conventional antibiotics gave rise to a search for new 

potent antimicrobial agents, with new mechanisms of action, as human therapeutics against drug resistant strains. In 

insects, humoral response to microbial infection includes de novo synthesis of antimicrobial peptides, which seem to 

be promising novel potential therapeutics. An earlier developed procedure for conversion of the analytical capillary 

zone electrophoresis (CZE) separations into preparative scale realized by free-flow zone electrophoresis (FFZE) [1] 

was applied to the purification of a component present in the fraction with antimicrobial activity isolated by RP- 

HPLC from the larvae hemolymph of fleshfly Neobellieria bullata. Dipeptide b-Ala-Tyr was identified to be the main 

component of this fraction by mass spectrometry [2]. CZE was used for qualitative and quantitative analysis of the 

isolated components before and after FFZE purification procedure. Besides b-Ala-Tyr, another minor component was 

found. By combination of pre-purification of that fraction by FFZE with subsequent RP-HPLC, complete separation 

of major b-Ala-Tyr and the minor component was achieved with the view of testing their antimicrobial activity. Minor 

component was identified to be b-D-fructopyranose-b-Ala-Tyr by mass spectrometry [3]. Derivatives of 

b-Ala-D,L-Tyr, i.e. b-Ala-D,L-Tyr-NH2, N-Ac-b-Ala-D,L-Tyr and b-D-fructopyranose-b-Ala-D,L-Tyr were synthesized 

and tested for antimicrobial activity [3]. For enantiopurity control of these compounds, the CZE methods have 

been developed using 2-hydroxypropyl-b-cyclodextrin (2-HP-b-CD) as a chiral selector. The best separation of 

enantiomers of b-Ala-D,L-Tyr and b-Ala-D,L-Tyr-NH2 was achieved in the background electrolyte (BGE) composed of 

32 mM Tris, 50 mM H3PO4, pH 2.5, and containing 20 mg/ml 2-HP-b-CD. Moreover, from the simultaneous analyses 

of isolated b-Ala-Tyr fraction and standards of b-Ala-D-Tyr and/or b-Ala-L-Tyr, it could be concluded that the major 

isolated dipeptide fraction contained b-Ala-L-Tyr isomer. The best resolution of N-Ac-b-Ala-D,L-Tyr enantiomers 

was achieved in BGE composed of 50 mM sodium tetraborate, pH 10, containing 60mg/ml 2-HP-b-CD. From the 

dependences of effective mobilities of the above compounds on the concentration of chiral selector in the BGE, 

the binding constants of their complexes with 2-HP-b-CD were determined. The work was supported by the GACR, 

grants no. 203/08/1428 and 203/09/0675, and by the ASCR, Research Project no. AV0Z40550506. Ms V. Liskova is 

thanked for her skilful technical assistance. [1] V. Kasicka, Z. Prusik, P. Sazelova, J. Jiracek, T. Barth, J. Chromatogr. 

A 796 (1998) 211. [2] A. Ciencialova, M. Mackova, M. Jarcevsky, B. Koutek, J. Jiracek, in: M. Flegel, M. Fridkin, C. 

Gilon, J. Slaninova (Eds.) Peptides 2004, Kenes Int., Geneva, 2005, p. 489. [3] A. Ciencialova, T. Neubauerova, M. 

Sanda, R. Sindelka, J. Cvacka, Z. Voburka, M. Budesinsky, V. Kasicka, P. Sazelova, V. Solinova, M. Mackova, B. 

Koutek, J. Jiracek, J. Peptide Sci. 14 (2008) 670. 


337


P04-013 CAPILLARY ELECTROPHORETIC ASSAY OF FLAVONOIDS AND PHENOLIC ACIDS IN PROPOLIS USING COMPLEX FORMATION 

WITH TUNGSTATE AND ON-LINE PRECONCENTRATION 

Siroka J., Petru K., Jac P., Polasek M. 

Charles University, Faculty of Pharmacy 

Corresponding author e-mail: jitka.siroka@faf.cuni.cz 

An original CZE method with UV detection has been applied to polyphenols profiling of different propolis extracts. 

For separation selectivity enhancement the addition of complex-forming tungstate agent into the background 

electrolyte (BGE) has been employed. The tungstate ions form negatively charged complexes with flavonoids/ 

phenolic acids comprising vicinal –OH groups in the molecule and these complexes differ in stability, charge and 

electrophoretic mobility. Due to unsatisfactory sensitivity of the UV detection of the separated tungstate complexes 

the application of an on-line preconcentration technique, i.e. large-volume sample stacking with polarity switching 

has been proposed. Optimum composition of the BGE was 5mM sodium tungstate, 50 mM N-(2-hydroxyethyl)- 

piperazine-2-(2-ethanesulfonic acid) (HEPES) of pH 7.4 and 25% v/v of methanol. The separation was performed in a 

60.2-cm uncoated fused-silica capillary (effective length 50cm, i.d. 75µm) with UV detection at 275nm. Large volume 

sample stacking procedure included sample injection at 150mBa for 99s, application of reversed polarity -30kV (for 

approximately 1.8min) and switching the polarity to positive +30kV for the separation of the analytes. The LOQ 

(S/N=10) values were determined for apigenin, rutin, hyperoside, quercetin, luteolin, chlorogenic, ferulic, p-coumaric 

and cinnamic acid. The LOQs ranged from 0.75µg/ml to 7.5µg/ml that compare well with those of UHPLC-UV [1]. 

[1] Z. Spacil, L. Novakova, P. Solich, Talanta 76, 2008, 189-199. Acknowledgement: Financial support by the Czech 

Ministry of Education (grant MSM 0021620822) and grant SVV 2010-261-001 are gratefully acknowledged. 

338 



P04-014 DETERMINATION OF HISTAMINE, PHENYLETHYLAMINE AND TYRAMINE IN RED WINES USING ON-LINE COUPLED CAPILLARY 

ISOTACHOPHORESIS CAPILLARY ZONE ELECTROPHORESIS WITH PHOTOMETRIC DETECTION 

Ginterova P. 1 , Marak J. 2 , Stanova A. 2 , Kaniansky D. 2 , Maier V. 1 , Sevcik J. 1 

1 

Palacký university in Olomouc 

2 

Comenius University in Bratislava 

Biogenic amines (BAs), namely histamine (HA), phenylethylamine (PEA) and tyramine (TA), are mainly produced in 

wine during alcoholic and malolactic fermentation of amino acids histidine, phenylalanine and tyrosine, respectively. 

Wine samples containing high concentration levels of BAs can cause direct or indirect health risks. Toxic levels 

of BAs depend on the tolerance of the individual human being. HA, PEA and TA are known to be associated with 

headaches, nausea and hypertension. 

The aim of this work was to develop an analytical method for the separation, identification and quantification of 

histamine, phenylethylamine and tyramine in red wine samples from different localities by using on-line coupled 

capillary isotachophoresis capillary zone electrophoresis (cITP-CZE) in hydrodynamically closed separation system 

with photometric detection. 

The separation of selected BAs was performed in a cationic mode by using automated electrophoretic analyzer EA 

202 A (Villa Labeco, Spisska Nova Ves, Slovak Republic). Firstly, optimization of electrolyte system for the ITP step 

consisting of a leading electrolyte and termination electrolyte was performed. An on-line sample preconcentration 

in the upper column (800 µm I.D., a total length was 140 mm) with contactless conductivity detector was performed 

in the ITP step. The optimization of a background electrolyte for the CZE mode was performed in the next step. All 

electrolytes contained hydroxyethylcellulose to suppress electroosmotic flow. The CZE separation was carried out 

in lower column (300 µm I.D, a total length was 160 mm) with contactless conductivity detector and photometric 

detector. Optimal wavelength for the detection of analytes by photometric detector was found. 

Acknowledgement: 

This work was supported by a grant from the Slovak Research and Development Agency (No.VVCE-0070-07) and the 

grants of Slovak Grant Agency, No`s.1/0882/09 and 1/0546/10. 


339


P04-016 STABILITY OF LINEAR POLYACRYLAMIDE COATED CAPILLARIES IN ACIDIC MEDIA IN THE PRESENCE OF ORGANIC 

MODIFIERS AND SURFACTANTS 

Anres P., Delaunay N., Gareil P. 

Chimie Paristech 

Since its first introduction in 1985 by Hjerten [1], linear polyacrylamide (LPA)-coated capillaries have been widely 

used because of the unique properties of LPA (i) to suppress electroosmotic flow (EOF) almost entirely and (ii) to 

reduce considerably adsorption of hydrophobic compounds such as proteins or surfactants thanks to its neutral and 

very hydrophilic character. These properties and the simplicity of the coating procedure made LPA-coated capillaries 

a prime choice for in-situ preconcentration techniques in capillary electrophoresis involving stacking and sweeping, 

where EOF suppression is required to enhance performances. Usually, with this kind of capillary, pH of background 

electrolytes are restricted to the 4-8 range because it seems that the stability of the coating is an issue for long 

life utilisation at pH below 4 and higher that 8. Indeed, in high acidic or basic conditions, acrylamide moieties can 

be hydrolysed into carboxylic groups, which may alter capillary properties regarding EOF and wall adsorption. 

However, to the best of our knowledge, no studies on the stability of capillaries in this pH range have been reported. 

Moreover, in MEKC and sweeping techniques organic modifiers are often used to tailor selectivity and no report 

is available on the stability of LPA-coated capillaries under such conditions. The aim of this work was to study the 

stability of LPA coated capillary with a view to their use with acidic background electrolytes (pH 2-4) in the presence 

of organic modifiers (acetonitrile and methanol), anionic and cationic surfactants (sodium dodecyl sulphate and 

hexadecyltrimethyl ammonium bromide), and long chain ionic liquid (dodecyldimethyl imidazolium tetrafluoroborate). 

To this end, capillaries were coated following the Hjerten’s procedure and allowed to be submitted for 70 hours to the 

different conditions studied while Vigh’s tests were performed every hour to follow the evolution of EOF. The results 

obtained allow concluding on the utilisation of this kind of capillary for preconcentration techniques in acidic media 

with organic modifiers and surfactants. This opens up new possibilities to implement field enhanced sample injection 

(FESI)-Sweeping-MEKC to preconcentrate compounds having pKas around 4-5, and next to separate them in their 

neutral form [2]. The use of such capillaries will, for sure, give better results in term of reproducibility, because the 

EOF is more stable comparing to non-coated capillaries at acidic pH. [1] S. Hjerten, M-D. Zhu, J. Chromatogr. 1985, 

346, 265. [2] J.P. Quirino, S. Terabe, Anal. Chem. 2000, 72, 1023. 

340 



P04-017 METABOLOMIC STUDY OF HUMAN HT29 COLON CANCER CELLS BY CE-MS. A COMPARATIVE STUDY OF METABOLITE 

PURIFICATION STRATEGIES 

Ibáñez C. 1 , Simó S. 1 , Rocamora L. 2 , Gomez A. 2 , Ferragut J.A. 2 , Cifuentes A. 1 

1 

Institute of Industrial Fermentations, Food Characterization, Madrid 

2 

Universidad Miguel Hernandez, Alacant 

Corresponding author e-mail: acifuentes@ifi.csic.es 

Capillary electrophoresis-mass spectrometry (CE-MS) is considered a complementary analytical tool to GC-MS 

and LC-MS for metabolomics since it provides fast separations with very high efficiencies and minimum sample 

volumes. CE-MS is particularly suitable for the separation of polar and charged compounds which might not be 

separated by LC or GC. In metabolomics, the presence of large molecular species such as proteins and/or high salt 

content, are potential interferences. Thus, when working with CE-MS, proteins can cause adsorption to the inner 

capillary wall, while high salt contents can lead important ESI ionization suppression and MS contamination. To 

avoid these interfering effects, sample preparation is often necessary for sensitive and reliable metabolite analysis. 

In the present work, a new CE-MS approach is presented to carry out the metabolome study of the cytoplasm 

fraction from human HT29 colon cancer cells. As a first step, four different metabolite purification strategies from 

HT29 cell culture cytoplasm were studied prior to its CE-MS analysis: two solid phase extractions (namely, using C18 

stationary phase and polystyrene-divinylbencene sorbent), and two procedures to remove proteins (precipitation 

with methanol and ultracentrifugation with a 3 kDa size-exclusion membrane). The four different metabolite 

purification procedures were compared in terms of number of extracted metabolites and CE-MS peak intensities. It 

was observed that the different treatments allowed the CE-MS analysis of a large number of metabolites (namely, 

each procedure allowed the analysis of more than 80 different compounds) with different sensitivity and, more 

interestingly, with different selectivity. The best results in terms of number of metabolites extracted from the 

cytoplasm were obtained using SPE with polystyrene-divinylbencene sorbent and methanol precipitation. It was 

observed that approximately 40% of the detected species were different among the different treatments, what 

highlights the importance of the selection of an adequate extraction methodology in metabolomic studies. Once the 

best purification strategy was selected, the effect of dietary polyphenols on human HT29 colon cancer cells is now 

under study at our laboratory following a complete Foodomics approach. 


341


P04-018 Development and optimization of peptides derivatization through thiol groups. Use in chemiluminescence detection 

in capillary electrophoresis 

Regueiro-Vilar O., Puerta A., Uriel C., Gómez A., de Frutos M., Diez-Masa J.C. 

Institute of Organic Chemistry (IGOC-CSIC), Madrid 

Corresponding author e-mail: diez-masa@iqog.csic.es 

Some glycoproteins have been described as biomarkers of different diseases. Changes in their glycoforms have 

been related to the pathological states. The determination of these glycoforms has some difficulties due to: i) 

complexity of the sample, ii) structural similarity between glycoforms, and iii) low concentration of many of the 

glycoproteins considered as biomarkers in human blood. 

Capillary electrophoresis (CE) is a suitable technique for analysis of glycoforms of intact glycoproteins but it 

is necessary to couple this efficient separation technique with a very sensitive detection to render it useful for 

biomarker analysis. Chemiluminescence (CL) is a simple and very sensitive detection method, though its use as 

detection technique requires a previous derivatization of the glycoproteins. However, derivatization of glycoproteins 

through the amino groups usually gives rise to multiple reaction products, spoiling the glycoforms separation in CE. 

Derivatization through thiol groups is a useful alternative. 

The aim of this work was to study the derivatization of glutathione through the thiol group with a luminol derivative for 

its analysis by CE with CL detection. The glutathione is used as a model system for polypetides analysis that could 

be of interest for the CE-CL analysis of biomarker glycoproteins. 

The chloroacetyl derivative of luminol (CAL) as derivatizing agent for polypeptides was synthesized in our laboratory. 

The selectivity of the reaction between amino and thiol groups of gluthatione and CAL was studied by Mass 

Spectrometry (MS). Our results show that with the optimized conditions for the derivatization reaction only the thiolderivative 

is obtained. 

Chemiluminescent emission of the glutathione derivatized with CAL reagent was compared with the one of luminol. 

We studied the influence of some parameters on the performance of CL reaction, including nature and concentration 

of the oxidants and the catalysts, and nature and pH value of the reaction buffer. In the optimized conditions, the 

quantum yield of the derivatized glutathione was only 50 times lower than the one of luminol. 

To couple CE and CL detection, it is necessary to use a post-column reactor where the oxidant is mixed with the 

derivatized plypeptides after separation. In this communication, a home-made sheath flow cuvette system was used 

as post-column reactor. The light produced inside the cuvette by the CL reaction was measured using an avalanche 

photodiode (APD) provided with a collection optics. Different light collection systems, such as microscope objectives 

of several numerical apertures, fiber optics systems, and APD direct exposure where compared in terms of detection 

sensitivity. 

Using glutathione as a model system, a complete methodology for peptide derivatization and CE analysis with 

CL detection is proposed, that could be extended to glycoform analysis of glycoproteins considered as potential 

biomarkers. 

The authors acknowledge the Spanish Ministry of Science and Innovation (Project CTQ2009-09399) and Comunidad 

de Madrid (Project S-GEN-0247-2006) for financial support. 

342 


P05 SUPERCRITICAL 

FLUID 


POSTER 

SESSIONS

POSTER SESSION 05 - SUPERCRITICAL FLUID CHROMATOGRAPHY 

P05-001 STUDY OF SEVERAL POLYSACCHARIDE BASED CHIRAL STATIONARY PHASES FOR THE ENANTIOSELECTIVE 

SEPARATION OF AN ACETAMIDE INTERMEDIATE BY USING SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) 

Toribio L., Nozal Mª J, Bernal J.L., Diego,J.C., Martin Mª T. 

University of Valladolid-Spain 

Corresponding author e-mail: ltoribio@qa.uva.es 

Since it is well known that a pair of enantiomers can display quite different activity and toxicity profiles, the 

pharmacological evaluation of each enantiomer and the enantiomeric purity of a drug are important tasks in 

drug development. Regulations do not only focus on the enantiomeric purity analysis of the active ingredient or 

finished form, but also to the chiral intermediates, in order to ensure the robustness of the synthesis process. As a 

consequence, chiral separation methods are subjects of growing interest in the pharmaceutical industry. Historically, 

HPLC has been the standard technique and the first choice for chiral analysis, however, the combined advantages 

of speed, efficient separations and environmental friendliness have made SFC the best choice for enantiomeric 

analysis in many laboratories. (4S-trans)-4-(ethylamino)-4-(N-acetamide)-5,6-dihydro-(6S)-methyl-4H-thieno- 

[2,3-b]thiopyran-7,7-dioxide is a chiral intermediate in the synthesis of an ophthalmic drug used for the treatment 

of glaucoma and ocular hypertension. The study of its enantiomeric separation by using SFC with different types 

of chiral stationary phases (CSPs) is presented in this work. The obtained results were highly successful, in all the 

studied cases the enantioresolution was higher than 2 and the analysis time close to 10 minutes. Acknowledgements 

The authors thank Gadea Pharmaceutical Group for financial support. Mª.T.M thanks the Spanish Ministry of Ciencia 

e Innovación the Ramon y Cajal contract. 

344 


P06 MULTIDIMENSIONAL 


POSTER 

SESSIONS

POSTER SESSION 06 - MULTIDIMENSIONAL CHROMATOGRAPHY 

P06-001 DEVELOPMENT OF A TWO-DIMENSIONAL COMPREHENSIVE HPLC-SYSTEM FOR THE ANALYSIS OF COMPLEX SAMPLES 

Haun J. 1 , Teutenberg T. 1 , Schmidt T.C. 2 

1 

Institute of Energy and Environmental Technology, R&D-Germany 

2 



The analysis of more and more complex samples, each containing hundreds of different constituents, becomes 

a challenge to an increasing extend. Although it is possible to achieve an additional mass/charge-separation by 

using high-resolution mass-spectrometric detectors in modern HPLC systems, these techniques often reach their 

limits if a multitude of components co-elute at the same retention time. In this case, the target analyte cannot be 

ionised to the needed extend and the signal intensity often decreases. Therefore, it is necessary to advance the 

separation of the constituents on side of the chromatographic system, which means before detection. In the past 

few years, comprehensive two-dimensional HPLC was found to be able to maximise the peak capacity via the 

approach of the orthogonality of two independent separation systems (e. g. size and hydrophobicity). However, the 

major disadvantages of these techniques are the high flow rates (up to 5 mL/min) which make it difficult to hyphenate 

mass-spectrometric detectors. Furthermore, the solvent consumption is very high. Therefore, the main goal is to 

develop a miniaturised comprehensive two-dimensional system based on capillary- and nano-HPLC. Regarding 

the miniaturisation it is important to choose the conditions in a way that the use of a mass-spectrometric detector 

becomes possible without using a flow splitter. The technical implementation is very difficult and complex, because 

parameters have to be correctly adjusted when changing from conventional HPLC to nano-HPLC. This concerns 

e. g. the maximum injection volume, which has to be adjusted to avoid an overload of the first dimension column. 

Moreover, it concerns the dead volumes which have to be reduced to minimise peak broadening. An Eksigent 

nanoLC ultra 2D system is used as a development platform. It includes the pumps and two 10-port valves for the 

modulation between the two separation systems. On this poster, the precise configuration of the system will be 

shown, which includes the whole modulation process and the developed capillary configuration. 

346 



P06-002 COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY—TIME-OF-FLIGHT MASS SPECTROMETRY FOR THE 

ANALYSIS OF ANTHROPOGENIC AND NATURALLY-PRODUCED ORGANOBROMINATED COMPOUNDS IN BLUEFIN TUNA 

FROM THE MEDITERRANEAN SEA 

Pena-Abaurrea M. 1 , Covaci A. 2 , Ramos L. 1 

1 

Institute of Organic Chemistry (IQOG-CSIC), Madrid 

2 


Corresponding author e-mail: l.ramos@iqog.csic.es 

Comprehensive two-dimensional gas chromatography (GC×GC) is a powerful separation technique that enhances 

detectability by combining different separation mechanisms in order to improve the overall separation efficiency 

provided by a one-dimensional GC separation. Combined with a selective mass detector, the complete GC×GC─MS 

system suitably enhances both the separation and the identification power to achieve unambiguous determination 

of the target compounds in the investigated sample [1]. Since recently, Time-of-flight mass spectrometry (ToF─MS) 

is the choice detector for GC×GC systems because of its accurate determinations specially for environmental 

applications. In fact, this tri-dimensional technique (result of two retention times and mass spectrum as identification 

tools) provides a definitive determination of the investigated pollutants in the presence of co-extracted matrix 

components [2]. Organobrominated compounds, such as polybrominated diphenyl ether (PBDEs) and structural 

analogues like the methoxylated PBDEs (MeO-PBDEs) have been recently studied due to their environmental 

relevance. Moreover, another 4000 naturally-produced organobromines have also been detected in the marine 

environment though their toxicological effects in the marine fauna have not yet deeply been studied. Among them, 

only few families have been measured at high concentrations: polybrominated hexahydroxanthene derivates 

(PBHDs), 2,4,6-tribromoanisole (TBA), a mixed halogenated compound (MHC-1) together with the above mentioned, 

MeO-PBDEs. This study evaluates the feasibility of GC×GC─ ToF MS for the identification of all organobrominated 

compounds, both anthropogenic and naturally-produced, extracted from muscle of bluefin tuna (Thunnus 

thynnus) from the Mediterranean Sea [3]. Various experimental parameters affecting the GC×GC separation have 

been optimised to achieve maximum separation of the different organobromines, both between different family 

homologues and the matrix components and between disimilar organobrominated analytes. Special attention has 

been paid to compounds previously analysed by one-dimensional GC─MS, for which coelutions were observed. 

Acknowledgments Authors thank MEC (Proj.: CTQ-2006-14993/BQU), MICINN (Proj.; AGL2009-09733) and CM 

(Proj.; S2009/AGR-1464) for financial support and Simoneta Corsolini for her kindly sample supply. MPA also 

thanks MEC for her predoctoral grant. References [1] Comprehensive two dimensional gas chromatography. Edit: L. 

Ramos. Elsevier, 2009; [2] SPJ. Van Leeuwen et al. J. Chromatogr. A 1186 (2008) 161-182; [3] M.Pena-Abaurrea et al. 

Chemosphere 76 (2009) 1477-1482 


347


P06-003 PRESSURIZED HOT WATER EXTRACTION, SUPERCRITICAL FLUID EXTRACTION AND GC X GC-TOFMS IN THE ANALYSIS 

OF FATTY ACIDS IN BROCCOLI FLORETS 

Bernal J. 1 , Arnaiz E. 2 , Bernal J.L. 2 , Toribio L. 2 , Kronholm J., Ruiz-Jiménez J. 3 , Hartonen K. 3 , Riekkola M.L. 3 

1 


2 

I.U.CINQUIMA, University of Valladolid 

3 


Corresponding author e-mail: jbernal@ifi.csic.es 

Brassicaceae family (Cruciferae) includes vegetables that are commonly grown and include broccoli, Brussels 

sprouts, cabbage, collards, etc. Broccoli was derived from a species of wild cabbage, Brassica oleracea, and 

its consumption is widely spread in Europe. Broccoli is a good source of health promoting compounds like 

glucosinolates, polyphenolic compounds, fatty acids, etc. Nowadays, the importance in the diet of functional food 

and nutraceuticals is growing constantly, so to determine the presence of these compounds in broccoli and their 

extraction could be of great interest. 

Because the polarity and selectivity of water can be varied by increasing temperature and because the Green 

Chemistry ideology is always recommendable, broccoli florets were extracted with pressurized hot water (PHW). In 

this work a new comprehensive gas chromatographic (GCxGC-TOFMS) method was developed for the determination 

of fatty acid methyl esters (FAMEs) after the extraction of broccoli samples by PHWE. Broccoli samples were 

also extracted with supercritical fluid extraction (SFE) and analyzed with conventional gas chromatography-mass 

spectrometry (GC-MS). 

The final method developed involved extraction of the samples with PHWE or SFE and a further liquid-liquid 

extraction step of the PHWE extracts to remove the water prior to the splitless injection of the sample into GCxGC- 

TOFMS or GC-MS (SFE extracts). The extracts were derivatized to fatty acid methyl esters. The separation and 

identification of fatty acids in GCxGC was achieved with a combination of a DB5-MS (20 m, 0.18 mm, 0.18 µm) and 

a Cyano (0.50 m, 0.1 mm, 0.10 µm) as the first and second GC columns, respectively. For the GC-MS analysis it was 

used a biscyanopropylsiloxane column (100m, 0.25mm, 0.20µm). 

The methods developed were successfully applied to the analysis of broccoli floret samples. 


The authors wish to thank the Junta de Castilla y León for the financial support (GIR127). J.B. would like to thank 

the CSIC and Spanish Ministry for the grants to support his stay in the Laboratory of Analytical Chemistry of the 

University of Helsinki. 

348 



P06-004 ANALYSIS OF CHLOROPARAFFINS USING COMPREHENSIVE TWO-DIMENSIONAL GAS CHROMATOGRAPHY 

Hofmeister S., de Koning S. 


Corresponding author e-mail: sjaak.dekoning@leco.de 

Recently there has been considerable interest in the analysis of chlorparaffines (CP) in environmental samples. CPs 

are alkane chains of several lengths (C10 – C30) substituted with width range of chlorine content (30 – 70%). The 

CPs can be devided into three groups based on their chain length; firstly, short chain CPs of C10 – C13, secondly, 

medium chain CPs of C14 – C17 and finally the long chain CPs of C17 – C30. The first group causes a high risk for th 

eenvironment and human resource. The analysis of CPs with gas chromatography is a challanging task, especially 

as these compounds do affect the analysis of polychlorinated biphenyls (PCBs). In this contribution the analysis of 

CPs, in combination with PCBs, with comprehenisive two-dimensional gas chromatography hyphenated with Timeof-Flight 

mass spectrometric detection (GC×GC–ToF MS) is evaluated. For qualification as well as quantification 

software based options like Classification Mass Spectral Featuring will be demonstrated. 


349


P06-005 APPLICATION OF MULTI-DIMENSIONAL CHROMATOGRAPHY TO THE SEPARATION AND IDENTIFICATION OF THE 

COMPONENTS OF FRESHWATER AND SEAWATER DERIVED DISSOLVED ORGANIC MATTER (DOM) 

Sandron S., Nesterenko E., Kelleher B., Paull B. 



Dissolved organic matter (DOM) in sea and freshwater represents one of the largest active organic carbon reservoirs 

on earth and is 

central to many environmental processes including heterotrophic bacterial activity, partial control of light penetration 

and exchange of gases on the sea surface. The chemical composition of DOM is extremely complex and is reported 

(1-3) to contain various classes of compounds: amino acids, organic acids, lipids, phosphonates, carboxyl-rich 

alicyclic molecules (CRAM) 

and carbohydrate like precursors. For environmental reasons (i.e. control and study of bacterial activity, affect of 

pollution on the eco-system and etc.) the investigation into the chemical composition of DOM is of prior importance. 

The main challenge in the study of DOM composition is separation of its components. In the work presented 

herein, for the separation of the compounds in the pre-dissolved in water DOM mixture, an off-line two-dimensional 

chromatography technique was used. The first dimension separation involved an anion-exchange chromatographic 

mode with suppressed conductivity detection for the separation of acids, and each of the separated peaks 

was manually collected and pre-concentrated by freeze drying. In the second dimension the pre-concentrated 

sample was restored in water and was analysed using reversed-phase liquid chromatography with tandem mass 

spectroscopy (ESI) detection. Each peak was analysed and MS/MS analysis was then performed on the two most 

intensive ions in each pattern. 

From the obtained patterns ATP and a number of amino acids were identified, demonstrating a hypothetical 

bacterial activity. Furthermore, n-phosphonomethyl glycine (glyphosate) has been detected, indicating herbicide 

contamination at this sampling point (lake site). In the higher molecular weight patterns a polymeric trend has been 

shown, demonstrating the presence of alkylic chains in more complex compounds. 

(1) R. Benner, J.D. Pakulski, M. McCarthy, J.I. Hedges, P.G. Hatcher, Bulk chemical characteristics of dissolved 

organic matter in the ocean, Science 255 (1992) 1561-1564 

(2) B.J. Dalzell, E.C. Minor, K.M. Mopper, Photodegradation of estuarine`dissolved organic matter: a multi-method 

assessment of DOM transformation, Organic Geochemistry 40 (2009) 243-257 

(3) M.P. Nawrocki, D. M. Karl, Dissolved ATP turnover in the Bransfield Strait, Antarctica during a spring bloom, 

Marine ecology progress series 57 (1989) 35-44 

350 



P06-006 TWO-DIMENSIONAL ION CHROMATOGRAPHY OF CATIONS 

Mc Gillicuddy N. 1 , Nesterenko E. 1 , Paull B. 1 , Nesterenko P.N. 2 

1 


2 



One-dimensional chromatography often does not provide adequate resolving power for many complex samples. One 

solution is multi-dimensional chromatography, which involves the exploitation of different separation mechanisms, 

and can therefore significantly improve peak resolution and selectivity. The choice of stationary phase depends on 

their orthogonality, meaning that the selectivity exhibited from the two phases used should be sufficiently diverse, 

and ideally oppositely matched. Two-dimensional chromatography, utilising ion exchange as one of the dimensions, 

has previously been used for various applications and analysis of complex samples. However, only a very limited 

number of studies using two ion exchange dimensions (2D-IC) have been completed [1], in this case utilising 

anion-exchange stationary phases on both dimensions. To-date no such study has been reported as applied to the 

analysis of cations. In the work presented herein, a 2D-IC approach for the separation of alkali, alkaline earth and 

transition metal cations has been developed. Two columns containing different stationary phases were connected 

via a simple switching valve configuration, which facilitates desired fraction collection, elution from the first column, 

and transport it to the second dimension column. The first dimension utilised chelation ion chromatography mode 

and the separation of metals was initially carried out on either polymer (Dionex Propac IMAC-10, 250 x 4 mm) and 

silica based iminodiacetic acid (IDA) functionalised columns. As expected, it was shown that separation of both 

alkali and alkaline earth metal cations was rather poor on both columns, while transition metals were well separated 

on both columns, with elution time significantly less on the polymer based column. On the second dimension a 

Phenomenex Onyx Monolith C18 (25 x 4.6 mm) column, dynamically modified with sodium dioctyl sulfosuccinate 

(DOSS) was used. The short DOSS-modified column provides the required rapid separation of the alkali and 

alkaline earth metals cations. Finally, the chelation and cation-exchange columns were used in an automated twodimensional 

arrangement for the separation of alkali, alkaline earth and transition metal cations using “heart cut” for 

trapping unresolved peaks prior the injection onto the second dimension column. 1. C.Johns, R.A. Shellie, C.A. Pohl, 

P.R. Haddad, Journal of Chromatography A, 1216 (2009) 6931–6937 


351


P06-007 PROBABILITY OF FAILURE OF THE WATERSHED ALGORITHM FOR PEAK DETECTION IN COMPREHENSIVE TWO- 

DIMENSIONAL CHROMATOGRAPHY 

Vivó-Truyols G. 1 , Janssen H.G. 2 

1 


2 

Unilever Research and Development Vlaardingen, Advanced Measurement and Data Modelling 

Corresponding author e-mail: g.vivotruyols@uva.nl 

A model to describe the signals obtained in two-dimensional chromatography was developed. The model was 

used to calculate the value of critical second-dimension retention time variability, D2tR,crit. Peaks showing an 

experimental variability in second dimension retention times above the critical value are wrongly detected when the 

so-called watershed algorithm is applied for peak detection. Several models to calculate D2tR,crit were developed: 

(a) exact model; (b) simplified model and (c) simple-modified model. Model (c) gave the best performance and 

allowed to deduce an analytical expression for the probability of failure of the watershed algorithm as a function of 

experimental D2tR, modulation time and peak width in the first and second dimension. It could be demonstrated 

that the probability of failure of the watershed algorithm under normal conditions in GCxGC is around 15-20%. Small 

changes of D2tR, modulation time and/or peak width in the first and second dimension could induce subtle changes 

in the probability of failure of the watershed algorithm. Theoretical equations were verified with experimental results 

from a diesel sample injected in GCxGC and were found to be in good agreement with the experiments. 

352 



P06-008 METABOLOMICS OF SMALL SAMPLES: MINIATURIZED SAMPLE-PREPARATION FOR GC×GC-TOFMS BASED METABOLITE 

ANALYSIS 

Kay L. 1 , Erban A. 2 , de Koning S. 1 , Kopka J. 2 

1 


2 

Max Planck Institut für Molekulare Pflanzenphysiologie , Willmitzer 

Corresponding author e-mail: lorraine.kay@lecouk.com 

Within the last years robust GC-MS metabolite fingerprinting and profiling platforms and routines were established 

for fresh plant material in a range of 50mg to 150mg basic raw material. Since the scientific progress shows a 

tendency towards observations of more specific cell-types and organs from plants, miniaturization during samplepreparation 

became necessary. We present a range of miniaturization methods during sample-preparation, such 

as manual tissue micro dissections, liquid micro sampling and laser-microdissection coupled to laser pressure 

catapulting of cyrosections, in short laser-microdissections (LMD). An outlook into the potential of GC×GC-TOFMS 

will be given, with focus on plant cyrosamples prepared with LMD 


353


P06-009 INTER-LABORATORY REPEATABILITY OF RAPID GC-TOFMS PLANT METABOLITE PROFILING AND GC-TOFMS SPATIAL 

METABOLITE ANALYSIS DURING MELON FRUIT DEVELOPMENT 

Kay L. 1 , Allwood W. 2 , de Koning S. 1 , Goodacre R. 2 

1 


2 

University of Manchester 


Within the EU Framework 6 META-PHOR (plant and food metabolomics) project (http://www.meta-phor.eu/), LECO, 

the University of Manchester and Max Plank Institute for Molecular Plant Physiology - Golm, have collaborated on a 

number of research projects focusing upon the strengths of GC-TOFMS based plant metabolomics. First the three 

groups wished to compare their three SOP’s for GC-TOFMS analysis of polar metabolites on a common sample 

set generated by a single technician within one laboratory. An evaluation of the pros and cons of each laboratories 

SOP was made as was a comparison of data from between the three laboratories. In our hands we found the 

inter-laboratory repeatability of GC-TOFMS analysis of the common sample sets was extremely promising with the 

processed datasets of the three laboratories producing near identical results when compared by classical Principle 

Component Analysis. In result of the comparison of the three laboratories SOP’s, a single analytical method was 

selected for all follow up GC-TOFMS experiments. One such experiment again performed between researchers from 

all three laboratories, studied spatial metabolite patterns in melon fruit throughout their development from immature 

fruit to commercial ripeness. Polar metabolite data from GC-TOFMS was combined with that from 1H-NMR and LC- 

MS, data based upon the analysis of VOC’s by SPME-GC-TOFMS and data based upon micronutrient content were 

also integrated thus providing many insights as to how the fruit develops through changes in primary and secondary 

metabolites and how these related to further changes in the VOC profiles. 

354 



P06-010 METABOLOMICS OF THE HYDROGEN PRODUCING ALGAE CHLAMYDOMONAS REINHARDTII USING GCXGC-TOFMS 

Keck M. 1 , Kay L. 2 , de Koning S. 2 

1 

University of Bielefeld 

2 



The microalgae Chlamydomonas reinhardtii is able to produce molecular hydrogen under specific conditions which 

is an important aspect with regard to renewable, CO2-free energy supply. In this study, we analysed the metabolite 

profiles of the high hydrogen producing strain Stm6Glc4 and the wild type cc406 (WT) before and during the 

hydrogen production phase. We have established GC×GC analysis coupled to fast TOFMS (LECO Pegasus IV) to 

analyse hydrophilic extracts of Chlamydomonas reinhardtii. GC×GC-TOFMS results in a good separation of these 

complex samples, which expands the chromatographic plane for coeluting compounds. Using the Pegasus 4D 

GC×GC-TOFMS together with the Statistical Compare feature of the LECO ChromaTOF software we were able to 

obtain a detailed view of metabolomic changes during hydrogen production. 


355

P07 HYPHENATED 

TECHNIQUES 

POSTER 

SESSIONS

POSTER SESSION 07 - HYPHENATED TECHNIQUES 

P07-001 DEVELOPMENT OF A NOVEL HYPHENATION SYSTEM BASED ON LIQUID CHROMATOGRAPHY, ISOTOPE RATIO MASS 

SPECTROMETRY AND RAMAN SPECTROSCOPY 

Wiese S. 1 , Teutenberg T. 1 , Jochmann M.A. 2 , Kujawinski D.M. 2 , Zhang L. 2 , Fischer B. 3 , Bettermann H. 3 

1 

Institute of Energy and Environmental Technology, R&D 

2 


3 

University of Heinrich-Heine 


Reliable authenticity control methods are of key importance to ensure both honest trading standards and 

consumers’ trust in food quality. The method of choice is compound-specific stable isotope analysis (CSIA) of target 

compounds. Although often presumed to be constant and stable, natural isotope abundance ratios show significant 

and characteristic variations when measured very accurately and precisely, which can be achieved by isotope 

ratio mass spectrometry (IRMS). Typically, magnetic sector mass spectrometers with fixed multiple detectors (one 

per isotope) are used. Complex compounds are reduced to simple molecules prior to measurement, for example, 

organic compounds are combusted to CO2, H2O and N2. For the determination of multiple compounds in a mixture, 

a separation is necessary prior to detection. Here, hyphenation to liquid chromatography (LC) would be an important 

addition to the well-established gas chromatography coupling. A problem arises because the mobile phase must 

be totally free from carbon for carbon isotope analysis. Hence, organic solvents that are typically used in LC have 

to be replaced by water. In order to enable the elution of polar and non-polar compounds in one chromatographic 

run, temperature programming has to be used instead of solvent programming. This technique, which is also 

known as high-temperature HPLC, is well understood and plays an important role for method optimization. What 

has to be considered is that a baseline separation is mandatory for non-biased isotope analysis since coelution 

may falsify the measured isotope ratios of a target compound. This procedure requires information concerning the 

structure of the separated analytes. The necessary structural information is obtained from Raman spectroscopy. 

One of the advantages of Raman spectroscopy is that it is not sensitive to water required as the eluent for liquid 

chromatography in LC-IRMS. In this project the Raman device is the on-line detector of high-temperature HPLC and 

links HPLC with IRMS. In this presentation, the basic concept of the novel hyphenation system will be presented. 


357


P07-002 DETERMINATION OF PRODUCTS OF HEMI(CELLULOSE) DEGRADATION BY GC/MS, PYROLYSIS-GC/MS AND CE/MS 

Bogolitsyna A. 1 , Borgards A. 2 , Rosenau T. 1 , Potthast A. 1 

1 


2 

Lenzing AG, Research and Development-Austria 

Corresponding author e-mail: anna.bogolitsyna@boku.ac.at 

The research is aimed at investigating the chemistry of hardwood polysaccharides, in particular cellulose and 

xylans, during degradation reactions with a focus on the scenarios after bleaching operations. The bleaching 

steps considered are different totally chlorine-free steps (TCF). Knowledge on reaction products and the fate and 

exact degradation mechanisms of (hemi)celluloses and residual lignin structures under alkaline conditions is still 

insufficient. The mixtures of degradation products of low-molecular weight carbohydrates under strongly alkaline 

and alkaline-oxidative conditions are very complex, as they are formed by a superposition of multiple fragmentation, 

rearrangement, and condensation reactions. Understanding the exact chemical structure of the products and their 

formation mechanisms is a prerequisite to in-depth understanding of process stream chemistry and mass balances, 

the identification and separation of possible valuable components, and the optimization of the effluent treatment 

systems. For separation of degradation products of (hemi)celluloses, an analytical methodology is required which 

shows a high sensitivity and robustness as well as the ability to simultaneously determine degradation products of 

carbohydrates and lignin. The task is rendered even more complicated by the complexity of the effluent mixture and 

the very low concentrations of its individual components. Generally, GC/MS is a suitable and sufficiently sensitive 

technique for the identification and quantification of such carbohydrate- and lignin- derived compounds. With 

carbohydrate degradation products being mainly aliphatic carboxylic mono- and di-acids and hydroxy-acids, the 

analysis by GC/MS usually requires a pre-column derivatization step. Sample derivatization is a time-consuming 

procedure which can cause some losses of volatile compounds during sample reduction and undesirable side 

reactions between sample components. In the recent years capillary electrophoresis (CE) has been established 

as a fast and suitable method for both the analysis of carbohydrates and their degradation products (aliphatic 

carboxylic acids) and the determination of lignin-derived compounds. It provides short analysis times and, at 

the same time, high separation efficiencies without a preliminary derivatization step. Coupling CE with a MSdetector 

allows achieving high selectivity and sensitivity together with providing information on analytes structure. 

Several derivatization methods for GC/MS have been tested and critically compared, applying trimethylsilylation, 

(trimethylsilyl)methylation or methylation of the respective reactive functionalities in the effluent mixture compounds. 

Also pyrolysis-GC/MS was used, either without sample derivatization or with simultaneous methylation. A 

comparison of the tested methods showed that the best separation of complex mixtures was obtained after 

methylation with diazomethane. This method allowed simultaneous determination of short- and long-chain aliphatic 

carboxylic acids and hydroxy-acids, aromatic compounds and carbohydrates with high sensitivity and efficiency 

of separation. The other esterification reagents (e.g. trimethylsilyl-diazomethane, TMS-DAM) as well as silylation 

reagents (e.g. N,O-bis(trimethylsilyl)trifluoroacetamide) allow separating mainly carboxylic acids but have difficulties 

with aromatic compounds. Pyrolysis-GC/MS provides a good knowledge on a composition of bleaching filtrates but 

gives only a principal overview of the sample content as it allows determination of the component fragments. The 

developed CE/MS method gives the possibility of the fast, selective and efficient separation of aliphatic carboxylic 

acids and phenolic compounds in one run without preliminary derivatization. 

358 



P07-003 ANALYSIS OF SYNTHETIC PHOSPHODIESTERASE INHIBITORS IN COUNTERFEIT PRODUCTS BY DATA DIRECTED LC/MS/MS, 

ACCURATE MASS MS/MS AND LC-UV 

Jones M.D., Twohig M. 

Waters Corporation-USA 

Corresponding author e-mail: Marian_Twohig@waters.com 

The ability to assess the authenticity of drug products provides confidence to the consumer on the safety of the 

medicine and protects the originators patent rights. The aim of this study is to develop analytical methods for the 

detection and characterisation of counterfeit drugs. The assay must be specific for the analytes and be sufficiently 

rapid to allow for fast sample screening. Methods: Legitimate samples of the three approved phosphodiesterase 

type-5 inhibitors (PDE-5), Sildenafil Citrate, Vardenafil Hydrochloride and Tadalafil tablet samples were purchased 

from a reputable US pharmaceutical wholesaler. “Brand” and counterfeit samples of the PDE-5 inhibitors were 

purchased online from Internet pharmacies. Sample were extracted with methanol, centrifuged and analysed by 

various analytical techniques. Chromatographic separations using 10 mM ammonium acetate and a 50:50 mixture 

of methanol/acetonitrile were preformed on a C18 column. The column was maintained at 60 deg C and a flow rate 

of 550µL/min was used. Mass Spectrometry experiments were carried out on a tandem quadrupole MS capable of 

acquiring simultaneous MS and MS/MS data. High resolution MS/MS experiments were carried out on a quadrupole 

Time of Flight mass spectrometer. Photo diode array data was collected simultaneously to provide additional 

information. The legitimate samples were compared with those obtained from internet pharmacies. Conclusions: It 

is estimated that 50% of the pharmaceutical products obtained on the internet are counterfeit1. During our study we 

encountered many counterfeit products including mixed counterfeits, those containing the active pharmaceutical 

ingredient (API) and another API, fraudulent counterfeits, containing the wrong API. Very different impurity profiles 

were obtained when authentic brand Sildenafil citrate samples were compared with equivalent “brand” products 

purchased from online pharmacies. Quantitation of the samples from the internet pharmacies and other suppliers 

of both brand and generic 100mg Sildenafil citrate tablets revealed the range of the sildenafil to be 59.6-mg 134.8 

mg per tablet. 1)The counterfeiting Superhighway http://www.eaasm.eu/Media_Centre/News/The_Counterfeiting_ 

Superhighway 


359


P07-004 DEVELOPMENT OF A DIRECT COUPLED HIGH-TEMPERATURE HPLC FOR THE FAST DETERMINATION OF ENZYMATIC 

REGULATION IN HOUSE DUST COMPONENTS USING AN ENZYMATIC ASSAY IN COMBINATION WITH MASS SPECTROMETRY 

Oeste K. 1 , Portner C. 1 , Teutenberg T. 1 , Letzel T. 2 

1 


2 

TU München, Competence Pool Weihenstephan 


The presented project is focused on the development of a fast and cost-efficient analytical method to detect 

enzymatic regulation in house dust, e. g. substances interacting with enzymes. House dust acts like a diffusive 

sampler for semi or low volatile organic compounds. Therefore, prevalent substances in house dust were used for 

method development. To detect regulatory active substances, this method combines separation by HPLC with a 

continuous-flow enzymatic assay and mass spectrometry [1, 2]. In order to maintain the enzymatic activity of the 

assay, a reduction of organic solvent in the mobile phase is necessary. Hence, high-temperature HPLC (HT-HPLC) 

is used to improve the chromatographic separation and to permit the biological function of the assay [3]. This setup 

also provides the opportunity to determine enzymatic regulation in food and feed. References: [1] A.R. de Boer et 

al. 2005. Anal. Chem. 77 (24), 7894–7900 [2] T. Letzel. 2008. Anal. Bioanal. Chem. 390, 257-261 [3] T. Teutenberg 

2010. High Temperature Liquid Chromatography: A User’s Guide for Method Development. RSC Chromatography 

Monographs 

360 



P07-005 ANALYSIS OF WAX ESTERS IN EDIBLE OILS BY AUTOMATED ON-LINE LC-GC COUPLING USING THE THROUGH OVEN 

TRANSFER ADSORPTION DESORPTION (TOTAD) INTERFACE 

Aragon A., Toledano R.M., Cortés J.M., Villén J., Vázquez A., 

Universidad de Castilla-La Mancha-Spain 

Corresponding author e-mail: ana.vazquez@uclm.es 

Vegetables oils are mainly composed of triglycerides and complex mixtures of minor compounds of different nature. 

These minor compounds can be divided into two groups: compounds derived from fatty acids, such as wax esters, 

and sterolic esters and other compounds which are not derived from fatty acids, such as sterols, hydrocarbons, 

tocopherols and others. The analysis of these minor constituents is essential as they are used as a reference for 

edible oil regulation and for the analytical assessment of its quality, origin, extraction method, refining procedure 

and possible adulteration of the oils. The Wax esters of analytical interest in vegetable oils are esters of fatty acids 

with aliphatic alcohols containing a total carbon atoms ranging from 40 to 46. In olive oils, the analysis of the wax 

esters is used for distinguishing among olive oils of different qualities such as cold pressed (extra virgin) and solvent 

extracted oils (pomace oil) [1]. The EU Official method [2] involves separation of the wax esters by silica gel column, 

collection of the corresponding fraction and further analysis by GC. It requires at least 3 hours. On-line coupling 

LC-GC separates the wax fraction by HPLC and automatically transferees it to the GC to be analyzed [3,4] avoiding 

the manipulation of the sample. Our research group has developed a new interface named TOTAD (Through Oven 

Transfer Adsorption Desorption) for the LVI of polar and non-polar solvents which is, consequently, suitable for the 

on-line coupling of LC-GC when LC is carried out in normal or reversed phase [5,6]. In the present work a simple, 

rapid and efficient method has been developed for the analysis of Wax Esters in Edible Oils by on line NPLC-GC 

using the TOTAD interface. The wax esters contents obtained with the described procedure were similar to that 

given as certified values by inter-labs certification study carried out by fourteen laboratories chosen from among 

those accredited by the International Olive Oil Council (IOOC). Since the number of operation needed is reduced, 

application of the new method to the routine analysis could be very helpful. REFERENCES 1. Nota, G.; Naviglio, D.; 

Romano, R.; Sabia, V.; Musso, S.S.; Importa, C. J. Agric. Food Chem. 1999, 47, 202-205 2. Comission Regulation 

(EC) Nº 702/2007 3. Grob, K.; Lafranchi, M.; Mariani, C. J. Chromatogr. 1989, 471, 397-405. 4. Grob, K.; Lafranchi, 

M.; Mariani, C. J. Am. Oil Chem. Soc. 1990, 67, 626-634. 5. Cortés, J.M.; Sánchez, R.; Díaz-Plaza, E.M.; Villén, J.; 

Vázquez, A. J. Agric. Food Chem. 2006, 54, 1997-2002. 6. Cortés, J.M.; Sánchez, R.; Villén, J.; Vázquez, A. J. Agric. 

Food Chem. 2006, 54, 6963-6968 


361


P07-006 AUTOMATED ANALYSIS OF WAX ESTER IN OLIVE BY ON-LINE NPLC-GC USING THE TOTAD INTERFACE: APPLICATION TO THE 

STUDY OF THE WAX ESTER CONTENT IN MONOVARIETY OLIVE OILS 

Lozano Y., Cortés J.M., Toledano R.M., Aragon A., Vázquez A., Villén J. 


Corresponding author e-mail: jesus.villen@uclm.es 

Olive Oil is mainly formed by triglycerides (95-99.7%) and by minor compounds (0.3-5%) that have a fundamental 

role, both from a nutritional and organoleptic and from an analytic point of view, as it is possible to detect the 

biological provenance and to classify product. The analysis of the wax esters is used to distinguish among olive oils 

of different qualityes such as cold pressed (extra virgin) and solvent extracted oils (pomace oil) (1). The European 

Union has established a legal limit for the wax esters C40 –C46 in extra virgin oil (250 mg/kg) and refined olive 

oils (350 mg/kg) (ECC 702/2007). Wax esters are compounds naturally present in olives; in particular, they are 

more abundant on the epicarp of the drupe and, during pressing, some of them are transferred to the oil. Wax 

ester concentrations lows are indicative of firm and hard olives, while the amount extracted is higher as softer and 

more degraded the olives are (2). The objective of this work was to study the influence of the variety of the olives 

in the composition of the wax ester of the virgin olive oil. The composition of the wax ester with 40, 42, 44 and 46 

carbon atoms of nine monovarietal virgin olive oils was studied. A total of 38 monovarietal virgin olive oil samples 

were analyzed. Six commercial monovarietal olive oil samples purchased in a local market were also analyzed. A 

simpler and faster procedure than the official one of the European Economic Community Regulation was used (3). 

Wax esters were analyzed by on line NPLC-GC using the TOTAD interface, developed by our research group (4, 5). 

This analytical method has been recently developed by Aragón et al (6). In this fully automated system, the oil with 

an internal standard is diluted and injected directly with no sample pre-treatment step other than a filtration into 

the HPLC. Wax ester are separated from other compounds of the oil in the LC system and the fraction containing 

the wax esters is automatically transferred to the GC by the TOTAD interface. A Statistical analysis was performed 

and conclusions are shown in the present work. 1. Amelio, M.; Rizzo, R.; Varazini, F. JAOCS 1998, 75, 527-530 

2. Biedermann, M.; Haase-Aschoff, P.; Grob. K. Eur. J. Lipid Sci. Technol. 2008, 110, 1084-1094 3. Comission 

Regulation (EC) Nº 702/2007 4. Pérez, M.; Alario, J.; Vázquez, A.; Villén, J. J. Microcol. Sep. 1999, 11, 582-589 5. 

Pérez, M.; Alario, J.; Vázquez, A.; Villén, J. Anal. Chem. 2000, 72, 846-852 6. Aragón et. al. Unpublished results. 

362 



P07-007 ANALYSIS OF FULLERENE NANOMATERIALS BY LIQUID CHROMATOGRAPHY/ATMOSPHERIC PRESSURE IONIZATION- 

ENHANCED RESOLUTION TANDEM MASS SPECTROMETRY 

Núñez O. 1 , Gallart-Ayala H. 1 , Martins C.P.B. 2 , Moyano E. 1 , Galceran M.T. 1 

1 


2 


Corresponding author e-mail: oscar.nunez@ub.edu 

The development, production, and use of nanomaterials are expected to increase rapidly over the next decade. 

The use of carbon-based nanoparticles such as the fullerene family, in electronic, biomedical and photovoltaic 

applications is growing [1], and some of them, namely C60 fullerene, are beginning to be used in personal care 

products. Since these materials enter into industrial and consumer use, it is inevitable that they will become 

dispersed into the environment. Therefore, it is essential to evaluate the risk of these materials on human and 

environmental health [2], although the lack of specific analytical methods for their detection and quantitation in 

environmental systems greatly limits these studies. For this reason, there is a need to develop effective, sensitive 

and reliable analytical methodologies for the analysis of these compounds in environmental samples. Liquid 

chromatography-mass spectrometry has been reported for the identification and quantitation of fullerenes in 

environmental samples such as waters [4] or water suspended materials [5], mainly using ESI or APCI ionization 

sources and focusing generally on C60 and C70 fullerenes. 

In this work, a fast liquid chromatography-enhanced resolution tandem mass spectrometry (hyperbolic triple 

quadrupole) methodology has been developed for the analysis of fullerene nanomaterials (C60 to C84). In order 

to achieve high efficiency and improve the chromatographic separation, porous shell and sub-2µm particle size 

columns with different stationary phases (C18, PFP, HILIC, phenyl hexyl, etc) were evaluated using toluene-methanol 

as mobile phase and several column temperatures. The use of different atmospheric pressure ionization sources 

(ESI, APCI and APPI) as well as dual source combinations (APPI/APCI, APPI/ESI) was evaluated and discussed. In 

general, mass spectra of fullerenes were dominated by the molecular ion [M]-•, although the oxides [MO] -• and 

[MO2] -• were also observed at low relative abundances. In order to fulfill with the identification requirements defined 

by EU Commission Decision 2002/657/EC, several scanning strategies were evaluated such as the use of enhanced 

resolution tandem mass spectrometry (Q1 or Q3 at 0.07 m/z units at full width half maximum) to achieve a mass 

resolution higher than 10.000 and the fragmentation of oxide ions to provide an additional transition for confirmation. 

Quality parameters such as limits of detection, limits of quantitation, linearity, precision and accuracy were 

established. Finally, the applicability of the method was evaluated by analyzing fullerenes in environmental samples. 

[1] Woodrow Wilson International Center for Scholars, Project on Emerging Nanotechnologies, 2007. http://www. 

nanotechproject.org/ 

[2] USEPA, U.S. Environmental Protection Agency, Nanotechnology White Paper, 2007. 

[3] D. Bouchard, X. Ma, J. Chromatogr. A 1203 (2008) 153-159. 

[4] Z. Chen, P. Westerhoff, P. Herckes, Environ. Toxicol. Chem. 27 (2008) 1852-1859. 

[5] M. Farré, S. Pérez, K. Gajda-Schrantz, V. Osorio, L. Kantiani, A. Ginebreda, D. Barceló, J. Hydrol. 383 (2010) 44-51. 


363


P07-008 UHPLC-MS/MS FOR THE ANALYSIS OF PHOTOINITIATORS IN PACKAGED FOOD: EVALUATION OF API SOURCES 

Gallart-Ayala H. 1 , Núñez O. 1 , Martins C.P.B. 2 , Moyano E. 1 , Galceran M.T. 1 

1 


2 


The alert for food contamination by ink photoinitiators arose in Europe in November 2005 when the Italian Food 

Control Authority detected the photoinitiator 2-isopropylthioxanthone (2-ITX) in baby milk at concentrations ranging 

from 120 to 300 µg/L. This led to the withdrawal of more than 30 million liters of milk from the market. Moreover, 

other photoinitiators residues such as 2-ethylhexyl-4-dimethylaminobenzoate (EHDAB) and benzophenone (BP) 

have also been found in food products. There is no regulation for this family of compounds in the EU but according 

to the European Food Safety Authority (EFSA) the presence of some of them could be considered as undesirable. 

In addition, the EU approved a Commission Regulation 2023/2006, which sets out the rules for good manufacturing 

practice of materials and articles intended to come into contact with food to prevent the contamination due to 

printing inks. 

In this work a high efficient and fast UHPLC-MS/MS method has been developed for the simultaneous analysis 

of eleven UV-ink photoinitiators in packaged food. A high efficient chromatographic separation (peak width < 8 s) 

was obtained in less than 2.5 min on a porous shell pentafluorophenylpropyl (PFP) column using methanol: formic 

acid -ammonium formate buffer as mobile phase at 850 μL min-1. The chromatographic system was coupled to a 

triple quadrupole mass spectrometer and different atmospheric pressure ionization sources (ESI, APCI, APPI and 

dual source combinations) operating in positive mode were evaluated in order to improve ionization efficiency and 

to reduce matrix effects. In general, the API single MS spectra were dominated by the [M+H]+ ion, but [M]+• was 

also observed when APPI was used and toluene added as a dopant. A different MS/MS fragmentation pattern was 

observed when [M+H]+ and [M]+• were selected as precursor ion. In order to improve the selectivity and sensitivity 

of the methodology, highly-selective reaction monitoring (H-SRM) operating at 0.1 m/z units at FWHM (full width 

half maximum) was used. Quality parameters such as limits of detection, limits of quantitation, linearity, precision 

and bias were evaluated. Finally, the UHPLC-MS/MS method was applied to the analysis of these compounds in 

packaged food samples and packaging materials at ppt levels after a simple and fast sample treatment based on 

QuEChERS methods. 

References 

- 2005 Chronology of Withdrawal of Nestlè and Other Liquid Milks www.ibfan.org/site2005/abm/paginas/articles/ 

arch_art/416-1.doc 

- A. Gil-Vergara, C. Blasco, Y. Pico, Anal. Bioanal. Chem. 389 (2007) 605. 

- Opinion of the Scientific Panel of Food Additives, Flavourings, Processing Aids and Materials in contact with Food; 

http://www.efsa.eu.int/science/afc/afc_opinions/catindex_en.html). 

- Commission Regulation (EC) No 2023/2006 

- H.Gallart-Ayala, E. Moyano, M.T. Galceran, J. Chroma. A 1208 (2008) 182. 

364 



P07-009 STABILITY-INDICATING ANALYSIS OF THE ACETYLCHOLINESTERASE INHIBITORS (-) HUPERZINE A, TACRINE AND 

GALANTAMINE 

Marques L.A., Maada I., Giera M., Kool J., de Kanter F., Lingeman H., Niessen W.M.A., Irth H. 


Stability analyses of drug substances are crucial for registration procedures. Degradation products have to be 

fully characterized not only in a chemical but also in a biological way. In the here presented study we investigated 

the stability of the three anti- Alzheimer’s drugs tacrine, (-) huperzine A and galantamine. Of these substances 

the first one is no longer in use because of its severe side effects, (-) huperzine A is in a clinical phase II trial 

and galantamine is used for the treatment of Alzheimer’s disease (AD). We investigated the stability of the three 

aforementioned substances according to ICH guidelines and determined the kinetics of the different degradation 

processes. All formed degradation products were chemically and biochemically characterized, elucidating their 

structure and defining their inhibitory effectiveness of inhibiting the target enzyme Acetylcholinesterase (AChE). 

In the case of tacrine a huge number of mono- and poly- oxygenated products were formed under the treatment 

with hydrogen peroxide. Most of the substances could be characterized by LC MS/MS analysis. The bioaffinity of 

the formed products was analyzed with an online continuous flow AChE bioassay. Under irradiation (-) huperzine A 

showed a transformation to a new photodegradation product, named photohuperzine A, which was isolated and fully 

characterized by spectroscopic methods. Photohuperzine A showed a 100 times lower activity against the target 

enzyme AChE than (-) huperzine A itself. Galantamine showed degradation under the irradiation with Xenon light 

(> 310 nm) and acid treatment; it proved basically stable under alkaline conditions and at higher temperatures. 


365


P07-010 THE USE OF RAPID AND PRECISE ELUTION PLATE AS EXTRACTION STEP FOLLOWED AS A PART OF HYPHENATED 

EXTRACTION METHOD BY HIG 

Snoblova M. 1 , Plaza M. 2 , Lojkova L. 1 , Valentova E. 1 , Vlcek J. 1 , Klejdus B. 1 

1 

Mendel University in Brno 

2 


Corresponding author e-mail: xsnoblov@node.mendelu.cz 

Development and aplication of µElution plate for the extraction of phenolic compounds from sea algae and 

their analysis using RRLC/MS/MS is described. The extraction and identification of phenolic compounds is 

presented from eight different sea algae samples, four brown algae (Cystoseira abies-marina, Sargassum vulgare, 

Sargassum muticum, Undaria pinnatifida) and four red algae (Hypnea spinella, Chondrus crispus, Halopytis 

incurvus, Porphyra spp.) via solid phase extraction (SPE) using Oasis µElution plate. Selected groups of benzoic 

acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acid), hydroxybenzaldehydes 

(4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde) and cinnamic acid derivatives (o-coumaric, p-coumaric, 

caffeic, ferulic, sinapic and chlorogenic acid) were investigated. 50 mg of algae with 80 % methanol mixture was 

extracted on Ika Ultra-Turrax® Tube Drive, filtered and evaporated. The residue was dissolved in 2 M HCl and 

hydrolyzed for 30 min. In hyphenated series of experiments, algae extracts from PLE and ultrasound-assisted 

extracts were used similarly. After passing through the extraction plate, the eluent was analyzed by rapid resolution 

liquid chromatography - tandem mass spectrometry with negative ion electrospray ionization using multiple 

reactions monitoring (MRM). Recoveries in range 96 – 100 % were obtained, with LOQs 0.01 – 2.1 pg/inj and LODs 

0.03 – 7.1 pg/inj, i.e. in the range of low µM. The µSPE enabled to avoid the evaporation step and pre-concentrate 

the analytes directly. The applied method allowed a simultaneous determination of phenols in less than 5 minutes. 

Thus, the analysis of different plant species containing trace amounts of polar phenols became possible. Phenolic 

compounds contained in algae were extracted using Oasis µElution plate for its increased sensitivity and 

pre-concentration effect – extract volume is only 500 µL. The Oasis MCX and SampliQ sorbents were selected: a 

mixed-mode sorbents, having both cation exchange and reversed-phase retention mechanisms. Condition the wells 

with 30 μL MeOH and then equilibrate with 30 μL water. Load 500 μL hydrolyzated algae sample; wash with 20 μL 

2% acetic acid/5 % methanol; elute with aqueous methanol solutions (fractions: 5 %, 10 %, 15 %, 20 % methanol, 

20 μL for each, all % are v/v %) with 2% NH4OH. The Oasis μElution plate eluates were injected directly, without 

dilution, onto the RRLC-MS/MS system. Robotic mechanism of the HPLC instrument is able to use the μElution 

plate trapping part directly and take samples from it. The work has been supported by grants No. 525/07/0338 

and 525/08/P540 from Czech Science Foundation and by the grant of the IGA AF Mendelu in Brno no. TP 1/2010. 

Merichel Plaza thanks CSIC for her I3P fellowship. 

366 



P07-011 SOLID PHASE MICRO EXTRACTION (SPME) OF OPIATES FROM URINE COUPLING WITH DESI-MS/MS DETECTION 

Kennedy J.H. 1 , Aurand C. 2 , Shirey R. 2 , Bell D.S. 2 , Wiseman J.M. 1 , Laughlin B. 1 , Sweeney B. 3, Gutierrez P. 4 , Ferrari R. 5 

1 

Prosolia, Inc, Research & Development-USA 

2 


3 

AIT Laboratories, Research & Development-USA 

4 


5 


Corresponding author e-mail: dave.bell@sial.com 

Urine testing for the presence of opiates and accurate quantitation is of critical importance for patients undergoing 

pain therapy treatment as well as possible substance abuse. Samples are typically screened using an auto analyser 

or ELISA reagent kit followed by confirmation testing by HPLC-MS/MS or GC-MS. Although automation exists, 

sample preparation and chromatography are time consuming. Recent advancements in solid phase micro extraction 

(SPME) and desorption electrospray ionization (DESI)–MS/MS make this combination of technologies applicable for 

direct analysis in matrix such as urine with minimal sample preparation. In this study, four common opiates were 

extracted from urine using SPME and analyzed directly by DESI-MS/MS. 

Urine samples were extracted using recently developed biocompatible SPME fibers coated with functionalized silica 

particles. After extraction, fibers were rinsed with water and secured in a prototype device for positioning the SPME 

fiber in the DESI spray. Analysis by scanning with a 1- D Automated DESI source coupled to a Thermo TSQ Quantum 

Discovery Max triple quadrupole mass spectrometer was completed in approximately 1 minute. 

The method was investigated over a concentration range of 10 to 2000 ng/mL for morphine, hydrocodone, 

oxycodone, oxymorphone, methadone and EDDP. Limit of Detection (LOD) and Lower Limit of Quantitation (LLOQ) 

values were comparable to those obtained by other techniques and were adequate for monitoring therapeutic levels 

of opiates as well possible abuse of these drugs. 

The approach of performing SPME-DESI-MS/MS demonstrated a unique approach for rapid analysis of biological 

samples. The results from SPME-DESI-MS/MS indicate that this methodology is suitable for direct screening of 

urine samples with minimal sample preparation and avoids the high cross reactivity of opiates in immunoassay 

techniques. The SPME-DESI-MS/MS combination provides a suitable method for combination for screening as well 

as quantitation of opiates in urine. 


367


P07-012 SIMULTANEOUS ANALYSIS OF CHLOROPHENOLS, NITROPHENOLS, ALKYLPHENOLS AND CRESOLS IN AGRICULTURAL 

SOILS BY GC-QQQ-MS/MS APPLYING A QUECHERS-BASED EXTRACTION APPROACH 

Padilla-Sánchez J.A., Plaza-Bolaños P., Romero-Gonzalez R., Garrido-Frenich A., Martínez Vidal J.L. 


Corresponding author e-mail: psj188@ual.es 

Phenolic compounds can be found in agricultural soils since they are present in phytosanitary products formulations 

or as transformation products of the active pesticides. This group comprises a variety of families, such as 

chlorophenols (CPs), nitrophenols (NTPs) or alkylphenols (APs). Some of them show high toxicity; estrogenic, 

anti-androgenic and vasodilatory activity. For these reasons, it is important to have analytical methodologies 

for the determination of a wide range of phenolic compounds in soils to determine their quality, especially for 

organic production areas. Currently, there are several extraction techniques reported in literature for the analysis 

of phenolic compounds, although most of them have been used for the extraction of individual groups of phenols, 

such as ultrasonic assisted extraction (USE), stir bar sorptive extraction (SBSE), Soxhlet extraction and pressurized 

liquid extraction (PLE). The wide polarity range of the aforementioned families makes difficult the performance of 

a simultaneous extraction. For the final determination, liquid chromatography (LC) and gas chromatography (GC) 

coupled to mass spectrometry detectors (MS) have been mainly applied. However, for the analysis by GC-MS, a 

derivatization step is necessary due to the high polarity of most of phenolic compounds. Several derivatization 

reagents have been used, such as bis(trimethylsilyl)trifluoroacetamide (BSTFA), pentafluorobenzyl chloride (PFBCl), 

pentafluorobenzyl bromide (PFBBr) or acetic acid anhydride (AAA) in basic medium. In this study, a QuEChERSbased 

procedure was developed, validated and applied to the analysis of real soil samples. The optimum results 

were obtained when 10 g of soil were extracted with 5 mL of water and 10 mL of acetonitrile (acetic acid 1%, v/v). 

Samples were shaken end-over-end for 1 h. Then, a liquid–liquid partition was formed by the addition of 1.7 g of 

sodium acetate, 4 g of sodium chloride and 6 g of anhydrous magnesium sulphate. To ensure the total remove 

of water, 1.5-mL aliquots of extract was transferred into a 2-mL polypropylene tube containing 0.75 g of MgSO4. 

A derivatization step prior to the final determination by GC coupled to a triple quadrupole analyzer operating in 

tandem MS (GC-QqQ-MS/MS) was performed using AAA and pyridine (Py). The optimized procedure was validated, 

obtaining average extraction recoveries in the range 69-103% (10 µg/kg), 65-98% (50 µg/kg), 76-112% (100 µg/kg) 

and 76–112% (300 µg/kg), with precision values RSD ≤ 22% (except for 4-chlorophenol) involving intra-day and 

inter-day studies at the same concentrations. Furthermore, 38 real soil samples were analyzed by the proposed 

method in order to assess its applicability. Some phenolic compounds (e.g. 2,4,6-trichlorophenol or 

4-tert-octylphenol) were found in the samples at trace levels (< 10 µg/kg). Acknowledgments The authors are 

grateful to Andalusian Regional Government (Regional Ministry of Innovation, Science and Enterprise)-FEDER for 

financial support (Project Ref. P07-AGR-02922). J.A.P.S. acknowledges financial support to the aforementioned 

project. P.P.B. acknowledges personal funding through Juan de la Cierva Program (Spanish Ministry of Science and 

Innovation-European Social Fund, SMSI-ESF). R.R.G. is also grateful for personal funding through Ramón y Cajal 

Program (SMSI-ESF). 

368 



P07-013 FAST AND SENSITIVE METHOD FOR THE ANALYSIS OF ESTROGENS IN WATER 

Lucci P., Núñez O., Moyano E., Galceran M.T. 


Corresponding author e-mail: mtgalceran@ub.edu 

Endocrine disrupting compounds (ECDs) are heterogeneous group of substances that may interact with the 

endocrine system of organisms. Estrogens are important members of the group of ECDs and they have been often 

identified as the major contributors to the endocrine-disrupting activity observed in aquatic environments [1]. They 

are excreted into the aquatic environment through human and animal urine and the use of natural and synthetic 

estrogens in medicine or in veterinary have caused the presence of several estrogens in aquatic ecosystems. 

Although the environmental concentrations of estrogens are very low, their adverse effect on the reproduction of 

wildlife and humans is not negligible [2]. To assess the ecological risk of these compounds, sensitive determination 

of estrogens in environmental water is needed. Several analytical methods have been developed to identify and 

quantify ECDs in water samples [3]. Currently, liquid chromatography coupled with tandem mass spectrometry 

is the most common approach. In the present study, a rapid analytical method for determination of seven 

natural and synthetic estrogens (estrone, 17β-estradiol, 17α-estradiol, estriol, 17α-ethinylestradiol, dienestrol and 

diethylstilbestrol) in water samples was developed using liquid chromatography–tandem mass spectrometry 

(LC–MS–MS). Chromatographic separations were carried out using a Fused Core Ascentis Express Phenyl-Hexyl 

HPLC column (2.7 μm, 10 cm × 2.1 mm i.d.) and isocratic elution with H2O/ACN/MeOH (51:44:5; v/v/v) operating 

at a flow rate of 450 μl/min and a temperature of 35C. The chromatographic separation of the selected estrogens 

was carried out in 2 min, representing a considerable reduction in run time compared to the LC method previously 

reported in bibliography [4-5]. Narrow peak width with good symmetry were obtained. Good separation was achieved 

with high resolving power (Rs > than 1.5) for 17α and 17β isomers of estradiol. Satisfactory results (Rs > than 1.3) 

have also been obtained for the separation of dienestrol and diethylstilbestrol. Tandem mass spectrometry analysis 

was performed with TSQ Quantum Ultra AM mass spectrometer (Thermo Fisher Scientific) by using multiple reaction 

monitoring (MRM). In order to evaluate the best ionization mode, the sensitivity of three different API sources (ESI, 

APCI, APPI) was assessed. Details on the operational consideration for such analysis as well as validation results for 

this study will be presented. ACKNOWLEDGEMENT This work has been supported by CARBOSORB project [(FP7 

Marie Curie Industry-Academia Partnerships and Pathways (IAPP)] REFERENCES [1]. M. Auriol, Y. Filali-Meknassi, 

R.D. Tyagi, C.D. Adams, R.Y. Surampalli, Process Biochem. 41:525–539 (2006). [2]. J.G Vos, E. Dybing, H.A. Greim, O. 

Ladefoged, C. Lambre, J.V. Tarazona, I. Brandt, A.D. Vethaak, Crit. Rev. Toxicol. 30: 71–133 (2000). [3]. R.D. Briciu, A. 

Kot-Wasik, J. Namiesnik, J Chromatogr Sci. 47: 127-39 (2009). [4]. C. Miège, P. Bados, C. Brosse, M. Coquery, Trends 

Anal. Chem. 28: 237-44 (2009). [5]. S.L. Rice, R.C. Hale, Anal. Chem. 81: 6716–24 (2009). 


369

P08 COLUMN 

TECHNOLOGY 

POSTER 

SESSIONS

POSTER SESSION 08 - COLUMN TECHNOLOGY 

P08-001 MICROBORE COLUMNS: A CONTRIBUTION TO GREEN CHEMISTRY 

Espuelas C. 1 , Ruth K. 2 , Hartmann T. 2 , Wille A. 2 

1 

Gomensoro S.A., Ion Chromatography-Spain 

2 


Industrie`s impact on the environment is steadily growing. Therefore, ecological approaches and sustainable 

procedures are needed. In response to the environmental challenges, several important laws such as the Clean Air 

and Water Act or the Pollution Prevention Act have been approved. While these acts rather focus on remediation 

in the sense that pollution is treated after it is formed, EPA`s green chemistry attempt aims at pollution prevention. 

It encourages the design of chemical products and processes that minimize the use and generation of hazardous 

substances. 

Microbore chromatography employs columns that have a smaller inner diameter (2 mm i.d.) than the normal bore 

columns (4 mm i.d.) and are thus operated with lower eluent flow rates (0.2 mL/min compared to 0.7…1.0 mL/min). 

Not least because of their decreased solvent consumption, they comply with EPA`s green chemistry philosophy. 

Moreover, environmentally benign carbonate/hydrogen carbonate solutions are important eluents for most anion 

separations. In the course of system miniaturization, not only columns got thinner, but also the suppressor module 

was scaled down, leading to reduced consumption of regenerant solution: for complete regeneration, as little as 

2 mL acid (1 mol/L) is sufficient and no exchange of cartridges or membranes is necessary. With 2 mm microbore 

columns and the adapted suppressor module, lower overall flow rates are achieved. Besides the ecological benefits, 

microbore columns require less frequent eluent preparation and thus improve accuracy and save time. Additionally, 

they get along with only limited sample material and can be perfectly interfaced with various inline detectors. 

This poster presents several applications that demonstrate the merits of a 2 mm i.d. column packed with a highcapacity 

anion exchange resin. 


371


P08-002 ON THE DEVIATIONS IN RETENTION TIMES AT INCREASING FLOW-RATE WITH CHROMOLITH COLUMNS 

García-Álvarez-Coque M.C., Pous-Torres S., Torres-Lapasió J.R., Ruiz-Ángel M.J. 



Silica-based monolithic columns are becoming usual in the analytical laboratories. So far, only the columns 

developed by the group of Nakanishi in cooperation with Merck KGaA (Darmstadt, Germany) are commercially 

available, under the trade names Chromolith and Onyx (Phenomenex, Torrance, CA, USA), licensed from Merck 

KGaA. These columns are made of a silica skeleton rod (a single piece), encapsulated within a poly(ether ether 

ketone) (PEEK) tube, tightly melted on the outer surface of the silica rod. The structural characteristics of these 

monoliths, and those of the conventional beds of microparticulate silica-based packing materials used in liquid 

chromatography, are very different. The most important features of Chromolith (Onyx) columns are their high 

external porosity resulting from the structure of the network of macropores (with a size of around 2.2 micrometers 

interconnected by channels through which the mobile phase flows), and the structure of the silica skeleton that 

consists in a network of small, thin threads of porous silica (mesopores). These structural features allow the 

combination of a low hydraulic resistance to the stream of mobile phase, and an enhancement in the rate of mass 

transfer of sample molecules. The volume of the channels (macropores) open for percolation of the mobile phase 

through the bed of stationary phase in monolithic columns is larger than the volume between the particles in packed 

columns. Also, the structure of the monolithic channels is less tortuous than the equivalent in packed columns. This 

allows flow-rates in the monoliths beyond those feasible for conventional microparticulate packed columns. Ideally, 

retention time versus reversed flow-rate plots should exhibit null intercepts, and slopes coinciding with the retention 

time at 1 ml/min. However, significant positive deviations of the intercepts correlating with the solute polarity were 

observed for several beta-blockers chromatographed with a Chromolith column in the 0.5–6 ml/min range, owing to 

the increased system pressure. Consequently, the retention factors depended slightly on the flow-rate. Chromoliths 

(packed in PEEK tubes) should suffer with pressure larger stress than microparticulate columns (packed in stainless 

steel tubes) and tend to swell and increase their volume. This would decrease the linear velocity inside the columns, 

and increase the retention at relatively low pressure (< 200 bar). In contrast, frictional heating, which is an issue 

for microparticulate columns, seems to be less significant for the highly permeable Chromoliths. The deviations 

associated to the perturbation of the retention equilibria by pressure should be smaller, owing to the size of the 

probe compounds and the working pressure. Independently of the reason of the deviations, the usefulness of the 

retention time versus reversed flow-rate plots to measure the deviations is here demonstrated. These plots allow 

an estimation of an apparent flow-rate and the correction of the retention times to the ideal behaviour, where the 

retention factors are independent of the flow-rate. 

The need for novel packing materials in both capillary liquid chromatography (CLC) and capillary 

electrochromatography (CEC) is apparent and the development towards more selective, application-oriented 

chromatographic phases is under development world-wide. In this study we have employed new synthesized metal 

oxide particles, functionalized or non-functionalized, as packing material for capillaries in LC and CEC. Especially in 

CEC the capillary packing procedure and the preparation of frits are challenging tasks, because packed capillaries 

have to withstand the high electric fields during the separation. In addition, to minimize bubble formation during 

the separation, the vials at the both ends of the capillary must generally be pressurized. The nanocasting approach 

has proven to be an excellent tool for preparing metal oxide materials with a controlled porosity and morphology. 

Since the morphology of the metal oxide replicas is dependent on the size and shape of the starting silica template, 

this technique has been adopted to prepare hierarchically porous monoliths [1] as well as chromatographic beads 

[2] consisting of fully crystalline Mn2O3, SnO2, ZrO2, and TiO2 phases. Nanocast SnO2 microspheres have 

shown excellent phosphopeptide enrichment capabilities [3]. The aim of this work was to study the applicability of 

polyethyleneimine-functionalized metal oxide materials to the separation of different analytes, and to clarify their 

ion-exchange/hydrophobic properties as packing materials in CLC and CEC. The effect of various pH-values, ionic 

strengths, types of buffers, and addition of organic solvents were investigated. The applicability of different types 

of frits on the performance of the separation will be demonstrated. Comparison of the techniques will be made with 

specific emphasis on the characteristic separation properties. 

[1] Smått, J.-H.; Weidenthaler, C.; Rosenholm, J.B.; Lindén,M., Chem. Mater. 2006, 18, 1443–1450 [2] Smått, J.-H.; 

Schüwer, N.; Järn, M.; Lindner, W.; Lindén, M., Microp. Mesop. Mater. 2008, 112, 308–318 [3] Sturm, M.; Leitner, A.; 

Smått, J.-H.; Lindén, M.; Lindner, W., Adv. Funct. Mater. 2008, 18, 2381–2389 

372 



P08-003 POLYETHYLENEIMINE-FUNCTIONALIZED METAL OXIDE MATERIALS FOR CAPILLARY LIQUID CHROMATOGRAPHY AND 

CAPILLARY ELECTROCHROMATOGRAPHY 

Bourdin D. 1 , Smått J-H. 2 , Sakeye M. 2 , Ruiz-Jiménez J. 1 , Baños-Pérez C. 1 , D’Orazio G. 3 , Kopperi M. 1 , Wiedmer S.K. 1 , Riekkola M-L. 1 , Fanali S. 3 

1 


2 

Åbo Akademi University 

3 

Institute of Chemical Methodologies, National Council of Research, Rome, Italy 

Corresponding author e-mail: marja-liisa.riekkola@helsinki.fi 

The need for novel packing materials in both capillary liquid chromatography (CLC) and capillary 

electrochromatography (CEC) is apparent and the development towards more selective, application-oriented 

chromatographic phases is under development world-wide. In this study we have employed new synthesized metal 

oxide particles, functionalized or non-functionalized, as packing material for capillaries in LC and CEC. Especially in 

CEC the capillary packing procedure and the preparation of frits are challenging tasks, because packed capillaries 

have to withstand the high electric fields during the separation. In addition, to minimize bubble formation during 

the separation, the vials at the both ends of the capillary must generally be pressurized. The nanocasting approach 

has proven to be an excellent tool for preparing metal oxide materials with a controlled porosity and morphology. 

Since the morphology of the metal oxide replicas is dependent on the size and shape of the starting silica template, 

this technique has been adopted to prepare hierarchically porous monoliths [1] as well as chromatographic beads 

[2] consisting of fully crystalline Mn2O3, SnO2, ZrO2, and TiO2 phases. Nanocast SnO2 microspheres have 

shown excellent phosphopeptide enrichment capabilities [3]. The aim of this work was to study the applicability of 

polyethyleneimine-functionalized metal oxide materials to the separation of different analytes, and to clarify their 

ion-exchange/hydrophobic properties as packing materials in CLC and CEC. The effect of various pH-values, ionic 

strengths, types of buffers, and addition of organic solvents were investigated. The applicability of different types 

of frits on the performance of the separation will be demonstrated. Comparison of the techniques will be made with 

specific emphasis on the characteristic separation properties. 

[1] Smått, J.-H.; Weidenthaler, C.; Rosenholm, J.B.; Lindén,M., Chem. Mater. 2006, 18, 1443–1450 [2] Smått, J.-H.; 

Schüwer, N.; Järn, M.; Lindner, W.; Lindén, M., Microp. Mesop. Mater. 2008, 112, 308–318 [3] Sturm, M.; Leitner, A.; 

Smått, J.-H.; Lindén, M.; Lindner, W., Adv. Funct. Mater. 2008, 18, 2381–2389 


373


P08-004 EFFECT OF MONOMER MIXTURE COMPOSITION ON SEPARATION OF SMALL MOLECULES USING POLY(DIVINYLBENZENE- 

CO-ETHYLVINYLBENZENE-CO-2-HYDROXYETHYL METHACRYLATE) MONOLITHIC ROD COLUMNS 

Smirnov K.N., Dyatchkov I.A., Telnov M.V., Pirogov A.V., Shpigun O.A., 


In recent years, porous polymer monoliths have been widely used as the stationary phases for the rapid high 

performance separation of biomacromolecules in gradient elution mode. The separation is provided by the high 

hydrodynamic permeability of monoliths, fast mass transfer kinetics in the column, and strong dependency of 

retention of multifunctional macromolecules on the mobile phase composition. These polymeric monoliths usually 

have low specific surface areas (< 50 m2/g). They are useless in the isocratic separation of low-molecular-weight 

organic compounds with similar properties. The development of polymer-based monolithic columns for the HPLC of 

small organic molecules is an actual problem. 

In the present study, poly(divinylbenzene-co-ethylvinylbenzene-co-2-hydroxyethyl methacrylate) monolithic rods 

were synthesized and applied to the reversed-phase HPLC separation of aromatic compounds. Monoliths were 

prepared via thermally initiated free-radical polymerization inside glass columns (150 mm × 3 mm i.d.). The reaction 

proceeded at 60°C for 22 h. 2,2’-Azo-bis-isobutironitrile was used as an initiator. Dodecanol-1 was used as a 

porogen. To provide covalent attachment of the monolith to the column wall, the surface of glass was modified with 

3-(trimethoxysilyl)propyl methacrylate. In order to compensate for the polymer shrinkage during the synthesis and 

prevent monolith from detachment from the wall, polymerization was conducted under the pressure of nitrogen. 

A series of monoliths with different monomer compositions was obtained. All the monoliths had high BET specific 

surface areas ranging from 380 to 490 m2/g. Increasing molar fraction of 2-hydroxyethyl methacrylate (HEMA) in the 

starting monomer mixture from 11 to 15% resulted in two orders of magnitude decrease in the column permeability 

(from 10−12 to 10−14 m2) and led to weaker retention of alkylbenzenes. Higher HEMA content was found to increase 

separation efficiency from 2000–5000 to 13000–19000 plates/m. The obtained columns were stable in water/ 

acetonitrile eluents containing up to 60% of water under pressures up to 100 bar. 

374 



P08-005 A DEVELOPMENT AND APPLICATION STUDY FOR ANION EXCHANGE+CATION EXCHANGE+NORMAL PHASE+REVERSED 

PHASE MULTI-MODE ODS COLUMN 

Yazawa I. 

Imtakt Corporation 

We have developed a novel multi-mode ODS column which provides anion exchange, cation exchange, normal 

phase, and reversed-phase. The technology consists of three types of ligands, including: ODS, anion, and cation 

ligands, and provides not only ion exchange and reversed phase separation mode, but also normal phase mode 

for polar compounds. This mode of separation is advantageous for various types of analysis, including: hydrophilic 

anions, cations, and drug metabolite analysis containing both ionic-hydrophobic and hydrophilic compounds. In 

addition, it can be useful for LC-MS by using low buffer concentration without ion pairing additives. This new 

multi-mode ODS technology offers different separation characteristics than conventional C18 phase - separations 

can be optimized by changing organic solvent concentration, pH, and / or ionic strength. This new technology has 

many opportunities to expand separations using C18 and other reversed / normal phase ligands. 


375


P08-006 COMPARISON OF FUSED CORE AND CONVENTIONAL PARTICLE HPLC COLUMNS FOR THE ANALYSIS OF ISOFLAVONES 


1 


2 


Corresponding author e-mail: manchon.noelia@inia.es 

Analysis of isoflavones is generally carried out by HPLC using different types of columns. The most used 

stationary phases are conventional reverse phase particle columns (3.5-5 µm) using mobile phases composed 

of water, acetonitrile and methanol with small amounts of acid. Analysis range between thirty and sixty minutes, 

which seriously limits sample output. Reducing particle size increases efficiencies but generates higher column 

backpressure, limiting flow rates and alternatives to reduce analysis time. In order to overcome these limitations, 

a new type of fused core particle columns has been developed. They are made of a solid kernel of silica (1.9 μm) 

covering with a layer of homogeneous porous material (0.35 μm) creating a total particle diameter of 2.6 μm. Because 

of particles are not fully porous like conventional particles the efficacy in chromatographic peaks are improved, 

allowing faster mass transfers and less effect of priority pathways (Eddy dispersion). Since this technology has only 

recently become available, there are no reports about the suitability and performance for the separation of natural 

products. Therefore, the objective of this work was the comparison of two different columns, a conventional particle 

and a fused core particle column, for the analysis of twelve main soy isoflavones (genistin, daidzin, glycitin and their 

respective acetyl, malonyl and aglycone forms) using different solvents and temperatures. The columns compared 

were XbridgeTM C18 (3.5 μm, 4.6x150 mm) and KinetexTM C18 (2.6 µm, 100 Å, 4.6x100 mm). HPLC analysis was 

carried out using water with 1% acetic acid (solvent A) and methanol 0.1% acetic acid or acetonitrile (solvent B) using 

different temperatures (25 and 30 ºC). The identification of isoflavones was carried out by comparison of retention 

times and UV spectra. Mean pressure reduction with the fused core particle column was 14 % when compared 

to the conventional particle column using the same flow rate. Also, due to the shorter column, analysis time was 

reduced for both methanol (18.5 %) and acetonitrile (30 % less) with the fused core particle column while maintaining 

resolution. Results also indicated that using the new technology it was possible to achieve a higher number of 

plates, lower peak width and improved peak symmetry. The narrower peaks are traduced into lower detection and 

quantitation levels. Moreover, the lower column backpressure may be explored to further reduce analysis time by 

increasing flow rate. Finally, the fused core showed remarkable inter and intra-day reproducibility. Therefore this new 

technology could be revolutionary way to faster isoflavones analysis in foods and to improve resolution, accuracy 

and sensitivity. 

376 



P08-007 A NEW RANGE OF HIGHLY RETENTIVE REVERSED-PHASE PACKINGS FOR LC AND LC/MS 

Faulkner W., Gartland J., Pereira L., Milton D. 


Highly retentive column packings based on high surface area silica supports can extend chromatographic 

performance even with small geometry columns. Short column lengths and small diameters can be used without 

compromising peak capacity or sensitivity. Increased analyte retention will also normally provide a better response 

in LC/API-MS, as the use of higher concentrations of organic modifier to facilitated elution improve nebulization and 

therefore sensitivity of the analysis. In the work presented in this poster, a new range of reversed-phase column 

materials based on a highly pure, high surface area silica and novel bonding technology is introduced. This high 

performance range of columns are available in different functionalities for selectivity screening or 

fine-tuning complex separations, and in a range of particles sizes down to sub-2μm to facilitate the development 

of fast, efficient and robust methods. The performance of these materials is demonstrated for reproducibility and 

stability and the phases are characterized for primary and secondary interactions. 


377


P08-008 EVALUATION OF TWO NEW IONIC LIQUIDS AS HIGH STABILITY AND SELECTIVE STATIONARY PHASES IN GAS 


González Álvarez J., Blanco Gomis D., Arias Abrodo P., Díaz Llorente D., Busto E., Ríos Lombardía N., Gotor Fernández V., Gutiérrez 

Álvarez M.D. 


Corresponding author e-mail: loly@uniovi.es 

Two ionic liquids (ILs), namely, (S,S)-1-butyl-3-(2’-hydroxy-cyclohexyl)-3H-imidazol-1-ium tetrafluoroborate and 

(S,S)-1-butyl-3-(2’-acetyl-cyclohexyl)-3H-imidazol-1-ium tetrafluoroborate have been employed as stationary phases 

in capillary gas chromatography. These new phases have demonstrated good column efficiency, wide operating 

temperature range and good thermal stability. Inverse Gas Chromatography (GC) analyses have been used to study 

the solvation properties of these ILs through a linear solvation energy model. The application of these ILs as new GC 

stationary phases was studied. These stationary phases exhibited unique selectivity for many organic substances, 

such as, alkanes, ketones, esters and aromatic compounds. 

The efficient separation of several mixtures containing compounds of different polarities and the good separation of 

the fatty acids methyl esters (FAMEs) and cis/trans isomers, indicate that they may be applicable as a new type of 

GC stationary phases. 

378 



P08-009 USE OF SCANNING CONTACTLESS CONDUCTIVITY DETECTION (SC4D) FOR THE VISUALISATION OF STATIONARY PHASE 

GRADIENTS IN CAPILLARY POLYMERIC MONOLITHIC ROD COLUMNS 

Currivan S., Connolly d., Paull B. 



The use of mobile phase and stationary phase gradients in separation processes is still an area within which some 

fundamental principles have yet to be fully understood. This is particularly the case for stationary phase gradients, 

be they gradients in charge, hydrophobicity, porosity or immobilised pH. In previous accounts of stationary phase 

gradient formation, both with particle packed and monolithic columns, the characterisation of the gradient could 

not be directly determined without the use of destructive methods, such as SEM and/or EDX, or through nonuniversal 

fluorescence based on-column visualisation methods. In the work presented here, sC4D was utilised for 

the direct measurement of the longitudinally varying stationary phase charge, originating from grafted ionic sites 

upon a monolithic polymer substrate. A variety of photo-grafted stationary phase gradients have been produced and 

characterised using sC4D, together with evaluation of retention factor (k) and efficiency, to understand the effects 

of a gradient stationary phase on chromatographic performance. For example the ion exchange chromatography 

of inorganic cations was explored on a gradient sulphonated monolith [1], whose charge distribution was fully 

characterised using sC4D. Additionally, a column consisting of a pH gradient was also formed and characterised 

using the sC4D method. A hydrophilic column (such as GMA-co-EDMA) was prepared and after amination reaction 

an ampholine solution was flushed through the monolith for a period of time. A voltage was applied slowly across 

the monolith and the reaction was completed at 60° C for 24 hours [2,3 ]. sC4D was applied here to visualise the 

distribution of ampholytes within the monolith. Physical and chromatographic results of both these investigations will 

be presented here. 1. Currivan, S.,et al, J. Sep. Sci., 2010, 33, 484 2. Yang, C., et al, Electrophoresis, 2004, 25, 1729 

3. Zhu, G., et al, Electrophoresis, 2006, 27, 3578 


379


P08-010 GOLD NANO-PARTICLE MODIFIED MONOLITHIC CAPILLARY COLUMNS FOR USE IN SELECTIVE MICRO-EXTRACTION OF 

THIOL GROUP CONTAINING COMPOUNDS 

Danilevics U. 1 , Walsh Z. 1 , Collins D. 1 , Nesterenko E.P. 1 , Paull B. 1 , Macka M. 2 

1 


2 



Monolithic columns have been shown to be an attractive alternative to conventional packed columns. The 

advantages of silica-based monoliths are high permeability, mechanical and thermal stability, and well studied 

surface functionalisation. 

Gold surfaces are known for their strong affinity for thiol groups, which has been exploited to create self assembled 

monolayers (SAMs) of thiol containing functional ligands. The broader aims of this project are to use gold nanoparticle 

modified inorganic monoliths as stationary phases for selectively trapping thiol group containing substances. 

In the work presented herein a novel method for the surface modification of the bare monolithic scaffold to create 

selective surfaces required for specific interactions was introduced. Thermal deposition of gold from a liquid 

organo-gold formulation (commercially used as gold paint for glass and porcelain) was used for creating gold nanoparticle 

coated silica monoliths. In the here developed procedure the diluted gold paint was first flushed through 

the monolithic silica capillary column. Afterwards a high temperature treatment was applied in order to create gold 

nano-particles on the silica surface after decomposing the organic matter in the paint and inducing the reduction of 

gold ions (Au3+ and Au+) to metallic gold (Au0). 

Gold nano-particle modified monoliths were characterised using Scanning Electron Microscopy (SEM), high 

resolution field emission SEM and optical microscopy, showing a coverage of the silica surface with gold nanoparticles 

of approx. 20-30 nm in diameter. Energy Dispersive X-ray (EDX) spectrometry was used to quantify amount 

of gold deposited onto the surface. The chromatographic behaviour and suitability of the obtained gold-modified 

silica monolithic microcolumns for selective extraction/trapping was tested using test compounds including thiols 

and peptides. 

380 



P08-012 HIGH CAPACITY GOLD NANO-PARTICLE MODIFIED MONOLITHIC STATIONARY PHASES FOR FLOW-THROUGH CATALYSIS 

OF SELECTED REDOX REACTIONS 

Floris P., Connolly D., Alwael H., Paull B. 



The use of gold nano-particles (AuNP) as a catalyst in electron transfer reactions has been well documented over 

recent years, however one of the main disadvantages of common catalytic reactions is that the AuNP catalyst 

is difficult to isolate from the product(s) after the completion of the reaction. A number of reports have therefore 

described the immobilisation of AuNP on solid supports such as silica, polymer resins or ion-exchange resins, 

to facilitate the easy recovery of the catalyst by filtration or centrifugation etc. Porous polymer monoliths (PPM) 

represent another potential support for flow-through catalysis due to their porous structure and interconnected flowpaths 

(pore diameter ~ 1-2 µm), thereby eliminating the need for centrifugation/filtration steps. This work represents 

the very first report of an AuNP-modified PPM for flow-through catalysis of selected redox conversions using 

monoliths fabricated both in pipette-tip and capillary (100 µm) formats for off-line or on-line operation, respectively. 

The methacrylate monoliths formed in both formats were modified with vinyl azlactone using photografting methods 

in a second step and then reacted with ethylenediamine to provide amine attachment sites for the subsequent 

immobilisation of AuNP. Citrate stabilised gold nanoparticles (20 nm) were flushed through each aminated polymer 

monolith resulting in covalent attachment of gold via the lone pair on the nitrogen (free amine). Field emission 

scanning electron microscopy was used to verify the high surface coverage of AuNP on the monoliths. The 

catalytic activity of AuNP immobilised on a polymer monolith in both tip and capillary format was demonstrated 

using selected redox reactions such as the reduction of Fe (III) to Fe (II) by sodium borohydride. A mixture of 

hexacyanoferrate (III) and sodium borohydride was pumped at 0.25 ml/hr using a syringe pump through two different 

monoliths in the tip format, one containing immobilised gold nanoparticles and the other having no gold present 

on the surface, but sharing the same polymer structure. The elimination of the absorption band at 420 nm which 

was indicative of the hexacyanoferreate (ІІІ) complex indicated complete reduction of Fe (III) to Fe (II). This AuNPmodified 

monolith in pipette-tip format has potential for off-line sample derivatisation prior to speciation studies by 

chromatographic means. In addition, the conversion of 4-nitrophenol to 4-aminophenol by sodium borohydride was 

also carried out on a 20 cm x 100 µm polymer monolith in a capillary format, by injecting nanolitre volumes onto the 

monolith, indicating that this AuNP-modified monolith could be operated completely on-line either before or after a 

separation column for pre- or post-column derivatisation. 


381


P08-013 PREPARATION AND CHARACTERISATION OF C-60 FULLERENE-MODIFIED GRAPHITISED-CARBON MONOLITHS FOR 

CHROMATOGRAPHIC APPLICATIONS 

He X., Paull B. 



In recent years monolithic graphitised and glassy carbon rods have been investigated as an alternative type of 

material to silica and organic polymers for application within high performance liquid chromatography. Potentially 

these carbon based materials have many advantages, such as thermal and hydrolytic stability and resistance to 

swelling. However, the main disadvantage of carbon monoliths reported up to date is a relatively low surface area 

and poor homogeneity of surface chemistry. Modification of the carbon monolithic skeleton with C-60 fullerenes 

which have a size of approximately 1 nm and very large surface area, can considerably increase the surface area 

of the prepared carbon monolith, and provide the unique selectivity previously demonstrated for fullerene modified 

silica gels, which suggest that the C60 bonded stationary phase should provide effective separation of polyaromatic 

hydrocarbons (PAH) and calixarene through π-π interactions. 1-2 

In the work presented herein, C-60-templated carbon monolithic rods were fabricated and characterised. The 

preparation of this carbon monolithic material consisted of five separated stages including synthesis of the C-60 

fullerenes immobilised 1.3-μm silica template, Impregnation of silica with a copolymer of a resorcinol/iron (III) 

complex and formaldehyde, carbonisation under inert atmosphere and removal of the silica beads and the catalyst 

using hydrofluoric acid. 4-5 To confirm the attachment of C60 fullerenes to silica template, the obtained product 

was characterised using scanning electron microscopy (SEM) field emission scanning electron microscopy (FE 

SEM), energy-dispersive X-ray (EDX) spectroscopy and Fourier transform infrared (FT IR) spectroscopy. All the 

above methods were used to confirm the covalent attachment of fullerenes to the silica surface. The prepared 

porous graphitic carbon monoliths, both nano-templated and blank (for comparison and reference purposes) were 

characterised by a number of methods. The morphology of the obtained carbonaceous rods was imaged by FE-SEM 

to reveal a highly interconnected bimodal porous structure. Also other methods SUCH as FT IR spectroscopy and 

EDX analysis were used to confirm the degree of removal of silica template after HF treatment. 

References: 

1. Jinno, K., Tanabe, K., Saito, Y., Nagashima, H., Analyst 1997, 122, 787 

2. Jinno, K., Tanabe, K., Saito, Y., Nagashima, H., Tregove, R.D., Anal. Commun. 1997, 34, 175 

3. Liang, C., Dai, S., Guiochon, G., Anal.Chem. 2003, 75, 4904 

4. Eltmimi, A.H., Barron, L., Rafferty, A., Hanrahan, J.P., Fedyanina, O., Nesterenko, E., Nesterenko P.N., Paull, B., J. 

Sep. Sci. 2010, 33, 1231 

382 



P08-015 CORE-SHELL STATIONARY PHASES USING ZIRCONIA CORES WITH POROUS NANODIAMOND SHELLS FOR REVERSED-PHASE 

HPLC 

Wiest L.A. 1 , Jensen D.S. 1 , Olsen R. 1 , Vail M.A. 2, Dadson A. 2 , Linford M.R. 1 

1 

Brigham Young University-Unites States 

2 

US Synthetic Corporation, Research and Development 


A new core-shell liquid chromatography phase for HPLC was created using 1.7 μm zirconium oxide (zirconia) cores 

with 0.5 μm porous nanodiamond shells. The resulting 3 cm column was analyzed with a test mixture containing 

four aromatic analytes: benzene, toluene, xylene and mesitylene, using a 1:1 acetonitrile/water mobile phase. All 

compounds were baseline separated, where the mesitylene peak had 41700 plates per meter with k’=8.1, a symmetry 

factor of 1.03, and a retention time of 7.1 minutes. The flow rate was 0.5 mL/min with a back pressure of 2200 psi 

(152 bar). The purpose in preparing this new stationary phase was to create a material with high stability at both 

very low and very high pH. This should allow proteins to be separated in their natural form. Analogous core-shell 

materials were prepared in an identical fashion on diamond core particles, where the resulting 

core-shell particles had 20 nm pores, which is ideal for peptide and small protein separations. For the zirconiadiamond 

core-shell materials, the separations were initially reproducible, but over an extended period of time the 

retention times decreased, resulting in chromatogram degradation. Initial results show that crosslinking the porous 

shell, and subsequent functionalization, all of which can be accomplished with straightforward chemistry, increases 

the phases’ mechanical stability and leads to an effective C18 column. 


383


POSTER 

SESSIONS

POSTER SESSION 09 - NANOTECHNOLOGY 

P09-001 ABSORPTION ENHANCEMENT ACHIEVED BY NANOSIZED ACTIVE PHARMACEUTICAL INGREDIENTS – PAMPA/HPLC 

EXPERIMENTS 

Rezacova A. 1 , Grunwaldova V. 1 , Oktabec Z. 1,2 , Kral V. 1,3 

1 

Zentiva k.s., Development-Czech Republic 

2 


3 

Institute of Chemical Technology 

Corresponding author e-mail: anna.rezacova@zentiva.cz 

An important question of current pharmaceutical industry is: how to achieve bioavailability for active compounds 

from BCS groups II and IV. These compounds are very hydrophobic and thus bad soluble and absorbable under 

physiological conditions. One of the solutions of this problem is nanotechnological approach. Kinetics as well as 

solubility is a function of particle size. Nanoparticles give us a clue to achieve required therapeutical concentrations 

even in case of very lipophilic substances. Parallel artificial membrane permeability assays (PAMPA) have become 

a very useful tool for predicting in vivo drug permeability and are well-suited as a ranking tool for the assessment of 

compounds with passive transport mechanisms. Use of the PAMPA assay allows compounds ranking into a low or 

high classification, using UV-VIS spectroscopy or HPLC [1]. Three samples (Glimepiride, Meloxicam and Valsartan) 

of nanoparticles were compared with samples containing the same micrometer-sized APIs. PAMPA plates were used 

according to the manual and consequently a HPLC method was used for determination of content of compound 

in acceptor/donor solution. All samples containing nanoparticles showed a higher permeability in a contrast to 

micrometer-sized APIs (see the attached Table). [1] http://www.bdbiosciences.com/, (21/05/2010). 


385


P09-002 CAN TEMPERATURE PLAY A ROLE ON NANO-LC COLUMN PACKING? 

Capriotti F., Palma P., Leonardis I., Famiglini G., Cappiello A. 

Università di Urbino, DiGeoTeCA-Italy 

Corresponding author e-mail: pierangela.palma@uniurb.it 

Nano-LC columns allow us to work with minute sample size and small volumetric flow-rates, therefore, they show 

an increase of sensitivity due to a reduced sample dilution. This is important for LC-MS coupling, in particular 

with concentration-sensitive detectors such as electrospray mass spectrometry. In this work we evaluated how 

temperature can affect the packing procedure of nano-LC columns, based on the slurry-packing technique, and 

their performance. Suspensions of packing material, C18 or C8 Pinnacle II stationary phases (Restek, Bellefonte, 

PA, USA), were prepared in a suitable mixture of solvents and were tested at different temperatures (room 

temperature, 50°C and 75°C). We observed that at higher temperatures the slurry sedimentation is slower. This is 

very interesting: in fact, it is important that the slurry is as homogeneous as possible during packing procedure to 

facilitate the process, avoiding the formation of obstructions. Using high temperature we could easily pack longer 

and more efficient columns (>15 cm). A lab-made frit was sinterized inside a fused silica capillary tubing using a 

suspension of silica and the heat of a flame for a few seconds. The performance of several columns was evaluated 

at different operating conditions through the calculation of the following parameters: capacity factor, k’; asymmetry 

factor, As; number of theoretical plates, N; Van Deemter plot. Long columns offer a high backpressure, therefore 

it was possible to test them only at 70°C and with H2O/CH3CN as mobile phase. Shorter columns were tested at 

different temperatures and with different mobile phases. The separation performance was evaluated with a test 

mixture (HPLC Reverse Phase Test Mix #1, Restek, Bellefonte, PA, USA) using an UV detector (Dionex UltiMate 

3000 Detector, Dionex, Sunnyvale, CA, USA). Real world applications were made using a long column (50 cm) on an 

LC-MS interface called Direct-EI. This interface was developed in our laboratory and it is based on the gas-phase 

ionization of molecules in classical electron ionization conditions. We compared our column with a commercial 

column to separate a test mixture. Data acquisition was carried out in selected ion monitoring (SIM) mode. We 

observed higher sensitivity and resolution with our column at 70° C, although time of analysis was longer due to the 

longer length of the column. 

386 



P09-003 GAS CHROMATOGRAPHY-MASS SPECTROMETRY DETERMINATION OF UV-FILTERS BY MAGNETIC ASSISTED CHEMICAL 

EXTRACTION WITH NANOPARTICLES IN ENVIRONMENTAL WATERS 

Román Falcó I.P. 1 , Chisvert A. 2 , Salvador A. 2 , Canals Hernández A. 1 

1 

Universidad de Alicante, Dpto. Química Analítica, Nutrición y Bromatología 

2 

Universidad de Valencia, Dpto. Química Analítca 

Analytical Chemistry, as others scientific fields, is deeply involved in the incorporation of nanoscience and 

nanotechnology. Nanomaterials are incorporated in spectroscopy, sample preparation/treatment, separation 

technique, electroanalysis and so on. This analytical chemistry trend has been discussed in several reviews1,2. 

Superparamagnetism is a special property magnetic nanoparticles, MNP (so called superparamegnetic 

nanoparticles) found among others in metal oxide nanoparticles arising due to nanometer size. These materials 

are used as extracting phase in magnetic assisted chemical separations (MACS). The surface modified MNPs 

show magnetic properties useful for magnetically assisted extraction purposes. Several surfactants and polymers 

coatings were used to confer multifunctional properties to nanoparticles. The surfactant can be physisorbed (e.g., 

cetyltrimethylammonium bromide or cetylpyridinium chloride) but also chemisorbed (e.g., alkyl carboxylates)3,4. 

The MNPs are presented as an interesting and promising alternative to miniaturize solid-phase extraction (SPE). The 

MNPs were used to extract and preconcentrate UV filters from water samples. CoFe2O4 magnetic nanoparticles 

were synthesized by coprecipitation with chemisorbed oleic acid on the surface. The nanometerials were 

characterized by TEM, XPS, N2-adsortion isotherms, TGA and FTIR spectrometry. The extraction procedure consists 

of: 75 mL of water sample, after salt addition up to 30% and adjusted to pH 3 were sonicated for 2 min with oleic 

acid-coated CoFe2O4 MNPs. in an ultrasound bath. Then the sample flask was vortex mixed for 2 min. The sample 

flask was placed over a strong B-Fe-Nd magnet for few minutes. Water was removed decanting the flask with the 

magnet in the bottom. Then UV-filters were eluted from the MNPs twice with 1.5 mL of hexane. Finally, both eluates 

are combined and in an eppendorf conical-bottom tube was evaporated under vacuum at room temperature. The 

residue was dissolved in 50 µL of BSTFA for derivatization of some analytes. The UV-filters were analyzed by gas 

chromatography-mass spectrometry. The produced MNPs show good capabilities for the UV-filters extraction. High 

enrichment factors were obtained for lipophilic UV-filters. Matrix effect was evaluated analysing tap water, river 

water and seawater. Acknowledgements: Financial support from the Spanish Government (projects n. PET2006- 

706-00, CTQ2009-12709 and CTQ2008-06730-C02-01) and the Regional Government of Valencia (Spain) (projects 

n. ACOMP07/053, ACOMP/2009/144, and A-04/09) is gratefully acknowledged. I.P.R.F. acknowledges his fellowship 

from “Caja de Ahorros del Mediterraneo (CAM)”. 1. M. Valcárcel & B. M. Simonet & S. Cárdenas; Anal Bioanal Chem 

391 (2008)1881-1887 2. A. Simón de Dios, M. E. Díaz-García; Anal. Chim. Acta 666 (2010) 1-22 3. Z. Peng, K. Hidajat, 

M. S. Uddin, Korean J. Chem. Eng. 20 (2003) 893-901. 4. A. Ballesteros-Gómez, S. Rubio. Anal. Chem. 81 (2009) 

9012-9020. 


387

P10 POLYMERS 

POSTER 

SESSIONS

POSTER SESSION 10 - POLYMERS 

P10-001 DETERMINATION OF HALOGENS BY COMBUSTION ION CHROMATOGRAPHY 

Espinosa M. 1 , Emmenegger C. 2 , Wille A. 2 

1 

Gomensoro S.A. 

2 


The acronym RoHs stands for the directive on the «Restriction of the use of certain hazardous substances in 

electrical and electronic equipment». In response to the decreasing lifetime and the skyrocketing production of 

electronic and electrical equipment, the above directive aims at limiting the use of six hazardous species (lead, 

mercury, cadmium, hexavalent chromium, polybrominated biphenyls and polybrominated diphenyl ethers) in 

manufacturing processes. In view of upcoming international regulations, a subject that will also gain paramount 

importance is the rapid and accurate monitoring of partly corrosive and toxic organohalogen compounds in 

challenging matrices such as coal, crude oil, naphtha, liquefied petroleum gas, etc. The most prominent techniques 

for halogen determination in these materials include inductively coupled plasma mass spectrometry (ICP-MS), ion 

selective electrodes (ISE) and ion chromatography (IC). However, the samples are often difficult to bring into solution 

and thus require complex and time-consuming sample preparation procedures. This problems can be overcome by 

using combustion IC: After an upstream thermal oxidative digestion, the liberated combustion gases are trapped 

in an aqueous absorbant that can be directly injected into the IC. Since combustion and absorption occur during 

the recording of the previous sample’s chromatogram, the overall analysis time is short. Whereas halogens are 

determined as fluoride, chloride, bromide or iodide, sulfur compounds are determined as sulfate. 

This presentation will highlight the applicability of automated combustion IC to the determination of trace 

halogenated and sulfur as well as phosphorous compounds in a wide variety of sample matrices. 


389


P10-002 CHARACTERIZATION AND DETERMINATION OF POLYVINYLALCOHOL BY NON-EQUILIBRIUM CAPILLARY 

ELECTROPHORESIS OF POLYMER-DYE EQUILIBRIUM MIXTURES 

Carrasco-Correa E.J., Beneito-Cambra M., Herrero-Martínez J.M., Ramis-Ramos G. 



Polyvinylalcohol (PVA) is a water soluble polymer with friendly properties in terms of safety, high biocompatibility and 

water solubility. PVA is widely used for a variety purposes, such as the manufacturing of cosmetics, pharmaceuticals 

and adhesives. In the last years, capillary zone electrophoresis (CZE) and capillary gel electrophoresis have been 

applied to the separation of polyelectrolytes. Although PVA has no electrical charge, it is capable of complexing 

cations, as Cu2+, and anionic dyes, as Congo Red (CR), giving rise to polycationic and polyanionic complexes, 

respectively. Further, PVA also forms polyanionic esters with borate and other inorganic anions. In this work, the 

formation of a PVA-borate-CR complex, and its application to the characterization and determination of the polymer 

by CZE, was described. The CZE behaviour of the complex was interpreted at the light of the theory of nonequilibrium 

capillary electrophoresis of equilibrium mixtures (NECEEM), which was formerly developed by Krylov 

et al. (J. Am. Chem. Soc. 2002, Analyst 2003, Anal. Chem. 2006, J. Biomol. Screen. 2006, Electrophoresis 2007) to 

characterize protein-dye complexes. A fused-silica capillary and background electrolytes (BGEs) containing either 

phosphate or borax were used. The electropherograms obtained in presence of borax were more reproducible than 

those obtained using phosphate; then, a borax buffer solution (pH = 9) was used as BGE. Mixtures of PVA and CR, 

also containing borax at pH 9, were hydrodynamically injected. PVA and its borate esters do not absorb in the UVvis; 

however, the PVA-borate-CR complex showed intense absorption in the visible region, and gave a band in the 

anionic migration region. At the same time, the complex underwent dissociation during migration in the BGE; thus, 

in the presence of an excess CR in the injected solutions, the electropherograms also showed a peak due to the 

initial equilibrium concentration of the free dye, plus an exponential region due to the dye released by the complex 

during migration. Independently from the molecular mass of PVA, saturation with the dye was observed at monomer/ 

dye molar ratios close to q = 4. The stability constant of the 4:1 complex was estimated to be logK ≈ 4.0. Calibration 

curves were obtained by increasing the PVA concentration while keeping a constant CR concentration. Linear 

relationships were observed when q < 4; however, shortly before reaching the saturation point (q = 4), the area of the 

PVA-dye band increased suddenly and the area of the peak due to the excess dye decreased. This behaviour was 

attributed to the higher stability of the complexes in the vicinity of the saturation point. Work to develop a CE method 

for the determination of PVA in industrial products is in progress. 


(M. Beneito-Cambra, University of Valencia and Químicas Oro); E. J. Carrasco-Correa thanks the University of 

Valencia for a grant. 

390 



P10-003 DETERMINATION OF FLUOROQUINOLONE ANTIMICROBIALS IN DRINKING AND AQUACULTURE WATER SAMPLES BY 

AUTOMATED ON-LINE 

Rodriguez E., Navarro-Villoslada F., Benito-Peña E., Moreno-Bondi M.C., Marazuela M.D. 


Corresponding author e-mail: mcmbondi@quim.ucm.es 

Pharmaceuticals represent a group of emerging chemicals of environmental concern [1]. Antimicrobials are an 

important group of pharmaceuticals, used both in human and veterinary medicine that have been detected in 

wastewaters, surface waters and drinking water, as well [2]. Many studies have demonstrated the incomplete removal 

of these pharmaceuticals during wastewater treatment and therefore, they can reach the surface and groundwater. 

In addition, antimicrobials are extensively used as feed additives in fish farms. Antimicrobials represent a cause 

of concern because of their potential contribution to the spread of antibiotic resistance in bacteria, thus having a 

negative impact on human health. Fluoroquinolones (FQs) are a group of antimicrobials broadly used nowadays in 

the treatment of both humans and food producing animals. The application of molecularly imprinted polymers (MIPs) 

as SPE sorbents (MISPE) offers improved selectivity in comparison with traditional SPE phases [3]. Briefly, MIPs are 

highly cross-linked synthetic polymers prepared in the presence of a template molecule. In this work, monodispersed 

MIP beads (average size 3 µm) have been prepared using enrofloxacin as template and, subsequently applied to 

the automated on-line preconcentration of six FQ antibiotics (enrofloxacin, norfloxacin, levofloxacin, ciprofloxacin, 

danofloxacin and sarafloxacin) in water samples, prior to their determination by liquid chromatography with 

fluorescence detection (MISPE-LC-FLD). Several parameters affecting the on-line MISPE method have been 

optimized including: pH and sample loading flow rate; composition, volume and flow rate of the washing and elution 

steps, breakthrough volume and loading capacity. The cross-reactivity study showed an excellent selectivity of the 

MIP for those FQs bearing a piperazine moiety, whereas other antibiotics, such as penicillins, or structurally related 

compounds were not retained by the polymer. The method has been successfully applied to the analysis of the 

selected antimicrobials in drinking and aquaculture water samples with limits of detection at the low ng/L level and 

recoveries ranging from 80 to 108% by percolating 25 mL of water sample. Acknowledgements The financial support 

of the Spanish Ministry of Science and Innovation (CTQ2009-14565-C03-03), UCM-Santander grant (GR58-08- 

910072) and the European Marie-Curie Program (MRTN-CT-2006-033873) is gratefully acknowledged. E.R.C thanks 

the Alban Programme of the European Union for a doctoral grant (No. E06D101058CO). References: [1] K. Kümmerer 

(ed.), “Pharmaceuticals in the Environment. Sources, Fate, Effects and Risk”, 3rd ed. Springer. Berlin (2008). [2] P.A. 

Segura, M. François, Ch. Gagnon, S. Sauvé, Environ. Health Perspect. 117 (2009) 675. [3] J. Haginaka, J. Sep. Sci. 32 

(2009) 1548. 


391


P10-004 CHARACTERIZATION OF CELLULOSE TREATED WITH ACETIC ACID VAPOUR –HAVING A LOOK INTO CLOSED OLD BOOKS 

Kostic M., Böhmdorfer S., Henniges U., Bogolitsyna A., Potthast A. 


Corresponding author e-mail: antje.potthast@boku.ac.at 

One of the main effects responsible for the ageing of historical and present-day papers is acidic hydrolysis. Acids 

contained in paper catalyze the cleavage of the glycosidic bond of cellulose, the essential component of paper. 

By this cleavage, the degree of polymerization of the biopolymer cellulose is substantially reduced, which has an 

extremely negative impact on the macroscopic properties of the damaged paper. Modern paper contains acids from 

the very beginning due to the production process, resulting in large-scale problems for libraries and conservators. 

Historical papers on the other hand are acid free after fabrication, but they still become acidic eventually and are 

damaged by hydrolysis. 

In our work, we investigated on the effect of vapour phase acetic acid on cellulose of different papers. Acetic acid is 

a common cause for cellulose degradation, since it is present in historical paper either as ink ingredient (as copper 

acetate pigment or as co-solvent) or as product of acid-catalysed hydrolysis of paper. It is expected that during 

natural ageing acetic acid accumulates in a book and leads to cellulose degradation. 

Different types of paper with or without copper acetate pigment were exposed to the vapour of glacial acetic acid for 

time periods ranging from hours to several weeks. To assess the damage caused by the acid, a combination of SEC, 

CE-MS, pyrolysis GC-MS and HPLC-MS was applied. Additionally, the extent of acetylation and oxidative damage of 

the treated cellulose was determined by both fluorescence and isotopic labeling prior to chromatographic analysis. 

We observed that all the samples were prone to increased hydrolysis, resulting in a reduced degree of 

polymerization of the cellulose chains. In the presence of copper pigment this effect was more pronounced. The 

treated cellulose and the oligomers that had formed during degradation were partially acetylated. The presence 

of cellulose acetate can account for certain known but not yet satisfyingly explained properties of historical paper 

both on a molecular level, eg. changed hydrogen bonding-pattern, and on a macroscopic level, eg. its increased 

hydrophobicity. Future research will focus on the direct detection of acetic acids and associated degradation 

products in historical books. 

392 



P10-005 IDENTIFICATION OF SURFACTANTS IN MODERN PAINTS BY USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

Osete-Cortina L., Doménech-Carbó M.T., Silva M.F. 

Technical University of Valencia, Instituto de Restauración del Patrimonio 

Corresponding author e-mail: tdomenec@crbc.upv.es 

Synthetic paints have become the most used materials in modern art since their development as artist´s materials 

in 1947, due to their suitable optical, physical and chemical properties [1]. The wide variety of formulations available 

makes them useful as binding media, adhesives or protective coatings. In these commercial formulations additives 

play an important role in the physical and chemical behavior of the paint. Especially, in acrylic and vinyl emulsion 

paints is important to mention the presence of surfactants as dispersion stabilizing agents. The identification of 

these compounds is essential because their loss in the painting during ageing or cleaning treatments is directly 

related to a significant modification in the optical and mechanical properties. However, the small concentration 

of surfactants in the paintings and their low thermal stability are the main drawbacks for their characterization 

by conventional separation techniques such as Gas Chromatography-Mass Spectrometry, besides, their higher 

affinity to aqueous media make them incompatible with the most part of derivatization reagents. Other techniques 

such as Pyrolysis-Gas Chromatography/Mass Spectrometry (Pyr-GC/MS) lead to a great extent of fragmentation 

and, consequently, the complexity of the pyrogram obtained increases, so that the identification of surfactants 

in this type of samples results more difficult. Currently, the hyphenation of analytical techniques such as two 

dimensional gas chromatography coupled to time-of flight mass spectrometry (GC x GC-(TOF)MS) or Temperature- 

Programmed Pyrolysis with Metastable Atom Bombardment Ionization Mass Spectrometry (TPPy/MAB-MS) 

[2-3] is the strategy most commonly used. Nevertheless, these techniques often are not easily available. Taking 

into account these considerations, the present work is focused on the development of a methodology for the 

characterization of surfactants present in modern paintings by using the more conventional hyphenated technique 

of Gas-Chromatography/Mass Spectrometry. For this purpose, several derivatization reagents have been evaluated 

in different reaction conditions and the results of these trials have been compared in order to determine the 

optimal analytical conditions. The results obtained in the present study lead to conclude that, although thermal 

decompositions take place in the GC injector making more difficult the identification of the surfactants, the use of the 

reagent trimethylsilylimidazole (TMSI) results in the effective derivatisation of these decomposition products that can 

be used as markers for the characterization of the additives analyzed. On the other hand, the combination of the two 

derivatising reagents TMSI and Ethyl Chloroformate (EFC), which acts in aqueous media, makes this methodology 

very useful for the analysis of aqueous extracts resulting from cleaning treatments applied in modern paints and for 

more complex samples in which other substances such as drying oils and proteins are present. Acknowledgments 

Financial support is acknowledged from the Spanish “I+D+I MEC” project CTQ2008-06727-C03-01/BQU supported 

by ERDEF funds, the “Generalitat Valenciana” I+D project ACOMP/2009/171 and the AP2006-3223 project ascribed 

to the program of predoctoral stages of researchers in Spanish universities from the Ministerio de Ciencia e 

Innovación (MICINN). References [1] T. J.S. Learner, Analysis of Modern Paints, The Getty Conservation Institute, 

2004, Los Ángeles. [2] J Chromatogr A, 1217(2010)749-754 [3] J. Am Soc Mass Spectrom 15 (2004) 1315-1319) 


393

P11ENANTHIOSEPARATIONS 

POSTER 

SESSIONS

POSTER SESSION 11 - ENANTHIOSEPARATIONS 

P11-001 COMPARATIVE ENANTIOSEPARATIONS OF PHARMACEUTICALS IN CAPILLARY ELECTROCHROMATOGRAPHY ON 

POLYSACCHARIDE-BASED CHIRAL STATIONARY PHASES CONTAINING SELECTORS WITH OR WITHOUT CHLORINATED 

DERIVATIVES 

Hendrickx A. 1 , Mangelings D. 1 , Chankvetadze B. 2 , Vander Heyden Y. 1 

1 


2 

Tbilisi State University 

Corresponding author e-mail: yvanvdh@vub.ac.be 

Chirality can lead to in vivo differences in pharmacokinetic, pharmacodynamic and toxicological properties of 

the stereoisomers. This is why EMA and FDA defined guidelines stating that the registration files of chiral drugs 

should contain methods that are able to separate and assay the enantiomers. Analytical techniques that are 

used for the separation of chiral molecules are, amongst others, high-pressure liquid chromatography (HPLC), 

capillary electrophoresis (CE), gas chromatography (GC), supercritical fluid chromatography (SFC) and capillary 

electrochromatography (CEC). The latter technique combines high selectivity and sample loading capacity 

due to the presence of a stationary phase, as in HPLC, with high efficiencies due to the use of an electrically 

driven mobile phase flow, as in CE. The screening conditions of an existing chiral strategy in CEC were tested 

for their applicability on four chlorine-containing polysaccharide-based stationary phases with cellulose tris(3- 

chloro-4-methylphenylcarbamate), amylose tris(5-chloro-2-methylphenylcarbamate), cellulose tris(4-chloro-3- 

methylphenylcarbamate) and cellulose tris(3,5-dichlorophenylcarbamate) selectors. The enantioselectivity of 

these phases is compared with those of the four phases used in the earlier defined strategy, namely cellulose 

tris (3,5-dimethylphenylcarbamate), amylose tris (3,5-dimethylphenylcarbamate), amylose tris ((S)-alphamethylbenzylcarbamate), 

and cellulose tris (4-methylbenzoate). A test set of 45 structurally diverse drug compounds 

was screened according to the conditions of the strategy. Furthermore different optimisation steps, which are the 

application of different voltages and mobile phase ACN-contents, were examined. The results of the screening and 

optimisation steps lead to different possibilities to upgrade the current strategy, resulting in improved success rates. 


395


P11-002 OPTIMIZATION OF DIRECT CHIRAL LC FOR ENANTIOMERIC SEPARATION OF ARYLPHENOXYPROPIONIC AND SAFENER 

HERBICIDES USING EXPERIMENTAL DESIGN METHODOLOGIES 

Magro-Moral J., Guillén-Casla V., Rosales-Conrado N., Pérez-Arribas L.V., León-González M.E., Polo-Díez L.M. 


Corresponding author e-mail: noerosales@quim.ucm.es 

Nowadays, about 25% of the pesticides used in agriculture industry are chiral compounds, which are produced 

and applied as racemates. Pesticide enantiomers frequently exhibit different bioactivity, toxicity and environmental 

behaviours. Therefore, to reduce the amount of manufactured agrochemicals and to prevent unnecessary enantiomer 

use causing adverse impact, only the active isomer should be employed. 

Arylphenoxypropionic acids (ArPPs) are a new class of herbicides widely used as esters for selective removal of 

most grass species from any nongrass crop. They are often applied together with safener herbicides to improve 

selectivity and to protect crop plants from herbicide damage. Some of them present chirality, the R-enantiomer 

showing greater herbicidal activity. Consequently, development of enantioselective analytical methods is of great 

interest for stereochemical purity evaluation. Direct chiral separation of ArPPs has been mainly performed using 

stationary phases such as phenylcarbamates and esters of cellulose, Pirkle-type and cyclodextrins. 

In this work, LC-UV methods have been developed for enantiomeric separation of two ArPPs [fluazifop-(R,S)-butyl 

and quizalofop-(R,S)-ethyl] and a safener herbicide [mefenpyr-(R,S)-diethyl], using a α 1-acid glycoprotein chiral 

column (AGP, 100 x 3.0 mm). Detection wavelength was set at 230 nm for fluazifop-butyl and quizalofop-ethyl and 

307 nm for mefenpyr-diethyl. Flow rate was maintained at 0.8 mL min-1 during the chromatographic run. 

Multifactorial experimental designs were done to optimize the mobile phase composition for the direct chiral 

separation. Experimental factors and ranges selected were propanol (5-10%), pH of 10 mM phosphate buffer 

(6.5-7.0) and temperature (15-25 ºC). Responses were expressed in terms of enantioresolution (Rs), enantioselectivity 

(α) and adjusted retention time (tr’) of the second eluted enantiomer. Mathematical models with R2>0.901 and 

standard error of estimation between 0.005-0.480 were obtained. Enantioresolution was significantly affected by 

2-propanol percentage in all cases. Temperature affected significantly Rs of fluazifop-butyl while buffer pH only 

affected significantly Rs of quizalofop-ethyl enantiomers. Similar effects were observed for α values. Regarding tr’, 

temperature and 2-propanol had a significant influence for all compounds. 

Multiple response analyses were carried out to determine the combination of experimental factors which 

simultaneously optimize Rs (≥1.0), α (≥1.1) and tr’ (minimum value). As a compromise, following mobile phase 

compositions and temperatures were selected: 2-propanol/10 mM phosphate buffer pH 7.0 (7:93 v/v) at 17 ºC for 

fluazifop-butyl; 2-propanol/10 mM phosphate buffer pH 7.0 (5:95 v/v) at 20 ºC for quizalofop-ethyl; and 

2-propanol/10 mM phosphate buffer pH 6.5 (9:91 v/v) at 15 ºC for mefenpyr-diethyl. Under these conditions, Rs 

between 0.8-1.3, a in the range 1.3-1.5 and tr’ between 8-22 min were expected and experimentally assessed. 

Analytical characteristics such as detection (LOD) and quantitation (LOQ) limits, linearity range and precision (% 

RSD) were established. LODs were in the range 0.020-0.060 mg mL-1. Good linearity was observed between 0.125 

and 40 mg mL-1 with R>0.995. Run-to-run (n=5) and day-to-day precision (N=15), estimated for tr’ and Rs, were 

< 5.7 % and < 4.8 % respectively. 

396 



P11-003 SEPARATION OF CIS-BIFENTHRIN ENANTIOMERS BY CYCLODEXTRIN MODIFIED MICELLAR ELECTROKINETIC 

CHROMATOGRAPHY. QUANTITATIVE ANALYSIS IN A COMMERCIAL INSECTICIDE FORMULATION 



Corresponding author e-mail: virginia.perezf@uah.es 

Pesticides in general and especially insecticides are considered the most important group of pollutants. About 25 % 

of them are chiral and this fact has important implications both in their biological activity and degradation patterns. 

Separation of chiral compounds is an interesting and challenging topic of research in many analytical chemistry 

areas, especially in pharmaceutical, biomedical, and environmental fields where pure enantiomeric forms are widely 

required. It is already well-known that enantiomers, in spite of their very similar structure, when exposed to an 

identical biological environment can show very different biological activity. Pyrethroids are synthetic insecticides 

widely employed for the control of insects in agriculture and around households. Bifenthrin (BF) contains two chiral 

centers and so four stereoisomers. However, only the cis diastereomers are employed in commercial formulations 

due to their higher insecticide activity compared with the trans ones. Furthermore, the toxicity of cis-BF against 

aquatic invertebrates was demonstrated to be enantioselective with the toxicity mainly residing in the 1R, cis-BF 

enantiomer being this isomer also the most persistent in soil, sediments and water after pesticide application. Taking 

into account that the insecticide activity is similar for the 1R and 1S enantiomers of cis-BF, the use of the racemic 

mixture is illogic, so commercial formulations elaborated with the pure enantiomer (1S) should be employed making 

necessary the development of chiral analytical methodologies for quality control. The first capillary electrophoresis 

(CE) method enabling the enantiomeric separation of the synthetic pyrethroid cis-BF was developed in this work. 

Cyclodextrin modified micellar electrokinetic chromatography was the CE mode employed for this purpose. The 

influence of several experimental parameters such as temperature, voltage, type and concentration of surfactant 

(chiral and achiral) and cyclodextrin was investigated. The use of the bile salt sodium cholate at a concentration 

of 100 mM in presence of 20 mM heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin enabled the separation of cis- 

BF enantiomers in less than 10 min and with a resolution of 2.8. The analytical characteristics of the developed 

methodology were evaluated allowing its application to the quantitation of cis-BF in a polyvalent commercial 

insecticide formulation. 


397


P11-004 CHIRAL SEPARATION OF METALAXYL AND BENALAXYL FUNGICIDES BY CD-EKC FOR THEIR SIMULTANEOUS DETERMINATION 

AND QUANTITATION WITH FOLPET IN COMMERCIAL FORMULATIONS. DETERMINATION OF ENANTIOMERIC IMPURITIES 



Corresponding author e-mail: virginia.perezf@uah.es 

Fungicides are a very important and diverse environmental and agricultural concern species. Their detection and 

determination in commercial formulations or environmental matrices such as soil, water, sediments, etc., require 

of high efficiency, selectivity and sensitivity methods. A significant number of these chemicals are chiral with the 

activity residing usually in one of the enantiomers, but they use to release into the environment as racemate and 

many times they are treated if they were single isomers. This fact can lead to wrong toxicological and degradation 

data, resulting in the need to investigate them separately. Metalaxyl and benalaxyl correspond to amide fungicides 

group. Their enantioselectivity has been widely studied demonstrating both the differences in their fungicidal 

activity and in their degradation pattern. Although both fungicides contain one chiral centre, all the fungicidal 

activity resides in the R-enantiomer being the other isomer almost completely inactive. Furthermore, when these 

two fungicides are exposed to a biotic environment, the preferential degradation of one of the enantiomers is clearly 

shown. In this situation when racemic metalaxyl is applied to most type of soils, the active enantiomer is faster 

degraded, resulting in residues enriched with S-metalaxyl (totally unneeded). However, in plants and some biological 

samples S-enantiomer of metalaxyl showed a faster degradation. For benalaxyl, just the same behavior is observed 

in soil but in tobacco, tomato, sugar beet and capsicum plants, the preferential absorption and degradation of 

S-enantiomer of benalaxyl resulted in an enrichment of the R-enantiomer residue in all vegetables. For these 

reasons there is necessary to develop new, rapid and simple methods for the determination of the enantiomers 

of these two fungicides. Furthermore, in commercial formulations metalaxyl and benalaxyl are usually mixed with 

other fungicides to enhance their effectiveness. Folpet is one of these fungicides so the new methods should 

allow also the determination of this compound. A screening of different parameters affecting the enantiomeric 

separation of metalaxyl and benalaxyl (nature and concentration of separation buffer, pH, and cyclodextrin type and 

concentration) was carried out to select the best conditions for the separation of these two amide fungicides. Using 

a 50 mM MES buffer (pH 6.5) the chiral separation of metalaxyl enantiomers was achieved in ~11 min using succ- 

-CD as chiral selector and in ~ 7.5 min for benalaxyl employing succ-β-CD. The developed methods were applied 

to the simultaneous determination of metalaxyl or benalaxyl and folpet in commercial formulation and a stacking 

strategy was applied to allow the determination of enantiomeric impurities. Several analytical characteristics, such 

as linearity, LODs, LOQs, precision, accuracy and selectivity were studied to assess the suitability of the developed 

methods for their application to the determination in commercial formulations and for determination of enantiomeric 

impurities. 

398 



P11-005 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ENANTIOSEPARATION OF AMINONAPHTHOL ANALOGS ON 

POLYSACCHARIDE-BASED CHIRAL STATIONARY PHASES 

Peter A., Aranyi A., Ilisz I., Pataj Z., Szatmári I., Fülöp F. 

University of Szeged-Hungary 

Corresponding author e-mail: apeter@chem.u-szeged.hu 

The search for new chiral ligands which can be efficiently applied in biology and asymmetric catalysis is currently 

a field of great interest in organic chemistry. The asymmetric addition of diethylzinc to aldehydes in the presence 

of catalytic amounts of chiral catalysts has attracted considerable attention [1]. The three-component modified 

Mannich reaction is a convenient method with which to prepare aminoalkyl-naphthols. Through the use of chiral 

amines, nonracemic aminonaphthol derivatives have been prepared which catalyze the asymmetric addition of 

diethylzinc to aryl aldehydes [2]. Since the asymmetric catalytic and biological activities of aminonaphthol analogs 

depend strongly on their stereochemistry, there is a clear need for analytical methods by which their configurations 

can be identified. The present paper describes normal-phase HPLC methods for the enantioseparation of new 

racemic aminonaphthol analogs possessing two chiral centers (Fig. 1). These HPLC methods rely on the use of 

tris-(3,5-dimethylphenyl)- or tris-(2-chloro,5-methylphenyl)carbamoylated cellulose- and amylose-based CSPs 

(Phenomenex Lux Cellulose-1 and 2 and Lux Amylose-2) The separation performances are compared, and the 

experimental data are utilized to discuss the effects of the mobile phase composition, the nature of the alcoholic 

modifier and the specific structural features of the analytes on the retention and separation. We are not aware of 

any previous published reports on the enantioseparation of aminonaphthol analogs possessing two chiral centers. 

[1] P. Knochel, R.D. Singer, Chem. Rev. 93 (1993) 2117. [2] I. Szatmári, F. Fülöp, Curr. Org. Synth. 1 (2004) 155. 

Acknowledgements A.P. is grateful to Gen-Lab Kft (Budapest, Hungary) and Phenomenex (Torrance, CA, USA) for 

the generous gift of the Lux Cellulose and Amylose columns. 


399


P11-006 STEREOSELECTIVE DETERMINATION OF BUFURALOL, 1’-OXOBUFURALOL AND 1’-HYDROXYBUFURALOL IN RAT LIVER 

MICROSOMAL FRACTION USING HOLLOW FIBER LIQUID PHASE MICROEXTRACTION AND HPLC 

Barth T., Simões R.A., Calixto L.A., Okano L.T., Pupo, M.T., Bonato P. S. 


Corresponding author e-mail: psbonato@fcfrp.usp.br 

Bufuralol (BF) is a chiral compound with non-selective β-adrenoceptor antagonist properties. The pharmacological 

activity of (S)-BF is approximately 100 times greater than that of the (R)-enantiomer. BF is biotransformed into many 

metabolites in human and animals. The 1’-hydroxybufuralol (1’-OH-BF) and 1’-oxobufuralol (1’-OXO-BF) are the major 

and active metabolites. The 1’-hydroxylation of bufuralol is mediated mainly by CYP2D6 cytochrome P450 isoform 

in human livers, and has been widely used to study the CYP2D6 genetic polymorphism in vitro. The aim of this work 

was to develop a method for the stereoselecive determination of BF, 1’-OH-BF and 1’-OXO-BF in rat liver microsomal 

fraction using hollow fiber liquid phase microextraction (HF-LPME) prior to HPLC analysis. In order to accomplish 

the extraction of these basic compounds, a three-phase HF-LPME sampling mode was selected. The analytes were 

extracted from aqueous alkaline solutions (donor phase) at pH 13 through the organic phase (n-octanol) immobilized 

in the pores of the hollow fibre into the acidic aqueous solution (acetic acid), as acceptor phase, present inside the 

lumen of the hollow fiber. The chiral resolution of BF, 1’-OH-BF and 1’-OXO-BF was carried out in a Chiralcel OD-H 

column at the normal phase mode with hexane : isopropanol : methanol: diethylamine (97.5:2.0:0.5:0.5, v/v/v/v), as 

mobile phase, and UV detection at 248 and 273 nm. The method was validated according to the literature and was 

linear over the concentration range of 100 – 5000 ng/mL for each enantiomer of BF and 1’-OXO-BF (r > 0.9967) and 

100 – 2500 ng/mL for each diastereomers of 1’-OH-BF (r > 0.9957) and presented limits of quantification of 100 ng/ 

mL for all analytes. The recoveries from 0.5 mL of rat liver microsomal fraction were in the range of 35 – 70%. Withinday 

and between-day precision and accuracy evaluated by RSDs and relative errors, respectively, were lower than 

15% for all analytes. Moreover the analytes were stable under biotransformation conditions and short term room 

temperatures assays. The validated method demonstrated acceptable results, being suitable for the application in 

biotransformation studies by rat liver microsomal fraction. Financial support: FAPESP, CNPq, CAPESç 

400 



P11-007 STEREOSELECTIVE HPLC DETERMINATION OF ISRADIPINE AND ITS MAIN METABOLITE IN RAT LIVER MICROSOMAL 

FRACTION USING HOLLOW-FIBER LIQUID-PHASE MICROEXTRACTION AS SAMPLE PREPARATION 

Simões R.A., Bonato P.S. 



Isradipine (ISR) is a dihydropyridine derivative calcium antagonist clinically used as an antihypertensive drug, and 

commercially available as a racemic mixture of its (+)-(S) and (-)-(R) enantiomers. The pharmacological effect is 

due primarily to the (+)-(S)-enantiomer. The kinetic disposition of ISR seems to be stereoselective, with higher 

plasmatic concentrations of the active enantiomer. CYP 3A4 was described to have a preponderant place in the 

biotransformation of ISR. The aim of this work was to develop a method for the stereoselective determination of 

ISR enantiomers and its main metabolite (MTB) in rat liver microsomal fraction using hollow-fiber liquid-phase 

microextraction (HF-LPME) for sample preparation. HF-LPME can be performed either in the three- or two-phase 

mode. For non-ionizable compounds, like ISR and MTB, two-phase mode is more appropriate. In this configuration, 

the target analytes are extracted from the aqueous sample into the acceptor organic solvent present both in the 

porous wall and inside the lumen of the hollow-fiber. In order to optimize the extraction of ISR and MTB from the 

microsomal incubation medium, several parameters which influence the extraction efficiency were evaluated. Such 

factors comprise: organic solvent type, extraction time, stirring rate and salt addition to the donor phase. The 

optimal HF-LPME condition was established as follows: 35 μL of hexyl acetate as the acceptor phase, 30 min of 

extraction, sample agitation at 1500 rpm and 10 cm fiber length. The chiral resolution of ISR and MTB was achieved 

using a stationary phase based on 3,5-dimethylphenylcarbamate derivative of amylose (Chiralpak AD column) 

and hexane/isopropanol/ethanol (94:04:02, v/v/v) as mobile phase, at a flow rate of 1.5 mL/min and UV detection 

at 225 and 325 nm. The method was validated according to the FDA guidelines. The method was linear over the 

concentration range of 50 – 2500 ng/mL for each enantiomer of ISR and 50 – 5000 ng/mL for MTB and presented 

limits of quantification of 50 ng/mL for all analytes. The mean absolute recoveries from 1 mL of rat liver microsomal 

fraction were 23% and 18% for MTB and each ISR enantiomers, respectively, with RSD values lower than 15%. 

The precision and accuracy of the method were tested by within-day (n=5) and between-day analysis (n=3) using 

microsomal incubation samples spiked with three concentrations of each ISR enantiomer and MTB. RSD and relative 

errors of less than 15% were obtained for all samples analyzed within-day and between-day. Furthermore, the 

analytes were stable after biotransformation conditions and short term room temperatures assays. The validated 

method demonstrated acceptable results, being suitable for the application in biotransformation studies by rat liver 

microsomal fraction. Financial support: CNPq, CAPES and FAPESP 


401


P11-008 ENANTIOSELECTIVE ANALYSIS OF ZOPICLONE AND ITS METABOLITES BY LIQUID CHROMATOGRAPHY/TANDEM MASS 

SPECTROMETRY 

Tonon M.A., Jabor V.A.P., Bonato P.S. 

Faculty of Pharmaceutical Sciences of Ribeirão Preto 

Zopiclone is a non-benzodiazepine hypnotic drug of the cyclopyrrolone class, indicated for the treatment of insomnia. 

It is extensively metabolized and the main metabolites are N-desmethyl zopiclone and zopiclone-N-oxide. Zopiclone 

is a chiral drug administered as a racemic mixture. It has also been reported that zopiclone pharmacokinetics is 

stereoselective after oral administration. Thus enantioselective methods for the analysis of zopiclone and its chiral 

metabolites in plasma are required as a tool in the study of kinetic disposition and therapeutic monitoring of this 

drug. A high-performance liquid chromatographic method with triple quadrupole mass spectrometry detection 

(LC-MS-MS) was developed and validated for the simultaneous quantification of zopiclone and its metabolites in 

rat plasma samples. These chiral analytes were separated using a stationary phases based on amylose derivative, 

Chiralpack ADR-H column, and ethanol-methanol-acetonitrile (50:45:5, v/v/v) plus 0.025% dietilamine as the mobile 

phase, at a flow-rate of 1.0 ml min-1. A post-column infusion of acetic acid 1% was delivered at a flow-rate of 0.25 

ml min-1 to improve the MS detection. The analytes were isolated from rat plasma by liquid-liquid extraction with 

dichloromethane-isopropanol (80:20, v/v) at pH adjusted at 6.0. Moclobemide was used as internal standard. All 

enantiomers were analyzed in 16 min and linear calibration curves over the concentration range of 7.5–500 ng 

mL-1 were obtained. The intra-day and inter-day accuracy and precision were lower than 15% for all analytes. The 

absolute recoveries were 74.6 and 75.7; 61.6 and 56.9; 72.5 and 70.7 for each enantiomer of zopiclone, N-desmethyl 

zopiclone and zopiclone-N-oxide respectively. This validated method was employed in a pilot study of kinetic 

disposition of ZO in rats. Financial support: CNPq, FAPESP 

402 



P11-009 ANALYSIS OF CHIRAL COMPOUNDS USING ONLINE HYPHENATION OF MASS SPECTROMETRY AND ACHIRAL 

ELECTROPHORETIC SEPARATION 

Ranc V., Knob R., Petr J., Sevcik J., Maier V. 

Palacky University Olomouc 

Corresponding author e-mail: vasekranc@mac.com 

Analysis of chiral compounds represents one of frequent tasks of analytical chemistry. This increasing tension for a 

development of chiral methods is given by a growing number of produced chiral drugs that have to be controlled. As 

analytical tools, chromatographic or electrophoretic approaches are usually used. Mass spectrometry could present 

an interesting alternative [1]. This work deals with a development of mass spectrometric chiral method based on the 

host-guest interactions [1]. The demonstration of application potential is based on the analysis of selected amino 

acids (Ala, Phe, and Val), L-Trp was selected as the chiral selector. Chiral discrimination is calculated using relative 

intensities of protonated host molecule and cluster containing host and guest molecule. It was shown that the ratio 

of these two relative intensities is proportional to the chiral composition of initial sample [2, 3]. The main aim of this 

work is to develop a chiral method based on the hyphenation of achiral electrophoretic separation with enantiomeric 

dicscimination using mass spectrometric approaches. Concerning elecrophoretical part, method for separation 

of selected amino acids was developed according to routine procedures. 0.75 mol/L formic acid in water was 

selected as background electrolyte. As a sheath liquid, water-methanol-formic acid (1:1:0.05, v/v/v) was used with 

an addition of L-Trp as chiral selector. Total concentration of L-Trp in sheath liquid was 1x10-3 mol/L. First, the study 

of selected experimental parameters was performed. The influence of sheath liquid composition and its flow rate 

were considered. From the selected parameters, concentration of the chiral selector plays a crucial role in the chiral 

discrimination. Quantification of chiral composition is based on 5 point calibration curves containing 0, 25, 50, 75 

and 100 % of corresponding D-Enantiomer for a determination of optical purity. Represented correlation coefficients 

are 0.986 for Alanine, 0.995 for Phenylalanine and 0.997 for Valine, respectively. This work was supported by the 

Research Project of the Ministry of Education of the Czech Republic No. MSM6198959216 and by a grant agency 

of Palacky University PRF_2010_028. 1. Sawada, M., Takai, Y., Yamada, H., Kaneda, T., Kamada, K., Mizooku, T., 

Hirose, K., Tobe, Y., Naemura, K.: Journal of the Chemical Society-Chemical Communications 1994, (21), 2497- 

2498 2. Sawada, M., Takai, Y., Yamada, H., Nishida, J., Kaneda, T., Arakawa, R., Okamoto, M., Hirose, K., Tanaka, 

T., Naemura, K.: Journal of the Chemical Society-Perkin Transactions 2 1998, (3), 701-710 3. Sawada, M., Takai, Y., 

Imamura, H., Yamada, H., Takahashi, S., Yamaoka, H., Hirose, K., Tobe, Y., Tanaka, J.: European Journal of Mass 

Spectrometry 2001, 7 (6), 447-459. 


403


P11-010 STUDY ON THE ENANTIOMERIC SEPARATION OF METHYL-SULFONYL METABOLITES OF PCBS 

Castro-Puyana M., Fernández M.A., González M.J., Gómara B. 


Corresponding author e-mail: bgomara@iqog.csic.es 

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants of great concern due to their 

persistency, bioaccumulation ability and toxicity. In a living organism, PCBs could be biotransformed through 

the mercapturic acid pathway to form the methyl-sulfonyl PCB metabolites (MeSO2-PCBs). Due to their lipophilic 

properties (similar to their precursor PCBs), their resistant to further metabolic degradation and their protein binding 

characteristics, MeSO2-PCBs tend to concentrate in lipid-containing tissues, especially fat and liver, turning them 

into a secondary class of contaminants of concern. Some of the MeSO2-PCB congeners, as well as their parent 

PCBs, exhibit axial chirality due to the asymmetric distribution of chlorine atoms on both rings. Multidimensional 

Gas Chromatography (MDGC) is a very powerful tool especially useful for the enantiomeric separation of chiral 

compounds since this technique takes advantage from the huge capacity given by two orthogonal chromatographic 

systems, one of them achiral and another one chiral. For this study, ten chiral MeSO2-PCB congeners were selected: 

3-MeSO2-CB91, 4-MeSO2-CB91, 3-MeSO2-CB95, 4-MeSO2-CB95, 3-MeSO2-CB149, 4-MeSO2-CB149, 3-MeSO2- 

CB132, 4-MeSO2-CB132, 3-MeSO2-CB174 and 4-MeSO2-CB174. To separate the MeSO2-PCB metabolites, a heartcut 

MDGC system was used. For this task, two Varian CP-3800 gas chromatographs, both equipped with electron 

capture detectors (ECD), and connected by a heated transfer line were employed. The heart-cuts were carried out by 

a Deans switching device, that rules the pressures and point the eluents forward the first or the second dimension. 

Different columns were studied to separate the MeSO2-PCBs enantiomers, always being the first dimension an 

achiral DB-5 column (5% phenyl 95% methyl polysiloxane, 30 m length × 0.25 mm i.d. and 0.25 µm film thickness), 

and testing three different chiral stationary phases as second dimension (Chirasil-Dex, BGB-172 and BGB-176SE). 

The best enantiomeric separation was obtained using the chiral phase BGB-176SE, which was able to separate up 

to six different MeSO2-PCB congeners. For the characterisation of the system, enantiomeric factors (EFs) were 

calculated as the area of the first eluted enantiomer divided by the sum of the areas of both enantiomers (values 

ranging between 0 and 1). The EF values obtained using pure standards confirmed that the instrumental method 

allows the robust enantiomeric separation of the MeSO2-PCB congeners, because the EFs calculated experimentally 

were in a very narrow range around 0.5 (corresponding to the racemic). This means that there were no alterations in 

their racemic composition through the instrumental system. Acknowledgements This study was supported by CSIC 

(project 200880I192) and CSIC-CM (project CCG07-CSIC/PPQ-2135) 

404 



P11-011 DETERMINATION OF CHIRAL ALPHA-HYDROXYCARBOXYLIC ACID IN FOOD BY CAPILLARY ELECTROPHORESIS WITH 

INDIRECT UV DETECTION 

Maier V., Znaleziona J., Ranc V., Petr J., Sevcik J. 


Corresponding author e-mail: vitezslav.maier@upol.cz 

Alpha hydroxycarboxylic acids (AHAs) exist in various food and cosmetic products and they are one of the 

main metabolites group in man metabolism. Enantiomeric separation of AHAs is very important analytical task 

owing to their difficult detection in commercial capillary electrophoresis that usually employed UV detection, 

moreover there exists less number of chiral selector suitable for their direct chiral resolution. Several methods 

for chiral discrimination of AHAs were published recently [1-3]. For direct chiral separation of AHAs by capillary 

electrophoresis namely cyclodextrins and ligand exchange were used [4,5]. Our group first described CE method 

using vancomycin as chiral selector for enantiomeric separation of D,L-lactic acid as model AHA by electrokinetic 

partial filling method in covalently coated capillary [6]. In this work the application of developed method for chiral 

separation of D,L-lactic acid with electrokinetic partial filling method using of vancomycin was applied on chiral 

separation of other AHAs (D,L-glyceric acid, D,L-lactic adic, D,L-malic acid and D,L-2-hydroxybutyric acid). Capillary 

electrophoresis with indirect UV detection employed 10 mM salicylate/L-His pH 4.5 as background electrolyte 

was used for simultaneous chiral separation of mentioned AHAs using of electrokinetic partial filling method of 

vancomycin cations from the injection solution containing background electrolyte with 20 mM vancomycin chloride. 

The chiral resolution of all separated AHAs were higher than 1.5. The developed method was applied for chiral 

separation and determination of studied AHAs in various food products (e.g. milk products, honey, wine, bear) with 

minimum sample pretreatment. [1] S. Kodama, A. Yamamoto, A. Matsunaga, T. Soga, K. Minoura, J. Chromatogr. A 

875 (2000) 371-377. [2] U. Meierhenrich, W. H.-P. Thiemann, H. Rosenbauer, J. Anal. Applied Pyrol. 60 (2001) 13-26. 

[3] K. Kim, J. Lee, D. Ha, J.H. Kim, J. Chromatogr. A 891 (2000) 257-266. [4] S. Kodama, A. Yamamoto, A. Matsunaga, 

Analyst 124 (1999) 55-59. [5] A. Desiderio, Z. Aturki, S. Fanali, Electrophoresis 22 (2001) 3286-3290. [6] V. Maier, 

J. Petr, R. Knob, J. Horaková, J. Sevcik, Electrophoresis 28 (2007) 1815-1822. Acknowledgements This work was 

financially supported by Ministry of Education of The Czech Republic (grant No. MSM6198959216) and by the the 

student project PRF_2010_028 of the Palacky University 


405


P11-012 REVERSAL OF ENANTIOMER MIGRATION ORDER USING POLYPYRROLE COATED CAPILLARIES 

Knob R., Znaleziona J., .Ginterova P., Ranc V., Petr J., Maier V., Sevcik J. 

Faculty of Science, Palacky University in Olomouc 

Corresponding author e-mail: rknob@seznam.cz 

Capillary electrophoresis presents frequently used technique in the analysis of chiral compounds. In contrast to 

liquid chromatography, CE offers advantages as possibility of employing wider range of chiral selectors, lower 

analysis costs, and faster method development utilizing various modes of CE. 

The reversal of migration order of enantiomers is also a significant aspect of chiral separations. Detection of an 

impurity presents a problem when a difference in enantiomers’ mobilities is small and when peak tailing occurs [1]. 

Therefore, it is advantageous to detect the impurity as the first peak. 

The reversal of migration order in CE can be achieved by two simplified approaches: by employment of chiral 

selector with different enantioselectivity [2], or by manipulation with the electroosmotic flow [3]. 

In presented work reversal of migration order of R,S-ibuprofen, D,L-tyrosine, and D,L 3,4 dihydroxyphenylalanine as 

model analytes was achieved using uncoated silica capillaries and capillaries with covalently bonded polypyrrole 

coating modified with dextran sulfate [4,5]. These capillaries exhibit strong cathodic electroosmotic flow even at 

low pH in contrast to uncoated silica capillaries. Sulfated-beta-cyclodextrin was employed as chiral selector and 

different polarity was applied in uncoated and polypyrrole coated capillaries. Migration order reversal was even 

obtained using the same background electrolyte in both capillaries. 

This work was financially supported by Ministry of Education of the Czech Republic (grant No. MSM6198959216) and 

by the the student project PRF_2010_028 of the Palacky University. 

References: 

1. T. Schmitt, H. Engelhardt, J. Chromatogr. A 697 (1995) 561. 

2. M. Yoshinaga, M. Tanaka, J. Chromatogr. A 679 (1994) 359. 

3. X.-W. Yao, D. Wu, F.E. Regnier, J. Chromatogr. 636 (1993) 21. 

4. R. Čabala, H. Frank, J. Chromatogr. A 1079 (2005) 344. 

5. R. Knob, S. Gerstmann, R. Čabala, H. Frank, Chromatographia 69 (2009) 1431. 

406 



P11-013 DETERMINATION OF D,L-LACTIC ACID IN SERUM FROM DIABETIC PATIENTS BY CE-ESI-MS 

Znaleziona J., Chrastina J., Ranc V., Petr J., Ginterova P., Sevcik J., Maier V. 


Corresponding author e-mail: jznaleziona@gmail.com 

Lactic acid plays an important role in several biochemical processes and is in mammals mainly composed of 

the L-lactic acid, which is produced under anaerobic conditions from pyruvic acid. Endogenous D-lactic acid is 

produced in the methanolic pathway of glucose from methylglyoxal and its amount is approximately 1% relative to 

L-lactic acid. The concentration level of D-lactic acid is significantly higher in the serum of diabetic patients. Normal 

concentration levels of D-lactic acid and L-lactic acid in serum are about 12 μM and 1 mM, respectively [1]. Several 

determination techniques of D- and L-lactic acid by LC and CE have been published but these methods employ 

derivatization steps and thus are time consuming [2,3]. A simpler and quicker method of simultaneous determination 

of D- and L-lactic acid would (could) be helpful for diabetes mellitus diagnosis. This work presents fast determination 

method of D- and L- lactic acid by using the capillary electrophoresis with electrospray mass spectrometry which 

takes 15 min. The analysis is performed in polyacrylamide coated capillary with 50 mM ammonium acetate (pH 

4.5) as background electrolyte, containing the vancomycin (7mM) as a chiral selector. To avoid the contamination 

of ion source of mass spectrometer, the partial filling technique up to 90 % of total capillary length was used. The 

linearity of developed method was in the concentration range 1 μM to 10 mM of D- and L-lactic acid, with correlation 

coefficients 0.995 and 0.997, respectively. The limits of detection were 1 μmol/L for D-lactic acid and for L-lactic 

acid. The method was applied to determine the concentrations of D- and L-lactic acid in diabetic patients’ serum 

and the obtained levels were compared with healthy volunteers’ serum after protein precipitation. This work was 

financially supported by Ministry of Education of The Czech Republic (grant No. MSM6198959216) and by the 

student project PRF_2010_028 of the Palacky University. References: [1] J.B. Ewaschuk, J.M. Naylor, G. A. Zello, J. 

Nutrition (2005) 1619-1625. [2] H. Hasegawa, T. Fukushima, J. Lee, K. Tsukamoto, K. Moriya, Y. Ono, K. Imai, Anal. 

Bioanal. Chem. 377 (2003) 886-891. [3] L. Saavedra, C. Barbas, J. Chromatogr. B 766 (2002) 235-242. 


407


P11-014 THE PILOT STUDY OF USING BOROMYCIN AS CHIRAL SELECTOR IN CAPILARY ELECTROPHORESIS AND DIRECT 

INJECTION MASS SPECTROMETRY – A COMPARATIVE STUDY 

Maier V., Ranc V., Svidrnoch M., Petr J., Sevcik J. 



Chiral recognition is one of the most important analytical tasks that have great impact on many part of science 

(e.g. pharmacology, medicine, biochemistry, molecular biology, etc.). Separation of enantiomers, configurational 

analysis and determination of enantiomers could be done by various analytical techniques such as GC, LC and CE 

namely [1]. In last decade, direct injection mass spectrometry was used for discrimination of enantiomers, too [2]. 

This work demonstrates the using of boromycin as a chiral discrimination agent in capillary zone electrophoresis 

and in direct injection electrospray mass spectrometry. Boromycin is a macrodiolide that exists as a hydrophobic 

Böeseken complex formed from boric acid and a chiral polyhydroxy macrocyclic ligand and it is insoluble in water, 

but slightly soluble in methanol and other organic solvents [3]. Selected chiral primary amines (D,L-noradrenaline, 

R,S-octopamine, R,S-tryptophanol, R,S-α-methylbenzylamine and R,S-2-amino-1-phenylethanol) were successfully 

separated using of boromycin as chiral selector in capillary zone electrophoresis in methanolic background 

electrolyte. All of studied primary amine enantiomers were separated with sufficient resolution values (Rs > 1.5) 

within 12 minutes. As a second part of this work, enantiomeric recognition ability of boromycin for mentioned chiral 

primary amines was studied using of direct injection electrospray mass spectrometry. Enantiomeric discrimination 

was based on the calculation of RPI (relative peak ratio) values for pure enantiomers for concrete selected amines. 

RPI value was calculated as a ratio of absolute intensity of protonated molecule of host molecule (boromycin) and 

absolute intensity of cluster containing boromycin and analyte (target enantiomer of selected amine). Obtained RPI 

values were compared for pure enantiomers of corresponding amines. RPI (chiral) values were calculated as ratios 

of of relative peak ratio values for pure R- and S- enantiomers, respectively. The higher difference from one means 

higher chiral selectivity for particular system. The observed RPI values, obtained using mass spectrometry were 

correlated with effective mobilities of separated enantiomers of chiral amines. The agreement between capillary 

electrophoresis data and mass spectrometry data was found. The enantiomers with lower effective mobility in 

capillary electrophoresis that means lower interaction with boromycin show lower interaction in mass spectrometry 

as well. [1] T.J. Ward, B.A. Baker, Anal. Chem. 80 (2008) 4363-4372. [2] M.G. Finn, Chirality 14 (2002) 534-540. [3] C. 

Wang, D.W. Armstrong, D.S. Risley, Anal. Chem. 79 (2007), 8125-8135. Acknowledgement This work was financially 

supported by Ministry of Education of The Czech Republic (grant No. MSM6198959216). 

408 



P11-015 COMPARATIVE CHIRAL HPLC METHODS FOR β-BLOCKERS USING DIFFERENT TYPES OF CHIRAL STATIONARY PHASES 

IN NORMAL PHASE AND POLAR PHASE ELUTION MODE 

Morante-Zarcero S., Sierra I. 

E.S.C.E.T., Universidad Rey Juan Carlos, Madrid 

Corresponding author e-mail: isabel.sierra@urjc.es 

In recent years, many pharmaceuticals including β-blockers are found both in waste and in surface waters as a result 

of excretion from human and veterinary use and due to an incomplete elimination in waste water treatment plants. 

Most of β-blockers are chiral and each enantiomer has a different pharmacological activity, potency and mode of 

action. S-enantiomer of atenolol and propanolol are more potent than their respective antipodes. Also, some of the 

biotransformation pathways for β-blockers in humans are stereoselective and recent ecotoxicological studies show 

that aquatic organisms are sensitive to these substances. As a result, chirality must be taken into consideration 

in understanding environmental fate and effects of this type of contaminants. For this reason, characterization of 

enantiomer composition in environmental waters is needed. Nowadays, direct liquid chromatography with chiral 

stationary phase (CSP) is the method of choice for enantiomer separation. In this work, we present comparative 

chiral HPLC methods for β-blockers (propanolol, metoprolol, pindolol y atenolol) using different types of CSPs 

including Chiralpak AD and a novel Lux Cellulose-1 based on polysaccharide, Chirobiotic T based on macrocyclic 

antibiotic and Sumichiral OA-4900 Pirkle type. All CSPs were used in both normal and polar phase elution modes. 

In polar phase mode, best results were obtained using Chiralpak AD and Chirobiotic T as CSP. All β-blockers were 

resolved using Chiralpak AD as CSP in a mobile phase composed by methanol/ethanol/ETA (50/50/0.3, v/v/v) but 

the chiral resolution obtained for atenolol was lower than 1. Nevertheless, using Chirobiotic T as CSP all compounds 

were resolved with a chiral resolution higher that 2 in different mobile phases. When the mobile phase was 

composed by methanol/ethanol/TEA (90/10/0.1, v/v/v) six β-blockers were resolved in the same analysis. Inversion 

of elution order was obtained for both CSPs. In normal phase mode, best results were obtained using Chiralpak 

AD and Lux Cellulose-1 as CSP. Using a mobile phase composed by n-hexane/ethanol (70/30, v/v) seven and eight 

β-blockers were resolved in the same analysis with Chiralpak AD and Lux-Cellulose1 as CSP, respectively. No 

inversion of elution order was obtained in this case. All tested conditions using Sumichiral OA-4900 as CSP failed to 

separate β-blockers. 


409


P11-016 GAS CHROMATOGRAPHY FOR SEPARATIONS OF CHIRAL COMPOUNDS USED IN PHARMACEUTICAL DEVELOPMENT 

PROJECTS 

Laniewski K. 

AstraZeneca R&D, Pharmaceutical Development, Department of Analytical Science-Sweden 

Corresponding author e-mail: krzysztof.laniewski@astrazeneca.com 

Manufacturing of drug substances requires strict control of impurities. When a chiral structure is a target compound, 

the control of a presence of an unwanted enantiomer is very important during a development of a process for 

synthesis of the desired substance. Gas chromatography (GC) is a very useful analytical technique to analyse 

organic impurities, because it provides high separation efficiency, universal detection by flame ionisation and rapid 

method development. Simple coupling of GC to several spectroscopic and spectrometric techniques facilitates a 

correct determination of desired analytes. This presentation will show an analytical approach for analysis of chiral 

compounds with respect to enantiomeric purity. Analyses using chiral stationary phases, procedures without 

and with derivatisations with e.g. acetic anhydride, trifluoroacetic anhydride and ethyl chloroformate prior to GC 

analyses, as well as formations of diastereomers using chiral derivatising reagents, e.g. (R)-(-)-α-methoxyphenyl 

acetic acid chloride and (R)-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride, followed by analyses using 

non-chiral GC columns will be presented and discussed. The examples and applications demonstrated relate to 

compounds in pharmaceutical analyses used as starting materials and intermediates in development projects. 

410 


P12 FAST 

SEPARATIONS 

POSTER 

SESSIONS

POSTER SESSION 12 - FAST SEPARATIONS 

P12-001 DETERMINATION OF 1,3-OCTANEDIOLS VIA 1,3-DIOXANES BY SOLID-PHASE MICROEXTRACTION AND HIGH-SPEED 

GAS CHROMATOGRAPHY 

Díaz Llorente D. 1 , Arias Abrodo P. 1 , Dapena de la Fuente E. 2 , Mangas Alonso J.J. 2 , Gutiérrez Álvarez M.D. 1 , Blanco Gomis D. 1 

1 


2 

SERIDA-Spain 

Corresponding author e-mail: piarab@uniovi.es 

A simple and fast method based on solid-phase microextraction (SPME) followed by high-speed gas 

chromatography (HSGC) was developed for the analysis of total 1,3-octanediols in apple juices by means of 

derivatization reaction to volatile 1,3-dioxanes. The figure shows the formation of 1,3-dioxanes from 1,3-diols and 

acetaldehyde. The derivatization reaction, SPME conditions, glycosidically bound fraction and 1,3-nonanediol as 

a surrogate standard were studied. The formation of 1,3-dioxanes from 1,3-diols was confirmed by GC-MS. The 

method was validated obtaining a regression coefficient (r2) of 0.9996, precisions between 0.3 and 9.8% and LOD of 

2.9 µgl-1. The method was applied to the analysis of twenty-one Asturian apple varieties finding a double reciprocal 

relationship between the concentrations of saturated and unsaturated 1,3 octanediol. 

412 



P12-002 MICRO COLUMN HPLC WITH DETECTION AT NANOSTRUCTURE 

Orinak A. 1 , Skantarova L. 2 , Orinakova R. 1 , Talian I. 2 , Fedorkova A. 2 

1 

University of P.J.Safarik in Kosice-Slovakia 

2 

Comenius University in Bratislava-Slovakia 

Corresponding author e-mail: andrej.orinak@upjs.sk 

The development of miniaturized chemical sensors is an increasingly active area of research. Adding vibrational 

spectroscopies to the arsenal of detection modes for microfluidics offers benefits afforded to structurally descriptive 

identification of separated analytes. Moreover, nanostructured materials, because of their high surface area, can 

provide critical enhancements in the performance of chemical microsensors [1]. Multifunctional nanostructured 

surfaces can play an important role in microhyphenation with different detection systems at nanostructure. In our 

research were used silver nanostructured films prepared by electrodeposition as coupling interfaces to simulate 

further integration into a chip. Multifunctionality of these substrates (analytical signal enhancement, separation 

function, pre-ionization function) were evaluated by surface enhanced Raman spectroscopy (SERS) as well 

secondary ion mass spectrometry (SIMS). Functional silver films were applied in hyphenation with micro column 

high performance liquid chromatography (uHPLC) where they served as analyte separated deposition and detection 

substrate. Eluate from uHPLC C18 column (ProteCol, SGE Analytical) has been continuously deposited onto linearly 

moved silver nanostructure to form unique deposition trace. Optimal mobile phase flow rate was established at 5 

uL/min and rate of substrate movement at 1.2-1.3mm/min.. Tested analyte was Rhodamine B and 6G, in mixture. As 

mobile phase was optimized acetonitrile/isopropanole/methanole – water, in different volumes. From SERS analyses 

we obtained million times signal enhancement while in SIMS three hundred times only. Separated compounds 

were well deposited onto silver substrate forming characteristic deposition trace for future spectral analysis 

and identification. Moreover, this silver films were found as good „ nanostructure initiator mass spectrometry“ 

substrates to improve ionization of complicated analytes without matrix application. New fragmentation ways were 

desribed in SIMS spectra. This research represents an introduction to integration of multifunctional nanostructures 

in miniaturised analytical systems that can lead to implementation in chip or sensor application. Advances in 

materials chemistry offer a range of nanostructured shapes and textures for building new biosensors. The use of 

microfluidisc for biofluid analysis gives a cheapper alternative to conventional techniques in diseases diagnosis. [1] 

Benkstein K.B.,Martinez C.J., Li G.,Meier D.C.,Montgomery CH.B., Semancik S.: Journal of Nanoparticle Research 

8(6)(2006)809-822. Acknowledgement The authors wish to thank Slovak grant agency VEGA for financial grants 

1/0134/10, 1/0043/08 and project APVV-0490-07 of the Slovak Research and Development Agency supporting this 

research project. 


413


P12-003 DETERMINATION OF BENZIMIDAZOLE COMPOUNDS IN FOOD BY UHPLC-MS/MS 

Martínez-Villalba A., Moyano E., Galceran M.T. 


Corresponding author e-mail: anna_martinez@ub.edu 

Parasitic infections are a main concern among livestock producers due to its frequent occurrence in intensive 

farming. For the treatment and prevention of helminthic parasites benzimidazolic compounds are routinely 

administered to farm animals at relatively high concentrations (milligrams per kg of body weight). These drugs 

undergo extensive metabolism and as a result, several metabolites in addition to the parent compound have been 

found in animal tissues. Since the structure of metabolites depend on the parent drug but also, on the tissue and 

the animal species, it is difficult to establish one residue marker for each drug. For this reason the current legislation 

establishes maximum residue levels (MRL) for the sum of the parent drug and its metabolites. In this work we 

have developed an ultra high performance liquid chromatography method coupled to tandem mass spectrometry 

(UHPLC-MS/MS) for the determination of 19 benzimidazoles (parent drugs and their metabolites). The optimal 

chromatographic separation was achieved using acetonitrile: 0.1% formic acid, pH 2.6 as mobile phase, 40ºC and 

gradient elution on a Fused-CoreTM C18 column. The separation provided high efficiencies (peak width 2-3 s) for all 

the compounds in less than 7 min and it was optimized to get good chromatographic resolution for fenbendazole/ 

ketotriclabendazole (needed to facilitate the polarity switching for MS detection) and for albendazole sulfone/5- 

hydroxymebendazole (two isobaric compounds). The applicability of two MS atmospheric pressure ionization 

sources, electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in positive and negative ionization 

modes was evaluated. Although positive-ESI was able to ionize most of the benzimidazoles [M+H]+, polarityswitching 

was necessary to detect ketotriclabendazole and triclabendazole in negative-APCI. CID-fragmentation 

by multiple-stage mass spectrometry (ion trap) and tandem mass spectrometry (triple quadrupole) was used to 

propose a common fragmentation pathway and to chose the most selective and sensitive transitions. The neutral 

loss of methanol was generally observed for all the compounds in addition to carbamate related fragmentations. For 

quantitative purposes highly-selective selected reaction monitoring (H-SRM, Q1 at 0.1 m/z unit FWHM) was used to 

provide maximum sensitivity and selectivity. The LC-MS/MS method developed was applied to the analysis of food 

samples after a fast and easy sample treatment based on QuEChERS (Quick Easy Cheap Effective Rugged and Safe) 

sample extraction approach. Under these conditions the method provided good performance in terms of linearity, 

repeatability, accuracy and limits of detection (low ppb levels). References: Q.A. McKellar, F. Jackson, Trends in 

Parasitology 20 (10) (2004) 456 M. Danaher, .O’Keeffe, J.D. Glennon, Analytica Chimica Acta 483 (2003) 313 M. 

Stamatakos C. Sargedi, Ch. Stefanaki, C. Safioleas, I. Matthaiopoulou, M. Safioleas, Parasitology International 58 

(2009) 115 

414 



P12-004 ULTRA-FAST ANALYSIS OF ISOFLAVONES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING FUSED CORE 

COLUMN TECHNOLOGY 


1 


2 

Universidad de Navarra, Dpto. Fisiología y Nutrición 

Corresponding author e-mail: manchon.noelia@inia.es 

The recent development of fused core technology in HPLC columns is enabling rapid and highly efficient separations. 

This technology generally consists of using techniques sol-gel to obtain a homogeneous porous layer on a solid 

core of silica. This allows a diffusion path much lower than the conventional columns with totally porous particles, 

because the inner core is solid fused silica, which is impenetrable by analytes, thus decreasing the resistance to 

mass transfer and narrower peaks. Furthermore, this material has an exceptionally narrow particle size distribution 

and high packing density compared with conventional materials, leading to smaller eddy diffusion, resulting in 

higher reproducibility and efficiency. This material can provide speed and efficiency similar to columns packed 

with sub-2 μm particles, but with reduced backpressures, due to this fact it is possible to use sub-2 μm columns 

without updating instrumentation for UHPLC. Therefore, the objective of this work was to develop an ultra-fast 

method for the determination of soy isoflavones taking advantage of fused core column technology. The isoflavones 

analyzed in this work are the main found in soybeans: genistin, daidzin, glycitin and their respective acetyl, malonyl 

and aglycone forms. They were identified by comparison of retention times and UV spectra using a PDA detector. 

The chromatographic analysis was performed using a core-shell column (KinetexTM C18 2.6 µm, 100Å, 100x4.6 

mm). Starting from a routine chromatographic method using water 0.1% acetic acid and acetonitrile as mobile 

phases, a step by step strategy was used to optimize temperature (25-50 ºC), flow rate (1.2-2.7 mL/min), mobile 

phase composition and equilibration time (1-5 min) in order to obtain an ultra-fast method of analysis. Increasing 

temperature resulted in a reduction of column backpressure (36.7 %) and retention time of all isoflavones (17-20 %). 

Temperatures above 35 ºC affected some isoflavones more than others to a point that changes in elution order were 

observed between Acetyl-glycitin and Malonyl-genistin. The best resolution and peak symmetry were observed at 

50 ºC. The use of higher temperatures also reduced peak width and consequently increased peak height, which 

allows lower detection and quantitation limits. The reduction of column backpressure caused by high temperatures 

also allowed to work with higher flow-rates and to reduce overall analysis time. The highest flow rate was limited 

by the system pressure limitations at 2.7 mL/min. The gradient was proportionally adjusted to the increase in flow 

rate. The optimized method allowed separation of all isoflavones in 5.8 min. The optimized equilibration time was 3 

min achieving high inter and intra-day variability lower than 0.5 %. The optimized method was successfully used for 

the analysis of different real samples with similar performance. Therefore the developed method can significantly 

increase sample throughout without compromising resolution, accuracy and sensitivity and could be used for wide 

range of applications. 


415


P12-005 MULTISYRINGE CHROMATOGRAPHIC SEPARATION (MSC) OF COPPER, CHROMIUM, AND NICKEL ON A DYNAMICALLY- 

COATED MONOLITHIC COLUMN WITH CHEMILUMINESCENCE DETECTION 

Diaz G. 1 , Horstkotte B. 2 , Maya F. 1 , Sanchez A.T. 2 , Duarte C.M. 2 , Cerdà V.1 

1 


2 

IMEDEA, Department of Global Change Research 

Corresponding author e-mail: victor.cerda@uib.es 

In this contribution we present the low-pressure separation of the transition metal ion Cu(II), Cr(III), and 

Ni(II) on a sodium dodecylsulfate (SDS) dynamically-coated octadecyl chromatographic monolithic column. 

Chemiluminescence detection was performed with post-column addition of luminol and hydrogen peroxide reagents. 

A home-made chemiluminescence flow cell was used in combination with a photomultiplier control device form 

Sciware SL (Palma de Mallorca, Spain). All solutions were driven using a multi-syringe pump device. In-situ column 

cleaning and conditioning was possible by the aspiration of the required solutions from a biocompatible selection 

valve. Chemical and physical parameters of the system such as eluent and reagent composition and pH, flow rate, 

and volumes of sample and water - used for sample stacking and gradient application – were thoroughly studied. 

The system featured high sensitivity with detection limits in the range of some tens of nanomol per liter and linear 

working ranges over one and a half orders of magnitude. Baseline separation in less than 10 min was possible 

using a 25 mm column and a combination of sodium oxalate, sodium chloride, and lithium hydroxide as eluent. The 

potential of soap-chromatography in combination with monolithic columns was successfully demonstrated with 

possible improvements by the use of longer columns for the separation of further metals. Acknowledgements The 

works was supported from the projects CTQ2007-64331 and CTM2008-01864-E/ANT funded by the MEC (Spanish 

Ministry of Education and Science). GD was financially supported by Carolina Foundation. BH was funded by a JAE 

Postdoctoral fellowship from CSIC. 

416 



P12-006 VERY FAST AND EFFICIENT SIZE-BASED SEPARATIONS OF POLYMERS AT ULTRA-HIGH PRESSURES 

Uliyanchenko E. 1 , Schoenmakers P.J. 1 , van der Waal S. 2 

1 

University of Amsterdam, Analytical-Chemistry Group, Van ‘t Hoff Institute for Molecular Sciences (HIMS) 

2 

DSM Resolve 

Corresponding author e-mail: e.uliyanchenko@uva.nl 

Using small (sub-2-μm) particles, Ultra-High-Pressure Liquid Chromatography (UHPLC) allows conducting fast 

and more efficient separations than conventional HPLC. UHPLC has proven to be a useful separation technique 

for different applications in life science, food, pharmaceutical and environmental analyses. However, little has 

been reported on the analysis of polymers with UHPLC, though UHPLC has great potential for such separations 

[1,2]. There are a few factors that limit the use of UHPLC for polymer separations. First, to provide information 

on the molecular-weight distribution one would need to use size-exclusion (SEC) columns with certain pore-size 

distributions. In UHPLC the pore size is limited by the stability of the packing material at ultra-high pressures. So 

far, there are no UHPLC SEC columns with large pore sizes. Additionaly, the narrow-bore columns, typically used for 

UHPLC to minimize the problem of heat dissipation, pose stringent requirements on the extra-column dispersion, 

especially for large (slowly diffusing) molecules. Moreover, UHPLC conditions generate high shear rates, which may 

affect polymer chains. In the present work we studied the feasibility of conducting efficient and very fast separations 

of polymers using wide-bore (4.6 mm ID) UPLC columns. The wider columns allow us to reduce the contribution of 

extra-column peak broadening to the total peak widths. Reliable SEC separations of polymers of molecular weights 

up to ca. 50 kDa may be performed within a few minutes at ultra-high pressures. Due to the small particles used in 

UHPLC it is possible to separate higher molecular weight polymers in the hydrodynamic-chromatography mode. 

These separations may be performed relatively rapidly and efficiently. 1. G. Stegeman, J.C. Kraak and H. Poppe, J. 

Chromatogr. 634 (1993) 149 2. S.-T. Popovici, W. Th. Kok and P. J. Schoenmakers, J. Chromatogr. A 1060 (2004) 237 


417


P12-007 COMBINING UHPLC WITH ADVANCED CHROMATOGRAPHIC TECHNIQUES FOR SELECT FOOD & BEVERAGE APPLICATIONS 

Steiner F., Fehrenbach T., Schmidt C., McLeod F., Martin M.M. 

Dionex Corporation, Product Marketing-Germany 

Corresponding author e-mail: frank.steiner@dionex.com 

Ultra High Performance Liquid Chromatography (UHPLC) is a rapidly growing technique that takes advantage of 

HPLC columns with sub-3 µm particles. It offers ultra-high resolution with maximum peak capacity and can also 

significantly fasten analyses by using higher linear velocities and shorter columns. This technology even enables 

to analyze medium complex samples in the sub-minute range so that any further attempt to increase analysis 

throughput definitely approaches physical and technical limits. Hence, the most improvements in laboratory 

workflows beyond that level can only be achieved by increasing automation and total system utilization time. This 

presentation describes technical solutions based on UHPLC in a dual system configuration using the example of 

selected food & beverage applications. It was investigated how UHPLC and advanced chromatographic techniques 

improve system utilization time compared to conventional LC. The significant enhancements in productivity are 

shown for the techniques of overlapping sample preparation, off-line column regeneration and parallel LC. The 

poster discusses the advantages of running two different applications on such a dual UHPLC system, either 

simultaneously or sequentially. This is illustrated by examples of the simultaneous determination of water- and 

fat-soluble vitamins and the automated subsequent analyses of water-soluble vitamins and soft drink additives. 

418 


P13 NEW 

DETECTION 

METHODS 

POSTER 

SESSIONS

POSTER SESSION 13 - NEW DETECTION METHODS 

P13-001 NEW ELECTROCHEMICAL DETECTOR FOR HPLC BASED IN SCREEN-PRINTED ELECTRODES: POLYPHENOLS ANALYSIS, 

ONE APLICATION 

Hevia D. 1 , Fanjul P. 2 , Hernandez D. 2 , Gómez-Cordovés C. 1 

1 

Instituto de Ciencia y Tecnología de los Alimentos y Nutrición (ICTAN)-CSIC 

2 

Dropsens S.L. 

Corresponding author e-mail: heviadavid@ifi.csic.es 

HPLC is one of the most important techniques to compounds analysis. In this technique, we can use several 

detectors based on different properties of molecules to analysis. In general, electrochemical detector (EC) has lower 

LOD than UV-Vis or fluorescence detector, but it has some problems such as the fact that the electrodes need to be 

electrochemically, chemically (cleaning by solvents) or mechanically (polishing) reconditioned to restore the original 

surface characteristics and this is sometimes a difficult and time-consuming procedure. 

In order to create a better electrochemical detector which is easy to work, the aim of this work is the design and 

optimization of a HPLC electrochemical detection based on disposable electrodes to analyze polyphenols of 

different nature. SPEs used in this work are based on a 4 mm diameter disk carbon working electrode, a carbon 

auxiliary electrode and a silver pseudo-reference electrode printed on an alumina substrate. These electrodes are 

fixed in a wall-jet flow cell and are easily connected to a potentiostat through a specific connector. The flow cell 

designed presents a wall-jet flow configuration that includes two pieces of polytetrafluoroethylene (PTFE) joined by 

a hinge and four magnetic bars. One of these pieces has inlet and outlet flow channels forming an angle of 30º. An 

o-ring in contact with the SPE delimits the electrochemical cell with a low volume. All the EC parts were put inside a 

Faraday box (box of lead) 

So as to optimizated chromatography conditions in polyphenols analysis, we did a preliminary study with only one 

compound (daphnetin). We used method previously described by Monagas et al 2007 with the next modifications. 

We observed that we need electrolites concentration but when we added more than 0,1 M the charge of peaks was 

less (loss of 10%). Another problem was organic modifier; we used MeOH or ACN without having any problems. We 

evaluated each percentages of ACN (until 50% ACN) and we used carbon nanotubes screen-printed because with 

it we can work for three days without any problems. The electrochemical potential used was 1.0 V because at this 

potential daphnetin showed the most response. The pH did not have any importance in the quantification of this 

compound. When we worked in a low flow 0,1 ml/min or similar, the transfer of charge was high and, therefore, the 

LOD/LOQ is low, but the peaks were width. Nevertheless, we meant to use this new system to analyse a high number 

of polyphenols in the same sample, we used flow at 1.0 ml/min. 

So, with this method we analyse 25 compound between 100 ng to 5 µg and in all of them we got a better correlation 

(r2>0.998). LOD/LOQ in some compounds were better in UV-Vis or fluorescen detector, but in other comopounds 

LOD/LOQ were better in EC. We concluded that we can use EC based on SPEs to analyse polyphenols because it 

is a good and cheap detector and we can put online after other detectors (DAD or fluorescence) thus increasing the 

analysis information. 

420 


POSTER SESSION 13 - NEW DETECTION METHODS 

P13-002 INVESTIGATION OF AN ENANTIOSELECTIVE SEPARATION PROCESS WITH SUSPENDED STATE HR/MAS NMR SPECTROSCOPY 

Yeman H., Albert K. 


Corresponding author e-mail: klaus.albert@uni-tuebingen.de 

In the world of separation processes, separations of racemic mixtures are considered of special interest. The 

separation of a racemate by HPLC is based on specific intermolecular interactions and requires a precise spatial 

and functional fit of the binding sites. It is the formation of a metastable diastereomeric complex with the chiral 

stationary phase (CSP) which results in tight binding of only one enantiomer.[1] A complete understanding of the 

complex interactions in the interphase between the stationary phase, analyte and mobile phase is of fundamental 

interest for further optimization.[2] A combination of various NMR techniques provides complementary information 

on the ligand, on the receptor, and on interactions possibly occurring between these. Solid-state NMR spectroscopy 

can directly provide information on the structure and dynamic features of the stationary phase including the 

immobilized selector. Suspended state High-Resolution/Magic Angle Spinning (HR/MAS) NMR offers the possibility 

to investigate interactions between an analyte and the CSP using the same solvents as in chromatography.[3] To 

gain a better understanding of the separation process a chiral separation was investigated by 1H saturation transfer 

difference (STD) HR/MAS NMR, spin-lattice relaxation time (T1) measurements and transferred Nuclear Overhauser 

Enhancement (trNOESY) NMR spectroscopy in the suspended-state. The results obtained by NMR can directly be 

correlated to chromatographic data.[4] Literature: [1] C. Hellriegel; U. Skogsberg, K. Albert, M. Lämmerhofer, N. M. 

Maier, W. Lindner, J. Am. Chem. Soc. 2004, 126, 3809-3816. [2] V. Friebolin; S. Marten, K. Albert, Mag. Res. Chem. 

2010, 48, 111-116. [3] S. Schauff; V. Friebolin, M.D. Grynbaum, C. Meyer, K. Albert, Anal. Chem. 2007, 79, 8323-8326. 

[4] H. Händel; E. Gesele, K. Gottschall, K. Albert, Angew. Chem. Int. Ed. 2003, 42, 438-442. 


421

P14 SPECIATION 

ANALYSIS 

POSTER 

SESSIONS

POSTER SESSION 14 - SPECIATION ANALYSIS 

P14-001 THE ALTERATION OF FRACTIONATION OF TRACE ELEMENTS SPECIES DUE TO BREAD MAKING 

Mestek O., Kominkova J., Koplik R., Santrucek J. 


Corresponding author e-mail: Oto.Mestek@vscht.cz 

Fractionation of Cu, Mo, Ni and Zn species in extracts of the raw material and baked bread was performed by on-line 

hyphenation of SEC/ICP-MS. An aqueous buffer solution (0.02 mol×l-1 Tris-HCl, pH=7.5) served for the extraction 

and as the mobile phase as well. Superdex HR 75 column (GE HealthCare) was applied for the sample fractionation 

and PE Elan DRC-e was used as the element-specific detector. Investigated samples were: the mixture for the home 

preparation of bread composed of wheat and rye flour (total content 2.83 µg/g Cu, 0.508 µg/g Mo, 0.229 µg/g Ni 

and 17.6 µg/g Zn) and crumb (1.61 µg/g Cu, 0.303 µg/g Mo, 0.145 µg/g Ni and 10.0 µg/g Zn) and crust (2.23 µg/g 

Cu, 0.431 µg/g Mo, 0.155 µg/g Ni and 13.0 µg/g Zn) of freshly baked bread. Almost all soluble Cu and Ni and major 

portion of Zn (> 80 %) in all analysed samples was bound in a fraction of M approx. 1-2 kDa. The remainder of Zn 

was found in a fraction of M approx. 4 kDa. Mo in all samples was located in a fraction of M approx. 3 kDa. The 

low-molecular weight chromatographic fractions richest in metals isolated from extracts of all samples were 

collected (Fractogel EMD Bio SEC, Merck) and subsequently the ligands of trace elements involved in these 

fractions were isolated by immobilised metal ions affinity chromatography (HiTrap Chelating column in Cu2+ cycle, 

GE HealthCare). After the acid hydrolysis the isolated ligands were analysed for the amino-acid composition by 

ion-exchange chromatography. Among other amino-acids a great deal of sulphur-containing amino-acids (cystein, 

carboxymethyl cystein) and dicarboxylic ones (aspartic and glutamic acid) were found. These types of amino-acids 

are known as units of strongly metal-chelating peptides. In parallel the isolated ligands were investigated by MALDI- 

MS. The desglycyl-phytochelatin PC6 was found in the raw material, while the samples of baked bread were free of 

this compound probably due to thermal decomposition or enzymatic degradation during rising of the dough. 

Speciation of the mercury in all investigated samples (0.013 µg/g in raw material, 0.011 µg/g in booth crumb and 

crust) was performed by RPC/ICP-MS hyphenation (Purospher® RP-8e, Merck) after extraction of mercury species 

into 1 % (v/v) mercaptoethanol – 1 mol/l HCl mixture. Only inorganic mercury was found in all extracts. 


423

POSTER SESSION 14 - SPECIATION ANALYSIS 

P14-002 DETERMINATION OF MERCURY SPECIES IN FOOD BY LIQUID CHROMATOGRAPHY - INDUCTIVELY COUPLED PLASMA 


Koplik R., Mestek O., Kominkova J. 


Corresponding author e-mail: Richard.Koplik@vscht.cz 

Mercury is a pollutant widely spread in all environmental compartments. The need for mercury speciation analysis 

in food materials was recognised in 1960s as a response to several fatal mass intoxications. Owing to mercury 

bioaccumulation and biomethylation in aquatic eco-systems the occurrence of methylmercury species has drawn 

most attention. Liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry allows 

an easy determination of mercury species as the detection is element-specific and very sensitive. Moreover, no 

derivatization is required. A good separation of mercuric (Hg2+), methylmercuric (MeHg+) and ethylmercuric (EtHg+) 

ions can be easily done by reversed-phase liquid chromatography using 2-mercaptoethanol (2ME)-containing mobile 

phase. A short chromatographic column (Purospher STAR RP-8e 75*4 mm, 3 µm) was applied. The best resolution of 

MeHg+ and Hg2+ was achieved using a buffered mobile phase with low methanol content (0.02 mol/l CH3COONH4 + 

0.2 % (V/V) 2-ME + 1 % (V/V) CH3OH). The effluent flow (0.8 ml/min) was combined with the flow of internal standard 

solution (Rh 50 ng/ml in 0.15 mol/l HNO3) and the mixed solution (1.2 ml/min) was delivered by the peristaltic pump 

to the nebulizer of the ICP-MS instrument. During analysis the signals of 202Hg and 103Rh were measured. Under 

these conditions the capacity factors of MeHg+ and Hg2+ are 4.7 and 6.5, respectively and the resolution is approx. 

2.4. The chromatographic run takes 16 min. Sample volume up to 200 µl can be injected without getting resolution 

worse. Repeated extraction by hydrochloric acid and 2-mercaptoethanol solution (1 mol/l HCl + 0.2 % (V/V) 2-ME) 

was applied for isolation of mercury species from food samples. Analyses of CRM 422 Cod Muscle, SRM 2976 

Mussel Tissue and SRM 1570 a Spinach Leaves reference materials proved the full recovery of mercury. On the other 

hand only 65 % of total mercury content was liberated from CRM 185 Bovine Liver by this extraction procedure. The 

method was applied for determination of mercury species in the samples of fish (both marine and freshwater species 

were selected) and some vegetables. The sum of MeHg+ (as the mass of Hg) and Hg2+ contents in fish samples 

ranged from 2 to 253 µg/kg. The prevailing mercury species in fish is methylmercury representing 84 to 99 % and 72 

to 96 of the total mercury content in marine and freshwater species, respectively. On the other hand the vegetable 

samples contain negligible amounts of MeHg+, while Hg2+ is the main species. 

424 


P15 

FOOD QUALITY 

AND SAFETY 

POSTER 

SESSIONS

POSTER SESSION 15 - FOOD QUALITY AND SAFETY 

P15-001 VALIDATION OF A CONFIRMATORY METHOD FOR DETERMINATION OF THYREOSTATS IN PIG URINE BY LC-MS/MS 

Taka T., Baras M.C., Bet Z.F.C. 

Lanagro-SP/Ministry of Agriculture-Brazil 

Thyreosthats are synthetic compounds, which inhibit thyroid activity and, when administered to food producing 

animals, increase weight gain mainly by water retention. Due to its detrimental effect over meat quality, and also 

following the discovery that some of these drugs may present carcinogenic activity, its use in food producing animals 

has been banned in several countries. 

In order to meet the necessity of Brazilian National Residues Control Plan to monitor illegal use of thyreostatic 

compounds in livestock, an LC-MS/MS method for determination of Tapazole (TAP), Thiouracil (TU), Methyl-thiouracil 

(MTU) and Propyl-thiouracil (PrTU) in pig urine was developed and validated. 

Firstly, samples were subjected to derivatization with 3-IBBr under pH 8.0 at 40°C for an hour. This step was followed 

by acidification and liquid-liquid extraction using ethyl-acetate:ciclohexane 80:20 solution. After evaporation, 

samples were resuspended in methanol:water 40:60 solution and centrifuged prior to instrumental analysis. 

Chromatographic separation was obtained using a C18 50x2.1mm column under 40°C temperature and mobiles 

phases consisting of 0.5% acetic acid aqueous solution (phase A) and methanol (phase B). Mass spectrometry 

was obtained using TSQ quantum access platform, MRM detection. It was necessary to combine both positive and 

negative ESI acquisition modes to achieve adequate results for all analytes. Fragmentation conditions and retention 

times (RT) are displayed in Table 1. 2-Mercaptobenzimidazole (MBI) was used as internal standard. 

Validation was carried out according to European Commission Decision 657/2002/EC. All determined values meet 

acceptability criteria. Repeatability, reproducibility, CCa, CCb and recovery results are presented in Table 2. As 

validation results imply, the developed method suits its confirmatory analysis purpose. In addition, it is a fast and 

simple method, which enables it to be used in routine. 

426 



P15-002 DETERMINATION OF POLYPHENOLS IN WINES BY LIQUID CHROMATOGRAPHY WITH SPECTROPHOTOMETRIC AND 

FLUORIMETRIC DETECTION. APPLICATION TO THE CHARACTERIZATION OF WINES 

Hernández Cassou S. 1 , Oliver R. 2 , Checa A. 1 , Saurina J. 1 

1 


2 


Corresponding author e-mail: xavi.saurina@ub.edu 

Wine features and quality depend on a wide variety of parameters including climatic and geological factors of 

production regions, grape varieties and maturation, technological practices, etc. Apart from sensory parameters, 

analytical methods are being increasingly applied to wine characterization. 

In this communication, a new HPLC method with UV-Vis and fluorescence detection is developed for the analysis of 

polyphenols in wines. Analytes are separated in a Synergy Hydro-RP C18 column using an elution gradient obtained 

from 9 mM H3PO4 aqueous solution and methanol as an organic modifier. UV detection is carried out at 280 nm and 

fluorescent detection at lexc = 260 nm and lem = 376 nm. The influence of variables such as pH, organic modifier 

percentage and, especially, the assessment of a proper elution gradient profile have been evaluated by experimental 

design. Additionally, multicriteria decision strategies have been utilized for achieving a reasonable compromise 

among relevant objectives to be considered such as number of compounds separated, resolution, analysis time, 

etc. Figures of merit including accuracy, precision, and detection limits have been established under selected 

experimental conditions using red wine samples. 

The method is applied to characterize red wines from different Spanish regions from the polyphenolic composition 

as a source of analytical data. Exploratory methods based on factor analysis are utilized to facilitate the extraction of 

relevant information dealing with quality, origin and winemaking practices. 


427


P15-003 GC-MS ANALYSIS OF CANNED FOOD PRODUCTS FOR BISPHENOL A 

Cao X-L. 

Health Canada, Food Research Division 

Corresponding author e-mail: xu-liang.cao@hc-sc.gc.ca 

A method based on solid phase extraction followed by derivatization and GC-MS analysis was validated for the 

determination of bisphenol A in canned food products. This method was used to analyse 78 canned food products 

for BPA. Concentrations of BPA in canned food products varied considerably among food types, but they were all 

below the specific migration limit of 0.6 mg/kg set by the EC Directive for BPA in food or food simulant.Canned tuna 

products had the highest BPA levels in general, with the average and maximum BPA levels of 137 and 534 ng/g, 

respectively. BPA levels in the concentrated soup products are considerably higher than those in the ready to serve 

soup products, with the average and maximum BPA levels of 105 and 189 ng/g for the concentrated soup compared 

to 15 and 34 ng/g for the ready to served soup. BPA levels in canned vegetable products are relatively low, about 

60% of the products had BPA levels less than 10 ng/g. Compared to the canned pure tomato products, BPA levels in 

canned tomato paste products are low. The average and maximum of BPA levels for the tomato paste products were 

1.1 and 2.1 ng/g, while they were 9.3 and 23 ng/g for the pure tomato products. 

428 



P15-004 UNCERTAINTY STUDY OF A MULTI-RESIDUE METHOD TO DETERMINE ORGANOCHLORINE PESTICIDES AND 

POLYCHLORINATED BIPHENYLS IN BOVINE FAT BY GAS CHROMATOGRAPHY - ELECTRON CAPTURE DETECTION 

Olivares I.R.B., Antunes Lopes F., Cordeiro Maia Saglioni E., Lopes Modro A., Hoffmann Kowalski C. 

National Laboratory of Ministry of Agriculture-Brazil 

Corresponding author e-mail: igorolivares@iqsc.usp.br 

The importance of quality in laboratories that analyze residue and contaminants in food has become essential 

because of the importance of analytical results and their impact in the society. These laboratories applied different 

tolls to reach the quality results, like validation process and uncertainty measurement. The validation process 

evaluate if the analytical method is fit for purpose and can be considered as an evidence of reliable results, and 

the uncertainty characterizes the dispersion of the quantity values being attributed to a measurand based on the 

information used. 

In recent years the declaration of estimated uncertainty of measurement has become an integral and essential part 

of analytical results. This measurement is necessary in order to establish the comparability of results obtained 

in different laboratories (with different uncertainties), and have been highlighted in Laboratory Quality Systems 

internationally recognized like ISO/IEC 17025:2005. 

The literature guides the application of two approaches to calculate the uncertainty: bottom-up (a hard calculation 

that consider all steps of the methodology) and top-down (a simplified method that consider the validation results). 

At first, these approaches can be used together to evaluated in detail all uncertainty sources. After this, is possible 

to identify the principal sources that can be used during the routine analysis in a simpler, faster and fit for purpose 

routine calculation. 

This study presents a detailed uncertainty calculation that considers a bottom-up and top-down approaches. The 

calculation was applied in a multi-residue method to determine organochlorine pesticides and polychlorinated 

biphenyls in bovine fat by gas chromatography - electron capture detection that was validated according to SANCO 

10684:2009. 

The results showed that the uncertainties associated with the extraction steps were not so important compared with 

uncertainties associated with the validation process. This way, in a multiresidue method described, the uncertainty 

calculated from validation process can be consider as a simpler, faster and fit for purpose uncertainty calculation. 


429


P15-005 VOLATILE CHARACTERIZATION OF MONOFLORAL AND MULTIFLORAL TYPES OF HONEYBEE-COLLECTED POLLENS BY 

GC-MS. INFLUENCE OF INDUSTRIAL PROCESSING IN THEIR VOLATILE FLAVOR CONSTITUENTS 

Domínguez-Valhondo D. 1 , García-Parra J. 1 , Bohoyo-Gil D. 1 , Hernández M.T. 1 , Bernalte M.J. 2 , Ayuso M.C. 2 , González-Gómez D. 1 

1 

Insituto Tecnológico Agroalimentario (INTAEX) 

2 


Corresponding author e-mail: david.gonzalezgo@juntaextremadura.net 

Honeybee-collected pollen constitutes a potential source of energy and proteins for human consumption. Indeed, 

the consumption of pollen products has drastically increased over the last decade. However, there are no many 

scientific studies regarding the chemical characterization of honeybee-collected pollen in relation to its organoleptic 

properties such as volatile compounds characterization. 

The aim of this study was to establish a gas chromatographic procedure to determine if the volatile fraction of 

honeybee-collected pollen was affected by the application of standard industrial thermal processes. Monofloral and 

multifloral honeybee-collected pollens were harvested using a pollen trap during 2009 in the region of Extremadura, 

a southwest area of Spain. Pollen samples were immediately stored at -80 C to avoid degradation through 

fermentation processes. In addition, industrial thermal processed pollen was given by local producers in order to 

evaluate how the industrial thermal process affected the volatile fraction. 

For the chromatographic separation and identification of the volatile fraction a gas chromatograph equipped with 

dynamic headspace concentrator coupled to an ion-trap mass spectrometer was used. Around 70 compounds were 

separated in a phenyl-methyl-siloxane 50 m chromatographic column and identified according to their mass spectra. 

The differences among the studied honeybee pollens, in terms on volatile profile, were studied by means of the 

analysis of variance and principal component analysis. 


Authors thank Junta de Extremadura of Spain (D.G. de Ciencia y Tecnología) and FEDER’s funds for the financial 

support (Project PDT09B002). Honeybee-collected pollen was provided by Euromiel S. Coop. de Segundo Grado 

(Mérida, Spain). 

430 



P15-006 ODOUR CHARACTERISATION OF DIFFERENT SPECIES/ORIGINS OF CURED VANILLA BEANS BY MEANS OF HS-SPME-GC- 

MS AND MS-BASED ELECTRONIC NOSE TECHNOLOGY 


Catholic University College Ghent 

Corresponding author e-mail: tim.vancaelenberg@kahosl.be 

The use of vanilla has a defining role in the flavour perception of confectionery food such as chocolates and bakery 

products. Nowadays, synthetically produced vanilla aromas (e.g. ethyl vanillin) are frequently used to obtain a 

vanilla-like flavour in food. Nevertheless, the complex aroma composition of natural vanilla beans is essential 

to produce high quality confectionery products. These vanilla pods are cultivated and cured in countries with a 

warm and humid climate. Because of the high demand from the market, the limited supply of quality beans and 

increased costs of production, last decennium vanilla became the third most expensive spice in the world. In 

consequence, food companies need to be supplied with a control tool to screen in a fast and objective way the 

flavour characteristics of the imported beans and to guarantee the quality standards of their final product. The 

aim of this investigation was to optimise a fast methodology to characterise and to evaluate the flavour differences 

between different species and origins of cured vanilla beans. In this study two vanilla species originating from 

different countries were investigated. Beans produced in Tahiti (Vanilla tahitensis M.), Madagascar (Vanilla 

planifolia A.), Uganda (Vanilla planifolia A.) and Papua New Guinea (Vanilla planifolia A.) were selected. All vanilla 

beans were cryogenically grinded to avoid loss of aroma volatiles before analysis. Chemical-analytical analysis 

of the vanilla beans was carried out by means of Headspace-Solid Phase Microextraction (HS-SPME) followed 

by Gas Chromatography-Mass Spectrometry (GC-MS). This time-consuming technique was compared to fast 

mass spectrometry-based electronic nose technology (MS-nose). The efficiency of both techniques for quality 

control of spices was already demonstrated in previous work (1). Data obtained by HS-SPME-GC-MS as well as 

by MS-nose technology were statistically treated and compared by using Principal Component Analysis (PCA). 

Furthermore, the contribution of some key odour components (e.g. vanillin, guaiacol and anisyl alcohol) to the total 

volatile amount could be related to the quality of the selected vanilla beans. In addition, a sensory ranking test with 

selected attributes (odour intensity, floral, creamy, woody and alcoholic) was carried out. Results obtained as well by 

chemical-analytical analysis as by sensory analysis were correlated using Partial Least Squares regression (PLS). In 

conclusion, MS-nose technology could be mentioned as a successful tool for fast and objective aroma classification 

of different species and origins of vanilla beans. Additionally, time-consuming HS-SPME-GC-MS and sensory 

analysis could be helpful to obtain detailed insight into the aroma composition of different vanilla beans. (1) Van 

Caelenberg T. and Dirinck P. An analytical approach for quality control of white pepper (Piper nigrum L.) using HS- 

SPME-GC-MS and MS-based electronic nose technology. In: Food for the Future - The Contribution of Chemistry 

to Improvement of Food Quality, Proceedings of the 15th European Conference on Food Chemistry (EURO FOOD 

CHEM XV), Copenhagen, Denmark, 2009, pp. 81-84. 


431


P15-007 EXTRACTION OF SELECTED FLAVONOIDS FROM VARIOUS BERRIES BY PRESSURIZED SOLVENTS 

Hohnova B. 1 , Stavikova L. 1 , Karasek P. 1 , Vespalcova M. 2 

1 


2 


Flavonoids are natural compounds widely distributed in plant kingdom. They are indispensable for human diet 

because, due to their antioxidant properties, they show protective effects against cancer, stroke and coronary heart 

diseases. Therefore, rapid and efficient procedures to extract antioxidants from plant materials are required for 

preparative and production purposes as well as prior to chromatographic analysis. The extraction by compressed 

liquids – Pressurized fluid extraction (PFE) and Pressurized hot water extraction (PHWE), present fast, effective and 

environmentally friendly extraction methods for the determination of flavonoids in various species of berries. 

PFE and PHWE followed by reversed phase high-performance liquid chromatography with UV-visible detection have 

been utilized for the determination of rutin and quercetin in the berries of less common fruit species growing in the 

Czech Republic (Sambucus nigra, Cornus mas, Hippophae rhamnoides, Aronis melanocarpa). The matrices were 

extracted by methanol or ethanol and subsequently by water at higher temperature 40-120 ºC and pressure 15 MPa 

during 15 minutes. The pressurized methanol was more effective solvent for the extraction of rutin and quercetin 

from the berries than pressurized ethanol; moreover, the most efficient extraction of selected flavonoids was 

achieved by pressurized water. 

Acknowledgement: 

The financial support of this work by the Czech Science Foundation (Projects GA203/08/1465 and GA203/08/1536) 

and by the Academy of Sciences of the Czech Republic (Institutional Research Plan No. AV0Z40310501) is gratefully 

acknowledged. 

432 



P15-008 EXTRACTION AND IDENTIFICATION OF FLAVONOIDS IN BRANCHES AND LEAVES OF DIFFERENT VARIETIES OF 

SAMBUCUS NIGRA 

Stavikova L. 1 , Grulichova H. 2 , Hohnova B. 1 , Karasek P. 1 , Vespalcova M. 2 

1 


2 


Corresponding author e-mail: stavikova@iach.cz 

The amount of rutin and quercetin in the extracts from the branches and leaves of wild Sambucus nigra and 

several cultivars of Sambucus nigra (Albida, Bohatka, Dana, Haschberg, and Körsör) was determined. All extracts 

were prepared by Pressurized Fluid Extraction (PFE – methanol was used as solvent) and Pressurized Hot Water 

Extraction (PHWE) at a pressure of 15 MPa and different temperatures, 120°C was used for the extraction of rutin 

and quercetin by PFE, 80°C and 100°C was used for the extraction of rutin and quercetin by PHWE, respectively. 

The identification and quantification of the above compounds by high-performance liquid chromatography with 

diode array detection (HPLC-DAD) based on SUPELCOSILTM LC 8DB (5μm; 250 x 4.6 mm) column separation was 

performed. Methanol: water: formic acid (36: 61.5: 2.5, pH 2.17 2.28) was used as a mobile phase. The flow rate was 

set at 0.7 ml/min and the wavelength was kept at 370 nm. Both compounds were determined and identified during 25 

minutes. 

PHWE extraction was more effective than PFE extraction. The largest amount of rutin was determined in the 

leaves of wild Sambucus nigra, 5.58 mg/g. The smallest amount of rutin was detected in Körsör leaves, 0.13 mg/g. 

Quercetin was not determined in any Sambucus nigra leaves. 

Both flavonoids were found in branches of wild Sambucus nigra. In wild Sambucus nigra branches, rutin was 

determined in a small amount while an important quantity was found in cultivated Sambucus nigra branches. The 

largest amount of rutin was found in Albida, 2.33 mg/g. Quercetin was not detected in any cultivated branches of 

Sambucus nigra. The largest amount of quercetin was contained in wild Sambucus nigra, 0.24 mg/g. 

Keywords: PFE, PHWE, HPLC, rutin, quercetin, Sambucus nigra. 

This work was supported by the Czech Science Foundation (GA203/08/1465 and GA203/08/1536) and by the 

Academy of Sciences of the Czech Republic (Institutional Research Plan AV0Z40310501). 


433


P15-009 CHARACTERIZATION OF NOVEL GALACTOOLIGOSACCHARIDES DERIVED FROM LACTULOSE 

Hernández-Hernández O. 1 , Moreno F.J. 2 , Montilla A. 2 , Clemente A. 3 , Olano A. 2 , Sanz M.L. 1 

1 


2 

Instituto de Fermentaciones Industriales-CIAL (CSIC), Madrid 

3 

Estación Experimental del Zaidín (CSIC), Granada 

Corresponding author e-mail: mlsanz@iqog.csic.es 

Galactooligosaccharides (GOS) are considered non-digestible carbohydrates with well-recognised prebiotic 

properties. They are synthesized from lactose by a transgalatosylation reaction, catalysed by β-galactosidase, giving 

rise to oligosaccharides of different glycosidic linkages and molecular weights. By using commercial enzymatic 

preparations (1, 2), novel GOS derived from lactulose have been obtained recently. In vitro fermentation studies 

of these carbohydrates with human faecal slurries have shown to promote the growth of bifidobacteria as well 

as the production of acetic acid (3). Several factors such as type of glycosidic linkage, monomeric composition 

and carbohydrate chain length are likely to affect the prebiotic activity of these oligosaccharides. Therefore, the 

molecular characterization of these oligosaccharides is critical in order to gain knowledge and understanding of 

structure-prebiotic function relationships for further exploitation. In this work, we have characterised novel GOS 

from lactulose by GC-MS, HPLC-ESI MS and MALDI-ToF MS. In adidition, chemical structures of GOS derived from 

lactose were studied for comparative purposes. Degrees of polymerization (DP) of GOS obtained from lactulose 

and lactose by transgalactosylation reactions catalysed by β-galactosidase from commercial enzymatic extracts 

were determined by MALDI-ToF MS, using 2,5-dihydroxybenzoic acid as matrix. HPLC-ESI MS analyses at positive 

mode were carried out using a graphitized charcoal column (100 mm x 2.1 mm, 3 µm; Hypercarb, Thermo Scientific) 

with H2O:methanol as mobile phase. GC-MS analyses were carried out using a HT5 column (25 m x 0.22 µm id x 

0.1 µm df) from SGE, coated with 5% phenyl polysiloxane-carborane following the method of Hernández et al. (4). 

Oligosaccharides were converted into their trimethylsilyl oximes previous their GC-MS analysis as described before 

(5). MALDI-ToF MS analyses of GOS from lactulose revealed the existence of oligosaccharides from DP2 to DP8. 

HPLC-MS analyses confirmed these data, being di- and trisaccharides the most abundant fractions. Galactosylgalactoses 

and galactosyl-fructoses were detected as major components in the disaccharide fraction, and glycosidic 

linkages 1-6 and 1-4 prevailed over the others. Further studies to evaluate their prebiotic activity are in progress. 

This work was financed by projects PIF-SIALOBIOTIC 200870F-010 funded by CSIC and POII10-0178-4685 funded 

by Junta de Comunidades de Castilla-La Mancha-FEDER. O. Hernández-Hernández thanks CSIC for a JAE Predoc 

grant. 1Cardelle-Cobas et al., J. Agric. Food Chem. 2008, 56, 3328–3333. 2Martínez-Villaluenga et al., J. Agric. Food 

Chem. 2008, 56, 557–563. 3Cardelle-Cobas et al., J. Dairy Res. 2009, 76, 1-9. 4Hernández et al. Int. Dairy J. 2009, 

19, 531–536. 6García-Baños et al., Chromatographia, 2000, 52, 221-224. 

434 



P15-010 DETERMINATION OF INOSITOLS AND OTHER LOW MOLECULAR WEIGHT CARBOHYDRATES IN DIFFERENT VEGETABLES 

EXTRACTS USING GC-MS 

Hernández-Hernández O., Ruiz-Aceituno L., Martínez-Castro I., Sanz M. L. 

Instituto de Quimica Organica General (CSIC) 


Vegetables are an essential source of energy characterized by the presence of sugars, mainly fructose, glucose and 

sucrose. Other minor components such as inositols, with important biological properties, can also be found. 

Inositols (cyclohexane-1,2,3,4,5,6-hexol) are present as minor components in plants with diverse molecular 

structures. The presence of these compounds has been reported in different vegetables such as grapes must (1), 

peanuts (2), fruits (3), etc. Inositols have shown to have positive health effect in humans. The oral intake of myoinositol 

can be used in the treatment of diabetic neuropathy (4); chiro-inositol have demonstrated to be effective 

against insulin resistance in muscle cells (5) and to have substantial beneficial effects for the treatment of different 

diseases such as: polycystic ovary syndrome, panic disorder, etc. (6,7). 

To the best of our knowledge, few works report the content of minor sugars and inositols in vegetables samples. 

Therefore, in this study a GC-MS method was used to determine the composition of low molecular weight 

carbohydrates (mainly inositols) in different vegetables extract. 

Extracts from 14 vegetables samples were obtained, using acetic acid (5% v/v) at 60ºC during 1 hour. Carbohydrates 

were converted to their trimethylsilyl oximes previous to their analysis. GC-MS was performed using a fused silica 

coated with SPB-1 column (30 m x 0.25 µm id x 0.25 µm df) from Supelco (Bellmonte, PA, USA), following the 

method of Sanz et al. (6). 

In general, fructose was the main carbohydrate detected in the analysed vegetables, followed by glucose, sucrose 

and galactose. Other di- and trisaccharides were also quantified in Dioscorea trifida, possibly due to the reserve of 

high molecular weight carbohydrates in this vegetable. Traces of sedoheptulose, glycosyl-glycerol and mannitol also 

were identified in most of samples studied. The major content of myo-inositol was found in purple lettuce (Lactuca 

sativa), followed by aubergine skin (Solanum melongena). Chiro-inositol was detected in the most of the species of 

the Lactuca and Cichorium genus, being the green lettuce the vegetable with highest content. Scyllo-inositol was 

also found in different vegetables, showing the highest values in the tuber sample (Dioscorea trifida). 

(1) Versini et al (1984). Vignevini, 11, 41-47. 

(2) Binder et al (1984). Carbohydrate Research, 129, 21-32. 

(3) Sanz et al (2004). Food Chemistry, 87, 325-328. 

(4) Clements et al (1980). The American Journal of Clinical Nutrition, 33, 1954-1967. 

(5) Yap et al (2009). Animal Cell Technology: Basic & Applied Aspects 15. 

(6) Benjamin et al (1995). Psychopharmacol Bull 31, 167-175. 

(7) Luorno et al (2002). Endocr Pract 86, 417-423. 

This work was financed by projects AGL2009-11909 (Ministerio de Ciencia e Innovación) and ANALISYC-II S2010/ 

AGR-1464 (Comunidad de Madrid). O. Hernández-Hernández thanks CSIC for a JAE Predoc grant. 


435


P15-011 A NOVEL MULTIPLEX PCR-CGE-LIF METHOD FOR THE ANALYSIS OF GENETICALLY MODIFIED YEASTS IN WINE SAMPLES 

Leon C. 1 , García-Cañas V. 1 , Gonzalez R. 2 , Cifuentes A. 1 

1 


2 

Institute of wine and grapevine science (CSIC) 

Corresponding author e-mail: virginia.garcia@ifi.csic.es 

The use of yeasts plays an important role in food science. Development of new yeast strains using recombinant 

DNA technology has been the most recent step used by wine microbiologists to improve specific properties in 

wine making. After the approval by the FDA and Health Canada of two genetically modified S. cerevisiae strains 

for wine making, transgenic yeasts could soon be extensively used in many other markets. The use of genetically 

modified organisms in food industry and agriculture is regulated by specific regulations in many countries. To verify 

the application of such regulations, it is necessary to develop analytical methodologies that can effectively detect 

genetically modified organisms in the food chain. In this work, a new analytical methodology based in the combined 

use of multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser induced fluorescence 

detection (CGE-LIF) has been developed to determine the presence of S. cerevisiae EKD-13, a genetically modified 

yeast strain, in non clarified wine samples. To achieve this, a DNA extraction procedure has been optimized for 

wine samples. A novel multiplex PCR system has been designed for the amplification of two transgenic sequences 

in the transgenic yeast strain EKD-13 and a reference gene in S. cerevisiae. CGE-LIF method, using bare-fused 

silica capillaries, was demonstrated very useful and informative for optimizing multiplex PCR parameters such as 

annealing temperature and primers concentration. Finally, sensitivity, accuracy and time of analysis of the CGE-LIF 

method developed at our laboratory using bare-fused silica capillaries was compared with a commercial CGE-LIF 

method using coated capillaries. Analysis with both separation methods demonstrated the good possibilities of this 

methodology to successfully determine the presence (or not) of transgenic yeast strain EKD-13 in different wine 

samples. 

436 



P15-012 SURVEY OF BISPHENOL A IN PLASTIC BOTTLES BY GAS CHROMATOGRAPHY TANDEM MASS SPECTROMETRY 

Mansilha C. 1 , Rocha S. 2 , Pinho C. 2 , Domingues V. 3 , Rebelo H. 1 , Gameiro P. 2 

1 

Instituto Nacional de Saúde Dr. Ricardo Jorge-Portugal 

2 

Requimte, Faculdade de Ciências Universidade Porto 

3 

Reqimte, Instituto Superior de Engenharia do Porto, Química 

Corresponding author e-mail: catarinamansilha@gmail.com 

Bisphenol A (BPA) is the common name for 2,2-(4,4’-dihydroxydiphenyl)propane, 4,4’-isopropylidenediphenol, or 

2,2’-bis(4-hydroxyphenyl)propane. It is one of many man-made chemicals classified as endocrine disruptor, acting 

as an estrogen receptor agonist with detrimental health impacts, especially for young children exposed during their 

critical stages of development [1-2]. BPA is deeply imbedded in the products of modern consumer society, being 

used for the production of industrial epoxies, polycarbonate plastics, fungicides, flame-retardants and antioxidants. 

Therefore, contamination is widespread in the environment leading to a myriad of exposures. BPA is also quite 

persistent as under normal conditions in the environment it does not readily degrade [3-4]. In the last several years, 

new scientific research on polycarbonate and other plastics has raised public concern about BPA exposure due to 

the quotidian use of such materials, including baby bottles, water bottles and food containers, returnable or not [1]. 

The aim of this study was to assess the evidence for BPA migration from different plastic containers of high density 

polyethylene (HDPE), polyethylene terephthalate (PETE) and polycarbonate in order to evaluate its concentration in 

the contents and, consequently, the exposition rate of population. Optimization and validation of a method based on 

a solid phase extraction (SPE) methodology coupled with gas chromatography tandem mass spectrometry (GC-MS), 

developed previously by our research group, was used to analyze water samples. For derivatization of the hydroxyl 

groups, 2,2,2-trifluoro-N-methyl-N-(trimethylsilyl)acetamide (MSTFA) was used. Matrix-induced chromatographic 

response enhancement was avoided by using matrix-standard calibration solutions and heteroscedasticity has been 

overtaken by a weighted least squares linear regression model application [5]. Deuterated bisphenol A was used 

as a quality control internal standard, so-called procedural or instrument internal standard, for process evaluation 

through recovery studies, being added in a constant amount to blanks, samples and calibration standards prior 

to extraction. Our studies have demonstrated that biologically active BPA was released from different plastic 

containers, including baby bottles, after simulated normal use. High temperatures, as microwave heating and 

exposure to boiling water, as well as sequences of washing and rinsing were also tested. 1. U.S. Food and Drug 

Administration, F., Update on Bisphenol A for Use in Food Contact Applications 2010. 2. Environment Canada, 

Proposed Risk Management Approach for Phenol, 4,4’-(1-methylethylidene) bis (Bisphenol A). 2008. 3. European 

Union Risk Assessment, R., 4,4’-isopropylidenediphenol (Bisphenol-A) CAS No: 80-05-7 Complete risk assessment. 

2010. 4. Carwile, J.L., et al., Polycarbonate Bottle Use and Urinary Bisphenol A Concentrations. Environmental 

Health Perspectives, 2009. 117(9): p. 1368-1372. 5. Mansilha, C., Melo, A., Rebelo, H., Ferreira, I.M.P.L.V.O., Pinho, 

O., Domingues, V., Pinho, C., Gameiro, P. (2010). Quantification of endocrine disruptors and pesticides in water by 

gas chromatography-tandem mass spectrometry. Method validation using weighted linear regression schemes. 

Journal of Chromatography A, doi: 10.1016/j.chroma.2010.05.005. 


437


P15-013 SEPARATION AND IDENTIFICATION OF TRITERPENES FROM PISTACIA LENTISCUS L. RESIN 

Nicholson T., Gradl M., Metzger M., Albert K. 

University of Tuebingen-Germany 

Corresponding author e-mail: tim.nicholson@uni-tuebingen.de 

The resinous exudates of Pistacia lentiscus L., commonly known as mastic and consisting of numerous triterpenoid 

components[1, 2], have long been valued as potent active ingredients for medicinal purposes. Earliest records for the 

medicinal application of mastic date back to ancient Egypt, where it was also used for embalming purposes. 

We applied solid-state cross polarisation magic angle spinning nuclear magnetic resonance spectroscopy (CP/ 

MAS-NMR) to obtain a unique fingerprint of mastic resin which enables the non-destructive detection of mastic or 

similar triterpenoid resins in unknown samples. In addition this method gave an overview of the functional groups 

of compounds present in the resin. For the further elucidation of the triterpene composition, a preparative HPLC 

method was developed and single compounds collected. The performance of the separation was monitored by GC- 

MS after derivatisation of the purified substances to both their trimethylsilyl (TMS) and methyl derivatives. Capillary 

NMR was used to generate 1H-, 1H-1H COSY-, HSQC-, HMBC- and 13C DEPT spectra for the identification of the 

purified substances. 

[1] G. A. van der Doelen et al. (1998) Analysis of fresh triterpenoid resins and aged triterenoid varnishes by highperformance 

liquid chromatography-atmospheric pressure chemical ionisation (tandem) mass spectrometry. 

Journal of Chromatography A, 809, 21-37 

[2] A. N. Assimopoulou, V. P. Papageorgiou (2005) GC-MS analysis of penta- and tetra-cyclic triterpenes from resins 

of Pistacia species. Part I and II. Biomedical Chromatography, 19, 285-311, 586-605 

438 



P15-014 DEVELOPMENT OF AN EXTRACTION AND PURIFICATION PROCEDURE FOR THE OBTAINMENT OF INOSITOL ENRICHED 

FRACTIONS FROM PINE NUTS 

Ruiz-Aceituno L., Rodríguez-Sánchez S. , Ramos L., Martínez-Castro I., Sanz M.L. 

Instituto de Química Orgánica General (CSIC) 


Inositols or 1,2,3,4,5,6-hexahydroxycyclohexanes are part of the cyclic family of carbohydrates, the most abundant 

in nature being myo- and chiro-inostiol. Myo-inositol is the best known, and it is present in nearly all living cells. It 

is a growth factor for microorganism, a lipotropic agent for animals and it is part of phosphatidilinositol, a frequent 

phospholipid in foods (1). It has been speculated that myo-inositol metabolism disorders have some influence in 

diabetic neurophaties and chronic renal failure (2). Chiro-inositol is present in citrus fruits (3) and soy molasses 

(4), whereas pinitol (methyl-chiro-inositol) is obtained from carob, pine nuts, clover and peanuts (5). Both inositols 

have shown to present an action similar to insulin and could be used for treatments connected with diabetes 

mellitus, obesity, atherosclerosis, etc. Chemical synthesis has been used for the production of inositols, although 

this process is still expensive. Consequently, different approaches have been followed for the extraction of these 

compounds from natural sources (6). However, the development of new procedures more economical and less time 

consuming is of great interest. Therefore, the aim of this work was to develop an extraction and purification method 

for the obtainment of inositol enriched fractions from pine nuts. As a first approach, the extraction procedure was 

optimized using different solvents (water, methanol and ethanol) under different conditions (temperature, ultrasonic 

agitation). The use of a non-polar solvent for the extraction of fats, solvent volume and time of extraction were also 

evaluated. To achieve exhaustive recovery, successive extractions from the same sample were carried out. Two 

different purification methods were further assayed: (i) samples were incubated with Saccharomyces cerevisiae, 

incubation time being optimised (0-24h); (ii) anion exchange chromatography using a strong base anion exchange 

resin in chloride form previously conditioned with 4mM NaOH. Analyses were performed by GC-MS using a 

capillary column coated with methylsilicone as stationary phase; carbohydrates were previously derivatised to their 

trimethylsilyl oximes (3). ESI MS and MALDI-TOF analyses were also carried out to evaluate the high molecular 

weight composition of the extracts. Myo-inositol, chiro-inositol, pinitol and glicosyl-inositols were detected in pine 

nut extracts. The best yields were obtained using water (60ºC, 2h) as extracting reagent in a ratio 1:10 (w:v). Both 

purification procedures were appropriate for the removal of mono- (mainly glucose and fructose) and disaccharides 

(sucrose). Further studies will be done to compare these procedures with more automatic methods. (1) Angyal, S.J. 

Adv. Carbohyd. Chem. 1959. Vol 14. 135-212. (2) Clements, et al.1980 Am. J. Clin. Nutr, 33, 1954-1967. (3) Sanz et al, 

2004. Food Chem 87, 325-328. (4) Saska et al. 1996, US Patent 5,482,631. (5) Binder and Haddon, 1984. Carbohydr. 

Res. 129, 21-32. This work was funded by projects AGL2009-11909 (Ministerio de Ciencia e Innovación) and 

ANALISYC-II S2010/AGR-1464 (Comunidad de Madrid). 


439


P15-015 FIRST ULTRA-PERFORMANCE LC BASED STRATEGY FOR PROFILING INTACT PROTEINS IN COMPLEX MATRICES. 

APPLICATION TO THE EVALUATION OF THE PERFORMANCE OF OLIVE (OLEA EUROPAEA L.) STONE PROTEINS FOR 

CULTIVAR FINGERPRINTING 

Esteve C. 1 , del Río C. 2 , Marina M.L. 1 , Concepción García M. 1 

1 


2 


Corresponding author e-mail: clara.esteve@uah.es 

There is a clear need for accelerating protein separations by HPLC. Different proposals have been developed 

including the use of perfusion and monolithic stationary phases. Nevertheless, these stationary phases, in some 

occasions, do not provide enough efficiency to resolve these huge molecules when they are present in complex 

matrices. Despite ultra-performance liquid chromatography (UPLC) columns have been successfully used for the 

efficient and rapid separation of small molecules, this is the first time sub-2 µm columns have been proposed for 

the separation of intact proteins in a real complex matrix: the olive stone. Olive proteins, mainly present in the olive 

stone, seem to have an important role in the processing and quality of olives and olive oil. Despite they have been 

very little studied, two different kinds of proteins have been documented: storage proteins and proteins associated 

to fatty bodies (oleosins). The main reason for this lack of information regarding olive proteins is due to their tricky 

extraction. Indeed, there is an intrinsic difficulty in the extraction of proteins from lipid matrices that, in the case of 

olive, has resulted in an absence of any methodology for the extraction of proteins from the whole olive stone. In this 

work, two different strategies were employed for the extraction of olive proteins: an enzymatic assisted extraction 

and a buffered extraction using a high-intensity ultrasonic probe. Moreover, different chromatographic columns, 

including conventional silica columns, a pellicular column, and perfusion and monolithic stationary phases, have 

been employed for the separation of olive proteins. Results were compared with these obtained with different 

sub-2 µm particle columns. UPLC columns were the only ones providing suitable protein profiles at a reasonable 

analysis time (15 min). These protein profiles were applied to the separation of olive stone proteins in different olive 

cultivars. Results demonstrated that the developed methodology enabled the differentiation among olive varieties 

by the direct observation of chromatograms, in some cases, or by using discriminant analysis. Thus, the proteins 

present in the olive stone could be considered suitable for cultivar fingerprinting of olives. 

440 



P15-016 SEPARATION OF PROTEINS FROM OLIVE OIL BY CAPILLARY ELECTROPHORESIS. APPLICATION TO THE 

DIFFERENTIATION OF MONOVARIETAL OLIVE OILS 



Corresponding author e-mail: cristina.montealegre@uah.es 

Olive oil is a very popular food due to its organoleptic properties and its nutritional value. Due to the increasing 

consumer demand of high-quality olive oils, production of monovarietal olive oils has increased over recent 

years, thanks to their distinctive character and characteristics of the olive variety from which they are elaborated. 

Since 2002, the Commission Regulation (EC No 1019/2002) controls the marketing standards for olive oils. 

However, the mixture of olive oils of different botanical origin is not mentioned here and regulations as well as 

analytical methodologies for the monovarietal olive oils control are currently needed. In addition, from the different 

components in olive oils, proteins are minor components that have never been used to differentiate olive oils in spite 

of being compounds of high informative value. In this context, it is remarkable the role of capillary electrophoresis 

(CE) as separation technique for proteins. Therefore, the aim of this work was the development of an analytical 

methodology enabling the separation of proteins from olive oils using CE and the pioneering use of protein profiles 

obtained by CE for the differentiation of monovarietal olive oils. An extraction procedure based on the precipitation 

of proteins in cold acetone and their isolation and solubilization in a hydro-organic medium prior to their in-capillary 

preconcentration in CE was the critical step in the method development since proteins are minor components of 

olive oils. Then, a CE separation using a basic medium in a dynamically coated capillary originated electrophoretic 

profiles with multiple peaks from which, seven of them, were identified as proteins due to the selective protein 

extraction procedure performed and the typical UV spectra of proteins (presenting absorption maxima at 210 nm 

for peptide bonds, at 254 nm for phenylalanine residues, and at 280 nm for tyrosine and tryptophan residues). The 

developed method was applied to the differentiation of monovarietal olive oils and, in the protein profiles obtained, 

three different regions (zone I, II, and III) were identified. Zone III enabled to distinguish easily between extra virgin 

olive oils made from the Arbequina variety and the Picual and Hojiblanca varieties. To classify the oils according 

to the three botanical varieties studied, multivariate methods were investigated. The discriminant function was 

significant at the 95% confidence level and showed an 89% prediction capability, enabling the correct classification 

of 16 of the 18 monovarietal olive oil samples studied. 


441


P15-017 A COMBINATION OF A SIMPLE EXTRACTION METHOD AND CAPILLARY ELECTROPHORESIS APPROACH FOR THE 

SEPARATION OF OLIVE PROTEINS 



Corresponding author e-mail: cristina.montealegre@uah.es 

Olive (Olea europaea) trees are among the oldest known cultivated trees in the world and are the most important 

oil producing crop in many Mediterranean countries. According to the oil content and the olive size, olive fruits 

can be used to produce olive oils or table olives. These table olives are highly appreciated for their good taste. 

Their industrial processing is restricted to only a few styles, each of which can be found in various commercial 

presentations, having the objective to remove the natural bitterness of this fruit caused by the glucoside oleuropein. 

The most common industrial processing are natural olives placed directly in brine, treated olives, and olives 

darkened by oxidation. Therefore, due to the numerous olive processes and washes, a possible effect in the olive 

characteristics and composition, specifically, in protein composition according to the olive treatment is expected. As 

a consequence, the aim of this work was to develop new extraction and capillary electrophoresis (CE) methodologies 

capable to separate proteins present in raw and treated olives. First, a new extraction method was developed 

to extract efficiently proteins from olive samples. The extraction solvent and two protein separation strategies 

(precipitation or filtration) were studied. The highest number and size of peaks was obtained by a chloroform/ 

methanol extraction followed by a protein precipitation in cold acetone. For protein separation in the olive protein 

extract, a CE method using an acid buffer was used. It consisted of a 1 M formic acid at pH 2.0, which provided 

a neutral inner capillary wall to avoid protein adsorption. Selected separation conditions ensured a positive net 

charge for proteins and a neutral charge for potential interferents as polyphenols. Under the developed separation 

conditions the analysis of 6 samples of raw olives (Picual, Hojiblanca, and Arbequina varieties from Jaen and Toledo) 

and 15 samples of industrial processed olives (Manzanilla and Gordal green treated olives and Cacereña olives 

darkened by oxidation) was performed. Interestingly, raw olives showed differences in protein profiles depending on 

the botanical variety of olives and the geographical region. Treated olives also showed differences in their protein 

profiles depending on the olive variety and treatment. In fact, a signal reduction in black olives compared with 

treated green olives was observed, as expected for the vigorously treatment for olives darkened by oxidation. 

442 



P15-018 DEVELOPMENT OF A CLEAN-UP PROCEDURE TO EXTRACT PROTEINS FROM THE OLIVE PULP. APPLICATION OF OLIVE 

PULP PROTEINS AS GENETIC FINGERPRINTERS IN THE OLIVE CROP 

Esteve C. 1 , del Río C. 2 , Marina M.L. 1 , García M.C. 1 

1 


2 


Corresponding author e-mail: clara.esteve@uah.es 

The huge genetic diversity of the olive cultivar (there are more than 2000 different olive cultivars) has made difficult 

the identification and classification of this crop. The interest for its correct classification is significant in order to 

protect its genetic diversity, to determine each variety authenticity, and to improve it genetically. Moreover, not all 

olive varieties present the same characteristics and, consequently, its quality depends greatly on the variety. Despite 

scientific community have developed methodologies based on plant morphological and agronomical characters 

and even based on molecular markers, none methodology have resulted totally satisfactory. Therefore, the aim of 

this work has been to develop a new strategy based on protein profiles. Olive proteins are mainly present in the 

olive stone. In a recent research, olive proteins from the olive stone were employed for the differentiation among 29 

olive different cultivars. Nevertheless, information extracted exclusively from the olive pulp is more reliable than the 

obtained from the whole olive, from the olive stone or from the olive seed. Indeed, genetic information expressed 

in the olive pulp corresponding to a certain olive cultivar comes only from the own olive cultivar. Nevertheless, the 

genetic information provided by the olive stone is not only provided by the own cultivar but from both progenitors: 

the mother which is the olive cultivar itself and the father which is any other variety for every olive in the olive tree. 

Extraction of olive proteins from the olive pulp is a difficult task. In fact, olive proteins are in a low concentration 

in comparison with other olive components specially lipids. Moreover, the olive pulp contains a high content of 

polyphenols and flavonoids that can greatly interfere in the extraction and detection of olive proteins. Our work was 

focused to the development of a methodology enabling the reliable extraction of olive proteins from olive drupes 

with a suitable elimination of interfering compounds. Eight different procedures were designed and evaluated for the 

extraction of proteins from the olive pulp. Protein extracts were injected into a chromatographic system employing a 

sub-2 μm particle column and UV and fluorescence detection. Protocols with no cleaning up step were not suitable 

for the removal of interfering compounds. The extraction procedure with a precleaning step followed by Tris/SDS/ 

DTT extraction and protein precipitation gave the most successful isolation of the proteins from the pulp. This 

methodology was applied to the separation of olive proteins in different olive cultivars. 


443


P15-019 STUDY OF VOLATILES PRODUCED BY PSEUDOMONAS SPP. ISOLATED FROM CORK USING HS-SPME-GC-MS 

Godayol A., Trias R., Prat C., Bañeras L., Anticó E. 


Corresponding author e-mail: enriqueta.antico@udg.edu 

The production of volatiles from microorganisms growing in different matrices is a well documented process and 

is usually associated to their metabolism. This phenomenon is particularly important when the microorganisms are 

present in the food stuff or in other materials that may be in contact with food. This is the case of cork stoppers, 

which will be used as closures for bottled wine. Recent studies have shown the diversity of microorganisms detected 

in cork material which can be found in the different stages of cork production. Additionally, the participation 

of fungi and bacteria in the production of selected volatiles bearing characteristic sensory attributes has been 

demonstrated for example in the case of the formation of tricholoroanisole (TCA) via the biomethylation of its 

precursor trichlorophenol (TCP). Alkyl pyrazines are a group of cyclic compounds which posses strong smell with a 

pronounced specificity depending on the substituent position. Alkyl pyrazines naturally occur in some vegetables 

and fruits (e.g. grapes from the variety Cabernet) but can also be formed due to microbial activity. For example, 

2-isopropyl-3-methoxy-pyrazine has been identified as a metabolic by-product of “Pseudomonas perolens” and 

Pseudomonas taetrolens. This pyrazine is responsible for off-flavour development in eggs, dairy products and 

fish, and has also been identified in some spoiled wines. In a previous work we have characterized seven different 

Pseudomonas isolates from cork. In the present study we investigate the participation of the different isolates 

in the formation of volatiles, in particular pyrazine metabolites in in vitro growing conditions with selected media 

compositions. The analytes were analyzed by gas chromatography and HS-SPME was essayed as an extraction and 

concentration technique. Several experimental conditions were tested, such as the type of SPME absorbent used 

and the extraction mode (e,g., from the head space of a Petri dish, in a vial taking a piece of the culture media and 

adding water together with NaCl, or growing the bacteria directly in a vial and exposing the fibre to the headspace). 

The compounds IBMP, IPMP and SBMP have been selected as model compounds to study their stability when they 

were added to non-inoculated growth media. Finally, several growing conditions, including the composition of the 

culture media and the incubation time were also tested. The GC-MS analysis of the volatiles from the headspace 

of the samples allowed the detection of several pyrazine derivatives which were identified from the MS spectra and 

from the Kovacs retention indexes. Acknowledgments: This study was financed by the MICINN (Spanish Ministry of 

Education and Science), projects CTM2008-06847-C02-02/TECNO and CGL2009-08338. The authors would like to 

thank Prof. S. Schulz for helping in the interpretation of MS spectra. 

444 



P15-020 INSECTICIDES EXTRACTION FROM BANANA LEAVES USING A MODIFIED QUECHERS METHOD 

González-Curbelo M.A., Hernández-Borges J., Ravelo-Pérez L.M., Rodríguez-Delgado M.A. 

Universidad de La Laguna (ULL) 

Corresponding author e-mail: mrguez@ull.es 

Bananas are one of the most important fruits produced and consumed around the world. In Spain, bananas are 

harvested in the Canary Islands, which is the prime banana producer among the European Union production regions, 

representing the most important crop of the islands in production terms [1]. Banana cultivars are probably one of the 

higher producers of organic materials such as leaves, which are not consumed by human beings but may be used 

for wrapping food in cooking or their employment as cattle and hogs feeding which is a direct and important way of 

consumption. Banana crops are frequently exposed to the attack of insects, nematodes and fungi. To fight against 

these diseases, organophosphorus pesticides (OPPs) are one of the most frequently employed worldwide. As a 

result, pesticide residues like the ones of OPPs can appear in banana leaves and, as a consequence, their residues 

could finally reach the consumer. Therefore, it is important to develop rugged and robust methodologies able to 

determine these compounds at very low concentrations in this kind of complex matrices. The QuEChERS method, 

standing for quick, easy, cheap, effective, rugged and safe and several modified versions have been successfully 

used to extract pesticides from a variety of foods, above all fruits and vegetables. The simplicity of the procedure 

and the ease of development make it very attractive for analytical chemists and a suitable standard method for 

pesticide residue analysis in other foods with proper validation. Therefore, the aim of this work was to study the 

application of the QuEChERS method as well as its modification for the extraction and preconcentration of a group 

of eight insecticides (seven of them OPPs, which are widely used in banana treatments) from banana leaves prior to 

their determination by gas chromatography (GC) with nitrogen-phosphorus detection. The developed method was 

also applied to investigate pesticide occurrence in different banana leave samples collected in different cultivars of 

the Canary Islands in order to ensure their safe consumption by animals. Confirmation of the presence or absence 

of pesticides was also carried out using GC-MS/MS. Bibliography [1] Galán-Saúco, V., & Cabrera-Cabrera, J. (2006). 

Proceedings of the XVII ACORBAT international meeting, Joinville, Brazil, 289-301. 


445


P15-021 VALIDATION OF A GC-MS METHOD FOR THE SIMULTANEOUS DETERMINATION OF EIGTH TRICHOTHECENES IN BARLEY SAMPLES 

Ibáñez-Vea M., Lizarraga E., González-Peñas E. 


Trichothecenes are toxic secondary metabolites produced by several fungi. The main trichothecene-producing 

fungi in Europe belong to the Fusarium genus. These toxins constitute a family of more than 170 metabolites, which 

can be classified into four categories (A, B, C and D), depending upon their chemical structure. Type-A and type-B 

trichothecenes are the most important ones due to their toxicity and their worldwide prevalence. Trichothecenes 

appear as natural contaminants in cereal grains such as wheat, barley, oat, maize, rice, and in derived products 

such as bread, malt and beer. In European agricultural commodities, type-A trichothecenes occur less frequently 

and at lower mean concentrations in comparison with DON (type B), although the type-A group has a higher toxicity 

than type-B. Due to the effects that several toxins may cause in human and animal health, it is very important to 

develop methods for simultaneous determination of several mycotoxins in a same foodstuff. In this project, a rapid 

and sensitive method for the simultaneous quantification of eight trichothecenes (DON, NIV, 3-ADON, 15-ADON, 

FUS-X, T-2, HT-2 and DAS) in barley samples by GC-MS is described. The procedure is based on the extraction 

of the toxins with a mixture of acetonitrile and water (84:16) and clean-up with Multisep® columns. The final 

extract was derivatized with PFPA and then analysed by GC-MS in SIM mode. The method was validated in barley 

according to selectivity, linearity, precision, accuracy, recovery and limits of detection and quantification. Mean 

recovery values ranged from 92.0 to 101.9%, with a mean relative standard deviation lower than 15%, although NIV 

showed a lower mean recovery (63.1%). This method has been successfully applied to the analysis of 44 barley 

samples collected during the 2007 harvest in Navarra (Spain). Abbreviations: deoxynivalenol (DON), nivalenol (NIV), 

3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenone-X (FUS-X), T-2 toxin (T-2), HT-2 

toxin (HT-2) and diacetoxiscirpenol (DAS). 

446 



P15-022 VALIDATION OF A UHPLC-FLD METHOD FOR THE SIMULTANEOUS DETERMINATION OF AFLATOXINS, OCHRATOXIN A 

AND ZEARALENONE IN BREAKFAST CEREALS 

Ibáñez-Vea M., Martínez R., González-Peñas E., Lizarraga E. 


Corresponding author e-mail: mivea@alumni.unav.es 

Mycotoxins are toxic secondary metabolites produced by several fungal species growing on many agricultural 

commodities and processed food, either in the field or during storage. Due to their toxicity and occurrence, the most 

important mycotoxins are aflatoxins (B1, G1, B2 and G2), ochratoxin A (OTA) and to a lesser extent, zearalenone 

(ZEA). 

With regard to their toxicity, these toxins represent a serious threat to both human and animal health. The 

International Agency for Research on Cancer has classified aflatoxin B1 and naturally-occurring mixtures of 

aflatoxins as human carcinogens (group 1), OTA as a possible carcinogen to humans (group 2B) and ZEA as not 

classifiable with regard to its carcinogenicity to humans (group 3). 

These toxins occur naturally in plant products such as cereals (mainly barley, maize, rye and wheat), nuts and dried 

fruit, and in their by-products (breakfast cereals, pasta, malt, beer,…). The European Commission has set maximum 

permitted levels of these toxins in different matrices. In breakfast cereals, the limits are: 2 µg kg-1 for aflatoxin B1 

and 4 µg kg-1 for the sum of AFB1, AFG1, AFB2 and AFG2; 3 µg kg-1 for OTA and 50 µg kg-1 for ZEA. 

The presence of several toxins in a foodstuff is very common, due to their fungal nature. Therefore, it is very 

important and necessary to develop and validate methods for the simultaneous analysis of several mycotoxins in 

different commodities. 

A fast and simple UHPLC-FLD method has been developed for the simultaneous determination of these toxins in 

breakfast cereals. The procedure is based on the extraction of the six mycotoxins with a mixture of acetonitrile and 

an aqueous solution of potassium chloride, and the purification of the extract with immunoaffinity columns before 

analysis. Detection of AFB1 and AFG1 is improved using a photochemical reaction. 

The method has been validated according to selectivity, linearity, precision, accuracy, recovery and limits of 

detection and quantification. The recovery and limits of detection and quantification values for the six mycotoxins 

are adequate for their analysis and fulfill the requirements established by the European Commission. This method 

has been successfully applied to the analysis of commercial samples of 25 breakfast cereals purchased in Navarra 

(Spain). 


447


P15-023 ANALYSIS OF 3-MCPD ESTERS IN EDIBLE OILS USING LARGE VOLUME INJECTION COUPLED TO COMPREHENSIVE GC×GC–TOF MS 

de Koning S. 1 , Zelinková Z. 2,3 , Hrnčiřík K. 2 , Janssen H-G. 2,4 

1 


2 


3 


4 



Recently there has been considerable interest in the formation of 3-chloropropane-1,2-diol (3-MCPD)-fatty acid 

esters during edible-oil processing. In model systems the 3-MCPD-esters have been shown to yield free 3-MCPD 

as a result of lipase activity. Because of the suspected toxicity of the free 3-MCPD, methods for the analysis of 

its fatty acid esters have recently been developed. The current methods for 3-MCPD ester analysis in edible oils 

and fats actually measure the total 3-MCPD content of the oil or fat after hydrolysis. The procedures consist of a 

number of subsequent steps starting with the hydrolysis, removal of the fatty acids (as their FAMEs), extraction of 

the free 3-MCPD with salting out, derivatisation with phenylboronic acid, preconcentration by solvent evaporation 

and finally GC-MS analysis. Deuterium labelled 3-MCPD-d5 or esters thereof, are used as internal standards. 

Potential problems in the procedure are degradation of the 3-MCPDs during (alkaline) hydrolysis resulting in high 

detection limits, and the formation of additional 3-MCPDs if chloride salts are used in the salting out extraction. 

In this work a feasibility study is presented that focuses on the use of comprehensive GC×GC–ToF MS with large 

volume injection for faster, more reliable and more sensitive 3-MCPD analysis in oils and fats. Sample aliquots up to 

25 µl are injected. As a result of this, the salting out step can be excluded from the sample preparation: low detection 

limits were obtained even at low extraction recoveries and thus the side reaction with the chloride can be eliminated. 

Additionally, the final preconcentration step could be skipped making the method faster and reducing the manual 

sample handling. A clear advantage of the use of comprehensive GC×GC–ToF MS is the substantially improved 

resolution of the GC separation leading to the elimination of interferences even at very low 3-MCPD levels. Also 

higher system stability is achieved due to the open-source design of the Time-of-Flight Mass Spectrometer. 

448 



P15-024 DETAILED CIS/TRANS FATTY ACID ANALYSIS OF EDIBLE OILS AND FATS USING COMPREHENSIVE GC×GC–FI 

de Koning S. 1 , Steenbergen H. 2 , Janssen H-G. 2,3 

1 


2 


3 



The determination of the fatty acid composition of edible oils and fats (as their methyl esters or FAMEs) is routinely 

performed in numerous food industries and laboratories all over the world. As long as no detailed information on the 

trans levels is required the analysis is rather straightforward. If trans-level analysis is desired, however, the analytical 

task starts to become more complex and long, highly polar GC columns with lengthy separations are needed with 

the interpretation of the chromatogram even than being a tedious process. The standard method for trans-FAME 

analysis uses a capillary GC separation on a 50 to 100 meter highly polar cyanopropyl column (e.g. CP-Sil 88). 

With this technique trans analysis is possible in relatively ‘simple’ samples such as untreated or mildly processed 

vegetable oils. For more complex samples, such as for example the analysis of fat blends incorporating fish oils, 

the task starts to become highly challenging and prone to (small) errors. In this contribution we will demonstrate the 

powerful characteristics of comprehensive GC×GC with FID detection for trans fatty acid analysis of commercial 

fat blends, processed oil samples and fish oils. Various column combinations for comprehensive GC×GC system 

are evaluated. Optimisation of the method is done by use of an Excel-based spreadsheet for GC×GC. Special 

attention is also paid to the methods for group-type integration available in the LECO ChromaTOF software. The 

results obtained with the new comprehensive GCxGC method are compared with those from the current standard 

GC methods, not only in terms of quantitative performance, but also with regard to operator time, reliability of peak 

identification etc. 


449


P15-025 USE OF LINEAR DISCRIMINANT ANALYSIS AND STEROL PROFILES ESTABLISHED BY HPLC-MS TO PREDICT THE GENETIC 

VARIETY OF EXTRA VIRGIN OLIVE OILS PRODUCED AT LA COMUNIDAD VALENCIANA, SPAIN 

Lerma-García M.J. 1 , Concha-Herrera V. 2 , Herrero-Martínez J.M. 1 , Simó-Alfonso E.F. 1 

1 

University of Valencia-Spain 

2 

Autonomous University of Zacatecas 

Corresponding author e-mail: m.jesus.lerma@uv.es 

Sterols are bioactive components occurring in all vegetable foods, constituting an important proportion of the 

non-saponificable fraction of extra virgin olive oil (EVOO). Sterol content depends on the kind of olive oil, but can 

also vary according to the genetic variety of EVOOs. For this reason, the evaluation of sterol profiles and total sterol 

contents constitute an excellent tool to assess oil authenticity and to check oil genuineness. Consequently, it is 

important to obtain fast and reliable methods to analyze these nutritionally important compounds. A method to 

determine sterol profiles of EVOOs by HPLC-MS has been developed. Sterol extracts were chromatographed on a 

dC18 Atlantis column (100 x 3 mm, 3 µm) with a gradient acetonitrile/water with positive-ion mode MS detection. 

Using linear discriminant analysis of the HPLC-MS data (extracted ion chromatograms), EVOO samples belonging 

to six genetic varieties which are commonly cultivated in the Comunitat Valenciana, Spain (Arbequina, Borriolenca, 

Canetera, Farga, Picual and Serrana) were correctly classified with an excellent resolution among all the pairs of 

categories with a prediction capability higher than 88%. 

Acknowledgements: Project CTQ2007-61445 (MEC of Spain and FEDER funds). M.J. Lerma-García thanks the 

Generalitat Valenciana for an FPI grant for PhD studies. V. Concha-Herrera thanks the University of Valencia for a 

contract. 

450 



P15-026 DEVELOPMENT AND COMPARISON OF HPLC METHODS FOR THE ANALYSIS OF PREBIOTIC OLIGOSACCHARIDES 

Brokl M., Hernández-Hernández O., Soria A.C., Sanz M.L. 


Prebiotics are non digestible carbohydrates which selectively stimulate the growth of probiotic bacteria. Different 

studies have assessed the influence of the chemical structure of these carbohydrates in their prebiotic properties, 

particularly their degree of polymerization (DP). Several authors have suggested that the optimal DP is in the range 

3-4 [1]; however, others have reported a good activity for oligosaccharides of DP 7 [2]. On the other hand, most of 

current studies have been focused on the determination of prebiotic properties and the analysis of their chemical 

structure is still scarce. High performance liquid chromatography (HPLC) is an appropriate technique for the analysis 

of oligosaccharides; different operation modes such as reverse phase (RP) or ion exchange being used. Highperformance 

anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) has been widely 

applied to the analysis of mono-, oligo- and polysaccharides. However, its chromatographic behavior has been 

found to be unpredictable [3] and its coupling to mass spectrometric detector requires specific instrumentation. 

Other stationary phases such as porous graphitized carbon (PGC), hydrophilic interaction chromatography (HILIC), 

etc have emerged as alternatives to RP or anion exchange columns for the analysis of polar compounds. However, 

although different applications to N- or O-glycans have already been developed using these columns, data on the 

analysis of neutral carbohydrates are still scarce. Therefore, the aim of this work was to optimize and compare 

different HPLC methods based on HPAEC-PAD, RPLC-ESI-MS, PGC-LC-ESI-MS and HILIC-ESI-MS for the analysis 

of neutral oligosaccharides. These procedures will be applied to the study of prebiotic carbohydrates. For this 

purpose, commercial standards of maltodextrins (DP 3-7), tetrasaccharides (isomaltotetraose, mannotetraose, 

cellotetraose, stachyose, nistose and galactotetraose) and pentasaccharides (mannopentaose, isomaltopentaose 

and verbascose) were used. Additionally, mixtures of prebiotic oligosaccharides (galacto-, fructo-, isomalto-, 

xylo- and gentiooligosaccharides) were also considered. As expected, HPAEC-PAD showed a good resolution of 

oligosaccharides with the same DP, although coelutions between DP4 and DP5 oligosaccharides occurred. It is 

worth noting the high retention experimented by maltodextrins as compared with other oligosaccharides of the 

same molecular weight. The use of a PGC column with a methanol:water gradient allowed the separation of most 

oligosaccharides under analysis. Several carbohydrates showed split peaks at 30ºC which became single peaks at 

80ºC. In contrast, this temperature increased peak overlapping. HILIC showed to be promising for oligosaccharide 

separation and RP was only found appropriate for the analysis of fructooligosaccharides. This work was supported 

by projects ANALISYC-II S2010/AGR-1464 (Comunidad de Madrid) and AGL2009-11909 (MICINN). M. Brokl thanks 

Comunidad de Madrid for a predoctoral contract and O. Hernández-Hernández thanks CSIC for a JAE Predoc grant. 

[1] H. Kaplan and R.W. Hutkins, (2000). Appl. Environ. Microbiol., 66, 2682. [2] M.L. Sanz et al, (2005). J. Agric. Food 

Chem., 53, 5911. [3] V. Morales et al, (2006). Chromatographia, 64(3/4), 233. 


451


P15-027 METHACRYLATE ESTER-BASED MONOLITHIC COLUMNS FOR NANO-LC SEPARATION OF TOCOPHEROLS AND 

TOCOTRIENOLS IN VEGETABLE OILS 

Lerma-García M.J. 1 , Cerretani L. 2 , Herrero-Martínez J.M. 1 , Bendini A. 2 , Simó-Alfonso E.F. 1 

1 


2 



Vegetable oils contain vitamin E, which is a mixture of tocopherols (Ts) and tocotrienols (T3s). Essentially, these 

compounds are a major lipid-soluble chain-breaking antioxidant that protects natural oils from oxidation and also 

prevent lipid peroxidation in biological membranes. For these reasons, it is necessary to recourse to analytical 

procedures to perform the identification and quantification of the individual components of vitamin E. Microscale 

separation techniques, such as capillary or nano-LC, are in the focus of modern analytical methods. The most 

important advantages of these systems over classical techniques are their better separation efficiencies, increase 

in sensitivity, and lower sample and reagent consumption. Then, a nano-LC method with UV-Vis detection using 

lauryl methacrylate ester-based monolithic columns was proposed to determine Ts and T3s in vegetable oils. The 

separation of Ts was optimized in terms of mobile phase composition on the basis of the best compromise between 

efficiency, resolution and analysis time. Using a mobile phase composed by acetonitrile/methanol/water, an excellent 

resolution between Ts was achieved within 18 min. The limits of detection were lower than 0.26 µg mL-1, being 

repeatability values of retention time and peak area below 0.15 and 3.1%, respectively. The method was applied to 

the quantification of Ts and T3s present in several vegetable oils from different botanical origins. 


Generalitat Valenciana for an FPI grant for PhD studies. L. Cerretani thanks the University of Valencia for a contract. 

452 



P15-028 FEASIBILITY OF ELECTROMIGRATION AND GAS CHROMATOGRAPHIC TECHNIQUES FOR ASSESSING GLYCOXIDATION OF 

PROTEINS 

Amigo-Benavent M., Montilla A., del Castillo M.D. 


Corresponding author e-mail: delcastillo@ifi.csic.es 

Advanced glycation end products (AGEs) or glycoxidation products are formed via Maillard reaction between 

reducing sugars and free amino groups of proteins, lipids and nucleic acids. These compounds are formed in foods 

and in vivo under physiologic conditions1 and are responsible for diabetes mellitus (DM) and aging complications2. 

One of the most representative AGEs is єN-carboxymethyl-lysine (CML)3. DM is one of the most important diseases 

in Western countries. Different compounds such as chlorogenic acid (CGA) from beverages (mate, coffe), several 

fruits and vegetables may possess antiglycating effect4. However, the mechanism of action of CGA against 

formation of Maillard reaction products in vitro and in vivo has not been established yet. The aim of this research 

was to develop a method to follow the glycoxidation of biological proteins and to evaluate the protective effect if so 

of chlorogenic acid against the formation of glycotoxins (carboxymethylated-protein or CML-protein). Glycoxidation 

model systems based on both human serum albumin (HSA) or human hemoglobulin (Hb) were performed as follow: 5 

mL of a protein solution (25 mg/mL of 0.2 M phosphate buffer pH 8) were treated with 360 µL of 0.3 M glyoxylic acid 

and 360 µL of 0.9 M sodium cyanoborohydride without (glycoxidation sample) and with addition of 100 µL of 0.4 mM 

CGA (antiglycoxidation sample) at 37ºC with constant stirring at 750 rpm up to 96 hours. Other controls based on 

protein and CGA alone were also undertaken. Glycoxidation was followed by SDS-PAGE, CZE5 and GC6 analysis. 

Data on SDS-PAGE, GC and CZE were compared. Results indicated the feasibility of the three analytical tools 

to look at protein glycoxidation. 1. Peppa et al. 2008 Hormones 7(2), 123-132. 2. Gugliucci et al. 2000. J America 

Osteopathic Association 100, 621-634. 3. Somoza et al. 2005 J Agric Food Chem 53, 8176-8182. 4. Gugliucci et 

al. 2009 Fitoterapia 80 (6), 339-344. 5. Ames et al. 2000. ACS Symposium Series 754; American Chemical Society. 

188-201. 6. Bosch et al. 2007 J Chromatogr B 860 (1), 69-77. Acknowledgements: These studies were supported by II 

Café, Salud y Nutrición Grant (Federación Española del Café and Federación Española de Nutrición) and Consolider 

Ingenio Programme project FUN-C-FOODCSD2007-00063. M. Amigo-Benavent thanks Danone Institute for the 

research fellowship. Authors also thank Lucia Soria for technical assistance. 


453


P15-029 ANALYSIS OF SOYBEAN BOWMAN BIRK INHIBITOR BY CAPILLARY ELECTROPHORESIS 

Amigo-Benavent M. 1 , Bravo L. 2 , del Castillo M.D. 1 

1 


2 

Instituto de Ciencia y Tecnología de los Alimentos y Nutrición, Metabolismo y Nutrición 

Corresponding author e-mail: mamigo@ifi.csic.es 

Soybean Bowman Birk inhibitors (BBI) are minor protein components of soybean with a molecular weight of about 

8000 Da. In the past decades, BBI inhibitors were considered as antinutrients because they inhibit digestive enzymes 

affecting the digestibility of proteins1. In recent years, BBI are being considered as promising anticancer agents of 

interest in food and biomedicine2,3. Different analytical tools have been used to identify soybean proteins but there 

is a lack of methods for routine analysis of BBI in foods. Since CZE analysis fulfils all the characteristics required to 

achieve this goal, its feasibility was assessed in the present work. Samples of standard BBI, soy flour, isolated soy 

protein and soy foodstuffs were analyzed by CZE. Sample preparation was improved to achieve accurate quantitative 

analysis of BBI. CZE conditions were optimized based on those methods previously described by others4,5. Results 

so far obtained suggest that CZE is an adequate inexpensive analytical tool for routine analysis of soy BBI protein. 1. 

Garcia et al., 1997. Crit Rev Food Sci Nutr 37, 361-391. 2. Clemente & Domoney, 2006 Curr Prot Pept Sci 7, 201-216. 

3. Losso, 2008. Crit Rev Food Sci Nutr 48, 94-118. 4. Jensen et al., 1996. J. Biochem. Biophys. Methods 33, 171-185. 

5. García-Ruiz et al., 2007. Electrophoresis 28, 2314-2323. Acknowledgements: These studies were supported by 

Consolider Ingenio Programme project FUN-C-FOOD CSD2007-00063. M. Amigo-Benavent thanks Danone Institute 

research fellowship. 

454 



P15-030 USE OF TRIGLYCERIDE PROFILES ESTABLISHED BY HPLC WITH UV-VIS DETECTION TO PREDICT THE BOTANICAL ORIGIN 

OF VEGETABLE OILS 

Lerma-García M.J. 1 , Lusardi R. 2 , Chiavaro E. 2 , Cerretani L. 3 , Ramis-Ramos G. 1 , Simó-Alfonso E.F. 1 

1 


2 

University of Parma 

3 



The determination of the botanical origin of high quality vegetable oils, as extra virgin olive oil (EVOO), is of interest 

in order to preserve the rights of the consumers and their confidence in the food market. High-quality oils are 

frequently adulterated with oils of lower price, including oils of other botanical species. Thus, quick, simple and 

reliable analytical methods are required for screening of quality oils. In this work, a method for the prediction of the 

botanical origin of vegetable oils, based on the determination of triglyceride profile by HPLC with UV-Vis detection 

has been developed. For this purpose, a 3% vegetable oil was dissolved in a mixture of acetonitrile/2-propanol/ 

hexane 2:2:1 (v/v/v) and chromatographed on a Kinetex C18 100A column (150 x 4.6 mm, 2.6 µm). The separation of 

triglycerides was optimized in terms of mobile phase composition and column temperature. The optimal separation 

conditions were achieved with a mobile phase composed by acetonitrile and n-pentanol at a column temperature 

of 10 ºC. The triglyceride peaks that were observed in the UV-Vis chromatograms were identified by mass 

spectrometry. Using UV-Vis detection, the ratios of areas of peak pairs were used as predictors. Linear discriminant 

analysis models were constructed and applied to the predict oil origin. These models were able to correctly classify 

vegetable oils coming from several botanical origins with an excellent resolution among all the category pairs. 


Generalitat Valenciana for an FPI grant for PhD studies. 


455


P15-031 USE OF UPLC-MS TO DETERMINE STEROL CONTENTS IN VEGETABLE OILS FROM DIFFERENT BOTANICAL ORIGINS 


1 


2 



Phytosterols (also called plant sterols) are bioactive compounds occurring in vegetable oils, constituting the greatest 

proportion of the unsaponifiable fraction. Their nature and quantitative distribution are characteristic of the original 

lipid source, which could be useful for the identification of the botanical origin of vegetable oils. For this reason, 

the determination of these minor components is of great value in establishing the oil genuineness and quality, 

having also a marked influence on typicality, flavour, aroma and shelf-life. In this way, the use of UPLC constitute an 

attractive way to analyze these compounds, because allows the application of high flow rates without compromising 

resolution, giving shorter analysis times than the conventional HPLC systems. A method for the determination of 

sterols in vegetable oils by UPLC with atmospheric pressure chemical ionization mass spectrometry detection has 

been developed. The separation of sterols was optimized in terms of mobile phase composition, column temperature 

and flow rate. The optimal conditions were achieved using an Acquity UPLC BEH C18 column (50 x 2.1 mm, 1.7 µm) 

with a mobile phase consistent of acetonitrile/water (0.01% acetic acid) using a linear gradient, at a flow rate of 0.8 

mL min-1 and column temperature of 10 ºC, giving a total analysis time below 5 min. The limits of detection were 

comprised between 0.03 and 0.07 µg mL-1, and repeatabilities for peak areas and retention times were better than 

5.0 and 0.4%, respectively. The content of main sterols present in several vegetable oils with different botanical 

origins was also established. 



456 



P15-032 PREDICTION OF THE GENETIC VARIETY OF EXTRA VIRGIN OLIVE OILS FROM LA COMUNITAT VALENCIANA, SPAIN, BY 

USING STEROL PROFILES ESTABLISHED BY UPLC-MS 


1 


2 



Along the last years, the consumption of extra virgin olive oils (EVOOs) has increased considerably in relation to 

the consumption of virgin and refined olive oils. Owing to its distinctive and peculiar intense taste, EVOOs obtained 

from some pure genetic varieties are highly appreciated. For this reason, simple and reliable analytical methods are 

required in order to authenticate the genetic variety of EVOOs. In this way, the use of UPLC, that combines the use 

of packings of small size range (1-2 µm) with ultra high pressure systems constitute an attractive way to analyze 

these compounds. It allows the application of high flow rates without compromising resolution, shortening analysis 

time compared to the conventional HPLC systems. A method to classify EVOOs according to their genetic variety 

using sterol profiles obtained by UPLC with atmospheric pressure chemical ionization mass spectrometry detection 

has been developed. The separation of sterols was optimized in terms of mobile phase composition, temperature 

and flow rate. The optimal conditions were achieved using an Acquity UPLC BEH C18 column (50 x 2.1 mm, 1.7 µm) 

with a gradient acetonitrile/water, giving a total analysis time below 5 min. The 11 sterol peaks observed in each 

sample were used to construct linear discriminant analysis (LDA) models. Ratios of the peak areas selected by pairs 

were used as predictors. With the sequential application of two LDA models and using a 95% probability, the EVOO 

samples belonging to seven genetic varieties were correctly classified with a prediction capability higher than 97%. 




457


P15-033 DETERMINATION OF 15+1 EU POLYCYCLIC AROMATIC HYDROCARBONS IN TOASTED CEREALS OF THE CANARY ISLANDS 

(“GOFIOS”) BY HPLC UTILIZING IONIC LIQUIDS AGGREGATES AS EFFECTIVE EXTRACTION SOLVENTS 

Germán-Hernández M., Pino V., Ayala J.H., Afonso A.M. 


Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds formed by two or more fused aromatic 

rings. They are widely known for their carcinogenic and mutagenic characteristics. Since 2005, the European 

Commission recommended the monitoring of fifteen PAHs, altogether with an additional PAH, in foods. These PAHs 

are commonly known as the 15 + 1 priority EU PAHs. These PAHs only have 8 hydrocarbons in common with the 

PAHs commonly monitored in environmental pollution studies following the rules of the Environmental Protection 

Agency of United States (US-EPA), which are commonly known as the 16 EPA. The diet of children in the Canary 

Islands includes a typical toasted cereal (“gofio”). The gofio is roasted flour. The possible presence of PAHs in gofios 

can come from the processing treatment of the cereals or by pollution of the cereal grain. There are no studies 

concerning the possible PAHs contents in gofios. This work shows the development of high performance liquid 

chromatographic (HPLC) method using fluorescence and UV-Vis detection for the determination of the 15 + 1 priority 

EU PAHs. The optimal chromatographic method utilizes a mixture of acetonitrile-water with a linear gradient elution 

changing from 70 to 100% of acetonitrile during 30 minutes (at 1 mL•min-1 flow), and then kept during 15 minutes 

at 100% of acetonitrile (with a flow of 2 mL•min-1). The obtained limit of detection (LOD) for the PAH monitored in 

UV-Vis detection (cyclopenta(c,d)pyrene) was 20.5 µg•L-1. The rest of PAHs presented LODs varying between 0.02 

and 8.72 µg•L-1 (fluorescence detection). This work also uses novel agents to extract PAHs from the gofio, instead of 

organic solvents commonly used in conventional PAHs extraction processes. Recent trends in analytical chemistry 

are shifted to the elimination or minimization of the organic solvent consumption in the extraction step, due to the 

known toxicity of organic solvents (environmental issue). The novel agents used in this work are ionic liquids (ILs) 

aggregates. ILs are basically salts with melting points below 100 ° C and negligible vapor pressure. Therefore, 

they are environmental-friendly. A group of ILs has shown to form aggregates in aqueous solution above a certain 

concentration. These ILs-aggregates can be used as solvents in extraction processes. The extraction processes 

can be accelerated by means of microwaves or ultrasounds. The IL phase containing the extracted PAHs can be 

subsequently injected in a HPLC without further clean-up steps to remove the IL. This work efficiently uses ionic 

liquid aggregates as extraction solvents for the 15 + 1 EU PAHs contained in gofios. 

458 



P15-034 USE OF DISPERSIVE LIQUID-LIQUID MICROEXTRACTION AS SAMPLE TREATMENT IN THE DETERMINATION OF 

CARBAMATES IN FRUIT JUICES BY SWEPING-MICELLAR ELECTROKINETIC CHROMATOGRAPHY 

Moreno-González D., García-Campaña A.M., Gámiz-Gracia L., Bosque-Sendra J.M. 


Corresponding author e-mail: dmoreno@ugr.es 

N-methylcarbamates (NMCs) are pesticides with chemical structure: R-O-C(O)-N(CH3)-R´, where R is an 

alcohol, an oxime or a phenol, and R´ is an hydrogen or a methyl group. They are increasingly used instead of 

organochlorine and organophosphorus pesticides, due to their broad spectrum of biological activity (insecticides, 

fungicides and herbicides) and lower environmental persistence. Their toxicity is mediated through the inhibition of 

acetylcholinesterase, being mortal at high dosages. Dispersive liquid-liquid microextraction (DLLME) is a very simple 

and rapid extraction technique based on a ternary component solvent system in which the extraction solvent and 

disperser solvent are rapidly injected into an aqueous sample by a syringe [1]. The mixture is then gently shaken 

and a cloudy solution (aqueous phase/disperser/extraction) is formed in the test tube. After centrifugation, the fine 

particles of extraction solvent are sedimented in the bottom of a conical test tube and collected with a syringe. The 

solvent is evaporated and the final extract is reconstituted in a proper solvent. In this communication we present 

the application of DLLME to the extraction of fourteen NMCs (oxamyl, methomyl, benomyl, carbendazim, asulam, 

baygon, carbofuran, aldicarb, carbaryl, promecarb, methiocarb, napropamid, fenoxycarb and carbosulfan) from 

juices and their determination by Micellar Electrokinetic Chromatography (MEKC) with diode array detection (DAD). 

To improve sensitivity, an on-capillary sample-concentration method based on sweeping has been developed. This 

approach is designed to focus the analytes into a narrow band within the capillary, thereby increasing the sample 

volume that can be injected without any loss of efficiency. It involves the accumulation of charged and neutral 

analytes by the pseudostationary phase that penetrates the sample zone (a buffer similar to the BGE but free of 

micelles) and sweeps the analytes, producing a focusing effect [2]. Separations were performed in a extended light 

path fused-silica capillary (64.5 cm × 50 µm I.D., 56 cm effective length); the separation buffer consisted of 100 mM 

borate and 50 mM SDS (pH 9.0), with 5% acetonitrile; the temperature of the capillary was kept constant at 27.5 

ºC and a voltage of 27 kV was applied (normal mode); the detection of the analytes was performed at a wavelength 

of 210 nm. Samples were introduced by hydrodynamic injection (35 mbar for 25 sec), dissolved in 100 mM borate 

(pH 9.0) with 5 % acetonitrile. Calibration curves were established for the studied compounds and detection 

limits ranging from 30 to 200 µg l-1 were obtained. The method is being applied to pineapple and banana juices. 

Acknowledgements: The “Junta de Andalucía” supported this work (Proyecto de Excelencia Ref: P07-AGR-03178) 

References [1] M. Reeze, Y. Assadi, M.R. Milani-Hosseini, E. Aghaee, F. Ahmadi, S. Berijani, J. Chromatogr. A 1116 

(2006) 1. [2] A.T. Aranas, M. Guidote Jr, J.P. Quirino, Anal. Bioanal. Chem 394 (2009) 175. 


459


P15-035 ANALYSIS OF OLIGOSACCHARIDES IN BEER VIA HPLC USING DIFFERENT STATIONARY PHASES 

Salplachta J., Flodrova D., Bobalova J., Cmelik R., 


Corresponding author e-mail: salplachta@iach.cz 

Carbohydrates in foods are important components often playing essential role, e.g., they are associated with taste 

and overall food quality. Moreover, they are also substrates for fermentation process. In brewing, carbohydrates 

represent major group of non-volatile compounds occurring in the final product – beer. Carbohydrates in beer 

are largely comprised of oligosaccharides consisting of 3–10 glucose units. As the beer characteristics (primarily 

sensory characteristics) are influenced by composition and concentration of oligosaccharides, beer carbohydrates 

analysis is of great interest. Closely related isomeric structures of oligosaccharides in real samples (like beer) 

make their determination and characterization quite challenging. Underivatized carbohydrates can be analyzed by 

high-performance liquid chromatography (HPLC) with refractive index detection, which is usually the technique of 

choice. In this respect, the aim of the present study was to develop a simple HPLC method for determination and 

characterization of oligosaccharides in beer. For this purpose, various stationary phases (especially HILIC-based, 

as well graphitized carbon) were evaluated in relation to separation efficiency of oligosaccharides. The optimized 

HPLC method was then applied to analysis of several beer samples. Data will be discussed in detail. Our results have 

revealed that the proposed HPLC-based approach seems to be convenient tool suitable for the determination and 

characterization of oligosaccharides in different types of beer. 

Acknowledgement 

This work was supported from the project No. 2B06037 of Ministry of Education, Youth and Sports, Czech Republic, 

and by the Institutional Research Plan AV0Z40310501. 

460 



P15-036 CAPILLARY ELECTROPHORESIS OF FREE FATTY ACIDS BY INDIRECT UV DETECTION: APPLICATION TO THE 

CLASSIFICATION OF VEGETABLE OILS ACCORDING TO THEIR BOTANICAL ORIGIN 

Escrig-Doménech A., Lerma-García M.J., Simó-Alfonso E.F., Herrero-Martínez J.M. 



Authentication of edible quality oils is of great importance from the point of view of both commercial value and 

health impact. The organoleptic properties, high nutritional value and health benefits of quality oils are related to the 

presence of many components, such as fatty acids, which concentration profiles differ according to the fruit variety. 

A relevant aspect of oil authenticity is the adulteration of quality oils by mixtures with oils from different botanical 

origin. Then, the evaluation of fatty acid profiles could be an excellent tool to assess oil authenticity. 

Traditionally, analysis of fatty acids has been approached spectroscopically or chromatographically, mainly by gas 

chromatography. However, a derivatization step is required. In this work, a capillary electrophoresis (CE) method 

with an alkaline buffer in the presence of an anionic chromophore (p-hydroxybenzoate or trimetoxibenzoate) for 

the indirect UV detection of fatty acids was developed. The effect of several buffer additives, such as neutral 

surfactants (derived from Brij family) and organic solvents on the fatty acid separation was examined. The optimized 

methodology was applied to obtain the fatty acids profiles of vegetables oils from different botanical origin. In 

addition, on the basis of fatty acid fingerprints obtained by CE, linear discriminant analysis (LDA) models were 

constructed in order to classify vegetable oils according to their botanical origin. 

Acknowledgements: Project CTQ2007-61445 (MEC and FEDER funds). M.J. L-G. thanks the Generalitat Valenciana 

for an FPI grant for PhD studies. 


461


P15-037 ACRYLATE ESTER-BASED MONOLITHIC COLUMNS FOR CAPILLARY ELECTROCHROMATOGRAPHY SEPARATION OF 

TRIACYLGLYCEROLS IN VEGETABLE OILS 

Medina-Escrivà L., Vergara-Barberán M., Lerma-García M.J., Simó-Alfonso E.F., Herrero-Martínez J.M. 



Edible vegetable oils consist predominantly of triacylglicerols (TAG) that generally follow a unique and typical 

pattern in the glycerol molecule, being their profile characteristic for the different botanical origin of the oils. The 

composition and structure of TAGs determine, to a large extent, the functionality of oils as food ingredients, and 

their physiological effects as components of the human diet. Due to these facts, the analysis of the TAG profiles is of 

great importance, but it is a challenging and complicated task. This is due to the great number of possible fatty acid 

combinations in the glycerol backbone, which generates an enormous number of individual TAGs. For this reason, 

the development and improvement of new analytical methods to determine these compounds is a major concern. 

Then, a capillary electrochromatogtraphy method with UV-Vis detection using octadecyl acrylate ester-based 

monolithic columns was proposed to separate TAGs in vegetable oils. The influence of pore size of the monolith and 

composition of the mobile phase were studied. The optimum monolith was obtained with a 9 wt% 1,4-butanediol 

in the polymerization mixture. A satisfactory separation between TAGs was achieved in less than 20 min with an 

60:40 (v/v) acetonitrile/2-propanol buffer containing 5 mM ammonium acetate. On the other hand, the TAGs profiles 

obtained were used to construct linear discriminant analysis models in order to classify the vegetable oils according 

to their botanical origin. 



462 



P15-038 ANALYSIS OF GLYCATED SODIUM CASEINATE PROTEINS BY MALDI-MS AND LC-ESI-MS 

Corzo-Martinez M. 1 , Galindo-Iranzo P. 2 , Lebrón-Aguilar R. 2 , Villamiel M. 1 , Moreno F.J. 1 

1 


2 

Institute of Physical Chemistry “Rocasolano” (CSIC), Madrid 

Corresponding author e-mail: j.moreno@ifi.csic.es 

Sodium caseinate (SC) is extensively used in the food industry as a functional ingredient due to its simple 

production, excellent nutritional value and versatile functional properties such as thickening, emulsifying and 

foaming properties. However, in spite of this, the food industry is moving towards the search of procedures to 

obtain new multifunctional ingredients. Over the past few years, there has been an increased interest in deliberately 

promoted Maillard reaction to obtain glycoconjugates with improved functionalities in relation to the initial proteins. 

Because of glycation-induced modifications in proteins can influence their functional properties, more precise and 

detailed information on structure of glycated proteins is essential to gain knowledge about their structure-function 

relationship. In this respect, the power of both matrix-assisted laser desorption/ionization mass spectrometry 

(MALDI-MS) and liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) for the 

monitoring and characterization of protein glycation has been demonstrated [1]. However, as far as we know, no 

studies on the advantages and disadvantages of both techniques for characterization of glycated SC have been 

carried out. Thus, the aim of this work was to compare the type of information that can be derived from the spectra 

of both MALDI-MS and LC-ESI-MS and determine the most suitable technique for the analysis of SC after its 

glycation via Maillard reaction with galactose and lactose. 

Following MALDI analyses, the less abundant k- and as2-caseins showed a low response, which impaired the 

accurate determination of the glycation degree of both proteins. Regarding the major caseins, as1- and b-casein, 

MALDI-MS spectra of glycated SC were characterized by an unique and broad Gaussian peak shape without good 

resolution, due to the great heterogeneity of the glycated forms of both caseins. 

LC-ESI-MS allowed the chromatographic separation of the four casein fractions, either in native or in glycated forms, 

within only nine minutes. ESI mass spectra were recorded in the negative ion mode due to the high content of SC in 

phosphorylated serine residues. As it occurred for MALDI, as2-casein also showed a low response by ESI, impairing 

its identification. Nevertheless, ESI-MS spectra corresponding to the unglycated and glycated forms of as1-, b- and 

k-casein were characterized by multiply charged molecular ions which allowed their identification, providing an 

accurate estimation of the number of carbohydrate molecules attached to each casein fraction. 

In conclusion, LC-ESI-MS was found to be a more efficient technique than MALDI-MS to determine the degree of 

glycation of SC. 


This work was supported by projects Consolider Ingenio 2010 FUN-C-FOOD CSD2007-00063 (MICINN) and ALIBIRD 

S2009/AGR-1469 (CAM). M. Corzo-Martínez thanks the CSIC for an I3P PhD-grant. 

References 

[1] Yeboah, F. K., and Yaylayan, V. A. (2001). Analysys of glycated proteins by mass spectrometry techniques: 

qualitative and quantitative aspects. Nahrung/Food, 45: 164-171. 


463


P15-039 STUDY OF THE CO-OCCURRENCE OF SIX OCHRATOXINS IN WINE 

Remiro R., González-Peñas E., Lizarraga E. 


Corresponding author e-mail: mgpenas@unav.es 

Ochratoxins are a family of toxic compounds produced by fungi of the genera Aspergillus and Penicillium that may 

occur as natural contaminants in different foods. Of these, the most important is ochratoxin A (OTA), whose presence 

in wine and grape juice has been reported in different countries, including Spain, which is one of the main wine 

producers in the world. In Europe, wine represents the second largest source of OTA intake for humans. 

Several OTA derivatives have been reported in reference literature, some of which are the result of OTA metabolism. 

Others, such as OTB (dechlorinated form of OTA) and OTC (ethyl ester of OTA), are also fungal metabolites. Under 

natural conditions, contamination of food with a mixture of fungal metabolites must be considered, and in fact, the 

co-occurrence of OTA and OTC in wine was described for the first time by Zimmerli and Dick in 1996. In order to 

avoid underestimating the total intake of ochratoxins, it is very important to determinate the co-occurrence of these 

compounds in wine, but the knowledge regarding the co-occurrence of OTA derivatives in wine is scarce. 

A previously validated HPLC-FLD analytical method capable of quantifying OTA, OTB and their methyl and ethyl 

esters in wine with low limits of detection has been successfully applied to 35 red wine samples belonging to the 

Navarra Designation of Origin. The presence of ochratoxins was confirmed using an LC/MS method. 

None of the samples analyzed exceeded the proposed European regulatory level of 2 μg/L for OTA (Commission 

Regulation (EC) Nº 1881/2006 of December 19, 2006). The presence of OTB, Me-OTA, Me-OTB, Ethyl-OTB and OTC 

in wine has been confirmed. OTA has been detected in 97.1% of the samples, whereas OTB, Me-OTA, Me-OTB, 

Ethyl-OTB and OTC were found in 100 %, 8.6 %, 82.9 %, 8.6 % and 45.7 % of the samples, respectively. 

464 



P15-040 CZE ANALYSIS OF COFFEE EXTRACTS POSSESSING ANTIOXIDANT AND ANTIBACTERIAL CAPACITIES 

Amigo-Benavent M., Mingo E., Martínez-Rodríguez A., del Castillo M.D. 

Instituto de Investigación en Ciencias de Alimentación, Microbiología y Biotecnología de los Alimentos 

Corresponding author e-mail: delcastillo@ifi.csic.es 

Coffee beverage has been associated with antibacterial and antioxidant capacities1. This investigation aimed 

at identifying and quantifying natural compounds in coffee that contribute to these properties. Coffee chemical 

compounds (chlorogenic acid) and aqueous extracts of green regular and decaffeinated Arabica and Robusta coffee 

beans were tested. Antioxidant properties of the coffee extracts were determined by TEAC2 and 2-deoxyribose3 

degradation assays. An extract from Vietnam Robusta green coffee beans possessing the highest antioxidant 

capacity among the samples, was selected and its antibacterial effect against Campylobacter jejuni, which 

is considered the most common causes of bacterial foodborne diarrheal disease throughout the world4, was 

determined. The minimal inhibitory concentration (MIC) of Vietnam Robusta green coffee against Campylobacter 

jejuni was performed. Chemical composition of the extracts was established by CZE5. Antioxidant and MIC results 

were correlated with the concentration of one of the major coffee compounds in the extracts. Chlorogenic acid, 

the most abundant phenol antioxidant naturally present in coffee extracts, showed bacteriostatic activity and 

antioxidant capacity against either ABTS•+ or hydroxyl radicals. In conclusion, CZE analysis allowed chemically 

characterize the green coffee extracts and identify chlorogenic6 acid as a main antioxidant and bacteriostatic 

compound of the extracts. To the best of our knowledge, this is the first time that is ascribed antibacterial activity 

against Campylobacter jejuni to coffee compounds and particularly to chlorogenic acid. Other major components 

of the extracts like trigonelline and caffeine may also exhibit both antibacterial and antioxidant properties and their 

contribution to the overall properties of the green coffee extracts are being investigated. 1. Del Castillo et al. 2005 

Eur Food Res Technol 221, 471-477. 2. Re et al. 1999. Free Rad Biol Med 26, 1231-1237. 3. Daglia et al. 2004. J Agric 

Food Chem 52, 1700-1704. 4. Levin R.E. 2007. Food Biotechnol. 21, 271-347. 5. Ames et al. 2000. ACS Symposium 

Series 754; American Chemical Society. 188-201. 6. Muthuswamy & Rupasinghe. 2007. J Food Agric Environment 5, 

81-85. Acknowledgements: These studies were supported by projects ALIBIRD-CM-S0505/AGR-0153 and AGL2009- 

07894. Authors also thank Lucia Soria for kind cooperation and Cafinsa, Fortaleza Company (Vitoria Spain) for 

provide the samples. 


465


P15-041 HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF STILBENES (CIS/TRANS-RESVERATROL AND 

CIS/TRANS-PICEID) IN HONEY 

Nozal MªJ. 1 , Martin MªT. 1 , Bernal J.L. 1 , Soto E. 1 , Bernal J. 2 

1 


2 

Institute of Industrial Fermentations(CSIC), Madrid 

Corresponding author e-mail: mjdnozal@qa.uva.es 

Resveratrol (3,4’,5-trihydroxystilbene) is a stilbene produced by plants in response to fungal infection or abiotic 

stresses such as those ones produced by heavy metal ions. Piceid (resveratrol-3-O-β-D-glucoside) is the main 

component of the Polygonum cuspidatum root, used in Japanese and Chinese folk medicine for the treatment of 

some cardiac ailments, like atherosclerosis or inflammation. Recent in vitro experiments have shown that resveratrol 

can inhibit the development of the microsporidian Encephalitazoon cunicoli. For this reason, nowadays, the potential 

of these natural compounds for controlling nosema infection in honeybees is being investigated. The aim of this 

study was to develop a high performance liquid chromatographic (HPLC) method with off-line isomerization for 

the analysis of residues of resveratrol, piceid and their isomers in honey samples produced by honeybees which 

were fed with resveratrol dietary supplement. The isolation of the analytes in honey samples has been performed 

by solid-phase extraction (SPE). Several SPE sorbents based in hydrophilic-lipophilic balance (Oasis HLB, 

Strata-X and Waters C18 Sep-Pack) have been tested to obtain the best recoveries. The trans-isomers were firstly 

phototransformed into their cis-isomers by UV radiation and then the target compounds were separated in a C18 

column by using isocratic elution, with a mobile phase which consisted of an acetonitrile and formic acid mixture 

at 30ºC. The detection was carried out with DAD, FLD and ESI-MS. The relation between signal intensity and the 

analytes concentration was linear over the concentration range from 0.03 to 10 mg L-1 and the LOD calculated for 

the trans- and cis-isomers of the studied compounds were close to 0.01 and 0.03 mg Kg-1 respectively. The method 

has been successfully applied to the analysis of honey samples. Acknowledgments The authors wish to thank the 

Spanish Ministry of Ciencia e Innovación and INIA for the financial support (project RTA2009-00105-CO2-02 ). 

E.Soto would like to thank MAEC-AECID for her grant. Mª. T. Martin and J. B. thanks the Spanish Ministry for her 

“Ramón y Cajal” and his “Juan de la Cierva” contracts. 

466 



P15-042 DETERMINATION OF TRYPTOPHAN, KYNURENINE, KYNURENIC AND XANTHURENIC ACIDS IN HONEY BY LIQUID 

CHROMATOGRAPHY (DAD, FLD AND APCI-MS) 

Nozal MªJ., Martin MªT., Toribio L., Soto E., Moncadas MªJ. 



Tryptophan (Trp) is an essential amino acid whose metabolism involves several different pathways. Kynurenine (Kyn) 

one of the metabolites and precursor of kynurenic acid (Kyna), an endogenous antagonist of ionotropic glutamate 

and nicotinic acethylcoline receptors, shows anticonvulsant and neuroprotective activity. Xanthurenic acid (HX), 

another metabolite, is suspected to be carcinogenic and it is known its relation with vitamin B6 metabolism. In 

this study, a liquid chromatography (LC) method was developed for the determination of Trp and its closely related 

metabolites in several honey types, in order to know their nutraceutical value. For this purpose, several LC detectors 

were used: diode array (DAD) fluorescence (FLD) or atmospheric pressure chemical ionization- mass spectrometric 

detection (APCI-MS). A solid phase extraction procedure with mixed-mode polymeric sorbent cartridges (Oasis 

MCX) has been employed for the isolation of compounds from honey samples, which were previously diluted with 

an aqueous solution of hydrochloric acid. Chromatographic separation was performed in isocratic mode on an polar 

endcapping C18 LC column (Synergi 4µ-Hydro-RP, 150 x 4 mm i.d.) using methanol and ammonium formate 20 mM 

in water, in proportion (93/7, v/v) as mobile phase. The proposed method has been applied to the analysis of honey 

samples from Ling, Lavender, Spanish lavender, Thyme, Rosemary, Heather, Sainfoin, Mixed flowers and Honeydew 

botanical origins. Although the use of conventional LC-DAD-FLD provides good results, some floral origins required 

the use of LC-APCI–MS to elucidate correctly some compound due to some matrix interferences. Average recoveries 

were influenced by the botanical origin of honey and the detection limits were in the range of 3 µg/g in LC-DAD, 

0.4µg/g in LC-FLD and 0.05 µg/g in LC-APCI-MS. Acknowledgments The authors wish to thank the Spanish Ministry 

of Ciencia e Innovación and INIA for the financial support (project RTA2009-00105-CO2-02). E. Soto thanks MAEC- 

AECID for her grant.M.T.M thanks Ministry of Ciencia e Innovación the Ramon y Cajal contract. 


467


P15-043 IDENTIFICATION OF BIOACTIVE PEPTIDES IN HYPOALLERGENIC INFANT MILK FORMULAS BY CAPILLARY 

ELECTROPHORESIS-MASS SPECTROMETRY 

Giménez E. , Català-Clariana S., Benavente F., Barbosa J., Sanz-Nebot V. 

University of Barcelona, Department of Analytical Chemistry. Nutrition and Food Safety Research Institute 

Milk proteins are precursors of many different bioactive peptides that may remain latent until being released by 

enzymatic proteolysis during gastrointestinal digestion or food processing. These bioactive peptides have been 

described as showing, among others, immunostimulating, antimicrobial, opioid, angiotensin-converting enzyme 

inhibition, mineral binding, antithrombotic or allergenic bioactivities [1]. However, identification and quantification 

of bioactive peptides in milk protein hydrolysates, fermented dairy products or, in general, in functional dairy 

foods with health-promoting or disease-preventing peptide ingredients is a challenging task because these highly 

complex samples can contain up to hundreds of different peptides at different levels of concentration [2]. This 

is the case of hypoallergenic infant milk formulas that are formulated from different types of bovine milk protein 

hydrolysates, in order to prevent protein allergy, which is the most common food allergy in the early childhood [3]. 

In this work, we have used capillary electrophoresis coupled to mass spectrometry (CE-MS) for the identification of 

bioactive peptides in different infant milk formulas. A sample clean-up pretreatment with a citrate buffer containing 

dithiothreitol and urea followed by solid-phase extraction (SPE) with different reversed-phase commercial cartridges 

was investigated to achieve optimum detection sensitivity in CE-MS. SPE with C18, StrataX and Oasis HLB 

cartridges allowed detection of the largest number of low molecular mass components, but combination of C18 and 

StrataX results was enough to achieve an excellent coverage of the studied milk. The monoisotopic molecular mass 

values of the low molecular mass components obtained by capillary electrophoresis ion-trap mass spectrometry 

(CE-IT-MS) allowed the tentative identification of nine bioactive sequences. Only the identity of five of them could 

be confirmed when accurate mass measurements were performed by capillary electrophoresis time-of-flight 

mass spectrometry (CE-TOF-MS), namely LKP, IPY, ALPM, PGPIHN and VAGTWY, which were reported to present 

angiotensin-converting enzyme (ACE) inhibitory and antimicrobial activity (only VAGTWY). [1] Meisel, H., Curr. Med. 

Chem., 2005, 12, 1905-1919 [2] Hernández-Ledesma, B., Amigo, L., Ramos, M., Recio, I., J. Chromatogr. A, 2004, 

1049, 107–114 [3] De Noni, I., Food Chem., 2008, 110, 897–903 

468 



P15-044 INFLUENCE OF VARIOUS STATIONARY PHASES ON HPLC SEPARATION OF GLYCATED INTACT BARLEY PROTEINS 

Flodrova D., Smetalova D., Salplachta J., . Bobalova J. 


Corresponding author e-mail: flodrova@iac.cz 

Lipid transfer proteins are closely related to various development processes in plants and to biotic/abiotic stresses. 

Recent studies show that ns-LTPs, especially their lipo/glycoprotein complexes are involved in different aspects 

of plant physiology and cell biology. Furthermore, LTPs have an effect on processing of plant products, mainly in 

cereal products. Barley non-specific transfer proteins are characterized by high thermal stability and resistance to 

proteolysis. These proteins remain intact during the malting process and represent one of the most important compounds 

in beer and foam. Plant ns-LTPs can be divided into two subfamilies, ns-LTP1 (~9 kDa) and ns-LTP2 (~7 kDa), prominent 

proteins found in barley grain and malt. Both proteins can occur in its various glycated isoforms or lipid-like adducts. 

In this respect, water soluble proteins from the barley malt were extracted and further separated by HPLC using 

several different stationary phases including reverse phases (C4, C8 and C18). Individual fractions were collected 

during each chromatographic runs by ProBot, directly spotted with matrix solution on a MALDI plate and then 

analyzed using MALDI-TOF/MS. Suitability of different stationary phases for the analysis of ns-LTPs and especially 

their glycated isoforms were then compared and discussed. Presented data confirm the utility of mass spectrometry 

coupled to HPLC for analysis of barley compounds undergoing glycation during malting. Acknowledgement: This 

work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (No.1M0570) and from the 

Institutional Research Plan AV0Z40310501. 


469


P15-045 CHARACTERIZATION OF ANTIOXIDANTS AND NEOANTIOXIDANTS PRESENT AFTER SUBCRITICAL WATER EXTRACTION 

OF ALGAE AND PLANTS 

Plaza M. 1 , Amigo-Benavent M. 1 , Ibáñez E. 1, del Castillo M.D. 1 , Herrero M. 2 

1 


2 


Corresponding author e-mail: mherrero@ifi.csic.es 

The subcritical water extraction (SWE) technique is being currently employed as convenient approach for the 

isolation of valuable components from plants. SWE is based on the extraction at high temperatures (100-374 ºC) and 

pressures high enough to maintain the water in the liquid state during the whole extraction procedure. As a result, 

this extraction technique provides shorter extraction times, is automated and does not require toxic organic solvents 

compared to other traditional extraction processes; furthermore, SWE involves extracting the sample in an oxygen 

and light-free environment ensuring the stability of labile compounds. Under SWE conditions the cellular structures 

of plant tissues can be broken releasing several compounds to the extra-cellular medium that are then dissolved in 

the hot liquid water. The released compounds may interact during the extraction process forming new compounds 

exhibiting different structure and properties compared to those corresponding to the original targets. Chemical 

reactions like Maillard and caramelization involving major components of vegetables might be favoured under SWE 

conditions. Previous studies indicated that the advanced products derived from these chemical reactions may exhibit 

antioxidant capacity. In this work, several SWE extraction conditions for the extraction of antioxidant compounds 

from different natural samples including algae (Phorphyra spp., Undaria pinnatifida and Cystoseira abies marina) 

and plants (rosemary, Rosmarinus officinalis L.) have been used. Two extraction temperatures were studied (namely, 

100 and 200 ºC) in order to fully characterize the extracts produced in the search for both native and neoformed 

antioxidants derived from Maillard and caramelization reactions. To do that, the extracts were ultrafiltrated by using 

centrifugal filter (3 kDa cut-off membrane); two fractions of each extract were obtained: low molecular weight fraction 

(LMW, < 3 kDa) and high molecular weight fraction (HMW, > 3 kDa). Afterwards, LMW fraction was submitted to a 

solid phase extraction (SPE) procedure using cation-exchange and reversed-phase cartridges, in order to separate 

in different fractions the potential compounds of low molecular weight derived from Maillard reaction (Amadori 

compounds) from the native phenolic antioxidants. The different fractions collected were characterized by 

UPLC-MS/MS. On the other hand, the HMW fraction was fractionated and characterized by FPLC-DAD using a size 

exclusion column. HMW fraction presented advanced Maillard reaction products. For all the separated fractions, 

antioxidant activity was measured by using ABTS and ORACFL assays. 

470 



P15-046 ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) ANALYSIS OF FREE AMINO ACIDS AND BIOGENIC AMINES IN 

DAIRY PRODUCTS 

Mayer H.K., Fiechter G., Fischer E. 

BOKU – University of Natural Resources and Applied Life Sciences Vienna 


In most cheese varieties, proteolysis occurring during maturation directly contributes to flavour and perhaps to 

off-flavour (e.g., bitterness) of cheese due to the formation of peptides and free amino acids (FAA). The relative 

proportions of individual amino acids are thought to be important for the development of the characteristic flavour 

of a cheese variety. Biogenic amines are produced in cheese by enzymatic decarboxylation of FAA. High levels 

of biogenic amines can result in food poisoning, and cases of histamine intoxication have occurred subsequent 

to the consumption of cheese. The objective of this study was to establish a pre-column derivatization method to 

determine free amino acids and biogenic amines in cheese. AccQ-Fluor derivatizing reagent (6-Aminoquinolyl-Nhydroxysuccinimidyl 

carbamate) was used to determine primary and secondary amino acids by ultra performance 

liquid chromatography (UPLC). A fast and reliable method was developed for the simultaneous determination of 19 

primary and secondary biogenic amines in cheese within nine minutes. Commercial milk and cheese samples from 

retail outlets in Vienna were analyzed referring to their free amino acid concentration as well as biogenic amine 

content. The highest total amount of free amino acids was found in original Parmigiano Reggiano (80-90g FAA/ 

kg cheese) and Grana Padano (60-70g/kg), whereas significant lower FAA concentrations were found in grated 

Parmesan samples. As compared to Parmesan, other cheese varieties showed a much lower FAA content (e.g., 

Gouda and Emmentaler about 11g/kg; Cheddar 7g/kg and Camembert 2g/kg). In milk samples, FAA concentrations 

were in the range 50-75 mg per litre. The biogenic amine content varied to a great extent, depending not only on 

the type of cheese (fresh, soft, semi-hard, hard, very hard), but also within a certain cheese variety. About 13.8% 

of samples had a histamine, and 22.4% had a tyramine content above 100mg/kg. The highest concentration of 

histamine was found in Tiroler Almkäse (1.160mg/kg) and Vorarlberger Bergkäse (397mg/kg), the highest amount 

of tyramine was found in Cantal, Olmützer Quargel and Tiroler Almkäse (about 470mg/kg). Moreover, 8.6% of 

samples had a putrescine or cadaverine content higher than 100mg/kg. The highest concentrations were detected 

in Olmützer Quargel (523mg putrescine and 748mg cadaverine per kg). Thus, the total concentration of biogenic 

amines in some samples was about 1.940mg/kg. In conclusion, the reliable UPLC analyses of free amino acid 

and biogenic amines in milk and cheese samples represent a valuable contribution to ensure the quality of dairy 

products in future. 


471


P15-047 EFFECTS OF HIGH PRESSURE PROCESSING ON FATTY ACID PROFILE IN VEGETABLES BEVERAGES 

Barba F.J., Esteve M.J., Frigola A. 


Corresponding author e-mail: maria.jose.esteve@uv.es 

Introduction 

Consumer demand for new products with good acceptance and high nutritional quality. The vegetables beverages 

can be ideal vehicles for newly discovered bioactive food ingredients targeting lifestyle. Vegetables beverage can 

contain in its formulation as a basic compound olive oil. In general, thermal processing is the selected method for 

improving the safety and shelf-life of vegetables beverages, however a big amount of desirable food consituents are 

destroyed during this treatment affecting nutritional value and sensory characteristics which determine consumer 

acceptance, therefore, alternative non thermal technologies like high pressure processing are being studied, 

because it is a posible alternative for inactivating microorganisms and enzymes without modifying nutritional or 

sensory characteristics. 

Aim 

Evaluation of fatty acid profile modifications in a vegetables beverage processed by high hydrostatic pressure. 

Materials and methods 

The sample is a vegetable beverage with the following composition: tomato (Lycopersicon esculentum Mill., 33%), 

green pepper (Capsicum annuum L., Italian pepper, 17%), green celery (Apium graveolens L., 8.5%), cucumber 

(Cucumis sativus L., 4%), onion (Allium cepa L., 4%), carrot (Daucus carota L., 4%), lemon (Citrus limon L, 1.7%), salt 

(1.7%), virgin olive oil (SOS Cuétara, S.A., Madrid, Spain, 0.8%), and water to 100%. 

The sample is introduced in bottles of PE-LD and is treated by different pressures (100-400 MPa for 5 minutes) in a 

high pressure unit EPSI NV, Walgoedstraot19 (Belgium). 

Fatty acid determination was stablished using gas chomatograhy. The GC system consisted in a Focus CG Thermo 

Finnigan system (Thermo Finnigan, Italy) with a split/splitless injector, a flame ionization detector, a CP-Wax 52CB 

capillary column (Varian, Spain) with a length of 30 m, 0.25mm internal diameter and 0.25 mm film thickness. 

Extraction and identification of fatty acids was stablished in accord to Zulueta et al. (2007). 

Results and discussion 

The percentage in the untreated vegetables beverage of saturated fatty acids (SFA) was 17.7%, monounsaturated 

fatty acids (MUFA) was 64.7%, and polyunsaturated fatty acids (PUFA) was 14.1%. Analysis of the variation in the 

SFA, MUFA and PUFA profiles in each field applied shows non-significant differences with treatment time for SFA, 

however a significant increase (p


P15-048 CHEMICAL COMPOSITION AND ANTIOXIDANT ACTIVITY OF PRESSURIZED LIQUID EXTRACTS FROM ROMANIAN 

AROMATIC PLANTS 

Miron T.L. 1 , Plaza M. 2 , Ibáñez E. 2 , Herrero M. 3 

1 

Faculty of Food Science and Engineering/”Dunarea de Jos” University, Department of Bioengineering–Romania 

2 


3 


Corresponding author e-mail: mherrero@ifi.csic.es 

Pressurized liquid extraction (PLE) is applied for extraction of natural compounds from three aromatic plants 

from Romania: oregano (Origanum vulgare), tarragon (Artemisia dracunculus) and wild thyme (Thymus serpyllum). 

Extractions were performed with water and ethanol at four different temperatures (namely, 50, 100, 150, 200 °C) for 

20 min. The antioxidant activities of the extracts were determined by using DPPH (1,1- diphenyl-2-picrylhydrazyl) 

radical scavenging and trolox equivalent antioxidant capacity (TEAC) in-vitro assays. Total phenolic content was 

determined spectrometrically according to the Folin-Ciocalteu procedure and calculated as gallic acid equivalents. 

Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to separate and identify the compounds 

present in the different plants extracts. The results showed that the extracts obtained using water as solvent 

possessed higher yield of extraction, total phenols content and antioxidant activities than the extracts produced 

with ethanol by PLE. Oregano was the richest plant in terms of amount of total phenolics as well as the most 

potent antioxidant. All the extracts were chemically characterized by LC-MS. Some compounds that are partially 

responsible for the antioxidant activities observed were identified. Besides, some interesting differences between 

the extracts produced with ethanol and the extracts produced with water could be observed. The main antioxidant 

identified in oregano was rosmarinic acid, although other important compounds were also identified, such as 

phenolic acids (protocatechuic, caffeic, chlorogenic and syringic acids) and flavonoids (luteolin-7-O-glucuronide). 

Wild thyme extracts’ composition was also characterized by the presence of several flavonoids (luteolin-7-Oflucoside, 

luteolin-7-O-glucuronide and apigenin-7-O-glucuronide) as well as rosmarinic acid and minor phenolic 

acids. On the other hand, in the tarragon extracts different caffeoylquinic acids and dicaffeolylquinic acids were 

identified as the main responsible for their antioxidant activity. The present study confirms that PLE offers a high 

potential for the extraction of natural compounds which could represent important sources for the production of 

food additives. 


473


P15-050 LC-MS; PROFILING AND QUALITY CONFIRMATION OF TEA SAMPLES FROM CAMELLIA SINENSIS & ASPALATHUS LINEARIS 

Tahrani A. 

University Heidelberg 

The invention of electrospray interface and the use of powerful mass spectrometric detectors in combination 

with liquid chromatography have revolutionized the field of analytical chemistry. For its inherent selectivity and 

sensitivity, LC-MS, has proven to be both fast and accurate as an analytical tool of choice on a routinely basis, and 

played a vital role to solve many problems related to nutraceuticals and food safety. Over the past few last years 

nutraceuticals started to gain a higher priority, and the safety and quality of food products are of growing concern 

for consumers, governments, and producers, specially in the European Union (EU), due to increasingly needs of 

imported nutritional raw material. Publishing what so called a White Paper on Food Safety the EU Commission 

seeked to ensure safe products along every step “from farm to fork”. LC–MS is widely employed for the analysis of 

nutraceuticals, since it is capable to provide a decisive information about a complex sample, enabling the screening, 

confirmation and quantitation of hundreds of components in one analysis step. These instruments are used to 

confirm nutraceuticals and food authenticity, production and processing proceuders, storage, transportation and 

labeling accuracy. Tea is known to be a very popularly consumed beverage in the world. It is defined as an infusion of 

herbs brewed with hot water and considered as one of the oldest nutraceutics. Green tea, obtained from the Camellia 

sinensis plant, has long been reported as having medicinal value. The medicinal activity of green tea extract is due to 

a variety of polyphenols most notably (-)-epigallocatechin-3-gallate (EGCG), which confirmed to be a dominant major 

compound in water infusions. On the other hand, leaves and fine stems, that obtained from Aspalathus linearis, are 

used for prapariation of rooibos tea. Fermented rooibos tea has been reported to show various biological activities. 

The chemoprotective properties and antimutagenic activity of unfermented rooibos, where also investigated. Diverse 

polyphenols have been identified and quantified in both fermented and unfermented rooibos tea. The main active 

compound in unfermented rooibos aqueous extract was aspalathin, a flavonoid glucoside. This poster is intended to 

show the application of LC-MS in monitoring tea samples form Camellia sinensis and Aspalathus linearis. 

474 



P15-051 SPME-GAS CHROMATOGRAPHY FOR LIPID OXIDATION EVALUATION DURING SHELF-LIFE OF INFANT FORMULAS 

García-Llatas G. 1 , Alegría A. 1 , Barberá R. 1 , Vasallo I. 2 , Lagarda M.J. 1 

1 


2 

Hero Institute for Infant Nutrition 

Corresponding author e-mail: guadalupe.garcia@uv.es 

INTRODUCTION During lipid oxidation (LO) of lipid-rich foods, which occurs in storage of infant formulas (IF), 

the hydroperoxides (from oxidation of unsaturated fatty acids), decompose, yielding a mixture of secondary LO 

products (alkanes, aldehydes, etc.) that introduce off-flavors, reduce nutritional value and shorten shelf-life. 

Saturated aldehydes have been identified by headspace-gas chromatography (HS-GC) and used to monitor LO, 

although they can be implied in different reactions, leading to their content variations during storage (Frankel, 1982; 

Zamora & Hidalgo, 2005). AIM Application of a SPME-GC methodology to evaluate lipid oxidation through volatiles 

determination during storage of IF MATERIAL AND METHODS A fish oil supplemented starter IF (IF-A) and a followup 

IF containing pre and probiotics (IF-B). Both samples were stored at room temperature in their commercial 

single-dose aluminium-fold pouches under inert atmosphere. For the analysis, performed every two months of their 

shelf-life (IF-A 18 months, IF-B 22 months) from fourth month onwards, a new package was opened for each sample. 

A previously validated method (García-Llatas et al., 2006) was applied. Briefly, 4 ml of 4% (w/v) reconstituted IF were 

introduced in a headspace glass vial, together with internal standard. Equilibration was performed at 37ºC during 

15 min and the HS extraction during 45 min with a SPME Carboxen/PDMS fiber. Then, the fiber was desorbed in the 

injector (250ºC, 5min) of a GC-FID, with an Equity 5 capillary column (30m x 0.53mm ID x 5mm) for quantification 

purposes. The oven was initially at 40 ºC (5min), increased to 100ºC at a rate of 4 ºC/min, then at 17ºC/min to 

220ºC (10min). Hydrogen was used as carrier gas and the FID temperature was 300ºC. Compounds were identified 

by using standards and by GC-MS analysis under similar conditions. RESULTS Saturated aldehydes (pentanal, 

hexanal, heptanal, octanal and nonanal) were identified in both samples as secondary products from LO. Propanal 

was also identified but not always at detectable levels. Hexanal was the main volatile ranging (ng/g sample) IF- 

A: 473 (t=8months) - 725 (t=14), IF-B: 190 (t=4) – 677 (t=8)), followed by pentanal. Heptanal, octanal and nonanal 

contents were lower than 100 ng/g during storage. Hexanal contents vary significantly from t=4 until the end of 

shelf-life in both samples, being higher in IF-A according to its fish oil enrichment. CONCLUSIONS Hexanal was the 

main aldehyde from LO detected in samples. Although its contents vary significantly during storage, there is not 

an accumulative tendency but fluctuating, probably due to their reaction with other IF components (as proteins) in 

the initiation of the Maillard reaction. ACKNOWLEDGEMENTS Study partially funded by Consolider Ingenio 2010 

program FUN-C-FOOD CSD2007-063 and Generalitat Valenciana (GVACOMP-2009-262). REFERENCES Frankel 

(1982) Prog Lipid Res 22, 1-33. García-Llatas et al. (2006) Food Chem 101, 1078–1086. Zamora and Hidalgo (2005) 

Crit Rev Food Sci Nutr 45, 49-59. 


475


P15-052 SENSITIVE METHOD FOR DETERMINATION OF MARINE BIOTOXINS IN FEEDING BIVALVES MOLLUSCS BY ULTRA- 

PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY 

Roca M., Carbonell E., Castillo M., Mancebo M.T. 


Corresponding author e-mail: carbonell_elv@gva.es 

Red tide is a natural phenomenon characterized by an increase in the concentration of some marine phytoplankton 

microorganisms that proliferate in large amounts under certain environmental conditions. Marine biotoxins are 

produced by some species of these microorganisms during harmful algal blooms and can accumulate in filter feeding 

bivalve molluscs. If eaten the concentrated toxins can cause a variety of illness in humans, and indeed are grouped 

according to the symptoms they may cause: diarrheic, amnesic and paralytic shellfish poisoning (DSP, ASP, and 

PSP) toxins. To ensure control over the presence of marine biotoxins in bivalve molluscs and shellfish, regulatory 

limits for consumer protection are laid down in EC regulation nº 853/2004. In the European Union the only currently 

prescribed official methods for the detection of these toxins are the mouse bioassays (MBA). These techniques are 

not specific to any one particular toxin, and are not quantitative, therefore its replacement by chemical analytical 

methods is necessary to improve the detection and monitoring of biotoxins. The aim of this study was to develop 

a sensitive, specific and multi-residue analytical method by ultra-performance liquid chromatography tandem 

mass spectrometry for the identification and quantification of some of the most representative biotoxins in bivalve 

molluscs. For this, it was carried out the optimization of detection conditions of analytes as well as the development 

of chromatographic separation by using different columns and mobile phases. 

476 



P15-053 DETERMINATION OF MELATONIN IN WINE AND PLANT EXTRACTS BY CAPILLARY ELECTROCHROMATOGRAPHY WITH 

IMMOBILIZED CARBOXYLIC MULTI-WALLED CARBON NANOTUBES AS STATIONARY PHASE 

Stege P.W. 1 , Sombra L.L. 1 , Wiedmer S.K. 2 , Messina G.A. 1 , Silva M.F. 3 

1 

University of San Luis. 

2 


3 

National University of Cuyo 

Corresponding author e-mail: pwstege@unsl.edu.ar 

The finding of melatonin, the often called ‘hormone of darkness’ in plants opens an interesting perspective 

associated to the plethora of health benefits related to the moderate consumption of red wine [1]. In the present 

work, the implementation of a new method for the determination of melatonin in complex food matrices by opentubular 

capillary electrochromatography (OT-CEC) with covalently immobilized carboxylic multi-walled carbon 

nanotubes (c-MWNT) as stationary phase is demonstrated. Applications of nanoparticles are of rising interest in 

separation science, due to their favorable surface-to-volume ratio as well as their applicability in miniaturization. 

A stationary phase with large surface area in combination with an electroosmotic flow-driven system has great 

potential in a highly efficient separation system [2]. The investigated samples comprised red and white wine, grape 

skin, and plant extracts of Salvia officinalis L. Various extractions methods were investigated, and the optimal 

parameters for the extraction were achieved at the following conditions: liquid extraction with 20 mL of methanol 

mediated by ultrasonication (200 W) for 7 min. at 15°C for 0.5 g of grape skin or dried herbs, or 10 mL of wine. 

The optimal conditions for the separation were 40 mM sodium tetraborate at pH 9.30, injection time was at 10 s at 

35 mbar, 15 kV, 18ºC (sample and capillary), and UV detection at 254 nm. The results obtained by OT-CEC were 

compared with those by CZE in terms of sensitivity. The results indicated higher resolution, better separation, and 

improved sensitivity in CEC, as compared with those obtained by CZE. In addition, highly repetitive and reproducible 

results between runs, days, and columns were demonstrated. The detection limit for melatonin was 0.01 ng mL-1. 

The method was successfully applied to the determination of melatonin in red and white wine, grape skin and plant 

extracts of Salvia officinalis L. The feasibility of using c-MWNT immobilized fused-silica capillary through covalent 

modification of bare capillaries for the determination of melatonin in complex food samples has been demonstrated. 

[1] Iriti, M., Rossoni, M., Faoro, F., J Sci Food Agric 2006, 86:1432–1438. [2] Sombra, L., Moliner-Martínez, Y., 

Cárdenas, S., Valcárcel, M., Electrophoresis, 2008, 29, 3850 – 3857. 


477


P15-054 LIQUID CHROMATOGRAPHY FOR THE SCREENING OF THIOURACYLS AND CORTICOSTEROIDS IN FARM ANIMALS 

Reig M. 1 , Toldrá F. 2 

1 

Instituto de Ingeniería de Alimentos para el Desarrollo (UPV), Technical University of Valencia 

2 

Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Valencia 

Corresponding author e-mail: ftoldra@iata.csic.es 

Liquid Chromatography for the Screening of Thiouracyls and Corticosteroids in Farm Animals Milagro Reig1 and Fidel 

Toldrá2 1Instituto de Ingeniería de Alimentos para el Desarrollo, Universidad Politécnica deValencia, Camino de Vera 

s/n, 46022 Valencia, Spain and 2Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Av. Agustín Escardino 7, 

46980 Paterna (Valencia), Spain Introduction The use of substances having hormonal or thyreostatic action as well as 

β-agonists is banned in the European Union. However, forbidden drugs may be added sometimes to feeds for illegal 

administration to farm animals. Low concentrations of these substances are known to increase weight gain and to 

improve feed conversion. Furthermore, these substances may also have a synergetic effect with other molecules. But 

they give poorer meat quality and can also exert potential toxic effects on consumers (Croubels et al., 2004). HPLC 

constitutes an adequate instrument for screening because it can be automated and computer-controlled (Toldrá and 

Reig, 2006). In this paper, the use of liquid chromatography for the screening and detection of residues of certain 

growth promoters in farm animals is presented and discussed. Materials and methods The studied methods are 

based on solid-phase extraction clean-up followed by filtration and injection into a reverse-phase high performance 

liquid chromatography (RP-HPLC) equipped with diode array detection (DAD) (Reig et al., 2006; Stolker et al, 2000). 

The examined substances are corticoids (dexamethasone) spiked in feed and antithiroideals (thiouracyls) spiked in 

urine. Results and discussion Dexamethasone, betamethasone and flumethasone are effectively separated through 

a C18 column (see figure 1), especially if an immunoaffinity column containing specific dexamethasone antibodies, is 

previously used (Reig et al., 2006). The separation is achieved in


P15-055 DETERMINATION OF PESTICIDE RESIDUES IN ANIMAL FAT USING GAS CHROMATOGRAPHY 

Castillo M., Gonzalez C., Miralles A. 


Corresponding author e-mail: gonzalez_carmac@gva.es 

Persistent pesticides were widely used in agriculture and industry for years and most of these compounds 

are banned at present. Due to their specific chemicals and physical properties, most of them are resistant to 

degradation and are fat soluble, facilitating their tendency to persist in the environment, bioaccumulate in the food 

chain, and concentrate in human and animal tissues. Although their use has been banned in developed countries for 

more than two decades, they are detected nowadays. Regulation (EC) Nº 396/2005 of the European Parliament have 

established maximun residue limits (MRLs) in fat animal in order to monitoring the safety food and to allow adoption 

measures in situations where food is likely to constitute a serious risk to human health, animal health or environment. 

Conventional methods for the análisis of pesticide residues in fatty samples, usually involve laborius and time 

consuming clean-up steps, including multiple digestión of extracts, acid digestión followed by liquid chromatography 

or gel permeation chromatography extraction (GPC) combined with adsorption liquid chromatography or solid-phase 

extraction and analytical determination by gas chromatography. A method has been developed and optimized for 

determination of residues of pesticides in fat samples within the national monitoring programme, whereby priority 

was given to simplify the clean-up, reduce use of toxic and harmful organic solvents and allow quantification and 

confirmation at ppb level. Homogenized fat samples were extracted using acetonitrile satured with hexane. The 

clean-up were done through fat precipitation by cooling the extract followed by dispersive solid-phase extraction 

with MgSO4, Primary Secondary Amine (PSA) and C18 endcaped. Pesticide residues were determined by GC-NPD 

and GC-ECD and were confirmed by GC-MS. This technique offers several advantages such as short clean-up time, 

high degree of automation, and high efficiency in the elimination of fats, which coupled to a sensitive and selective 

determination by GC with MS detection. The developed method is sensitive, quick, easy and allow the multiresidue 

determination of 37 pesticides including organoclorine, organophosphorus and pyrethroid pesticides. 


479


P15-056 DETERMINATION OF POLYPHENOLS IN WINE BY CAPILLARY ZONE ELECTROPHORESIS 

Franquet H., Núñez O., Saurina X., Hernández S., Puignou L. 


Corresponding author e-mail: helenchan00@gmail.com 

Moderate consumption of wine has been associated with reduced risk of cardiovascular diseases and cancer, as 

well as with several beneficial effects on the immune system and cognitive functions [1]. These health-promoting 

properties have been associated with the presence of phenolic compounds, in particular with the presence of 

flavonoids and the stilbene resveratrol. Other phenolic compounds, such as phenolic acids, catechins and other 

flavonoids play an important role in wine quality, contributing in flavor and color properties, especially on red 

wines [2]. The determination of the quantitative composition and factors affecting the composition of these active 

substances, using reliable methods, is considered at the moment a priority. Reversed-phase liquid chromatography 

(LC) with UV detection or coupled to mass spectrometry (LC-MS) have been the method of choice for the analysis 

of phenolic compounds in wines [3,4]. Lately, capillary electrophoresis (CE) techniques have been increasingly 

proposed as an alternative to LC methodologies using also UV detection [5,6] or couple to mass spectrometry (CE- 

MS) [7]. However, in most of these works no more than 10 polyphenols are usually analyzed. The aim of this work is 

to develop a capillary zone electrophoresis (CZE) method using UV detection for the separation and determination of 

20 polyphenols and phenolic acids in wine samples. For this purpose, a 30 mM sodium borate buffer solution at pH 

9.2 containing 5% isopropanol was proposed as background electrolyte. Electrophoretic separation was performed 

using uncoated fused-silica capillaries of 57 cm (50 cm effective length) x 75 µm I.D., and applying a capillary voltage 

of -25 kV. Pressure-assisted (0.5 psi) electrophoretic separation was also proposed in order to reduce total analysis 

time. Capillary temperature was maintained at 25 oC and hydrodynamic injection (10 s, 0.5 psi) was used for sample 

introduction. Quality parameters such as limits of detection (0.3-2.5 mg/L), limits of quantitation, linearity (r2>0.995) 

and run-to-run and day-to-day precisions at two concentration levels (RSD values lower than 15%) were obtained. 

The applicability of the proposed CZE method was evaluated by analyzing polyphenols and phenolic compounds 

in several wine samples. Finally, in order to enhance sensitivity, some in-line preconcentration procedures such 

as field-amplified sample injection were evaluated. [1] E. Nurk, H. Refsum, C.A. Drevon, G.S. Tell, H.A. Nygaard, 

K. Engedal, A.D. Smith, J. Nutr. 139 (2009) 120-127. [2] A.G.H. Lea, P. Bridle, C.F. Timberlake, V.L. Singleton, Am. 

J. Enol. Vitic. 30 (1979) 289-300. [3] L. Jaitz, K. Siegl, R. Eder, G. Rak, L. Abrnco, G. Koellensperger, S. Hann, 

Food Chem. 122 (2010) 366-372 [4] A.M. Tarola, V. Giannetti, Chromatographia 65 (2007) 367-371. [5] R.G. Peres, 

G.A. Micke, M.F.M. Tavares, D.B. Rodriguez-Amaya, J. Sep. Sci 32 (2009) 3822. [6] L.A. Katsova, A.V. Alekseeva, 

J. Anal. Chem. 63 (2008) 1024-1033. [7] G. Vanhoenacker, A. De Villiers, K. Lazou, D. De Keukeleire, P. Sandra 

Chromatographia 54 (2001) 309-315. 

480 



P15-057 FATTY ACID CONTENTS IN FRESH AND PROCESSED FISH CONSUMMED IN VALENCIA (SPAIN) 

Ubillús F. 1 , Lagarda M.J. 2 , Farré R. 2 

1 

University of Piura (Perú) 

2 


INTRODUCTION 

Fish and/or seafood intake is recommended due the positive effects on human health of their omega-3 fatty acid 

contents. The traditional Spanish Mediterranean diet is rich in olive oil that contributes to monounsaturated fatty 

acid intake but also in fish and seafood that contribute to its beneficial effects. 

AIM 

To determine, by gas chromatography, the fatty acid contents in fresh or processed fish frequently consumed in 

Valencian Community, useful to know their contribution to omega-3 fatty acid intake. 


Fourteen samples of fresh or processed fish were bought in markets of Valencia (Spain). Samples were dried and 

homogenised and an adaptation of Folch’s method1 was applied to a weight of dry sample (containing, at least 

20 mg of fat). Methyl esters were obtained by transmethylation using acetyl chloride (80ºC/1h) and extracted with 

hexane2. Internal standards (tripentadecanoine and tricosanoic acid) were added prior to transmethylation. 

A Perkin-Elmer Autosystem XL (Perkin-Elmer, Norwalk, USA) gas chromatograph coupled with a flameionization 

detector (FID) and with a 100 m x 0.25 mm i.d. SP-2560 fused silica capillary column, phase poly (80% 

biscyanopropyl/20% cyanopropylphenyl siloxane) (Supelco, Bellefonte, USA) was used for FAME analysis. The oven 

temperature/time rates were 80 ºC/ for 5 min,10 ºC/min to 160 ºC for 5 min, 6 ºC/min to 200 ºC for 20 min and 5 ºC/ 

min to 230 ºC for 25 min. Detector temperatures = 250 ºC. PTV Injector = 70 ºC to 250 ºC in ballistic mode (split 

70:1) Hydrogen (Air Liquid, Madrid, Spain) was used as a carrier gas (22 psig). The response was monitored with 

Turbochrom workstation software (Perkin-Elmer, Norwalk, USA). 

Fatty acids were identified and quantified by using calibration curves with fatty acids standards 

RESULTS 

In the analysed samples fat content is highly variable, in accordance with data reported in food composition tables3. 

The highest v-3 fatty acid contents were found in fresh salmon (C18:3 = 1.532; C20:5 = 0.539; C22:6 = 0.817 g/100g 

fresh sample), followed by sardines (C18:2 = 0.509; C20:5 = 0.712; C22:6 = 0.603 g/100g fresh sample) and the 

lowest to fresh anchovy (C18:2 = 0.005; C20:5 = 0.017; C22:6 = 0.047 g/100g fresh sample). 

In all analysed samples, the ratio between v-6 and v-3 is lower than 1, except in fish canned in oil (such as tuna in 

olive oil, or in a seed oil) with the exception of sardines in olive oil (v-6/ v-3 = 0.424). 

CONCLUSIONS 

There is a high variability in the fat and fatty acid contents of fish. Sardines (fresh or canned in olive oil) due to their 

contribution in omega-3 and the ratio between v-6 and v-3, in addition to the economic factor, constituted a good 

source of v-3 

REFERENCES 

1 Folch, J., Lees, M., Sloane, S.G.H. (1958) J Biol Chem 226: 497-509 

2 Lepage, G. and Roy, C.C. (1986) J Lipid Res 27: 114-120 

3 Souci, S.W., Fachmann, W., Kraut, H. (2000) Stuttgart: Medpharm Scientific 


481


P15-058 EFFECT OF PH ON CHROMATOGRAPHIC RETENTION OF MEAT POLAR COMPOUNDS USING FOUR DIFFERENT HILIC 


Aristoy M-C. 1 , Mora L. 1 , Toldrá F. 1 , Reig M. 2 

1 


2 


Corresponding author e-mail: mcaristoy@iata.csic.es 

Introduction In HILIC, mobile phase forms a water-rich layer on the surface of the highly polar stationary phase 

creating a liquid-liquid extraction system [1, 2]. The analytes possessing higher polarity will have a higher affinity to 

the stationary aqueous layer than the analytes possessing weaker polarity and so will show higher retention times 

in the chromatographic separation [3]. In this way, mobile phase pH influences on polarity by affecting ionization of 

analytes, and thus their retention characteristics. A total of four columns with four different polar stationary phases 

(bare silica, amide and zwitterionic groups bonded to both porous silica, and polymeric beads) were selected to 

study the effect of mobile phase pH on retention characteristics of some polar compounds typically found in meat. 

Materials and methods Atlantis Silica column (Waters), TSKgel Amide 80 (Tosoh Bioscience) and ZIC HILIC and ZIC 

pHILIC (SeQuant). Mobile phases consisted of 10 mM ammonium acetate in water adjusted at different pH values 

(solvent A), and acetonitrile (solvent B). The separation conditions were a linear gradient of organic solvent (B) from 

80% to 20% in 20 minutes. 10 mL of standards mixture (2.5 mM) were injected into the HPLC system. Results and 

discussion Mobile phase pH has a significant impact on retention and selectivity in HILIC by influencing solute 

ionization. In this study, pH was adjusted to 3.5, 6.0, 7.8, and 9 to manipulate selectivity. In all silica based columns, 

pH 3.5 offered the worst selectivity for the compounds under study. These conditions on polymeric column results 

in excessive retention, so higher pH conditions were necessary in order to elute the analytes. Neutral pH values (pH 

6.0 and pH 7.8) offered good retention times and kept the best resolution for all compounds. Retention times in all 

columns remained essentially unchanged at pH 6 and pH 7.8. Polymeric column was also tested at pH 9 and also 

no differences in retention were observed. Results are shown in Figure 2. Acidic mobile phase pH conditions mainly 

charge basic and phosphorylated groups, whereas higher pH values, both acid and basic groups are charged. As 

it is shown in Figure 2, at low pH conditions the retention of basic compounds such as CAR and ANS was higher 

than in neutral/basic pH conditions in which these compounds eluted earlier. Conclusion HILIC chromatography 

is a reliable method to separate meat compounds. Polarity and ionizability characteristics of these compounds at 

different pH conditions together with stationary phase interactions influence on retention behaviour of all compounds 

under study. Chromatographic conditions tested at 6 or higher pH values, show good results in all columns. 

Acknowledgments Grant AGL2007-65379-C02-01 MICINN (Spain) and FPU scholarship from MEC are acknowledged. 

Work under Unidad Asociada IIAD-IATA. References [1] A.J.Alpert, Journal of Chromatography, 499 (1990) 177. 

[2] C.T.Mant, L.H.Kondejewski and R.S.Hodges, Journal of Chromatography A, 816 (1998) 79. [3] L.Kovalova, 

C.S.McArdell and J.Hollender, Journal of Chromatography A, 1216 (2009) 1100. 

482 



P15-059 EVALUATION OF PHYTOESTROGEN CONTENT OF SOY-BASED NUTRITIONAL SUPPLEMENTS VIA ULTRA PERFORMANCE 


Fiechter G., Raba B., Jungmayr A., Mayer H.K. 



As intrinsic to Leguminosae with particular regard to soybean, isoflavones comprise a class of weak-estrogenic, 

polyphenolic phyto-chemicals that are mainly credited for their health promoting effects especially associated 

with certain hormone dependent cancers, cardiovascular diseases and osteoporosis. Concerning their biomedical 

attributes, a vast multitude of commercial soy-based nutritional supplements has been widely emphasized as a 

complementary alternative to hormone replacement therapy, promising safe remedies for alleviating menopausal 

symptoms. However, prevalent nutritional information tend to label solely the total isoflavone contents, whereas 

the specific isoflavone conjugates as well as the applied calculation basis, whether referring to bioactive aglycone 

equivalents or the conjugated glycoside forms, often remain unclear. Hence, commercially available soy-based 

nutritional supplements were characterized via a newly established ultra performance liquid chromatography (UPLC) 

method, on both their native conjugated isoflavone spectra subsequent direct extraction, as well as on quantitative 

amounts derived as total aglycones after enzymatic hydrolysis utilizing Helix pomatia juice. Capitalizing on sub-2 

µm particles, the established RP-UPLC technique facilitated an efficient chromatographic separation of all 12 soy 

intrinsic isoflavone forms within 10 minutes, thus demonstrating vast improvements compared to conventional HPLC 

techniques in particular regarding enhanced resolution, sensitivity and speed. Derived native isoflavone profiles 

after solvent extraction implied a certain variability, comprising conjugated forms, especially glycosides, as the 

predominant isoflavonic constituents throughout the majority of supplements, whereas only two samples indicated 

the more bioavailable free aglycones as prevailing compounds. Concerning possible instabilities as well as interconversions 

especially of esterified isoflavone glycosides during sample preparation, thereby implicating variable 

profiles, an enzymatic hydrolysis towards stable aglycones ensures a more accurate option for quantification. 

Incubation with beta-glucuronidase, inherent in Helix pomatia juice, in consideration of adequate organic 

solvent concentration to maintain isoflavone solubility without inhibiting enzyme activity, yielded in a complete 

de-conjugation, indicating exclusively aglycone residues with no traces of glycoside- or esterified conjugates. 

Moreover, the robust quantification as total aglycones subsequent to enzymatic hydrolysis unexceptionally yielded 

negative deviations referring to the labeled specifications, thus implying that stated amounts were typically 

calculated on basis of the high molecular isoflavone conjugates. Hence, incorporation of those higher molecular 

weights consequently yielded in considerable higher virtual isoflavone contents. This in fact will complicate any 

consumer decision that is basically relying on the labeled content itself, hence pointing out the urgent need for more 

transparent specifications and a uniform basis for calculating the labeled isoflavone contents, thus permitting better 

comparability of these nutritional supplements. 


483


P15-060 DETERMINATION OF PESTICIDE RESIDUES IN APPLE BY GC/MS AND LC/MS 

Banic Simicic J., Stankov V., Marošanovic B. 

SP Laboratory, Instrumental analysis-Serbia 

Corresponding author e-mail: splaboratorija@soyaprotein.com 

SP Laboratory was formed in 1980. In 2001 it was accredited (ISO/IEC 17025). It is a commercial laboratory for food, 

feed and environmental sample analysis. SP Laboratory has accreditation for determination of pesticide residues 

in fruits and vegetables. In Serbia there is a high production of fruits and vegetables specially apple varieties. The 

leading apple varieties is Idared with 50% of all apple production. 

From the summer of 2009 and early 2010 more than 100 commercial samples of apple made in Serbia were analyzed 

on pesticide residues. Determination of pesticide residues in fresh and frozen samples were analyzed by GC/ 

MS (GC 6890N/MS 5975 by Agilent with RTLPest 3 library) and LC/MS (Ultimate 3000/MSQ by Dionex). Sample 

preparation was done by dispersive SPE-QuEChERS-method (EN 15662 :2008). [1] 

Different varieties of apple samples (Idared , Golden delicious , Granny Smith, Breaburn, Golden delicious reinders, 

Jonagold,Fuji,Red delicious) were used for data processing. 30% of analyzed samples were positive on pesticide 

residues. In these samples 24 different pesticide residues were determined (Boscalid, Brompropylate, Captan, 

Carbendazim, Difenoconazole, Diphenylamine, Flusilazole, Flutriafol, Kresoxim-methyl, Myclobutanil, Penconazole, 

Phosalone, Phosmet, Propargite, Propiconazole, Pyrimethanil, Spiroxamine, Tebuconazole, Tebufenpyrad, 

Tetraconazole, Triadimefon, Triadimenol, Trifloxystrobin and Vinclozoline). Idared varieties had the highest number 

of pesticide residues. Other varieties such as Granny Smith and Fuji had the lowes number of pesticide residues 

(only two). The most frequent pesticide residue was Captan. Only in 2 samples (breaburn and idared) we have found 

pesticide residues (captan, boscalid, flutriafol) concentracion higher than Serbian and EU legislation. Almost all of 

analyzed samples, which were positive on pesticide residues,had pesticide residues concentracion in accordance 

with legislation. 

Reference: 

[1] EN 15662 :2008 Foods of plant origin – Determination of pesticide residues using GC-MS and/or LC-MS/MS 

following acetonitrile extraction/partitioning and clean-up by dispersive SPE-QuEChERS-method 

484 



P15-061 “SOYMILK” IN RELATION TO COW MILK 

Raba B., Fiechter G., Schreiner M., Mayer H.K. 



“Soymilk“ is a beverage made from soybeans [Glycine max (L.) Merr.] and has become an important factor in human 

nutrition, due to the absence of cholesterol and lactose. The composition of “soymilk“ is different from bovine milk 

in many aspects, such as fatty acid profiles, fat, protein as well as soluble sugar content. Furthermore, “soymilk” 

has a significant content of phyto-estrogens (isoflavones) that are inexistent in cow milk, and plays an important 

role in human nutrition by now. The major objective of this study was to establish an appropriate methodology to 

determine the selected composition of “soymilk”, and to compare “soymilk” with cow milk. For the fatty acid profiles, 

“soymilk“ was lyophilized, derivatized by transmethylation and analysed via GC-FID. Cow milk was extracted, 

subjected to alkaline transmethylation to minimize loss of short-chained fatty acids and also analysed by GC-FID. As 

expected, “soymilk“ was rich in omega-3-fatty acids (e.g., alpha-linolenic acid), whereas short-chained fatty acids 

were not present. In contrast, a high percentage of saturated and mono-unsaturated fatty acids were detected in 

cow milk. Fat content of both milks was analysed by the method of Röse-Gottlieb, a gravimetric reference method. 

The main result was that cow milk showed a higher fat content than “soymilk“. Protein content was analysed using 

the Kjeldahl method. While cow milk had a protein content of approximately 3.5%, “soymilk“ contained around 4.8% 

protein. Moreover, minor differences in non-protein-nitrogen (NPN) content could be observed. Soluble sugars were 

determined by HPLC-RID using a mixture of acetonitrile and water as mobile phase. The applied chromatographic 

system provided an efficient separation of five sugars (glucose, fructose, sucrose, raffinose and stachyose), of which 

sucrose represented the main component. In “soymilk”, probiotic sugars were found at appreciable levels, but were 

not present in cow milk. For lactose-intolerant persons, “soymilk” is a good alternative to cow milk, which has a high 

lactose content of about 4.7%. Regarding phyto-estrogens, 40 mg/100 g were found in “soymilk”. The isoflavone 

composition was characterised via a newly established ultra performance liquid chromatography (UPLC) method. 

Quantitative amounts were determined as total aglycones after enzymatic hydrolysis utilizing beta-glucuronidase 

from Helix pomatia juice. Many research studies have indicated that consumption of functional foods, that are rich in 

isoflavones, are associated with a wide variety of health benefits, including prevention of breast and prostate cancer, 

cardiovascular diseases and reduced symptoms of diabetes and postmenopausal bone loss. These functional 

foods include also ”soymilk“. In general, “soymilk” turned out to be a valuable food and may be regarded as an 

option in human nutrition, in particular in certain cases (e.g., lactose intolerance). However, taking into account 

recent literature, “soymilk” can definitively not be considered as an adequate substitute for bovine milk, especially 

regarding nutritional needs for young children. 


485


P15-062 RAPID HPLC METHOD FOR THE SCREENING OF CARBADOX IN FEED AND MEAT 

Batlle N. 1 , Reig M. 2 , Aristoy M-C. 1 , Toldrá F. 1 

1 

Instituto de Agroquímica y Tecnología de Alimentos, Valencia 

2 


Introduction The use of antibiotics in animal production may contribute to an increase in the human exposure, 

and therefore to the development of antibiotic-resistant pathogens and increased allergies due to its presence 

in foods [1]. The control of the absence of these substances in foods was regulated in the Directive 96/23/EC on 

measures to monitor residues in live animals and animal products. Decision 2002/657/EC [2] provided rules for the 

analytical methods to be used in testing of official samples. Due to the large number of samples to be analyzed, it 

is necessary to develop effective screening methodologies. The objective of this work was to develop a rapid HPLC 

method for the screening of carbadox in feed and its metabolite quinoxaline 2-carboxilic acid (QCA) in meat. This 

method implied the use of new columns with packaging of lower size and shorter lengths. UV detection instead of 

mass spectrometry makes these analysis available for modest laboratories. Materials and methods The method was 

validated using fortified feeds with different concentrations of carbadox. The procedure for the extraction and HPLC 

analysis of carbadox from feed and its metabolite from meat are schematised in figures 1 and 2, respectively. Results 

and discussion Analysis of CBX in feed: The recovery and repeatability data are shown in table 1. The decision limit 

(CCα) was found to be 5 μg/g and the detection capability (CCβ) was 6 μg/g. Analysis of the metabolite QCA in meat: 

The optimised methodology was based on Sin et al. [3]. The methanol percentage and pH of the mobile phase were 

adjusted to improve the selectivity of the separation. Ten control blanks were analysed to check the specificity and 

any potential matrix interferences. Chromatograms of the meat matrix and a fortified meat sample (10 μg/Kg of QCA) 

are shown in figure 3. The recovery and repeatability data for the metabolite are shown in table 2. CCα and CCß for 

QCA were 2.24 μg/Kg and 2.26 μg/Kg, respectively. Conclusion HPLC methods discriminate well the presence of 

carbadox in feed or its QCA metabolite in meat based on the use of particular extraction procedures and separation 

methods. These techniques show a good alternative to routine screening analysis of such antibiotic in feed and its 

metabolite in meat. Acknowledgements Grants A05/08 and A-01/09 from the Platform on Research for Food Safety, 

Centro Superior de Investigación en Salud Pública CSISP, Conselleria de Sanidad (Valencia, Spain) is acknowledged. 

Work prepared within Unidad Asociada IAD (UPV)-IATA (CSIC) framework. References [1] Reig, M. & Toldrá, F. (2009) 

Veterinary drugs and growth promoters residues in meat and processed meats. En: Safety of meat and processed 

meats (F. Toldrá, Editor), Springer, New York, USA, pp. 365-390. [2] Commission Decission 2002/657/EEC of 17 

August 2002 implementing Council Directive 96/23/EC concerning the performance of the analytical methods and 

the interpretation of results. Official Journal of European Community L 221: 8, 2002. [3] Sin, D.W.M., Chung, L.P.K., 

Lai, M.M.C., Siu, S.M.P., Tang, H.P.O. (2004) Anal. Chim. Acta 508, 147-158. 

486 




487


P15-063 DETERMINATION OF COCCIDIOSTATS IN MILK BY LC-MS-MS 

Nász S., Eke Z. 



Coccidiostats are feed additives to prevent and treat coccidiosis, an infectious disease caused by protozoa 

belonging to the genus Eimeria. Two groups of coccidiostats are known: chemically produced ones including 

Halofuginone, Diclazuril, Nicarbazin, Decoquinate, Robenidine and polyether ionophores including Lasalocid, 

Monensin, Narasin, Salinomycin, Maduramicin, Semduramicin. These compounds are licensed as feed additives 

in the European Union according to Commission Regulation 1831/2003/EC. Because of the unavoidable carry-over 

of these substances in non-target feed maximum levels in food of animal origin were specified for coccidiostats 

in Commission Regulation 124/2009/EC. We developed an HPLC-MS-MS method which is able to determine the 

coccidiostats in milk matrix even at low concentration level and meets the requirements of Regulation 124/2009/ 

EC. Sample cleanup for the determination of pharmaceutical compounds from milk is typically performed by matrix 

solid phase dispersion (MSPD) or - after extraction by organic solvent - by solid phase extraction (SPE). Taking into 

consideration the low volatility of anticoccidial drugs and that they can be ionized easily by electrospray, therefore 

an LC-MS technique is preferred for their measurements. In our study a reversed phase liquid chromatographictandem 

mass spectrometric method with a simple solvent extraction and purification on SPE is presented. The main 

steps of the method include addition of organic solvent to milk samples, centrifugation, removal of matrix by SPE, 

evaporation and HPLC-MS-MS determination. Coccidiostats licensed by EU, chemically produced and ionophores 

as well, can be determined and quantified reliably at μg/kg concentration level in milk by the developed method. 

488 



P15-064 EXTRACTION OF BIOACTIVE COMPOUNDS FROM BY-PRODUCTS OF THE OLIVE OIL INDUSTRY 

Temirzoda T.N. 1 , Plaza M. 1 , Segura-Carretero A. 2 , Herrero M. 3 , Ibáñez E. 4 

1 

Instituto de Fermentaciones Industriales-CSIC-Spain 

2 


3 


Corresponding author e-mail: elena@ifi.csic.es 

Olive oil industry generates various types of by-products being the most significant ones olive leaves, olive-mill 

wastewater (alpechín) and olive oil cake (orujo) or “alperujo”, which is produced when two-phase extraction process 

is used. There are numerous published scientific articles dealing with the chemical composition of olives, olive oil, 

olive-mill wastewater and olive oil cake. In contrast, only few works have been dedicated to the separation and 

identification of bioactive compounds in olive leaf. Olive leaves can reach 10% of the total weight of olives arriving 

to the mill, thus being an interesting potential source for bioactive compounds. In this work, pressurized liquid 

extraction (PLE) is explored as a potential useful advanced extraction technique for the attainment of interesting 

bioactive compounds from Olea Europea L. leaves. Ethanol and water were employed as solvents. These two 

solvents have the advantage of being safe for their use in the food industry as well as environmentally clean, in 

contrast to the most used toxic organic solvents. Four different extraction temperatures were studied (namely 

50, 100, 150 and 200 ºC) maintaining 20 min as the extraction time, in order to determine the best possible PLE 

extraction conditions to produce phenolic compounds-rich extracts. The extracts obtained were characterized 

in terms of total phenols composition and their antioxidant capacity was determined. Two in-vitro assays were 

employed to this aim, namely DPPH (1,1- diphenyl-2-picrylhydrazyl) radical scavenging and trolox equivalent 

antioxidant capacity (TEAC) assays. Results showed that the extracts obtained with water at 200 ºC contained 

the highest amount of total phenols. Besides, at these conditions the highest antioxidant activities were also 

produced. Moreover, the PLE extracts produced were chemically characterized by using LC-MS. Important phenolic 

compounds partially responsible for the antioxidant capacities showed by the extracts were identified. Among them, 

the most important were oleuropein, tyrosol as well as other flavonoids. Besides, a new ultra-performance liquid 

chromatography-tandem mass spectrometry method (UPLC-MS/MS) was applied for the quantification of some 

interesting phenolic compounds in these extracts. 


489


P15-065 EFFECT OF SEVERAL DOPANTS ON THE DETERMINATION OF CAROTENOIDS BY LIQUID CHROMATOGRAPHY/ 

ATMOSPHERIC-PRESSURE PHOTOIONIZATION/MASS SPECTROMETRY 

Rivera S., Vilaró F., Canela R. 

University of Lleida 

Corresponding author e-mail: canela@quimica.udl.cat 

Carotenoid analysis of foods is inherently difficult not only for the huges number of these compounds usually present 

in the samples and their instability but since some carotenoids co-elute. Consequently, many researchers have 

complemented the identification of carotenoid using mass spectrometers equipped with electrospray ionization 

(MS-ESI) or atmospheric pressure chemical ionization (MS-APCI) sources. Recently, atmospheric pressure 

photoionization ionization (APPI) has been introduced as a new ionization method for liquid chromatography-mass 

spectrometry analysis (LC-MS). This new ionization technique is specially suitable for nonpolar compounds. The aim 

of our experiment was to analysis two carotenes and four xantophylls by APPI studying the effect of four dopants on 

their ionization. UHPLC/MS analyses were carried out using an ACQUITY Ultra Performance LC system (Waters, 

Milford, MA, USA). Detection was carried out using an Acquity TQD tandem-quadrupole MS equipped with a 

ZSpray atmospheric pressure ionization interface. UHPLC chromatographic separations were performed on 

reversed phase column ACQUITY UPLC® BEH 300 Å C18, 1.7 μm, 2.1 × 150 mm (Waters, Milford, MA) and a gradient 

system with the mobile phase consisting of solvent A: acetonitrile: methanol: (70:30, v/v) and solvent B: H2O 100 

% at a flow rate of 0,35 mL/min. The gradient programme used was: initial 80 % A and 20 % B; linear gradient to 

100% A in 3 min; hold for 9 min, return to initial conditions in 0,1 min, followed by equilibration for 2 min. The most 

abundant APPI–MS/MS transition for each compound was monitored in the multiple reaction monitoring (MRM) 

mode to obtain the highest quantitative sensitivity. Direct infusion of standard solutions in acetonitrile:methanol: 

(70:30, v/v) for each product was used to determine the best conditions for each compound. The most intense 

signal at optimized cone voltages, energy collisions and other instrument parameters were individually investigated. 

Additionally, the six compounds indicated above were analysed by UHPLC-MS/MS-APPI and four dopants were 

tested trying to improve the ionization and enhance the signal of the carotenoids. Acetone, toluene, anisole and 

chlorobenzene were used as dopants. Positive ionization was used observing that chlorobenzene was the best 

dopant increasing up to 5 fold the chromatographic signals of the carotenoids. 

490 



P15-066 A GC-MS METHOD FOR THE EVALUATION OF 3-DEOXYGLUCOSONE AND GLUCOSONE CONTENTS IN STORED HONEYS 

AND ARTISANAL MUSTS SYRUPS 

Ruiz-Matute A.I. 1 , Hernández-Hernández O. 1 , Castro-Vázquez L. 2 , Sanz M.L. 1 , Martínez-Castro I. 1 

1 

Instituto de Quimica Organica General(CSIC)-Spain 

2 

University of Castilla La Mancha 

Corresponding author e-mail: iqomc16@iqog.csic.es 

α-Dicarbonyl compounds such as 3-deoxyglucosone (3-DG) and glucosone are reactive intermediates formed in the 

Maillard reaction and degradation of sugars. These compounds have been described to have a significant clinical 

factor in microvascular and macrovascular implications and in metabolic diseases (1). 

Different analytical methods have been reported for the study of a-dicarbonyl compounds, the most common being 

the pre-labelled high performance liquid chromatographic (HPLC) methods (2). However, quantitative data for these 

compounds in food products is scarce (1). 

Gas chromatography (GC) is a powerful technique for the analysis of carbohydrates and their derivatives. Therefore, 

in the present work, the optimization of a GC method was carried out to achieve an appropriate separation between 

3-DG, glucosone and monosaccharides (previously converted into their trimethylsilyloximes) in different food 

samples with high sugar content. The effect of time and temperature in the production of these compounds was also 

evaluated by this method. 

A 25 m x 0.25 mm i.d. x 0.25 mm film thickness fused silica column coated with SPB-1 (crosslinked methyl silicone) 

was used. The optimised temperature program consisted in holding 180 °C for 3 min then programmed to 200 °C at 

a heating rate of 1 °C min-1 and finally heated at 5 °C min-1 to 300 °C for 10 min. 

A study of 3-DG and glucosone formation during Maillard reaction and sugar caramelization was performed using 

model systems of glucose, fructose and galactose conjugated with N-α-acetyl-lysine via Maillard reaction, under 

controlled conditions. The evolution of both compounds was also studied in honey submitted to different storage 

conditions: 10, 20, and 40 °C for 12 months and to an accelerated storage: 80ºC 1, 2 and 4 hours. Artisanal must 

syrups were prepared and the formation of 3-DG and glucosone was monitorized during the procedure. 

The optimized GC-MS method allowed the simultaneous analysis of 3-DG, glucosone and monosaccharides 

separated with a good resolution. A similar behavior was observed in all the samples under study: 3-DG increases 

with temperature and time of storage while glucosone practically keeps constant or slightly decreases. 

References: 

1. Lo et al., Food Chem. 107 (2008) 1099-1105. 

2. Marceau and Yaylayan, J. Agric. Food Chem. 57 (2009) 10837-10844. 

This work was financed by projects PIF-SIALOBIOTIC 200870F-010 (CSIC) and ANALISYC-II S2010/AGR-1464 

(Comunidad de Madrid). O. Hernández thanks CSIC for a JAE-Predoc grant. 


491


P15-067 LC-DAD-ESI-TOF MS METHOD FOR THE DETERMINATION OF PHENOLIC METABOLITES AND SOME OTHER POLAR 

CONSTITUENTS FROM AVOCADO (PERSEA AMERICANA) 



In this communication, we describe the development of an analytical method which is able to carry out an efficient 

and rapid analysis of phenolic compounds and other polar constituents from avocado (Persea americana). To achieve 

that, we used high performance liquid chromatography (HPLC) coupled to different detection systems, such as diode 

array detector (DAD) and mass spectrometry (MS), by using as interface an electrospray ionization (ESI) and a time 

of flight (TOF) as analyzer. Separation and detection conditions were optimized by using a standard mix containing 

39 compounds belonging to phenolic acids and different categories of flavonoids; analytes which could be 

potentially present in the polar extract of fruits. Optimum LC separation was achieved on a Zorbax Eclipse Plus C18 

analytical column (4.6 x 150 mm, 1.8 micrometers particle size) by gradient elution with water + acetic acid (0.5%) 

and acetonitrile as mobile phases, at a flow rate of 1.6 mL/min. The detection was carried out by ultraviolet-visible 

absorption at 240, 280 and 320, 440, 520 and 640 nm, and by ESI-TOF MS (in positive and negative polarity), in 

order to facilitate the identification of the compounds under study. The developed method was applied to the study 

of three different varieties of avocado (Hass, Lamb-Hass and Rugoro). 17 compounds were identified unequivocally 

with standards and apart from that, about other 25 analytes (depending on the variety under study) were tentatively 

identified taking into account the accuracy and isotopic information provided by TOF MS. Figure 1. UV profiles (240 

and 280 nm) of the standard mixture of 39 phenolic compounds and Extract Ion Chromatograms (EICs) obtained 

when the optima parameters for the separation and detection were used. Areas of elution of phenolic acids and 

related compounds, flavones, flavanols, flavanones and flavonols are defined. Peaks identification: 1, gallic acid; 2, 

protocatechuic acid; 3, gentisic acid; 4, 4-hydroxybenzoic acid; 5, chlorogenic acid; 6, catechin; 7, vanillic acid; 8, 

caffeic acid; 9, syringic acid; 10, homovanillic acid; 11, epicatechin; 12, vanillin; 13, p-coumaric acid; 14, ferulic acid; 

15, ellagic acid; 16, sinapinic acid; 17, rutin; 18, 3-hydroxycinnamic acid; 19, taxifolin; 20, benzoic acid; 21, narirutin; 

22, naringin; 23, quercetin-3-O-gluc-6´´-acet; 24, myricetin; 25, neohesperidin; 26, trans-cinnamic acid; 27, quercetin; 

28, kaempferol; 29, laricitrin; 30, poncirin; 31, naringenin; 32, apigenin; 33, luteolin; 34, isorhamnetin; 35, chrysin; 36, 

pinocembrin; 37, galangin; 38, kaempferide; 39, pinocembrin-7-methyleter. 

492 



P15-068 PROFILING PHENOLIC ACIDS FROM AVOCADO (PERSEA AMERICANA) WITH A SENSITIVE CE-UV METHOD 



Corresponding author e-mail: elenahf@ugr.es 

Phenolic acids are a subclass of a larger category of metabolites commonly referred to as “phenolic compounds”. 

Phenolic acids are quite important, since they have been associated with colour, sensory qualities, and nutritional 

and antioxidant properties of foods. Fruits and vegetables produced in Mediterranean basin are rich in phenolic 

compounds. In the current study, we focused on avocado fruit (Persea americana) because it is an important crop 

of the tropical coast of Granada (Spain) and is increasingly valued by consumers, not only for its unique flavour and 

texture, but also for its reported health benefits. Herewith we present the development of a powerful CE-UV method 

able to detect and quantify an important number of phenolic acids in avocado, in particular 21 phenolic acids 

(benzoic and cinnamic phenolic acids). Different extraction systems were evaluated to isolate the compounds under 

study from the apolar matrix they belong to, by checking the recovery percentage for each analyte and the number 

of compounds that were extracted. As far as CE separation is concerned, we made an exhaustive optimization of all 

the variables involved (type of buffer, ionic strength, pH, polarity, voltage, injection time, detection wavelength, etc.). 

All the results are discussed in depth in this communication and the analytical parameters achieved using the final 

optimal conditions are shown. Finally, the new method was used for profiling the phenolic acids present in different 

varieties of avocado fruit (Lamb Hass, Rugoro, Hass, among others). A qualitative and quantitative comparison was 

carried out looking for potential varietal markers. Figura 1. Electrophoretic profile of an extract from avocado fruit 

(Lamb Hass variety) obtained with the following conditions: 25 mM sodium tetraborate buffer, pH 9.3, capillary sizes 

50 µm i.d. x 50 cm effective length, voltage 25 kV, injection time 10 s, normal polarity, temperature 25ºC. Detection 

wavelength was set at 280 nm. 


493


P15-069 DEVELOPMENT AND OPTIMIZATION OF A METHOD FOR DEOXYNIVALENOL DETERMINATION IN PAPRIKA USING LIQUID 

CHROMATOGRAPHY-TRIPLE QUADRUPOLE-MASS SPECTROMETRY 



Trichothecenes are secondary metabolites produced by several fungal genera, include certain species of 

Myrothecium, Trichoderma, Cephalosporium, and Stachybotrys, but mainly by Fusarium species. Fusarium spp is 

worldwide-found in plant debris and crop plants. Growth of Fusarium species and toxin production can occur at 

relatively low temperatures on agricultural commodities in the field or during storage. 

Trichothecenes have a varying degree of cytotoxic potency and a sesquiterpenoid ring structure, which can be 

classified according to the presence or absence of characteristic functional groups. All trichothecenes contain an 

epoxide at the C12,13 position, which is responsible for their toxicological activity. Cool-wet conditions during the 

flowering period are described as stimulating Fusarium infection. 

Deoxynivalenol (DON), also known as vomitoxin, is the most commonly found trichothecene all over the world and is 

associated with various adverse health effects in animals and humans. The most prominent toxic effects of DON are 

growth retardation and immunotoxic effects. It is mainly produced by Fusarium graminearum and F. culmorum. DON 

appears predominantly in wheat, corn, rye, rice and barley and also in some spices. 

In Spain, paprika has a distinct smokey flavor and aroma, as it is dried by smoking, typically using oak wood. Paprika 

is a key ingredient in several Spanish sausage products, as well as much Spanish cooking. Smoked paprika can be 

found in varying intensities from sweet and mild, medium hot, or very hot and spicy. 

An exploratory investigation was carried out in paprika for the determination of DON. For this purpose, a liquid 

chromatography-mass spectrometry technique was used, utilizing a triple quadrupole mass spectrometer of the 

latest generation. 

The method has been successfully validated in-house and was used to determine the occurrence of DON in samples 

of paprika. 

The optimized method consisted of an extraction with acetonitrile-water (84:16, v/v) solution followed by a solidphase 

extraction clean-up process in a cartridge made of different adsorbing compounds. After solvent evaporation 

in N2 stream the residue was dissolved and DON was separated and determined by LC-MS/MS. The limit of 

detection for this method was 0.014µg DON/g paprika sample and the recovery rate for a paprika sample spiked with 

0.1 µg DON/g was 119%. 

Acknowledgements: the authors wish to thank financial support from FEDER and Spanish Government “Ministerio de 

Ciencia e Innovación” (Project AGL2007–66416–C05–01/ALI, and two research grants). 

494 



P15-070 DETERMINATION OF HT-2 TOXIN AND T-2 TOXIN IN PAPRIKA BY CAPILLARY GAS CHROMATOGRAPHY WITH ELECTRON 

CAPTURE DETECTION 


University of Valencia-Spain 

The trichothecenes are a large family of chemically related toxins produced, particularly, by moulds belonging to the 

genus Fusarium and, to a lesser extent, by Myrotecium, Trichoderma, Cephalosporium, Verticimonosporium, and 

Stachybotrys. 

Their common chemical structure is based on a tetracyclic sesquiterpene nucleus, which is characterized by an 

oxane ring, a double bond in the 9,10 position, and a stable epoxide ring in the 12,13 position; many of the toxic 

properties of these mycotoxins have been attributed to the epoxy group. 

Depending on their functional groups, the trichothecenes have been classified into four groups. Type-A 

trichothecenes are the least polar of the four groups. 

T-2 and HT-2 toxins are type-A trichothecene mycotoxins produced by several Fusarium species, mainly F. 

sporotrichioides, F. poae, F. equiseti and F. acuminatum. 

T-2 toxin is a potent inhibitor of DNA, RNA and protein synthesis, and shows immunosuppressive and cytotoxic 

effects. The acute toxicity of T-2 and HT-2 toxins is quite similar. In vivo T-2 toxin is rapidly metabolized by 

deacetylation to HT-2 toxin and, consequently, the toxicity of T-2 toxin in vivo might partly be attributed to HT-2 

toxin. The EU is studying legislative limits for these two mycotoxins. 

These mycotoxins may contaminate a variety of cereal grains, such as wheat, maize, oats, barley, rice and in some 

spices such as pepper, especially in cold climate regions or during wet storage conditions. T-2 and HT-2 toxins can 

also be found in by- products intended for direct human consumption. 

Paprika is a spice made from the grinding of dried fruits of Capsicum annuum. In many cuisines, it is used to add 

color and flavor to dishes. There are different types of paprika from sweet to spicy (hot). 

There is a need to develop sensitive and accurate analytical methods for determining these mycotoxins in paprika to 

properly assess the relevant risk of human exposure. Different methods, including gas-chromatographic and liquidchromatographic 

methods for the determination of type-A trichothecenes in cereals have been developed but there 

are few methods for the determination of HT-2 and T-2 in paprika. 

The aim of this work was to develop and optimize an analytical method for HT-2 y T-2 in paprika using capillary 

gas chromatography with electronic capture detection. The method that gave the best recoveries involved an 

extraction of the sample with acetonitrile-water, clean-up by solid-phase extraction on a cartridge made of different 

sorbent materials followed by a further purification in an immunoaffinity column. which was specific for the two 

toxins. The eluate, after solvent change, was derivatized with pentafluoropropionic anhydride and injected into the 

chromatographic system. The limits of detection for T-2 and HT-2 toxins were 0.007 and 0.004 µg/g, respectively, 

and the recovery rates for paprika spiked with 1.0 0 µg toxin/g were 71.1% and 80.1% for HT-2 and T-2 toxins, 

respectively. 

Acknowledgements: the authors wish to thank financial support from FEDER and Spanish Government “Ministerio de 

Ciencia e Innovación” (Project AGL2007–66416–C05–01/ALI, and two research grants). 


495


P15-071 NITRATES IN BABY FOOD WITH FRUITS OR VEGETABLES BY ION CHROMATOGRAPHY 

Stankov V., Marosanovic B., Bognar A. 

SP Laboratory, Instrumental analysis Dpt-Serbia 

Corresponding author e-mail: splaboratorija@soyaprotein.com 

Nitrates are formed from fertilizers and decaying plants. They are found in the air, soil, water and food (particularly in 

vegetables and fruits) and they are produced naturally within the human body. Once taken into the body, nitrates may 

be converted to the more toxic nitrites. The condition where the nitrite reacts with the iron and hemoglobin is known 

as methemoglobinemia (often called ‘’blue baby syndrome’’). In SP Laboratory, determination of nitrates in baby 

food with fruits and vegetables was done by ICS-1000 system with UV Detector by Dionex, USA. Separation and 

identification of nitrates was done with analytical column AS14 and guard column AG14. Mobile phase was 3.5mM 

Na2CO3/1mM NaHCO3 buffer system. Sample preparation of fruits and vegetables is based on sample clarification 

by Carrez I (K4[Fe(CN)6]*3H2O) and Carrez II (ZnSO4*7H2O) reagents. Borax, also known as sodium tetraborate, 

(Na2B4O7•10H2O) is also added to the samples as emulgator to obtain a stable emulsion. [1] Validation parameters 

for the nitrate ion (NO3-): range of calibration curve 2.5-25mg/l, range of method 0.01-250mg/kg, recovery 96.41- 

104.8%, repeatibility 0.004-1.372%, reproduction 0.011-5.35%, precision 0.011-5.410%, uncertainty of measurement 

5.32-8.10% and LoQ 0.01mg/kg. During the last year, we analyzed nitrates in more than 300 samples of baby foods 

for infants and young children with fruits or vegetables. Samples with vegetables contained higher amounts of 

nitrate compared to samples with fruits. 15% of all samples were without nitrates (


P15-072 MINERAL CONTENTS OF THE RESTAURANT FOODS AND PROCESSED FOODS IN SOUTH KOREA 

Kang S-H., Kim K-C., Kim J-B., Kim D-H., Aum H-S., Jin S-N., Yoon M-H., Lee J-B., Park Y-B. 


Corresponding author e-mail: yongbae@gg.go.kr 

The dietary lifestyle has been influenced by globalization and industrialization in South Korea. According to the 

korean national health and nutrition examination survey, the consumption of restaurant foods and processed 

foods has also increased. The objective of this study was to investigate the mineral (Na, Ca, K, Fe, P) contents in 

restaurant and processed foods and construct the database for public health. 360 samples of restaurant foods 

and 313 samples of processed foods were collected from six provinces in South Korea. The five mineral (Na, Ca, K, 

Fe, P) contents of restaurant and processed foods were analyzed by Inductively Coupled Plasma-Optical Emission 

Spectrometers (ICP-OES) after nitric acid digestion by microwave. In restaurant foods, seasoned vegetables (1314.21 

± 272.40 mg/100g), braised foods (684.57 ± 110.71 mg/100g), and kimchies (633.52 ± 65.36 mg/100g) showed 

relatively high sodium contents. Highly detected potassium and calcium contents were 334.37 ± 52.36 mg/100g and 

59.50 ± 20.73 mg/100g respectively in kimchies. Grilled and fried foods showed high phosphorus contents (176.39 ± 

153.86 mg/100g), and steamed foods presented the highest iron contents (20.03 ± 10.71 mg/kg). In processed foods, 

sodium was highly detected in sauces, and meat & fish foods, and the contents were 1478.63±1058.17 mg/100g, 

602.84±255.76 mg/100g, respectively. Oils and fats foods presented relatively high potassium (653.42 ± 44.80 

mg/100g) and phosphorus contents (372.84 ± 25.05 mg/100g). Sodium, which could be caused by osteoporosis or 

hyperpiesia, was detected at high concentration in Gyeonggi-Do and Chungcheong-Do among the six sampling 

provinces. In general, restaurant foods such as korean stew, Chinese food had higher sodium contents than 

processed foods. Therefore, the development of low sodium contents recipes will be needed for public health. Key 

words : sodium, potassium, calcium, phosphorus, iron, restaurant food, processed foods 


497


P15-073 TOTAL SUGAR CONTENTS OF THE RESTAURANT FOODS AND PROCESSED FOODS IN SOUTH KOREA 

Yong-Bae P., Suk-Ho K., Ki-Cheol K., Jung-Boem K., Dae-Hwan K., Hee-Suk A., Sun-Nam J., Mi-Hye Y., Jong-Bok L., 


Corresponding author e-mail: yongbae@gg.go.kr 

The obesity problem has been emerged by the consumption of restaurant foods and processed foods which 

contained the high sugar contents according to the Korean national health and nutrition examination survey in South 

Korea. The aim of this study was to examine the total sugar contents (fructose, glucose, sucrose, lactose, maltose) 

in restaurant and processed foods, and construct the database for public health. 360 sample of restaurant foods 

and 313 sample of processed foods were collected from six provinces in South Korea. HPLC-RID(High Performance 

Liquid Chromatography-Refractive Index Detector) was used to determine the total sugar contents of restaurant 

foods and processed foods. Results of recovery tests were above 89%. Detection limits of sugar ranged from 0.01% 

to 0.05%. Correlation coefficient(r2) of calibration curve was 0.999 in total sugar. In restaurant foods, hard-boiled 

foods(24.33 ± 7.24 g/100g), seasoned vegetables (19.53 ± 8.03 g/100g), and breads and snacks (13.15 ± 6.44 g/100g) 

showed relatively high total sugar contents. Highly detected fructose and glucose contents were 4.15 ± 1.60 g/100g 

and 5.70 ± 3.43 g/100g respectively in seasoned vegetables. breads and snacks showed high sucrose(5.67 ± 4.61 

g/100g) and lactose(0.94 ± 0.55 g/100g) contents, and hard-boiled foods presented the highest maltose contents 

(13.98 ± 2.89 g/100g). In processed foods, jams and sauces contained the highest amount of sugar. Other processed 

foods with high total sugar contents included sweet corn, and beef boiled down in soy sauce. Proceeding from 

this fact, the development of low sugar contents recipes will be needed for public health. Key words : total sugar 

contents, fructose, glucose, sucrose, lactose, maltose, restaurant food, processed foods 

498 



P15-074 EFFECTS OF ELECTRON-BEAM IRRADIATION ON THE FORMATION OF CHOLESTEROL OXIDE PRODUCTS IN RAW PORK 

LOIN BY GC-MS. 

Lozada-Castro J.J. 1 , Santos-Delgado MªJ. 2 , Polo-Díez L.M. 2 

1 

Nariño University, Chemistry Sciences-Colombia 

2 


Corresponding author e-mail: mjsantos@quim.ucm.es 

Cholesterol in the human diet comes mainly from animal food sources and is essential for some organism functions. 

Cholesterol can be oxidized in the presence of oxygen, light, heat, radiation, free radicals and so on; its oxidation 

products form a group of cholesterol oxides (COPs) whose structure is similar to that of cholesterol but which can 

be potentially more harmful to health than cholesterol. Consequently, the interest regarding the toxicity of COPs 

has increased markedly in recent years, as they may be linked to the development of atherosclerosis, cancer and 

coronary heart diseases, as well as modification of the membrane functions., Electron-beam (E-beam) irradiation 

can be used to minimize or eliminate the microbial contamination of RTE food in order to lengthen its life; however, 

the irradiation process could accelerate oxidation reactions and it may produce undesirable compounds such as 

cholesterol oxidation products (COPs). COP content has been determined by using different analytical techniques, 

HPLC and GC being the most widely used for food analysis in view of the following; a variety of high sensitivity 

detectors is available, identifications are easier by MS and peak capacity is higher than for HPLC, although a 

previous derivatization reaction is required. Silylation is often used, Tri-Sil being a useful reagent. In this work, 

COPs were identified by GC-MS in vacuum-packed raw loin samples (both non irradiated and E-beam irradiated 

at 1.5 kGy). The samples were prepared by using accelerated solvent extraction (ASE) and clean up by solid phase 

extraction (SPE) with C18 cartridges; then, COPs were determined after Tri-Sil derivatization; the method had to 

be optimized and reaction products were identified using the NIST Library spectra by establishing how many silyl 

groups they contained. New COPs were formed and others changed in concentration during the radiation process. 


499


P15-076 DETERMINATION OF TRIGONELLINE IN SEEDS AND VEGETABLE OILS BY CAPILLARY ELECTROPHORESIS AS A NOVEL 

MARKER FOR THE DETECTION OF ADULTERATIONS IN OLIVE OILS 

Sánchez-Hernández L. 1 , Puchalska P. 2 , García-Ruiz C. 1 , Crego A.L. 1 , Marina M.L. 1 

1 


2 

Warsaw University, Department of Chemistry 

Corresponding author e-mail: laura.sanchezh@uah.es 

Trigonelline (N-methyl nicotinic acid) is an alkaloid belonging to the group of pyridine betaines possessing a 

quaternary amino group. Several health properties of trigonelline such as hypoglycemic, hypocholesterolemic 

effects, antitumor, antimigraine, or antiseptic have been reported [1]. A new capillary electrophoresis methodology 

with UV detection is presented enabling the determination of trigonelline in seeds and vegetable oils. The 

electrophoretic conditions were optimized and an in-capillary sample stacking preconcentration strategy was 

carried out in order to increase detection sensitivity. Under the optimized conditions, baseline separation among 

trigonelline peak and the other sample peaks was obtained. Analytical characteristics of the method showed its good 

performance in terms of linearity (r > 0.999), precision (relative standard deviations < 5%), and limits of detection (up 

to 0.9 µM or 1 ng/g for oils). The developed method was applied to the analysis of soya and sunflower seeds, three 

varieties of olives, and vegetable oils (sunflower, soya, and olive). Trigonelline was determined in soya and sunflower 

seeds and their respective oils while it was not detected in olive nor olive oils. Different mixtures of extra virgin olive 

oil with seed oils were analyzed detecting between 10-20 % of seed oils in olive oils. As a consequence, trigonelline 

is proposed in this work as a novel marker for the detection of adulterations of olive oils with other vegetable oils 

such as soya and sunflower oils. Literature: [1] Duke, J.A. Handbook of Medicinal Spices; Ed.; CRC Press, New York, 

2001; p. 297. 

500 



P15-077 A CE-ITMS2 METHODOLOGY FOR THE DETERMINATION OF NON-PROTEIN AMINO ACIDS IN VEGETABLE OILS AS NOVEL 

MARKERS FOR THE DETECTION OF ADULTERATIONS IN OLIVE OILS 

Sánchez-Hernández L., Crego A.L., Marina M.L. 


Corresponding author e-mail: laura.sanchezh@uah.es 

A new analytical methodology based on capillary electrophoresis-ion trap tandem mass spectrometry (CE-ITMS2) 

is proposed enabling the identification and determination of six non-protein amino acids in vegetable oils. The 

developed methodology is based on a previous derivatization of the amino acids with butanol and their separation 

by CE using acidic conditions followed by on-line coupling to an ion trap analyzer for MS2 detection established 

through an electrospray-coaxial sheath flow interface. The electrophoretic and interface parameters were optimized 

obtaining the separation of all compounds in less than 15 min with resolutions higher than 2.5. The method was 

validated by assessing its precision, LODs and LOQs and linearity range and applied to the identification of the 

selected non-protein amino acids in soya oils, sunflower oils, maize oils and extra virgin olive oils. MS2 experiments 

performed the fingerprint fragmentation of these compounds allowing to corroborate the presence of some nonprotein 

amino acids in seed oils but not in olive oils. The amino acid contents were determined and different mixtures 

of extra virgin olive oil with seed oils were prepared and analyzed. The results showed the high potential of the 

method due to the possibility of using these compounds as novel markers for the detection of adulterations of extra 

virgin olive oils with seed oils. Thus, the developed method could be considered a simple, rapid and reliable method 

for the quality evaluation of extra virgin olive oils permitting its authentication. 


501


P15-078 CHARACTERIZATION OF BIRCH EXTRACTS BY CHROMATOGRAPHIC AND MASS SPECTROMETRIC TECHNIQUES 

Soria A.C., Rodríguez-Sánchez S., Sanz M.L., Sanz J., Martínez-Castro I. 

Inst. Química Orgánica General (CSIC), Madrid 

Corresponding author e-mail: acsoria@iqog.csic.es 

Nowadays, the increasing trend towards the use of foods or supplements containing bioactive compounds from 

natural sources has promoted the search for strategies to obtain plant extracts enriched with these compounds, 

the analytical characterization of these complex mixtures being a prior requirement. The most common species of 

birch, Betula pendula or Silver birch, is a deciduous tree belonging to the genus Betula in the Betulaceae family, and 

growing in temperate climates of North America, Europe and Asia. B. pendula has been described to possess a wide 

number of biological activities including anti-microbial, anti-inflammatory, antioxidant, anti-cancer, anti-HIV, etc 

(1, 2). These properties are related to its chemical composition including, among others, flavonoids, tannins, 

glycosides, triterpenoids, etc (3, 4). Despite several papers having dealt with the analysis of certain of these 

compounds (3, 5), no comprehensive analytical characterization by chromatographic and mass spectrometric 

techniques of birch extracts of different polarity has been yet carried out. The aims of this study have been: (i) 

optimization of extraction conditions of B. Pendula bark; (ii) characterization by GC-MS of the extracted compounds, 

after conversion into their volatile derivatives; and (iii) global characterization of extracts composition by direct EI- 

MS. Ground birch bark samples (1 g) were extracted using 6 mL of different solvents (H2O, methanol, H2O : methanol 

(50:50, v/v), H2O : acetone (50:50, v/v)), prior to their conversion into trimethylsilyl oximes according to Sanz et al. (6). 

All extracts presented a similar qualitative composition, water and methanol extracts showing the highest yield (386- 

455 mg g-1 dry matter). Homogenization with Ultraturrax showed no noticeable improvement on extraction. Common 

compounds such as soluble carbohydrates and polyalcohols (glucose, fructose, galactose, sucrose and other 

disaccharides, mannitol and myo-inositol) and triterpenoids, polyphenols and glycosides typically present in Betula 

extracts such as betuloside, betulin, chatequin, β-sitosterol, salidroside, etc were determined by GC-MS. Direct EI- 

MS data allowed the characterization of low volatility compounds not identified by GC-MS such as lupeol. This work 

has been funded by projects ANALISYC-II (S2010/AGR-1464) and PRONAOS (CDTI, CENIT-2008 1004). 1.Sami, A., 

Taru, M., Salme, K., Jari, Y.-K. 2006. European Journal of Pharmaceutical Sciences, 29, 1-13. 2.Co, M., Koskela, P., 

Eklund-Akergren, P., Srinivas, K., King, J.W., Sjöber, P.J.R., Turner, C. 2009. Green Chemistry, 11, 668-674. 3.Baser 

K.H.C., Demirci, B. 2007. Arkivoc, 7, 335-348. 4.Krasutsky, P.A. 2006. Natural Products Reports, 23, 919-942. 

5.Vainiotalo, P., Julkunen-Tiitto, R., Juntheikki, M.R., Reichardt, P., Auriola, S. 1991. Journal of Chromatography, 547, 

367-376. 6.Sanz, M.L., Sanz, J., Martínez-Castro, I. 2004. Journal of Chromatography A, 1059, 143-148. 

502 



P15-079 DETERMINATION OF ANTICARCINOGENIC PROTEINS IN SOYBEAN 

Anta-Saiz L., Marina MªL., García MªC. 


Corresponding author e-mail: concepcion.garcia@uah.es 

There are numerous studies demonstrating a direct association between the ingestion of soybean and low cancer 

incidence, particularly breast, colon, and prostate cancer. Moreover, there are also evidences associating this 

anticarcinogenic activity with the presence of Bowman-Birk inhibitor and lectin. Soybean Bowman-Birk inhibitor 

(BBI) is a polypeptide of 71 amino acids and a molecular weight of 8 kDa that is capable of inhibiting proteases 

involved during the initiation and propagation of cancer. By other hand, soybean lectin is a tetrameric protein with 

a molecular weight of 110-120 kDa (four subunits of 30 kDa each) capable of binding to cancer cell membranes or 

their receptors causing cytotoxicity and inhibition of tumor growth. Nevertheless, both soybean BBI and lectin have 

also been associated with nutritional disorders. These premises make necessary the determination and control of 

BBI and lectin in soybean and soybean derived products. This work has been focused to the development of an 

analytical method enabling the rapid and simultaneous determination of BBI and lectin in soybean. Two different 

strategies were designed for the extraction of BBI and lectin from soybean: extraction of soybean proteins using a 

Tris-HCl buffer followed by isolation of BBI and lectin by the isoelectric precipitation of other soybean proteins or 

by the direct extraction of BBI and lectin using an acetate buffer. The effect of the previous soybean defating on the 

extraction of BBI and lectin was also studied. Moreover, the possibility of using a high-intensity focalized ultrasonic 

probe for accelerating the extraction was explored and an optimization of the extraction time and ultrasound 

amplitude was performed. The extracts obtained were analysed by SDS-PAGE and RP-HPLC-ESI-MS for the correct 

identification of BBI and lectin in soybean. Finally, a chromatographic methodology using a perfusion column and 

UV detection was optimized for the rapid determination of BBI and lectin in soybean. After evaluating its analytical 

characteristics (linearity, precision, and recovery), the method was applied to the quantitation of these proteins in 

different soybean cultivars. 


503


P15-080 DETERMINATION OF FLUOROQUINOLONES OF VETERINARY USE IN FOODS BY CAPILLARY HPLC WITH LASER INDUCED 

FLUORESCENCE DETECTION. COMPARISON OF METHODOLOGIES FOR SAMPLE TREATMENT 

Lombardo-Agüí M., Hernández-Mesa M., Gámiz-Gracia L., Cruces-Blanco C., García-Campaña A.M. 


Corresponding author e-mail: amgarcia@ugr.es 

Quinolones are one of the most important groups of synthetic antibiotics used for the treatment of veterinary 

diseases in food-producing animals such as cows, pigs, chicken, etc. The widespread administration of these 

antibiotics represents a potential risk to human health, as they can cause allergic hypersensitivity reactions and 

resistant bacteria. Thus, European Union has set Maximum Residue Limits (MRLs) for these antibiotics at the low 

µg/L or µg/kg levels in foodstuffs of animal origin by means of the Commission Regulation (EU) N. 37/2010. The 

development of extraction and analysis methods that ensure these antibiotics residues are not present at harmful 

levels for human in foods is mandatory. Different sample treatments, as solvent extraction, pressurized liquid 

extraction (PLE) and solid phase extraction (SPE), have been applied depending on the sample characteristics and 

the different quinolone residues found. However, simpler and more efficient extraction systems are demanding. 

The use of molecularly imprinted polymers (MIPs) and QuEChERS have been recently introduced for the extraction 

of fluoroquinolones. MIPs are synthetic materials with artificially generated recognition sites able to specifically 

capture target molecules. This is an alternative for a simpler and efficient cleanup process previous to the 

analysis of quinolones. On the other hand, QuEChERS is a very simple methodology that involves minimum steps, 

being very effective for the cleanup of complex samples. In the present work, commercial MIPs (SupelMIPTM 

SPE) and QuEChERS (Agilent SampliQ QuEChERS kits) have been evaluated and compared for the extraction of 

fluoroquinolones. For this purpose, a capillary HPLC method has been developed for the determination of the eight 

quinolones of veterinary use (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, 

Oxolinic acid and flumequine). This technique shows several advantages compared to analytical HPLC, such as 

better resolution, lower detection limits and lower solvent consumption. Also a very selective and sensitive detection 

is achieved using laser induced fluorescence (LIF) coupled with capillary HPLC by means of a 325 nm He-Cd laser. 

The separation was carried out in about 13 minutes using a Luna C18 column (150 x 0.3 mm, 5 µm) and a mobile 

phase of 0.01 M ammonium citrate pH 4.75: acetonitrile with 0.01 M ammonium citrate, in gradient mode. The flow 

rate was 15 µl/min and the temperature of the column was kept at 35°C. An injection volume of 8 µl was used. Under 

these optimum conditions, calibration curves were established, obtaining limits of detection between 0.08 and 0.88 

µg/l. Acknowledgements: Projects AGL2007-64313/ALI (MICINN) and P08-AGR-4268 (Excellence Project, Junta de 

Andalucía) supported this work. Manuel Lombardo Agüí thanks the University of Granada for a predoctoral grant 

(Plan Propio UGR). 

504 



P15-081 DETERMINATION OF SOYMETIDE IN DIFFERENT SOYBEAN DERIVED FOODS BY CAPILLARY-HPLC 

Domínguez-Vega E. 1 , Kotkowska O. 2 , García MªC. 1 , Crego A.L. 1 , Marina MªL. 1 

1 


2 

University of Warsaw 

Corresponding author e-mail: elena.dominguezv@uah.es 

Traditionally, the dietetic value of a protein was evaluated by its nutritional quality which was based on the 

bioavailability of essential amino acids and its digestibility. Another item to take into account now is the possibility of 

generating peptides with important biological activities. This fact has made food to be in the spotlight in the search 

of this kind of therapeutic peptides. Soybean is a leguminous with high protein content and that constitutes one 

of the most consumed foods in the world. In addition to be a very cheap source of proteins, soybean consumption 

has always been associated with health benefits. Several research works have been focused on the study of the 

presence of bioactive peptides in soybean. Soymetide (MITLAIPVNKPGR) is a peptide present in soybean exhibiting 

immunostimulating properties. Soymetide is released by trypsin digestion of soybean b-conglycinin, which is one of 

the major components of soybean proteins. Once demonstrated the capability of soymetide to improve physiological 

functions in immunodepresive patients, there is a clear need for its determination in soybean products for human 

consumption. 

This work proposes a new methodology based on an accelerated trypsin digestion followed by a capillary-HPLC 

peptide separation to determine soymetide in soybean dairy products: powdered milks and infant formulas. Trypsin 

digestion was performed in only 30 min using a high-intensity ultrasonic probe. Two different capillary columns 

were tested and different parameters were optimized in each case. The use of a 300 mm column with a new particle 

design (fused-core technology) enabled a suitable separation of soymetide from other peptides in less than 15 min. 

Different analytical characteristics were evaluated: selectivity, linearity, existence of matrix interferences, precision, 

and limits of detection and quantitation. The method was applied to the determination of soymetide content in 

different soybean dairy products for human consumption. Soymetide contents were compared with the observed for 

the soybean sources (soybeans, soybean flour, and soybean proteins isolate) employed in the manufacture of these 

soybean foodstuffs. 


505


P15-082 HIGH INTENSITY ULTRASONIC ASSISTED ENZYMATIC DIGESTION AND CAPILLARY-HPLC FOR THE PEPTIDE MAPPING OF 

SOYBEAN PROTEINS 

Domínguez-Vega E., García MªC., Crego A.L., Marina MªL. 



Soybean is well known as a source of vegetable proteins (48-50 %) with high nutritional value, functional properties, 

and low cost. Characterization of soybean proteins has been performed from protein profiles obtained using 

HPLC. Digestion of total proteome and analysis of resulting peptide profiles constitutes an alternative to protein 

analysis and in addition can proportionate new information about less abundant proteins that can be overlooked 

by conventional methods of protein analysis or about protein modifications as a consequence of food processing. 

However, peptide profiling, in general, involves the use of tedious and time consuming digestion protocols. Moreover, 

digestion of total proteome provides thousands of peptides and its analysis requires highly efficient and sensitive 

separations which are difficult to obtain by using conventional columns. The aim of this work was to optimize a 

fast, effective, and sensitive methodology for the enzymatic digestion of soybean proteins and the profiling of the 

resulting peptides by capillary-HPLC. 

This work proposes, for the first time, the use of a high intensity ultrasonic probe to accelerate the tryptic digestion 

of soybean proteins. Different digestion parameters were optimized: protein extracting solution, reduction and 

alkylation conditions (time, concentration, and temperature), trypsin:protein ratio, and ultrasonic conditions 

(sonication amplitude and time). Separation of peptide profiles was carried out by capillary-HPLC. The effect of the 

variation of chromatographic conditions (elution gradient, column temperature, and injection volume) on peptide 

separation was also studied using two capillary columns with different column diameters and particle sizes. 

Moreover, samples were focused at the top of the column in order to obtain an increasing sensitivity without loss 

of efficiency. This method was successfully applied to the profiling of soybean peptides from transgenic and nontransgenic 

soybeans and from different pigmented beans commercialized as soybeans. 

506 



P15-083 DETERMINATION OF CHLORAMPHENICOL RELATED COMPOUNDS IN FOOD MATRICES BY FAST LIQUID 


Alechaga E., Moyano E., Galceran M.T. 


Corresponding author e-mail: elida.alechaga@ub.edu 

Chloramphenicol (CAP) is a wide-spectrum antibiotic active against gram-positive, gram-negative and anaerobic 

bacteria widely used in human and veterinary medicine because of its availability and low cost, until serious side 

effects on bone marrow were observed. For this reason, CAP was banned in both the USA and EU in 1994 and new 

synthetic analogues, thiamphenicol (TAP) and florfenicol (FF), were developed to be used in veterinary medicine. 

The European Union established maximum residue levels (MRL) for TAP (50 ng/g) and for the sum of FF and its 

metabolites (expressed as florfenicol-amine, FFA) (100-2500 ng/g) in foodstuffs of animal origin (Council Regulation 

2377/90/EC), while for CAP, since it is a banned compound, a minimum required performance level (MRPL) of 0.3 

ng/g was defined. In this study, a fast, sensitive and selective method based on liquid chromatography coupled 

to tandem mass spectrometry (LC-MS/MS) has been developed for the determination of CAP and its related 

compounds (TAP, FF and FFA) in a variety of animal-derived foods. The chromatographic separation was carried out 

on a Fused Core Phenyl-Hexyl column with methanol:acetic acid–ammonium acetate buffer (5 mM, pH 5) as a mobile 

phase, achieving high chromatographic efficiencies (wb < 10 seconds) and short analysis time (less than 2 minutes). 

In regard to mass spectrometry, different Atmospheric Pressure Ionisation (API) sources, Electrospray (ESI), Heated- 

Electrospray (H-ESI) and Atmospheric Pressure Chemical Ionisation (APCI), were compared in terms of sensibility 

and matrix effect. The best results were obtained with the H-ESI source in positive mode for FFA and negative 

mode for CAP, TAP and FF. Tandem mass spectrometry in highly selective-selected reaction monitoring (H-SRM) 

was employed to enhance sensitivity and selectivity. For CAP and TAP transitions due to the loss of CHOCl and the 

cleavage of the alkyl branch were followed, whereas for FF, the neutral loss of HF from the two main peaks of the 

isotopic cluster was preferred. The LC-MS/MS method showed good performance in terms of linearity, precision 

and accuracy, providing limits of quantitation below ppb level. To assess the applicability of the method, several 

food samples of animal origin (fish and meat muscle, eggs and prawns), including a reference material (prawns) for 

CAP, were analysed. - S. Zhang, Z. Liu, X. Guo, L. Cheng, Z. Wang, and J. Shen. J. Chromatogr., B: Anal. Technol. 

Biomed. Life Sci. 875[2], 399-404. 2008. - S. A. Tittlemier, J. Van De Riet, G. Burns, R. Potter, C. Murphy, W. 

Rourke, H. Pearce, and G. Dufresne. Food Addit. Contam. 24[1], 14-20. 2007. - R. Yamada, M. Kozono, T. Ohmori, 

F. Morimatsu, and M. Kitayama. Biosci., Biotechnol., Biochem. 70[1], 54-65. 2006. - J. Van de Riet, R. A. Potter, M. 

Christie-Fougere, and B. G. Burns. J.AOAC Int. 86[3], 510-514. 2003. 


507


P15-084 PHENOLIC COMPOUNDS IN WHEAT CULTIVAR GRAINS 

Rodríguez Rodríguez E. 1 , Díaz Romero C. 1 , Afonso Morales D. 2 , Hernández Rodríguez L. 1 

1 


2 

Exmo. Cabildo Insular de Tenerife, Agricultural Biodiversity Conservation Center from Tenerife 

Corresponding author e-mail: emrguez@ull.es 

Wheat is an important component of the human diet and therefore it may be an important source of phenolic 

antioxidants. Epidemiological studies suggest that consumption of whole grain and bran may help to prevent 

cardiovascular diseases, diabetes and certain forms of cancer. These beneficial health effects are usually attributed 

to the presence of dietary fibre and bioactive secondary metabolites, including phenolic acids. There are a 

considerable variation of the contents of phenolic compounds and flavonoids in the literature. This could be due to 

several factors including the methods of milling, agricultural practice, climatic conditions and cultivar. The aim of 

the present study was to determine the phenolic compounds (hydroxybenzoic acids, hydroxycinnamic acids and 

flavonoids) in the grains of different wheat cultivars in order to obtain an approach in the characterization of the 

phenolic profile of this cereal and contribute information to the producers to the selection of cultivars. A correlation 

study was carried out to find out the relationships between the analyzed phenolic compounds. 

Three hydroxybenzoic acids: p-hydroxybenzoic acid, vanillic acid and syringic acid; six hydroxycinnamic acids: 

p-coumaric acid, ferulic acid and four ferulic acid derivatives [(E,E)-4,4´-dihydroxy-3,5´-dimethoxy-β,3´-bicinnamic 

acid (8-5´diFA), (E,E)-4,4´-dihydroxy-5,5´-dimethoxy-3,3´-bicinnamic acid (5-5´diFA), (Z)-β-{4-[(E)-2-carboxyvinyl]- 

2-methoxy-phenoxy}-4-hydroxy-3-methoxycinnamic acid (8-O-4´ diFA) and trans-5-[(E)-2-carboxyvinyl]-2-(4- 

hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (8-5´diFA benzofuran form)]; and a 

flavonoid (apigenin) were identified and quantified in 34 accessions corresponding to 19 cultivars of wheat applying 

HPLC coupled to diode array detector. Considerable differences between the wheat cultivars were observed in 

the phenolic contents. Some cultivars (Colorado, Del País, Barbilla, Jallado, Raspinegro Canario and Plaganudo) 

could be selected according to the high levels of phenolic compounds. Ferulic acid was the major phenolic acid 

compound followed by syringic and p-hydroxybenzoic acids. The proportion of ferulic acid present as dimeric 

forms ranged from 4.2 to 8.6 % across all of the wheat cultivars analyzed. Apigenin, p-hydroxybenzoic and syringic 

acids did not show significant correlations. Many correlations between the determined hydroxycinnamic acids were 

observed. Highly significant correlations between the hydroxicinnamic acids: p-coumaric acid, ferulic acid and all the 

diferulic acid derivatives were observed. The four diferulic acid derivatives can be estimated known the ferulic acid 

concentration. 

In conclusion, the major phenolic profile of the wheat grain is defined by three hydroxybenzoic acids 

(p-hydroxybenzoic acid, vanillic acid and syringic acid), six hydroxycinnamic acids (p-coumaric acid, ferulic acid 

and four ferulic acid derivatives), and a flavonoid (apigenin). It is confirmed an important variation in the phenolic 

acids and flavonoids between wheat cultivars, which suggests the selection and development of wheat cultivars with 

higher levels of phenolics as a useful tool to improve the public health. In this sense the Colorado, Del País, Barbilla, 

Jallado, Raspinegro Canario and Plaganudo cultivars could previously be selected due to the high levels of phenolic 

compounds. 

508 



P15-085 EVALUATION OF RELEVANT TOF-MS PARAMETERS IN LC-MS SCREENING FULL SCAN METHODS FOR PESTICIDE 

RESIDUE ANALYSIS 

Mezcua M., Malato O., Lozano A., Agüera A., Fernandez-Alba A.R. 


EVALUATION OF RELEVANT TOF-MS PARAMETERS IN LC-MS SCREENING FULL SCAN METHODS FOR PESTICIDE 

RESIDUE ANALYSIS M. Mezcua, O. Malato, A. Lozano, A. Agüera, and Amadeo R. Fernández-Alba. Pesticide 

Residue Research Group. European Reference Laboratory EURL. University of Almeria. 04120 (Spain). mmezcua@ 

ual.es An automatic screening method based in full scan analysis by LC-TOF-MS is used for the analysis of 100 

samples of fruits and vegetables from countries outside the European Union. The automatic screening method 

uses a database which includes 300 pesticides, their corresponding fragments and their isotopic signals. Two 

instruments with two different resolution powers (15000 and 7500) were considered to evaluate their capability to 

resolve the ions included in the database of those present in the matrix. Four different matrices, pepper, rice, garlic 

and cauliflower were evaluated. It has been demonstrated that these resolutions are enough to resolve interferences 

away from signals of interest in the studied matrices. Several parameters involved in the automatic screening method 

were carefully studied to avoid false positives and negatives in the studied samples. These parameters are: peak 

filter (number of counts of chromatographic peaks) and search criteria (retention time window and error window). 

Semi-quantification of positive samples was performed by LC-TOF-MS. Only two calibration points were used with 

this purpose, obtained by using solvent solutions of the corresponding pesticides. The quantification results were 

compared with those obtained by a LC-QqQ-MS/MS system, proving that differences achieved were lower than 

50% in most of the cases. From a total of 100 samples analysed, 80 of them were positives, 189 pesticides were 

detected and the average of pesticides per sample was 2.3. A maximum of 9 pesticides were detected per sample. 

90 pesticides were found in concentrations lower than 10 μg/Kg, 50 pesticides in concentrations between 10 mg/ 

Kg and 100 μg/Kg and 46 pesticides in concentrations higher than 100 μg/Kg. The samples was extracted following 

the QuEChERs method for further analysis in the LC systems. Acknowledgements: The authors acknowledge funding 

support from the Junta de Andalucía (Regional Government of Andalusia, Spain) (Project ref. AGR-4047). 


509


P15-086 LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY FOR PERFORMING CONFIRMATORY ANALYSIS OF 

ANTBACTERILAS IN ANIMAL-FOOD PRODUCTS 

Blasco C., Masia A., Morillas F., Pico Y. 


Corresponding author e-mail: cristina.blasco@uv.es 

In modern systems of livestock breeding, veterinary drugs are employed for therapeutic, metaphylactic, 

prophylactic and growth promotion purposes [1] and [2] and are administered as feed additives or via the drinking 

water. The occurrence of unwanted residues in edible products can be the result of illegal use, in the cases of 

prohibited medicines, or of failure to respect the proper withdrawal times before butchering, in the cases of 

permitted medicines. Adverse effects for consumer health are connected with the intrinsic toxicity of a drug 

and its metabolites; some chemotherapeutics are also suspected of being carcinogenic, but the main concern 

is the selection of resistant bacteria that, through the food chain, can be transferred to humans [3] A novel rapid 

multiresidue/multiclass procedure with liquid chromatography tandem mass spectrometry (LC–MS/MS) detection 

has been developed to screen for the presence of veterinary drug residues in animal tissues. The method uses 

a new sample preparation procedure loosely based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and 

Safe) methodology. Validation to date has been restricted to chicken muscle and has been performed according 

to European Commission guidelines [COMMISSION DECISION 2002/657/EC] for nitroimidazoles, sulphonamides, 

fluoroquinolones, quinolones, ionophores and dinitrocarbanilide. The method was applied to sixteen antibacterials 

including 5 quinolones (ciprofloxacin, enrofloxacin, flumequine, norfloxacin, ofloxacin), 1 penicillin (penicillin V), 1 

macrolide (spiramycin) and 9 sulfonamides (sulfachlorpyridazine, sulfadiazine, sulfadimethoxine, sulfaguanidine, 

sulfamethoxazole, sulfamethoxypyridazine, sulfapyridine, sulfaquinolaxine and sulfathiazole). These antibacterials 

are examples of some of the major classes of veterinary drugs. The LC- MS/MS coupled to QuEChERS method for 

screening and confirmation of antibiotics residues in muscle tissue samples represents an exceptionally effective 

method with the advantages of requiring only small samples sizes and solvent volumes and providing satisfactory 

recovery, repeatability and reproducibility References: 1. Andreu V., Blasco C., PicoY. Trends Anal. Chem., 26 (2007) 

534. 2. Blasco, C., Torres, C.M. & Picó, Y. TRAC Trends Anal. Chem. 26 (2007) 895. 3. A.E. Van den Bogaard and E.E. 

Stobberingh, Int. J. Antimicrob. Agents 14 (2000) 327. 

510 



P15-087 CHARACTERIZATION OF 4-O-β-D- GLUCOSIDE OF P-COUMARIC ACID IN MUSTS OF. CV ALBARIñO (VITIS VINíFERA L) 

Zamuz S., Vilanova M., Masa A. 

Mision Bioloxica de Galicia (CSIC) 

Corresponding author e-mail: amasa@mbg.cesga.es 

Phenolic compounds are frequently used as chemical markers in chemotaxonomy. Moreover, some phenolics 

are antioxidants contributing to a reduction in the risk of cardiovascular diseases, hyperthension and cancer (1). 

Initially antioxidant activity was principally associated with stilbenes and flavonoids, but evidence is now increasing 

that hydroxycinnamates and their derivatives are similarly actives. The hydroxycinnamic acid derivatives are the 

major group of phenolic compounds in white musts and wines and Baderschneider and Winterhalter (2) reported 

that the radical scavenging capacity of these derivatives does not differ significantly from that of other phenolic 

antioxidants. However, and in spite of the importance of this phenolic compounds, there is little information about 

these compounds in musts; it is especially true for musts from Galician white cultivars. In this study we report the 

characterization of 4-O-β-D-glucoside of p-coumaric acid in musts of the white cv Albariño, the most important 

white cultivar growing in Galicia (northwest Spain). This compound was previously reported in white German wines 

but, as far as we know, it was identified for the first time in white musts. The extraction of phenolic compounds was 

carried out according to the procedure described previously (3) and the extracts were used for the reversed-phase 

HPLC-DAD analysis. Peak of retention time 8.1 (peak A) showed an UV spectrum with an absorbance maximum in 

294 nm that suggest an hydroxycinnamic acid-sugar derivative. Extracts were repeatedly injected (twenty times) in 

HPLC and the fraction corresponding to the 7-9 minutes (that contains this chromatographic peak) collected using a 

Waters WFC III fraction collector. Fractions were mixed, evaporated to dryness in a vacuum evaporator, resolubilized 

with 0.5 mL of methanol/water (1:1, v/v) and repeatedly injected (ten times) in HPLC with new chromatographic 

conditions for the best separation of peak A. Peak A was collected, mixed and characterized by spectrophotometric 

and chromatographic methods by comparison with standard. Further evidence for the structure of the compound 

corresponding to peak A was by analyzing the products of the alkaline and enzymatic hydrolysis (4). Aglycone 

was identified as p-coumaric acid by comparison with an authentic standard and sugar as glucose according to 

Markham (4). 1.- Li, H.; Wang, X.; Li, P. and Wang, H. (2009). Phenolic compounds and antioxidant properties of 

selected China wines. Food Chemistry 112: 454-460. 2.- Baderschneider, B. and Winterhalter, P. (2001). Isolation 

and Characterization of Novel Benzoates, Cinnamates, Flavonoids, and Lignans from Riesling Wine and Screening 

for Antioxidant Activity. J. Agric. Food Chem. 49 (6): 2788-2798. 3.- Masa, A.; Vilanova, M. And Pomar, F. (2007). 

Varietal differences among the flavonoid profiles of white grape cultivars studied by high-performance liquid 

chromatography. J. Chromatogr. A 1164: 291-297. 4.- Markham, K.R. (1982). Techniques of Flavonoid Identification. 

Academic Press, London. 


511


P15-088 RAPID METHODOLOGY FOR THE DETECTION OF OLIVE OIL ADULTERATIONS WITH SEED OILS USING FLOW INJECTION 

ELECTROSPRAY IONIZATION WITH TRIPLE QUADRUPOLE MASS SPECTROMETRY 

Sánchez-Hernández L., Castro-Rubio F., Nozal L., Novella J.L., Marina MªL., Crego A.L. 


Corresponding author e-mail: antonio.crego@uah.es 

A new screening method is proposed to automate the monitoring of non-protein amino acids and betaines in different 

types of oils (soya, sunflower, maize and olive). The screening consists of a continuous-flow system connected 

directly to the MS/MS equipment (electrospray ionization coupled to triple quadrupole mass spectrometer). The 

sample treatment consists of a derivatization with butanol under acidic conditions previous to tandem experiments 

in positive ion mode in an analysis time less than 2 min. The proposed method provided easy discrimination power 

to determine the presence of several non-protein amino acids and betaines in the studied oils. The presence of 

some betaines and non-protein amino acids in seed oils but not in olive oils was corroborated enabling to propose 

these compounds as markers for the detection of adulteration of olive oils. Different mixtures of extra virgin olive 

oil with seed oils were analyzed and their reliable typification via these markers for the detection of adulterations 

in extra virgin olive oils was achieved. The usefulness of the rapid and sensitive screening method by tandem 

mass spectrometry was demonstrated showing sample handling simplicity and enabling the elimination of the 

chromatographic step. Moreover, the method supposes a fast and applicable procedure for analytical laboratories 

for olive oil authentication which is high demanded due to its wide consumption and healthy benefits. 

512 



P15-089 DETERMINATION OF WATER-SOLUBLE VITAMINS IN HONEY USING TWO RP-HPLCMETHODS: WITHOUT AND WITH CTAB 

IN THE MOBILE PHASE 

León Ruiz V. 1 , Vera S. 2 , González-Porto A.V. 1 , San Andrés M.P. 2 

1 

Junta de Comunidades de Castilla-La Mancha, Centro Agrario de Marchamalo 

2 


INTRODUCTION 

The determination of different nutrients in honey samples is very important for their characterization in the different 

origin denominations. In the Castilla-La Mancha honeys it is studied the vitamin composition with this purpose in 

different honey sources. 

EXPERIMENTAL 

Chromatographic system used in this work was a Perkin-Elmer equipped with a pump model 250, a thermostatic 

oven Jet-Stream Plus from Knauer, an injector Rheodyne valve with 20 µL and a programmable UVVIS detector 

model 785A form Applied Biosystems and a fluorescence detector Pelkin-Elmer 200 with an interface Perkin-Elmer 

950A. Stationary phase was a C18 column µBondapack 10 µm, 150x3.9 mm from Waters. 

RESULTS 

The work has been realized in three parts. Firstly, the mobile phase used for the separation of vitamins was 

composed by H2SO4 0.01% at different pH ranged between 2.5 and 3.7. In this part it was studied the influence of a 

little quantity of an organic solvent (methanol, ethanol, n-propanol and n-butanol) and the temperature from 25ºC to 

40ºC. In this case, the best separation for the vitamins C, nicotinic acid, nicotinamide, B1, B5 and B6 was obtained 

without organic solvent with pH 3.5 and 25ºC. Vitamin B6 was detected by UV and fluorescence. This mobile phase 

is not appropriate to elute in an adequate time the vitamins B2 and B9. 

The second mobile phases tested for vitamin’s separation were those which contain an organic solvent at 

percentages lower than 10%. In this case, the better conditions obtained were at the same mobile phase 

composition as before but with a 2% methanol. The only one advantage over the previous case in a shorter total 

analyse time. 

The last mobile phases tested were formed in presence of a cationic surfactant as hexadecyltrimethylammonium 

bromide (CTAB). The concentration added to the mobile phase was ranged between 10 and 20 mM and in all 

cases using a 2% of a short-chain alcohol (Methanol, n-Propanol and n-Butanol). The best composition found was 

CTAB 10mM/H2SO4 0.01%/MeOH 2%. The influence of pH was also studied and its value is critical to obtain the 

separation and modify the retention order of the compounds. The pH chosen was 2.75 which give us a total analysis 

time of 6.5 minutes for the separation of vitamins B1, B3H, C, B3N and B2. In this case is not possible to separate 

the vitamin B5 and therefore, both methods can be complementary. 

The analytical characteristics of the method with CTAB mobile phase gives detection limits lower than 1 mg/L with 

UV detection and lower than 0.5 mg/L with fluorescence detection. The robustness was lower than 5% in all cases. 

In different honey types and honeydew samples, it was applied the method and in same honeys, it was found 

vitamins C, B3H and B2. 


513


P15-090 SAMPLE PREPARATION METHOD DEVELOPMENT OF MELAMINE AND CYANURIC ACID IN SOME STOCK FARM PRODUCTS 

FOR THE ISOTOPE DILUTION GC-MS 

OH CH. 1 , Kim SH. 1 , Cheon SH. 1 , Yoon JE. 1 , Hahm JH. 1 , Lee HJ. 1 , Roh JS. 2 , Kim KS. 3 , Park JW. 4 , Woon JH. 4 

1 

Semyung University-South Korea 

2 

HAITAI Confectionery & Foods Co., Ltd.-Safety Guarantee Institute-South Korea 

3 

Chosun University, Dept. of Food & Nutrition-South Korea 

4 

National Veterinary Research & Quarnatine Service, NVRQS-South Korea 

The recall of 170 tons of tainted milk powder in the northern region of Ningxia, China (Feb., 2010) remind us the 

disastrous melamine (MEL) tainted milk products crisis, 2008. Melamine co-exposure with cyanuric acid can 

induce acute MEL – cyanurate crystal nephropathy, which can lead to renal failure at much lower doses than if 

either compound were ingested alone. The stock farm products are the difficult matrices due to the high contents 

of fat and protein for the melamine analysis. Liquid chromatograph with tandem mass spectrometer (LC-MSMS) 

is recommended for the analysis of melamine. However LC-MSMS is still not common to ordinary food analysis 

laboratory due to the high expense. Therefore the sample preparation method of the cheese, pork and Ham was 

developed for the GC-MS analysis of MEL and cyanuric acid (CYA). Each 1g of the sample was defatted with 

dichloromethane by ultra-sonication for an hour, extraction with methanol was followed. After centrifugation 

and filtration, part of methanol extract was acquired for the trimethylsilylation (optimized in this study). For the 

quantitative analysis, 4-chloro-2,6-diamininopyrimidine (CDAP) and isotopically labeled (IL) melamine and cyanuric 

acid were used as internal standards. Most of quantitative results by IS, CDAP was relatively poor than the results by 

IL-MEL & CYA. The recovery range of MEL & CYA (at 1ppm in the sample) was from 70 to 120%. The quantitative loss 

pattern of CDAP and MEL & CYA might be quite different during the above sample preparation steps. The isotope 

dilution technique might be necessary for the quantitation of MEL & CYA from the stock farm products tried in this 

study 

514 



P15-091 QUALITY INDICATORS IN CARROTS BLANCHED BY ULTRASOUND TECHNIQUES 

Gamboa-Santos J. 1 , Montilla A. 1 , Soria A.C. 2 , Villamiel M. 1 

1 

Instituto de Investigación en Ciencias de Alimentación (CIAL) 

2 

Instituto de Química Orgánica General (IQOG), Madrid 

Corresponding author e-mail: mvillamiel@ifi.csic.es 

Carrots (Daucus carota L.) are one of the most important root vegetables for their nutrient content. However, carrots 

are seasonal in nature and to extend their shelf-life, industrial processing (dehydration, freezing, etc) is usually 

carried out. Conventional blanching is one of the most commonly used pretreatments prior to processing because 

of their ability to deactivate the enzymes (peroxidase (POD), pectinmethylesterase (PME)) responsible for carrot 

quality deterioration. However, blanching, depending on the conditions, can negatively affect the quality of carrots. 

Thus, losses of nutrient such as sugars, polyphenols, vitamins, etc may take place due to leaching and heat-induced 

changes derived from the initial stages of the Maillard reaction. In this sense, alternative blanching methods such 

as sonication have been described to be advantageous in the case of mushrooms and cauliflower(1). In addition, the 

increasing consumer’s demand of premium quality products has promoted the study of different quality indicators in 

blanched processed vegetables(2). In spite of all these papers, no works on the application of ultrasound for carrot 

blanching have been carried out. 

The present study was undertaken to optimize the operating conditions (temperature, time and ultrasonic power) in 

the ultrasonic (US) blanching of carrots to preserve their nutritional and functional properties. Selected conditions 

were chosen on the basis of the evaluation of different quality parameters (carbohydrates, 2-furoymethyl lysine, 

polyphenols). Comparison with conventional blanching was also studied. 

Optimization of sonication process included the study of different sample geometries (slices of 2 and 4 mm 

thickness) and blanching equipments: ultrasonic probe (BRANSON) operating at 0.26W/cm3 and 0.13W/cm3: 15, 30 

and 60 minutes and ultrasonic bath (SONICA EP 2200) operating at 0.04W/cm3: 40 and 60°C, 30 and 60 minutes, 

were tested. For comparison purposes, the conventional blanching conditions were: 60°C, 40 minutes; 95°C, 5 

minutes; boiling, 1 minute; steam, 2 minutes. 

The quality indicators evaluated were: deactivation of POD and PME determined by colorimetric and titrimetric 

methods respectively; carbohydrate content by GC-FID; concentration of 2-furoylmethyl lysine quantified by ion-pair 

RP-HPLC; antioxidant activity determined by the ORAC method and total polyphenol content determined by the 

Folin-Ciocalteau method. 

US bath did not show differences over enzymatic deactivation and the different quality indicators evaluated. 

On the contrary, ultrasound probe had an important effect over enzymes. Losses of nutrients (polyphenols and 

carbohydrates) were very important in conventional blanching and minor when ultrasound probe or steam blanching 

were the selected treatments. These results seem to indicate the usefulness of ultrasonic probe as an effective 

method for enzymatic inactivation with minor changes in carrot quality. 

This work has been funded by Ministry of Science and Innovation of Spain (project AGL2007-63462), Consolider 

(CSD2007-00063 INGENIO 2010) and CYTED (P109AC0302). JG-S thanks CSIC for a predoctoral grant. 

(1)Jambrak et al. (2007) J. Food Engin. 81: 88-97. 

(2)Soria et al. (2009) J. Sci. Food Agric. 89: 267–273. 


515


P15-092 PRODUCTION OF AFLATOXINS BY ASPERGILLUS PARASITICUS CECT 2681 ON RICE STERILIZED BY TWO DIFFERENT 

METHODS 

Cuadrench-Tripiana A., Navajas H., Agut M., Comellas L. 

Ramon Llull University, Barcelona 

Corresponding author e-mail: lluis.comellas@iqs.es 

Aflatoxins B1, G1, B2 and G2 are fungal secondary metabolites produced by some Aspergillus strains. Aflatoxins 

are the stronger natural carcinogens and their main target organ is the liver. The International Agency for Research 

on Cancer (IARC) has classified aflatoxin B1 in the Group I as a human carcinogen and G1, B2 and G2 in the Group 

2B as possible carcinogens to humans. Then mycotoxins occur widely in vegetable products like cereals, fruits, 

dried fruits, beer and meat products of animals fed with contaminated feed. In this work, Aspergillus parasiticus 

CECT 2681 has been grown in sterilized rice during 7 days at 27ºC. It has been reported that aflatoxins B1, B2, G1 

and G2 can be produced by this strain. Two diferent techniques have been used to sterilize the rice before the strain 

inoculation; autoclaving (121ºC, 30 min) and UV light (254 nm, room temperature, 3 hours). The aim of this work 

is to compare the aflatoxins production using rice sterilized by both techniques. To determine the mycotoxigenic 

capacity of Aspergillus parasiticus, 5g of rice with 5 ml distilled water were sterilized by autoclaving in a 100ml 

erlenmeyer flask. At the same time, another 100 ml erlenmeyer flask with 5g of rice and 5ml distilled water were 

sterilized with UV light. Then, flasks were inoculated with a 6 mm culture disk of the fungal strain and incubated at 

27ºC for 7 days. After this, each culture was extracted twice with 45 ml of mixture of methanol: water (90:10). Then 

in a 100ml flask an aliquot of 5 ml of the extract was mixtured with 6,5 ml of water. Finally, each sample was filtrated. 

The chromatographic analysis was made with a UHPLC-UV method. It can be sumerized that in all cases the total 

aflatoxins production in autoclaved rice is four times higher than the aflatoxins production using UV sterilized rice. 

516 



P15-093 GCXGC-ITD-MS CHIRAL ANALYSIS OF FLAVOURS 

Guadayol M. 1 , Vendrell E. 1 , Viscasillas C. 1 , Caixach J. 2 , Collgrós F. 1 

1 

Dallant, S.A., Functional Food Research Department 

2 

IDÆA-CSIC, Mass Spectrometry Laboratory, Barcelona 

Corresponding author e-mail: fcollgros@dallant.com 

Introduction Many natural flavour compounds are chiral and both enantiomeric compounds can show different 

properties like taste, smell or biological activity. One way to determinate the authenticity of these flavours is to 

analyse the enantiomeric purity of chiral substances since they have distinctive enantiomeric ratios depending 

on its origin. The detection of using synthetic flavours in food and beverages is a key point that have placed more 

importance since the Regulation EC 1334/2008 of 16 December 2008 on flavourings and certain food ingredients 

with flavouring properties for use in and on food (L354/34 on 31.12.2008) has been published. Some techniques 

have been used to analyse chiral compounds, but most of them are very complicated and don’t work with complex 

matrices. GCxGC-ITD-MS is a novel, simple and effective technique to evaluate the enantiomeric excess in 

complex mixtures. The aim of this study was to develop a methodology using GCxGC-ITD-MS to determinate 

different kinds of chiral flavouring substances alone or in a complex food flavour. Material and methods Chiral 

compounds were injected directly while a headspace-solid-phase microextraction (HS- SPME) coated with 

polydimethylsiloxane (PDMS, 100μm thickness) was used to collect and concentrate flavours from food matrices. 

Analyses were performed on two Varian Model 3800 gas chromatographs, which were connected together by 

means of a heated transfer-line. The first chromatograph is equipped with FID detector and DEANS Valco valveless 

system. The second chromatograph is connected to an ion trap mass analyser with a liquid CO2 capillary cold 

trap. The dual column set used comprised a VF-1ms ,15m x 0,25mm i.d, 0,25μm thickness VF1-ms coated column 

(Varian, CP8907) in the first dimension coupled to a 30m, 0.25 mm id, 0.25 μm thickness Ciclolsil-B coated column 

(J&W, 112-6632 for the second dimension through a deactivated fused silica tubing. All the spectra were recorded 

in the electron impact mode at an ionisation voltage of 70eV an a scan speed m/z=30-200 in 0,5 second. Results 

and discussion The results demonstrate that HS-SPME combined with GCxGC–ITD-MS is a good technique to 

separate flavouring substances alone or in a complex food flavour like essential oils or flavour fruit juices. Every 

chiral analysed compound needed a specific temperature program and a specific valve program, especially in a real 

matrix. 50 compounds were analysed with four sorts of chiral columns. Only Cyclosil-B was capable of separating 

35 flavour compounds with Rs>1 despite having different structures. These chiral compounds belong to varied 

families like lactones (δ-hexalactone, γ-undecalactone, γ-dodecalactone), esters (ethyl 2-methylbutanoate, methyl 

2-methylbutanoate, 3-octylacetate), terpenes (limonene, α-pinene, β-pinene), terpene alcohols (terpinen-4-ol, 

linalool, menthol), alcohols (3-hexanol, 2-methylbutanoll) or acids (2-methyl butanoic acid) In conclusion, GCxGC- 

ITD-MS showed to be a useful technique to research the natural origin of a food and beverage flavour and a powerful 

tool for any quality and safety control purposes. 


517


P15-094 OBTENTION AND CHARACTERIZATION OF GLYCOSYLATED DERIVATIVES OF LOW MOLECULAR WEIGHT CHITOSAN 

THROUGH AMIDE FORMATION 

Ruiz-Matute A.I., Cardelle-Cobas A., Montilla A., Olano A., Corzo N., 


Corresponding author e-mail: acardelle@ifi.csic.es 

Chitosans are cationic biopolymers composed of β (1→4) linked residues of D-glucosamine and 

N-acetyl-D-glucosamine which have been described to have broad applications in medicine, cosmetics, foods and 

agriculture (1). However, their use is limited since they are only soluble in acidic medium (pH


P15-095 FORMATION OF CHITOSAN DERIVATIVES BY REDUCTIVE ALKYLATION WITH MODIFIED FUNCTIONAL PROPERTIES 

Cardelle-Cobas A., Ruiz-Matute A.I., García-Bermejo A.B., Montilla A., Corzo N., Olano A. 


Corresponding author e-mail: anaruizm@ifi.csic.es 

Chitosan is a natural polymer which presents various biological activities, such as antitumor, cholesterol-lowering, 

antibacterial and antifugal effects and it has been demostred than these properties depend largely on its number 

of free amino groups in the molecule and on its molecular weight and origin. Due its characteristics, chitosan is 

used in such diverse fields as technology, foods, cosmetics, medicine, biotechnology, agriculture and the paper 

industry. However, despite this large number of properties presented by chitosan, its applications are limited by its 

insolubility at neutral and basic pH. Chitosan is soluble in acid solutions at pH below its pka (


P15-096 USE OF DIFFERENT ANALYTICAL TECHNIQUES TO EVALUATE CHITOSAN-CARBOHYDRATE COMPLEXES FORMATION VIA 

MAILLARD REACTION 

García-Bermejo A.B., Cardelle-Cobas A., Ruiz-Matute A.I., Olano A., Corzo N., 


Corresponding author e-mail: anbegab@ifi.csic.es 

In recent years, chitosan has received much attention because it can be an alternative to synthetic polymers in many 

technological processes, presenting very interesting features such as its biocompatibility and biodegradability. 

However, chitosan is insoluble at neutral and basic pHs which constitute a limitation on certain applications on food. 

Chitosan is the only polysaccharide in nature owing cationic properties due to the presence of amino groups in its 

molecule and it is known that their different properties depend largely on its amino groups, molecular weight and 

origin. One of the most employed strategies is the introduction of carbohydrate residues in the chitosan molecule, 

which improves its hydrofile character of chitosan increasing the solubility(1). The Maillard reaction takes place by 

condensation of the carbonyl group of reducing sugars, aldehydes or ketoses and amino groups of amino acids, 

peptides, proteins or nitrogenous compounds and is a major reaction that takes place during the processing 

and storage of foods. One of the most sensitive methods for evaluating the early steps of Maillard reaction is the 

determination of Amadori compounds(1) through of furoyl-methyl amino compounds determination. The presence 

of free amino groups in chitosan makes it a potential compound to be involved in this reaction. The objective of this 

work has been to assess Maillard reaction development and evolution during storage of dried chitosan-carbohydrate 

mixtures. Glycosylation mixtures were prepared by freeze drying a solution containing lactose and chitosan 

previously purified in our laboratory. Storage assays were performed under vacuum in a desiccator at 60ºC for 11 

days and a water activity of 0.65 (NaNO2). Samples were taken at different times during 11 days and then hydrolyzed 

under conditions previously reported in the literature(1). Ion pair RP-HPLC-UV analyses of hydrolyzates were carried 

out in a C18 column (250 x 4.6 mm i.d.) at room temperature using 20% acetonitrile, 0.2% formic acid and 5 mM 

sodium heptanesulfonic acid solution as mobile phase. Flow rate was 1.2mL/min and wavelength used was 280nm(2). 

HPAEC-PAD, SEC-HPLC and FT-IR were used to quantify unreacted lactose, molecular weight of chitosan fractions 

and determine functional groups, respectively. RP-HPLC-UV profiles of hydrolyzates of stored chitosan-carbohydrate 

mixtures showed the presence of an unknown and major peak with less retention time than 2-furoyl-methyl lysine 

(furosine) used as standard. During storage, a maximum value of this compound was reached after 3 days and then 

decreased with time. HPAEC-PAD analysis of lactose showed a decrease with the time of storage, corresponding 

to the increase observed of unknown compound. SEC-HPLC analyses showed molecular weight changes during 

formation of chitosan-carbohydrate complexes. Also, a modification of the characteristic bands of amino groups 

of chitosan in the FT-IR spectra was observed. These results show the usefulness of this analytical method to 

evaluate the progress of Maillard reaction. Acknowledgments: This work was financed by projects AGL 2008-00941/ 

ALI, Alibird-CM-P 2009/AGR 1469 and CONSOLIDER Ingenio 2010-Func-CFood CSD 2007 00063. Bibliography: (1) 

Moreno et al., Food Research International 39 (2006) 891–897;(2)Delgado T. et al., Chromatographia 33 (1992) 374- 

376. 

520 



P15-097 SEPARATION OF PROANTHOCYANIDINS FROM FOOD SUPPLEMENTS USING DIFFERENT HPLC COLUMNS 

Smrke S., Vovk I., Simonovska B. 


Corresponding author e-mail: irena.vovk@ki.si 

In the past decade there has been a great deal of interest in proanthocyanidins (PAs), especially oligomeric PAs 

because of their health benefits and recently a variety of food supplements produced from plant material containing 

PAs have emerged in the market. PAs are naturaly occuring flavan-3-ols that are present in a wide variety of plants 

and foods. Naturally most abundant monomers are (+)-catechin and (-)-epicatechin. Mixtures of procyanidins from 

plants consist of chains of monomeric units most commonly linked through C-4 to C-8 interflavan bonds, in some 

cases procyanidins are linked through C-4 to C-6 bonds (both are called type-B PAs) and double linked heterooligomers 

also exist (type-A PAs). Because of the huge variety of compounds chromatographic separation and both 

qualitative and quantitative work are very difficult. 

We have developed two HPLC methods one for a rapid and simple quantification of (+)-catechin and (-)-epicatechin 

and another for separation of oligomeric PAs. For the determination of (+)-catechin and (-)-epicatechin in food 

supplements samples were extracted in 70% aqueous acetone and if further purification was needed because 

of interference of polymeric PAs, samples were purified using only liquid-liquid extraction. The separation was 

achieved using a Luna PFP column and detection of compounds was carried out using a fluorescence detector. The 

advantage of PFP column over the conventional C18 are shorter retention times because of better retention factors 

of (-)-epicatechin and procyanidin B2. 

Hydrophilic interaction chromatography is well known for the separation of PAs according to the degree of 

polymerization (DP), however, our studies of Luna HILIC column have shown also some separation of compounds 

of the same DP providing more information about the structural diversity of oligomeric PAs. Even better separation 

capacity was achieved by means of off-line 2-dimensional chromatography where fractions of the same DP collected 

from the separation run on a HILIC column were further separated on C18 or PFP columns. 


521


P15-098 TLC-MS METHOD FOR DETECTION OF NEOXANTHIN, VIOLAXANTHIN, LUTEIN AND BETA-CAROTENE 

Cernelic K. 1,2 , Simonovska B. 2 , Vovk I. 1,2 , Albreht A. 2 

1 

EN-FIST Centre of Excellence, Ljubljana 

2 



Over 600 known carotenoids are divided into oxygenated xanthophylls such as lutein, zeaxanthin, violaxanthin, 

neoxanthin and hydrocarbon carotenes such as beta-carotene, alpha-carotene and lycopene. Due to their structures 

(several possible isomers, instability, etc.) their extraction, isolation, identification and determination represent a big 

challenge. As strong antioxidants they might have also generally a beneficial effect on human health as has been 

shown by the epidemiological studies. 

The aim of our study was to develop a method based on thin-layer chromatography–mass spectrometry (TLC-MS) for 

the detection of the main carotenoids in spinach: neoxanthin, violaxanthin, lutein and beta-carotene. Two different 

types of silica gel 60 sorbents (C18 RP and CN) and several developing solvents were successfully used for the 

separation of the studied compounds from the extract prepared from 1 g of lyophilized spinach (equal to 12.67 g of 

fresh material) extracted with 25 ml of ethanol for one hour at a room temperature. All the plates were pre-washed 

with methanol before the application of the samples and were developed ascending in a normal saturated chamber. 

Two TLC methods using the HPTLC C18 RP plate developed in methanol : acetone : n-hexane (70:20:15 v/v/v) and 

the HPTLC CN plate developed in chloroform : n-hexane : methanol (35:60:5, v/v/v) were selected for the TLC-MS 

studies. In the case of the separation on the HPTLC C18 RP plates followed by extraction of the carotenoids by 

means of TLC-MS interface (Camag) using methanol as an eluent and adding 1% of an ethanoic acid after elution, 

their identification was achieved with APCI-MS (lutein m/z =551 - [MH +-H2O], violaxanthin m/z =601 - [MH+], 

neoxanthin m/z =583 - [MH+-H2O], and beta-carotene m/z = 537 - [MH+]). Unfortunately, due to the interfering noise 

appearing at the extraction of the studied compounds from the HPTLC CN plates by means of TLC-MS interface, 

it was not possible to detect the studied compounds by MS, but they were successfully identified in the classical 

way by scraping the sorbent, followed by extraction of compounds and direct injection into the mass detector. The 

confirmation of violaxanthin and neoxanthin on both sorbents was made also with vapors of HCl, where the bend 

with violaxanthin in a few seconds changed color from yellow to blue, as the evidence of the presence of diepoxides 

and the band with neoxanthin changed color to green, as the evidence of monoepoxides. 

522 



P15-099 TLC-MS DETECTION OF LECITHIN AND PROANTHOCYANIDINS IN CHOCOLATE 

Glavnik V., Simonovska B., Vovk I., Albreht A. 



Thin-layer chromatography (TLC) is inexpensive, quick, and useful technique for screening and quantification of 

complex mixtures. Until now, many desorption ion sources (MALDI, IR, FAB, etc.) were employed for compound 

identification from the plates but these ion sources could not be coupled to LC system. Recently Camag has 

launched a TLC-MS interface, a device that on-line extracts analyte from various TLC/HPTLC plates using suitable 

solvent and LC pump and feeds extracts into MS detector with APCI, APPI or ESI ionisation sources. 

Lecithin is a complex mixture of phospholipids (phosphatidylcholine (PC), phosphatidylinositol, 

phosphatidylethanolamine (PE)), which is added to chocolate during the manufacturing process to give a smooth and 

fluid consistency to the chocolate. Chocolate is also a good source of plant-derivated compounds having antioxidant 

properties namely proanthocyanidins which consist of flavanols (monomeric units). The main flavanol in chocolate is 

(-)-epicatechin. Chocolate also contains considerable amount of (-)-epicatechin dimer, procyanidin B2. 

Separation of phospholipids was performed on pre-washed HPTLC silica gel 60 plates using chloroform-methanolwater 

(65:25:4, v/v/v) as a developing solvent in a saturated twin-trough chamber, while proanthocyanidins were 

separated on cellulose plates using 1-propanol-water-acetic acid (20:80:1, v/v/v) as a developing solvent in a 

horizontal chamber. Mass spectra were scanned on a Finnigan LCQ mass spectrometer equipped with electrospray 

ionization source in a positive mode for lecithin and in a negative mode for (-)-epicatechin and procyandin B2. 

Methanol was applied as eluent for both groups of compounds. Characteristic m/z values were 760 and 782 for 

lecithin standard while 289 and 578 m/z values were typical for (-)-epicatechin and procyanidin B2 standards, 

respectively. All these m/z values accept 760 were also characteristic for chocolate sample. The reason for 

discrepancy in the case of lecithin is in the source of lecithin. In the chocolate is mainly soybean lecithin while 

in used standard, lecithin is from egg yolk. TLC-MS technique can be very useful for detection or identification 

of proanthocyanidins and lecithin but requires higher applications of the analytes than derivatization with 

4-dimethylaminocinnamaldehyde for proanthocyanidins and 5% molybdatophosphoric acid for lecithin. Therefore, 

quantification of investigated compounds should be performed with derivatization of the plates. 


523


P15-100 RAPID ANALYSIS OF 5-NITROIMIDAZOLES AND THEIR HYDROXY METABOLITES IN ANIMAL TISSUE BY LC-MS/MS 

León N., Igualada C., Moragues F., Roca M. 

Conselleria Sanitat, Public Health Research Center (CSISP), Valencia 

Corresponding author e-mail: leon_nur@gva.es 

5-Nitroimidazoles are veterinary drugs widely used for the treatment and prevention of bacterial and protozoal 

diseases in poultry besides for swine dysentery. However, it has been reported that 5-nitroimidazoles show 

mutagenic, carcinogenic and toxic properties. Dimetridazole (DMZ), metronidazole (MNZ) and Ronidazole (RNZ) are 

included in the table 2 (prohibited substances) of the Commission Regulation nº 37/2010. Ipronidazole (IPZ) and the 

hydroxymetabolites (HMMNI, MNZOH and IPZOH) are not licensed as veterinary drugs and are considered forbidden 

compounds. In consequence, any presence of the 5-nitroimidazoles and their metabolites in animal products has to 

be considered as a violation of the regulations. A MRPL of 3 μg/kg is recommended in the CRL Guidance Paper 

(7 Dec 2007) for each nitroimidazole and also for their metabolites in animals products. A rapid, sensitive and reliable 

multiresidue method by liquid chromatography tandem mass spectrometry (LC-MS/MS) for the determination of four 

5-nitroimidazoles (MNZ, DMZ, RNZ, IPZ), and three of their metabolites (HMMNI, MNZ-OH and IPZ-OH) in animal 

tissue, was developed. It was validated in accordance with the Commission Decision 2002/657/EC. The method was 

based on an organic extraction with acetonitrile followed by centrifugation for 10 min at 4000 rpm. The supernant 

was collected and clean-up with hexane and evaporated to dryness. The determination was achieved by LC-MS/MS 

using electrospray source in positive ion mode. The combination of a fast clean-up with LC-MS/MS has proved to be 

a suitable and rapid strategy for the surveillance of nitroimidazoles in animal tissue. 

524 



P15-101 DETERMINATION OF PBDES IN FISH BY GAS CHROMATOGAPHY-TRIPLE QUADRUPOLE MASS SPECTROMETRY AND 

ESTIMATION OF THE DIETARY INTAKE IN THE POPULATION OF VALENCIA (SPAIN) 

Pardo O. 1 , Beser M.I. 2 , Yusa V. 2 , Beltran J. 3 

1 

CSISP, Food Safety 

2 

CSISP, Public Health Laboratory 

3 

Univesity Jaume I, Castelló de la Plana 

Corresponding author e-mail: pardo_olg@gva.es 

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in plastics and textile coatings, 

and these compounds have been recognized as ubiquitous environmental contaminants [1]. PBDEs are chemicals 

of concern because their rapidly increasing levels both in humans and in the environment and for their potential for 

cancer and others health effects [2]. The European Food Safety Authority has requested recently data on levels of 

those compounds in foodstuffs in addressing the human exposure assessment of BFRs [3], due to diet is the main 

route for human exposure to PBDEs. Since fish is an important part of the human diet [4], the possible impact on 

human exposure has to be closely monitored. This has been shown in a recent studies [5, 6], which indicated that 

fish contributed significantly to the human dietary exposure to PBDEs. The analytical methodology consists on an 

extraction step by pressurized liquid extraction (PLE) at 100 ºC and 1500 psi with n-hexane:dichloromethane (1:1, 

v/v) as extraction solvent followed by two cleanup steps, the first by gel permeation chromatography (GPC) and a 

second using solid phase extraction with silica columns. Gas chromatography coupled to triple quadrupole tandem 

mass spectrometry (GC-MS/MS) with electron impact (EI) and working in selected reaction monitoring (SRM) mode 

has been selected in order to analyze the selected compounds. Collision energy and injector settings have been 

optimized for all compounds. In order to fulfill the EFSA requirements PBDEs congeners #28, 47, 99, 100, 153, 154, 

183 and 209 and the PBB congener 153 have been determined in 123 fish samples purchased in Valencian markets 

in order to estimate the toxicological risk associated with their intake. Among analyzed compound, BDE-47 and 

BDE-99 showed the highest levels (averages of 361.1 and 307.5 ng/kg of wet weight respectively). The estimated 

dietary intake of PBDEs and BBD-153 due to fish consumption for a standard male adult of 68.5 kg body weight has 

been found to be 26.6 ng/day which is lower to that reported in a 2008 survey made in Catalonia, Spain. References 

[1] Y. Ashizuka, R. Nakagawa, K. Tobiishi, T. Hori, T. Iida. J. Agric. Food Chem. 53 (2005) 3807. [2] L.S. Birnbaum, D.F. 

Staskal, Environ. Health Perspect. 112 (2004) 9. [3] EFSA Journal (2009) Request data on BFR levels in foodstuffs. 

[4] J.L. Domingo, A. Bocio, Environ. Int. 33 (2007) 397. [5] C. Thomsen, H.K. Knutsen, V.H. Liane, M. Froshaug, H.E. 

Kvalem, M. Haugen, Mol. Nutr. Food Res. 52 (2008) 228. [6] J.L. Domingo, R. Martí_cid, V. Castell, J. M. Llobet. 

Toxicology 248 (2008) 25. 


525


P15-102 DETERMINATION OF OCHRATOXIN A BY CAPILLARY HPLC WITH LASER INDUCED FLUORESCENCE DETECTION USING 

DISPERSIVE LIQUID-LIQUID MICROEXTRATION 

Arroyo-Manzanares N., Gámiz-Gracia L., García-Campaña A.M. 


Corresponding author e-mail: amgarcia@ugr.es 

Ochratoxin A (OTA) is a toxic secondary metabolite, naturally produced by several fungi (Penicillium and Aspergillus 

species), with carcinogenic, nephrotoxic, teratogenic, immunotoxic, and possibly neurotoxic properties. OTA is 

considered as a possible human carcinogen and maximum residue limits (MRLs) for this compound have been 

established by the EU in several foods (Directive (CE) Nº 1881/2006). As an example, the MRL in wine has been 

set at 2 µg Kg-1. Dispersive liquid–liquid microextraction (DLLME) is a novel environmentally friendly samplepreparation 

technique, showing important advantages because it is fast, inexpensive, easy to operate with a high 

enrichment factor and consumes low volume of organic solvent. This method is based on a ternary component 

solvent system in which the extraction solvent and disperser solvent are rapidly injected into the aqueous sample, 

containing the analytes, by a syringe. The mixture is then gently shaken and a cloudy solution (water/disperser 

solvent/extraction solvent) is formed in the test tube. After centrifugation, the fines particles of extraction solvent 

are sedimented in the bottom of a conical test tube and the analytes in the sedimented phase can be determined by 

different techniques [1 ,2]. In this communication, capillary HPLC coupled to laser-induced fluorescence detection 

(LIF, He-Cd laser, excitation 325 nm) is proposed for the determination of OTA in wine samples after previous 

extraction of the analyte by DLLME. A SDS micellar solution has been used in the mobile phase to increase the 

fluorescence intensity inherent to OTA. Chromatographic conditions have been established as follows: Luna C18 

column (150×0.5 mm, 5 µm); a mobile phase consisting on water (2% acetic acid, 0.2 M SDS): methanol (30:70, v/v) 

at a flow rate of 14 µL/min; a temperature of 40ºC; and an injection volume of 1.2 µL. Under these conditions a limit 

of detection in the low ng L-1 range (3×S/N) was obtained. DLLME conditions were optimized by using experimental 

designs, obtaining the following values: 5 mL of sample solution, 660 µL of chloroform as extraction solvent, 940 µL 

of acetonitrile as disperser solvent and 5% (w/v) of NaCl. The sedimented phase was evaporated to near dryness 

using a gentle stream of N2 and reconstituted with 1 mL of methanol. The solution was filtered and injected into the 

chromatographic system. Under these conditions, a very clean extract was obtained, with recoveries about 95%. 

Thus, DLLME has shown to be a very selective and efficient extraction procedure, which combined with the highly 

sensitivity of the capillary HPLC-LIF system could provide a very useful method for the analysis of OTA in wine, being 

also an alternative to the expensive immunoaffinity columns commonly used for extraction of mycotoxins. To our 

knowledge, this is the first time that this methodology is used for mycotoxin determination. Acknowledgements: The 

“Junta de Andalucía” supported this work (Proyecto de Excelencia Ref: P07-AGR-03178). [1] M. Rezaee, Y. Yamini, M. 

Faraji, J. Chromatogr. A, 1217 (2010) 2342–2357. [2] C. Bosh Ojeda, F. Sánchez Rojas, Chromatographia, 69 (2009) 

1-11. 

526 



P15-103: APPLICATION OF PRE-COLUMN DERIVATIZATION WITH 2,3-NAPHTALENEDIALDEHYDE AND RP-HPLC-FL FOR 

THE ANALYSIS OF GLUTATHIONE AND ITS PRECURSOR G-GLUTAMYL-CYSTEINE IN WINES AND MODEL WINES 

SUPPLEMENTED WITH OENOLOGICAL INACTIVE YEAST PREPARATIONS 

Andujar-Ortiz I., Rodríguez-Bencomo J.J., Moreno-Arribas, M.V., Del Pozo-Bayón M.A. 

Instituto de Investigación en Ciencias de la Alimentación (CIAL) (CSIC-UAM), Biotecnología y Microbiología 

Glutathione (GSH) is a thiol containing tripeptide (L-γ-glutamyl-L-cysteinyglycine) that can be present in musts and 

wines. Its importance for wine quality is associated to its strong antioxidant properties helping in the stabilisation 

of some aroma components and in reducing the browning of musts and wines. Due to its antioxidant activity, in 

recent years different Inactive Dry Yeast Preparations (IDYs) rich in GSH are being employed in winemaking in order 

to prevent wine oxidation. However, there are not scientific evidences about the amounts of biological GSH from 

IDYs that can be released into the wines. Therefore, this study aimed to develop a HPLC methodology to determine 

reduced GSH, oxidized GSH (GSSG) and its precursor γ-glutamylcysteine (γ-GC). This methodology was applied to 

quantify GSH and related compounds in model wines supplemented with 8 IDYs. Finally, the evolution of GSH during 

the production and shelf-life of a wine supplemented with a GSH enriched IDY has been performed. 

Two RP-HPLC-FL methods were developed to determine GSH, GSSG and γ-GC by adapting several protocols 

from the literature (1, 2). Total GSH and γ-GC were determined by using a precolumn derivatization with 

2,3-naphtalenedialdehyde (NDA) and borate buffer pH 9.2 containing 1% of ethanethiol. Determination of reduced 

GSH was carried out in similar conditions, with the exception of borate buffer pH 9.2, that was prepared containing 

dithiothreitol 0.5 mM instead of ethanethiol. The GSSG concentration was obtained subtracting the reduced GSH 

level from the total GSH. To know the release of GSH from IDY preparations, eight different types of IDYs supplied 

by three different manufactures were used. IDY preparations were added into the model wines at the same dose 

recommended by manufacturers (0.3 g/L). In addition, two types of rose wines, a control wine and an IDY wine 

(supplemented with an GSH rich IDY preparation) were industrially manufactured. 

The method showed good analytical characteristics for the quantification of γ-GC, GSH and GSSH. For instance, 

the method showed a reproducibility above 6% (n=25) and a linear range between 0-20 mg/L with a R2=0.9982 for 

the determination of total GSH. The results showed that those IDYs recommended to avoid wine oxidation (therefore 

including glutathione in their composition) released different amounts of GSH and γ-GC depending on the type of 

IDY (on the provider type). In addition, other types of IDY without specific applications to act as antioxidants, also 

released small amounts of GSH, likely coming from the yeast cytoplasm. Variations in the evolution of the GSH 

during the winemaking and shelf life of rose wines produced with the GSH enriched IDY was also found, which could 

have important implications for the aroma and colour of the wine. 

References 

1. Lavigne, V., Pons, A., Dubordieu, D. 2007. J. Chrom. A. 1139. 130. 

2. Marchand, S., de Revel, G. 2010. Anal. Chim. Acta. 660. 158. 


527

POSTER 

SESSIONS 

P16 

CLINIC AND 

PHARMACEUTICS

POSTER SESSION 16 - CLINICS AND PHARMACEUTICS 

P16-001 SUITABILITY OF MICELLAR LIQUID CHROMATOGRAPHY FOR DOPING CONTROL 


1 


2 



Ergogenic aids are commonly used, misused and abused in sport, to produce a broad scale of effects: endurance 

during competition, reduction of body weight, aggressiveness, mental concentration and physical strength, delaying 

fatigue and pain desensitisation. The use of pharmacologically active substances to improve performance in sport 

goes back centuries but has increased in the past 40 years. In an article entitled “The toxic torch of the modern 

Olympic Games”, Prendergast et al. (Vet. Hum. Toxicol. 45 (2003) 97–102) state that the quest for greatness has 

driven many athletes and coaches to push for unfair advantages by the use of performance-enhancing (ergogenic) 

substances, commonly referred to as “doping”. The issue of doping control in sport involves the development of: 

(i) reliable analytical procedures and efficient strategies to process (ii) a large number of samples, (iii) for a variety 

of doping agents, (iv) in a short period of time (during sports events). For this purpose, methods needing little 

or no sample preparation are of great interest for screening purposes. Reversed-phase liquid chromatographic 

techniques with aqueous-organic mobile phases and mass spectrometry or diode-array spectrometric detection 

yield satisfactory results for the identification of prohibited substances in sport. However, time-consuming 

sample pre-treatment steps are required, which reduces the sample throughput. The potentiality of micellar liquid 

chromatography (MLC) with hybrid mobile phases containing sodium dodecyl sulphate above its critical micellar 

concentration (CMC) and an organic solvent is here discussed. The surfactant solubilises the protein components 

of urine, serum and plasma samples, which permits their direct injection into the chromatographic system. Only 

dilution and filtering of the samples may be required. This increases the sample throughput, with improved accuracy 

and precision. The elution of drugs in a wide range of polarities allows that most MLC analyses are performed in 

isocratic mode, with short retention times and good selectivity. The limits of detection with MLC making direct 

injection of the physiological sample are often similar to those usually reported for other chromatographic methods, 

which allows the detection of the prohibited substances at least 24–48 h after being administered. However, the 

injection of a large number of untreated samples, or a large volume of sample (> 20 uL) can damage the packing 

material, shortening the life of the column, or force its frequent regeneration. Two of the most important groups 

(anabolic steroids and diuretics), prohibited at all times (in and out of competition), have been mainly considered, 

while methods for all groups of substances prohibited only in competition and in some particular sports (stimulants, 

narcotics, glucocorticoids and beta-blockers) have been developed. Methods for doping substances in horse races 

(local anesthetics, anticonvulsant agents, bronchodilators and calcium channel blockers) have been also developed. 


529


P16-002 DETERMINATION OF ETHOXYDOL IN PLASMA BY MICROEMULSION LIQUID CHROMATOGRAPHY 

Pirogov A.V., Pashkova E.B., Shpigun O.A., 


Corresponding author e-mail: e_pashkova@list.ru 

Ethoxydol (3-hydroxy, 6-methyl, 2-ethylpyridine malate) is a new antihypoxant, showing dose related antiarrhythmic 

effect. In comparison with widely used Propranolol and Mexidol it prevents the development of ventricular fibrillation 

and supposed to be more effective and harmless. Thus, the problem of the determination of ethoxydol in blood 

plasma is of great importance for clinical trials. The technique of microemulsion liquid chromatography (MELC) was 

first reported in 1992 and has been successfully used for the analysis of different pharmaceuticals since then. It 

allows to shorten the time of the analysis in an isocratic mode and to simplify the procedure of sample pretreatment. 

In current work a new selective and simple technique of the determination of ethoxydol in blood plasma by 

microemulsion liquid chromatography with a fluorescent detection was developed. The separation was performed 

on a reversed-phase column Phenomenex Gemini using oil-in-water microemulsion as a mobile phase. The time of 

the analysis was 10 minutes in an isocratic mode. The suggested way of the sample pretreatment allowed to stabilize 

etoxydol in plasma. The samples were diluted twice with the microemulsion and then the plasma proteins were 

precipitated. In comparison with other techniques (one-step protein precipitation, liquid or solid-phase extraction) 

the suggested scheme turned to be simple, quantitative (the recovery was about 98%) and rapid. Absorbance and 

fluorescence spectra of ethoxydol were studied, and it was found that the maximum in the fluorescence spectrum 

was displaced on 40 nm in the microemulsion media in comparison with water-organic solution. The detection limit 

at the selected conditions (297/399 nm) was 50 ng/ml what was 6 times lower that in RP-HPLC mode. The suggested 

technique was successfully applied in clinical trials. 

530 



P16-003 ADME(T) PARAMETERS OF FUSED MANNICH KETONE AND ISOCHROMANONE MOLECULAR LIBRARIES COLLECTED BY 

SEPARATION METHODS 

Huszar M. 1 , Varga A. 1 , Horvath A. 1 , Vantus T. 1 , Lorand T. 2 , Agocs A. 2 , Keri G. 1 , Idei M. 1 

1 

Semmelweis University, 

2 

University of Pécs,, 

Corresponding author e-mail: igazgato@tki.office.mta.hu 

Molecular libraries (such as tetralones, aurones, chromanones, Mannich ketones) containing structurally related 

compounds, have been previously investigated. Recently [1] we presented the relationship between antiproliferative 

activity and lipophilicity in the case of isochromanone molecules and we also showed the importance of ADME(T) 

characterisation and structure-activity relationship in drug research. As an extension of these works further study 

was performed on a 17 member isochromanone and 12 member fused Mannich ketone libraries. In the present study 

the members of the two libraries were characterised by computer calculated and chromatographically measured 

lipophilicity values, biological efficiency and permeability data. Permeability was determined by the fast, effective 

and inexpensive method, called parallel artificial membrane permeability assay (PAMPA). Interaction between the 

molecules and the biological membrane was measured by immobilized artificial membrane (IAM) chromatography 

column which provided additional data for structure-activity relationship studies. The presence of the 

phosphatidylcholine groups of the IAM column makes it possible to mimic biological membrane and its interaction 

with the analysed drug molecule. Compared to the normal reverse phased column, which gives good insight into the 

lipophilicity properties of the measured compounds, this column provided information about permeability and the 

so called “phospholipophilicity”. We found linear correlation between the lipophilicity data (measured by RP-HPLC) 

and the permeability or phospholipophilicity (measured by IAM-HPLC). We also studied the relationship between 

the biological efficiency and the chemical properties, such as lipophilicity, permeability or phospholipophilicity 

to gain a complete database used for the selection of biologically effective and uneffective drug candidates. 

Acknowledgement: This work was supported by the NKTH-ANR MuKIT grant. [1] Huszar, M.; Varga, A.; Horvath, A.; 

Lorand, T.; Agocs, A.; Idei, M.; Mandl, J.; Vantus, T;; Keri, G.: Curr. Med. Chem., 2010, 17, 321-333. 


531


P16-004 MULTIMARKERS SCREENING OF VARIOUS BODY FLUIDS FOR MONITORING OXIDATIVE STRESS DISEASE 

Syslova K. 1 , Kacer P. 1 , Kuzma M. 2 , Vlckova S. 3 , Lebedova J. 3 , Fenclova Z. 3 , Pelclova D. 3 

1 

Institute of Chemical Technology-ICT Prague-Czech Republic 

2 

Institute of Microbiology, Laboratory of Molecular Structure Characterization-Czech Republic 

3 

1st Medical Faculty, Charles University-Czech Republic 

Corresponding author e-mail: Kamila.Syslova@vscht.cz 

Oxidative stress can be defined as a body imbalance between the production of reactive oxygen species (ROS) 

and the biological system ability to readily detoxify the reactive intermediates or easily repair the resulting damage. 

ROS generated near cell membranes oxidize membrane phospholipids (membrane lipid peroxidation), which can 

be further transformed in a chain reaction. Endogenously generated aldehydic lipid peroxidation products are 

malondialdehyde, a,b-unsaturated aldehydes (mainly 4-hydroxynonenal and 4-hydroxyhexenal) and saturated 

aldehydes (hexanal, heptanal and nonanal). Aldehydes are formed by lipid peroxidation of v-6 (arachidonic acid, 

linoleic acid) and v-3 (oleic acid) polyunsaturated fatty acids. The aldehydes (biomarkers) quantification in various 

body fluids (exhaled breath condensate, plasma and urine) represents a remarkable tool for the diagnostics of 

oxidative stress induced diseases. 

The work presents a new method for the determination of aldehyde-biomarkers in body fluids based on LC-ESI/ 

MS/MS. Malondialdehyde, 4-hydroxynonenal and saturated aldehydes (n-C6 to C13 aldehydes) were quantified 

after derivatization with Girard’s reagent T. LC-ESI-MS/MS, operated in neutral loss (NL) mode, was used for its 

exceptionally high degree of selectivity and sensitivity. The combination of mass spectrometry detection with 

separation techniques (HPLC) enabled retaining the analytes of interest from the solvent front, while avoiding a coelution 

of salts and endogenous matrix components that could suppress the ionization of the analytes during the 

electrospray-ionization (ESI). 

The developed method unequivocally enabled a parallel determination of several oxidative stress biomarkers at only 

one analysis run. The method was optimized and validated. Finally, the method was tested on real clinical samples 

collected from patients with different disorders induced by oxidative stress (silicosis, asbestosis) and the results 

were compared to the control group of healthy subjects. Acknowledgement: The work was supported by the Ministry 

of Health of the Czech Republic (Grant NS 10298-3/2009), and the Ministry of Education, Youth and Sports of the 

Czech Republic (Grant CEZ: MSM 223 100001) and Grant Agency of the Czech Republic (Grant GA CR 203/08/H032). 

532 



P16-005 DEVELOPMENT AND VALIDATION OF A RAPID HPLC METHOD FOR RELATED SUBSTANCES OF LASW1338 IN A FUSED- 

CORE COLUMN. IMPORTANCE OF THE GRADIENT DWELL VOLUME 

Carrera F., Serra C., Julià M., Ebri I., Dulsat J.F. 

Laboratorios Almirall, Analysis R&D 

Corresponding author e-mail: francesc.carrera@almirall.com 

LASW1338 is a poly-aromatic retinoid that can be used in dermatology as a potent agent for the treatment of 

psoriasis due to its antiproliferative activity. As a new active pharmaceutical ingredient, the development and 

validation of analytical methods to control assay and impurities in the drug substance is a must. The analysis of 

related substances by HPLC traditionally demands long methods which today are being replaced by ultra high 

pressure LC methods (UPLC, UHPLC, rrLC…). In addition, there is the possibility of imitating an UPLC method by 

using a fused-core column in a conventional HPLC system. In this work, a rapid (12 minutes) method has been 

developed to separate LASW1338 and its potential impurities and intermediates (up to 10) by using a fused-core 

(or core-shell) column. As the .range of polarities of such a family of compounds is quite wide, the use of a strong 

gradient of mobile phases and flow rate has been a must. The same chromatographic method is also used for the 

assay determination. Both methods (for related substances and for assay), have been validated: specificity, detection 

and quantification limits, linearity, range, system precision, method precision, accuracy, stability of solutions and 

robustness have been evaluated. Both methods have shown to be valid for their purpose. Once the methods had 

been developed and validated, their applicability in several LC systems has been studied: different brands (Waters, 

Agilent and Thermo), different concepts (mix at high or low pressure) and different generations (HPLC or UPLC) 

have been tested. The gradient dwell volume, also called gradient delay volume, of each instrument has shown to 

be crucial to keep the selectivity of the method. If a rapid LC gradient method is intended to be used in different 

systems, the gradient dwell volume of each system should be previously evaluated, and the gradient profile should 

be changed accordingly. In other words, the validation performed will be only valid if the method is applied to 

systems with similar gradient dwell volumes. 


533


P16-006 ADJUSTING SELECTIVITY WITH COLUMN CHEMISTRY IN LC/MS 

Pereira L., Faulkner W., Barattini V., Milton D. 

Thermo Fisher Scientific, Chromatography Consumables & Speciality Products 

Changes in reversed phase HPLC (RP-LC) selectivity are generally obtained by changing the mobile phase 

composition or column packing. However, solvent and buffer type choices are restricted in LC/MS since these need 

to balance the chromatographic separation requirements with the ionization efficiency. In LC/MS with Atmospheric 

Pressure Ionization (API), solution and gas phase chemistries need to be optimized in order to maximize ionization. 

Generic mobile phases, which provide good detection signal are generally chosen first, and therefore the analyst 

is left with another parameter, column chemistry, to adjust the selectivity of the LC separation. High performance 

column packings in a range of functionalities for selectivity screening or fine-tuning complex separations are 

commercially available. Stationary phases having highly pure silica as a support and reproducible, robust bonded 

phase give high performance with MS compatible generic mobile phases. In this poster the selectivity changes that 

can be obtained with alkyl chain, polar endcapped, phenyl, phenyl/hexyl chemistries when using generic mobile 

phases are illustrated for basic, acidic and neutral analytes. The columns used in this study use proprietary bonding 

technology to facilitate the best possible performance even with weakly buffered mobile phases, typical of LC/MS 

applications. This approach for stationary phase screening significantly reduces method development time. 

534 



P16-007 SELECTIVE SOLID PHASE EXTRACTION OF COCAINE FROM BIOLOGICAL SAMPLES USING MOLECULARLY IMPRINTED 

POLYMERS 


UMR PECSA 7195 CNRS - UPMC - ESPCI Paris Tech 

In trace analysis, the study of biological samples such as hair or urine requires sample pre-treatment to clean-up 

the sample matrix. The large choice of available sorbents for solid phase extraction (SPE) makes it a highly versatile 

method for this purpose. However, the retention of target analytes results from non-selective interactions with the 

stationary phase that may lead to the co-extraction of interfering substances. Therefore, selective sorbents based 

on a molecular recognition mechanism can be developed such as molecularly imprinted polymers (MIPs). MIPs are 

polymeric materials with specific recognition sites designed for a target molecule. 

In this work, a MIP was made for the specific recognition of cocaine and its main metabolite, benzoylecgonine. The 

synthesis of the imprinted material was carried out by the assembly of an acidic monomer and a cross-linker around 

the template molecule (cocaine) in an aprotic and slightly polar solvent. The subsequent photochemically initialized 

radical polymerization provided a highly crosslinked rigid material. An extraction profile in pure organic medium 

(acetonitrile) was then studied. Therefore, a selective extraction procedure was carefully developed and optimized. 

The capacity of the imprinted material was determined, and an extraction procedure was successfully applied to the 

analysis of spiked hair samples. Indeed, an extraction recovery of 88 % was obtained for cocaine on the MIP while 

0% was extracted on the non imprinted polymer. 

Finally, an extraction procedure was developed in aqueous media. Since cocaine is rapidly metabolized in human 

body to benzoylecgonine, this metabolite is still determined in both blood and urine. Consequently, the retention 

on the MIP of the principal metabolite of cocaine was also studied. A strong and selective retention was already 

obtained for its extraction in pure organic media. Hence, the extraction procedure was optimized in aqueous media 

and applied to the clean-up of spiked urine samples. The support will be evaluated to other complex samples such 

as blood to improve the sensitivity of the analysis. 


535


P16-008 HILIC CHROMATOGRAPHY A TOOL FOR PHARMACEUTICAL QUALITY CONTROL OF POLAR COMPOUNDS 

Rupérez F.J., Fernández-Fidalgo C., Martínez-Alcázar M.P., Barbas C., García A. 

Universidad CEU-San Pablo, CEMBIO, Madrid 

Corresponding author e-mail: ruperez@ceu.es 

One of today’s promising new developments in pharmaceutical industries is related to reformulate existing drugs 

to develop line extensions for OTC products. Safety concerns require testing to be done to ensure the absence 

of degradation products at an established level during stability of the pharmaceutical formulations. A new orally 

disintegrating formulation with the association of acetaminophen (paracetamol) and phenylephrine hydrochloride 

has been launched to the market and its quality control is a challenge. These actives, as usual components of 

preparations against the common cold, have long been studied, as well as their degradation products [1-3]. 

Nevertheless, new formulations mean new excipients with possibly new interactions and degradation products. In 

the analytical point of view this is not an easy task due to the imbalance of the analytes in the formulation (1000 

mg acetaminophen vs 8.2 mg phenylephrine), with very different polarities. In addition this new formulations 

contain numerous excipients some of them polar and UV absorbing, such as saccharine and flavours, with similar 

chromatographic behaviour to impurities and/or degradation products. Hydrophilic Interaction Chromatography 

(HILIC) is still under research, but it is rapidly catching on as a method for analyzing polar compounds, that are 

poorly resolved by reverse-phase liquid chromatography. It can offer great advantages in the field of pharmaceutical 

analysis due to the polar nature of compounds and its suitability for MS analysis due to the use of a high ratio of 

organic solvent, mainly acetonitrile. In order to explore the applicability of HILIC mode to the analysis of this new 

dosage form, four different columns were tested: Atlantis HILIC (Waters), Luna HILIC (Phenomenex), TSKGel Amide 

(Tosoh) and Supelcosil LC-Si (Supelco). The best results were achieved with the Type A Silica column (Supelcosil) 

where the main impurity of acetaminophen, 4-aminophenol, was successfully separated with 30% aqueous phase 

(25 mM acetic acid pH 3.0) in acetonitrile. During stability tests (forced degradation) two unknown impurities 

with similar spectra to phenylephrine appeared and the method was optimised (pH 5.0, 75% acetonitrile) for their 

separation and transferability to mass spectrometry (1200 HPLC, 6520 QTOF from Agilent), where a third degradation 

compound was detected. With data from MS and MS/MS, structures of the degradation products have been 

proposed, which were not previously described in Pharmacopoeias, corresponding to interactions of phenylephrine 

with excipients. [1] Marin, A; Espada, A; Vidal, P; Barbas, C. Major degradation product identified in several 

pharmaceutical formulations against the common cold. Anal. Chem. 2005, 77(2): 471-477 [2] Marin, A; Barbas, C. 

LC/MS for the degradation profiling of cough-cold products under forced conditions. J. Pharm. Biomed. Anal. 2004, 

35(5): 1035-1045 [3] Marin, A; Garcia, E; Garcia, A; Barbas, C. Validation of a HPLC quantification of acetaminophen, 

phenylephrine and chlorpheniramine in pharmaceutical formulations: capsules and sachets. J. Pharm. Biomed. Anal. 

2002, 29(4):701-714 

536 



P16-009 DETERMINATION OF ACIDITY CONSTANTS BY THE CE INTERNAL STANDARD METHOD: ACIDIC INTERNAL STANDARDS 

Cabot J.M., Fuguet E., Ràfols C., Bosch, E., Rosés, M., 

University of Barcelona, Química Analítica 

Corresponding author e-mail: marti@apolo.qui.ub.es 

The determination of acidity constants is of main interest, specially in the pharmaceutical industry, since many 

potential drugs are weak acids or bases, and their use in further studies strongly depend on the value of certain 

physicochemical parameters such as the acidity constant, hydrophobicity, and solubility. Several methods to 

determine acidity constants have been described in the literature. Among them, capillary electrophoresis offers 

several advantages since the presence of impurities is not so important and only low amounts of sample are 

required. However, the classical CE method implies the measurement of the mobility of the substance of interest 

at several pH values, by the preparation of adequate buffers at constant ionic strength in different pH ranges. The 

proposed methodology is based on the use of an internal standard of similar pKa value as the analyte. It has all the 

advantages of CE as method for pKa determination, but in this case the number of measured points is lower and 

no pH measurement of the buffer solutions is required. In this work the fast CE method has been applied to the 

determination of acidity constants, and a reference list of acidic internal standards is proposed. [1] E. Fuguet, C. 

Ràfols, E. Bosch, M. Rosés, J. Chromatogr. A 1216, 2009, 3646-3651 


537


P16-010 CHARACTERIZATION OF VACCINE ADJUVANT COMPONENTS BY MS AND HPLC-MS 

Cotte J.F., Sonnery S., Martial F., Dubayle J., Dalençon F., Talaga P., Haensler J., Adam O. 

Sanofi Pasteur, Research Department 

Corresponding author e-mail: jean-francois.cotte@sanofipasteur.com 

Sanofi Pasteur, the vaccines division of sanofi-aventis group, is the largest company in the world entirely devoted to 

human vaccines. Bacteria, viruses and parasites are for the human population a source of more and more resistant 

enemies. In this respect, vaccines turn out to be very interesting weapons for fighting against infections. 

There are several types of vaccines according to the nature of the antigens entering their composition. It is often 

necessary to formulate these antigens with an adjuvant able to increase and drive the specific immune response. 

Many kinds of adjuvant candidates exist, including but no limited to aluminum salts, liposomes and emulsions. 

Among the adjuvant formulations developed by Sanofi Pasteur, the oil-in-water emulsion-based adjuvant, is a 

promising family [1]. 

Oil-in-water emulsions can be considered as dispersed systems, with an oily phase dispersed in an aqueous phase, 

and stabilized by surfactants. Among the emulsions evaluated, some are stabilized by two surfactants, a hydrophilic 

one belonging to the family of polyethoxylated alcohol and a hydrophobic one from the sorbitan oleate’s family. 

The work presented in this poster concerns the use of Mass Spectrometry (MS) and High Performance Liquid 

Chromatography-Mass Spectrometry (HPLC-MS) for both the characterization and the quantification of the two 

surfactants used in the emulsions. MS was indeed a powerful analytical tool for complex mixture characterization, 

such as surfactants. 

First part of the work is about MS characterization of the two surfactants. Determination of fatty alcohols and 

ethylene oxide distribution for the polyethoxylated alcohol, and nature of fatty acids for sorbitan oleate, have been 

performed. 

Then, a quantification method for the two surfactants has been successfully developed after optimization of HPLC- 

MS analytical conditions and selection of some relevant ions. 

[1] Vogel, F., Caillet, C., Kuster, I., Haensler, J. 2009. Emulsion-based adjuvants for influenza vaccines. Expert Rev. 

Vaccines, 8, 483-492. 

538 



P16-011 SIMULTANEOUS DETERMINATION OF DEXPHANTENOL, MEPYRAMINE MALEATE AND LIDOCAINE HYDROCHLORIDE IN 

COMBINED PHARMACEUTICAL GELS BY CAPILLARY ELECTROPHORESIS 

Basmakci G., Satana H.E., Yarimkaya S., Ertas N., Goger N. 

Gazi University, Analytical Chemistry 

Corresponding author e-mail: ngoger@gazi.edu.tr 

A new capillary electrophoresis method was developed to analyze pharmaceutical preparations containing ternary 

combination of dexphantenol, mepyramine and lidocaine simultaneously. Best results were obtained by using 20 

mM pH 3,0 phosphate buffer as the background electrolyte. The separation was performed through a fused-silica 

capillary (75 µm internal diameter, 50 cm total length, 41 cm effective length) at 20˚C with the application of 5 

seconds of hydrodynamic injection at 50 mbar pressure and potential of 30 kV. Detection wavelength was 200 nm. 

Under these conditions, the migration times were found to be 7.363 min for dexphantenol, 2.457 min for mepyramine 

and 3.520 min for lidocaine. Linearity ranges for the method were 25- 200 µg/mL for dexphantenol, 7,5- 60 µg/mL for 

mepyramine and 7,5- 60 µg/mL for lidocaine. Limit of detection values were found as 3,06 µg/mL for dexphantenol, 

0,76 µg/mL for mepyramine and 1,75 µg/mL for lidocaine. According to the validation study, it was proved that the 

developed method was accurate, precise, sensitive, specific and repeatable. Also the results were compared with an 

HPLC method for determination of these active substances. 


539


P16-012 ADVANCES IN STATIONARY PHASE CHEMISTRY FOR LC METHODS DEVELOPMENT 



Method developers employ several tools to achieve optimum separations for their compounds of interest, including 

different columns, mobile phases, and software packages. Ideally, the optimum method is obtained using the 

minimum number of screening runs possible. The most efficient method development approach is to cover as much 

of the selectivity space as possible by testing multiple columns and mobile phases prior to optimization, which 

in turn can be performed manually or with software assistance. In this poster, we investigate the influence of the 

stationary phase on selectivity, peak capacity, and retention time drift while using generic method development 

gradients. Special attention is paid to the performance of columns in low ionic strength mobile phases (i.e., formic 

acid and ammonium hydroxide) for challenging mixtures of basic, acidic, and neutral analytes. Columns giving the 

widest selectivity differences are incorporated into a novel methods development strategy that employs new high 

pressure LC instrumentation and integrated optimization software. Truly orthogonal options for efficient methods 

development are now available with the new UPLC system, which incorporates a quaternary mixing pump and flowthrough 

needle injector that allows sequential methods development for both reversed-phase and HILIC modes 

without the need to manually switch wash solvents. 

540 



P16-013 HYDROPHYLIC LIPOPHYLIC INTERACTION CHROMATOGRAPHY (HILIC) OF CITICOLINE USING A SILICA COLUMN 

Guermouche S. 

USTHB 

In HILIC, the stationary phase (here, silica) adsorbs a thin layer of water from the mobile phase. Separation is 

hypothesized to occur based on analyte partitioning between the stagnant adsorbed aqueous layer and the less 

polar, dynamic, mostly organic mobile phase. Retention can be regulated by factors that affect the polarity of the 

mobile phase, with higher ionic strength or water content decreasing partitioning into the adsorbed aqueous layer 

and accelerating elution. Selectivity can be regulated by addition of different organic solvents. HILIC can be applied 

especially to molecules with a low partition coefficient log Pow. In this work, a simple and specific hydrophilic 

interaction liquid chromatography (HILIC) procedure for the quantification of Citicoline in finished dosage forms has 

been developed. The method is based on hydrophilic interaction of the analyte with silica. Analysis was made on 

silica column (Atlantis Hilic, 50x4.6mm, 5µ particle, Waters, USA). Mobile phase was constituted of formate buffer 0.1 

M, pH 3 and acetonitrile (30/70, v/v). Temperature was fixed to 30°C, flow rate to 0.5 mL.min-1. Detection was carried 

out in UV at 235 nm using a diode array detector (991 PDA, Waters, USA). Validation of the selected procedure was 

made by determining the classic parameters. Good linearity was found in the concentration range studied in the 

presence and the absence of the excipients. Convenient repeatability and intermediate precision was obtained with 

a respective RSD of 1.96 and 1.10%. Accuracy was 99.37%. The proposed method was then applied to quantify 

citicoline in several pharmaceutical forms. 


541


P16-014 ENDOCRINE DISRUPTOR CHEMICALS (EDCS) IN BIOMEDICAL DEVICES: GAS CHROMATOGRAPHIC-MASS 

SPECTROMETRIC METHOD FOR THE DETERMINATION OF BISPHENOL A AND PHTHALATE ESTERS IN NASOGASTRIC 

TUBES AND FEEDING SYRINGES 

Vela F. 1 , Ballesteros O. 1 , Zafra-Gomez A. 1 , Jiménez-Díaz I. 1 , Navalón A. 1 , Fernández M.F. 1,2 , Olea N. 1,2 , Vílchez J.L. 1 

1 


2 

San Cecilio University Hospital 

Corresponding author e-mail: oballest@ugr.es 

Phthalate esters and bisphenol A (BPA) belongs to a group of compounds commonly called endocrine disrupting 

chemicals (EDCs) which covers a wide range of synthetic and natural substances able to alter the normal hormone 

function of wildlife and humans, consequently causing adverse health effects [1,2]. Those compounds are used 

in different materials as plastics and plasticizers, glasses, cosmetics, etc. Because of their harmful effects, the 

evaluation of the fate and the presence in environment and biological samples of these chemicals is necessary in 

order to avoid human exposition. A gas chromatography-mass spectrometric method for the qualitative analysis of 

the presence of free BPA, its chlorinated derivatives and seven phthalates (dimethylphthalate, DMP; diethylphthalate, 

DEP; dipropylphthalate, DPP; dibutylphthalate, DBP; butylbenzylphthalate, BBP; bis-2-ethylhexylphthalate, BEHP 

and dioctylphthalate, DOP) in nasogastric tubes and baby feeding syringes is proposed. The procedure involves the 

extraction of compounds from the samples with dichlorometane (shaking for six hours), followed by a clean-up step 

by centrifugation and finally the silylation of bisphenol A and chlorinated derivatives prior to their detection. The 

method was satisfactorily applied for the identification of the target compounds in several nasogastric tubes and 

feeding syringes. The results demonstrated the presence of BEHP in nasogastric tubes and BPA in syringes. Further 

studies will be developed in order to evaluate the possible transference of these compounds due to the exposition of 

patients to these devices. [1] Fernández MF, Olmos B, Granada A, López-Espinosa MJ, Molina-Molina JM, Fernández 

JM, Cruz M, Olea-Serrano F, Olea N (2007) Environ Health Perspect 115:8-14. [2] Bosquiazzo VL, Varayoud J, Muñoz 

de Toro M, Luque EH, Ramos JG (2010) Biol Reprod 82(1):86-95. 

542 



P16-015 DETERMINATION OF VITAMINS A-ACETATE, D2 A E-ACETATE IN OINTMENT SAMPLES BY HPLC USING MONOLITHIC 

COLUMN 

Zakova P., Sklenarova H., Solich P. 


Corresponding author e-mail: Petra.Zakova@faf.cuni.cz 

The presented study deals with HPLC method for the separation and simultaneous determination of vitamins 

A-acetate (retinol-acetate), vitamin D2 (ergocalciferol) and E-acetate (tocopherol-acetate). In this case vitamin 

E acetate was used as internal standard. Particle based and monolithic column with different lengths (25 - 50 mm) 

and various internal diameters (2 – 4.6 mm) were tested. Acetonitrile, methanol and mixture of acetonitrile, 

methanol and water were tested as mobile phases. Separation was carried out under following conditions: 

20 µL sample volume, Onyx Monolithic C18 column (50 x 4.6 mm) with 5 mm monolithic precolumn, mobile 

phase acetonitrile:methanol:water 49:49:2 (v/v/v), flow rate 2 mL min-1. Detection was observed at two different 

wavelengths 265 nm (D), 290 nm (A and E). The described method was validated using several validation parameters: 

peak asymmetry, resolution, number of theoretical plates, height equivalent of theoretical plate and repeatability. 

Extraction method for topical ointment containing vitamins A-acetate and D2 was optimized. The isolation procedure 

was developed on the basis of methods for analysis of topical preparations routinely used in our laboratory. Many 

various extraction media (acetonitrile, methanol, ethanol, hexane, chloroform, isopropyl-alcohol and acetone) and 

time of individual extraction steps were tested. Finally, methanol was chosen, because of lower placebo background 

using this extraction agent. Keywords: extraction, HPLC, cholecalciferol, monolithic column, Onyx, retinol-acetate, 

separation, tocopherol-acetate The authors gratefully acknowledge financial support of the Czech Ministry of 

Education, MSM 0021620822; Grant Agency of the Charles University, No. 34609/2009; the grant SVV-2010-261-001 

and Zentiva a.s. 


543


P16-016 COMBINATION OF MONOLITHS AND MODERN TECHNOLOGIES FOR FAST HPLC ANALYSIS IN CLINICAL RESEARCH 


1 


2 


3 


Corresponding author e-mail: lenkakrcmova@seznam.cz 

High-Performance Liquid Chromatography is a powerful analytical tool that has been extensively used for the 

analysis of wide range of compounds from different matrices. The heart of each HPLC method is the column, 

which enables separation of compounds based upon selectivity and column performance. Monolithic columns 

are a relatively new format of stationary phases for HPLC and much remains to be done, recent achievements 

open new vistas for the preparation of an entirely new class of columns, also called “stationary phases of fourth 

generation,” their tolerance to high flow rates, and their rapid speed of chromatographic separations that can be 

achieved at acceptable back pressures, make the monolithic column format superior in biomedical applications 

to the more common columns packed with beads. The application of monolithic columns into the clinical practice 

can reduce the amount of time and resources usually dispended during the analysis of significant number samples 

using the common HPLC columns. Connection of monoliths with modern instrumentation (Rack Changer-special 

HPLC auto sampler with micro titration plates, switching valve...) and new fast sample preparation techniques 

(extraction manifold for micro titration plates, ultracentrifugation...) allows big advantage in biomedical analysis. 

Miniaturization is the modern trend in bioanalysis especially in sample preparation. These modern technologies 

allow shorter analysis and sample preparation times which enable to measure large sequences. Small amount of 

samples and solvents contribute to protect the environment. In order to monitor cancer patients, concentrations 

of different biologically active compounds are monitored in blood, urine and other biological fluids. Neopterin 

(NP) is an unconjugated pteridine produced from guanosine triphosphate in increased quantities as a measure 

of oxidative stress elicited by the immune system. Increased urinary neopterin concentration has been reported 

in patients with various primary tumors, including epithelial ovarian carcinoma or metastatic breast carcinoma. 

As NP concentration decrease after successful therapy, but increase when the disease progresses, a method for 

clinical therapy monitoring of urinary neopterin is needed. Creatinine is a major breakdown product of mammalian 

metabolism produced in muscle tissue and eliminated in urine. The urinary creatinine concentration is often 

used to correct urinary excretion of other chemical substances due to urine dilution effects, since the excretion 

of creatinine is reasonably constant throughout the day. Vitamin E is a major antioxidant in serum. The term 

vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of 

chemoprevention. It is suggested that alpha-tocopherol may be valuable in the prevention and therapy for certain 

types of cancer. The vitamins A (retinol) and E (alpha-tocopherol) serve as reducing agents being able to inactivate 

the toxic effects of free radicals and protect the organism against oxidative stress. In this work the new developed 

methods for the simultaneous determination of fat-soluble vitamins, its metabolites and neopterin in various human 

fluids by the means of high performance liquid chromatography using monolithic column technology with modern 

HPLC equipment and new extraction techniques are presented. Supported by: MSM0021620822, MZO00179906, 

MSM0021620820, GAUK124809/2010 and Zentiva 

544 



P16-017 TROUBLESHOOTING OF SIMULTANEOUS DETERMINATION OF NEOPTERIN, CREATININE, KYNURENINE AND TRYPTOPHAN 

IN VARIOUS HUMAN BIOLOGICAL FLUIDS 


1 


2 


3 


Corresponding author e-mail: lenkakrcmova@seznam.cz 

Increased neopterin concentrations in body-fluids, such as serum or urine, are connected with diseases linked with 

cellular immune reaction, e.g. viral infections, including HIV infection and infections by intracellulary living bacteria 

or parasites, autoimmune diseases, inflammatory diseases, rejection episodes following organ transplantation and 

certain malignant diseases. In all these different diseases the cellular immune system is involved in the pathogenesis 

and/or affected by the underlying disease process, and neopterin concentrations are very closely linked with the 

progression of these diseases. Therefore it is of interest for laboratory diagnosis to measure the degree of activation 

of the human immune system. This is possible in an easy but specific way by the determination of neopterin 

concentrations. [1] Neopterin is a small aromatic molecule strongly fluorescing in its fully oxidized form, and 

therefore can be measured with high sensitivity by using its native fluorescence. It is hydrophilic substance, which 

is more soluble in water than in organic solvents, and hence cannot be extracted by such solvents. Neopterin has to 

be protected from light because of its degradation and simultaneous measurement of creatinine is also necessary 

for correction of physiological variations of urine concentrations. Creatinine is a breakdown product of creatine 

phosphate in muscle. The loss of water molecule from creatine results in the formation of creatinine. Kynurenine is a 

major metabolite of the amino acid tryptophan used in the production of niacin. Accelerated tryptophan degradation 

is observed in diseases and disorders concomitant with cellular immune activation, like infectious, autoimmune and 

malignant diseases, as well as during pregnancy. Therefore, changes in tryptophan metabolism could contribute 

to the modulation of oxidative stress in tissues and to alterations of glutamate receptor stimulation. It can occur 

in neurodegenerative disorders, such as Parkison’s disease, Huntington’s and Alzheimer’s disease, in stroke, in 

epilepsy, in multiple sclerosis and in amyotrophic lateral sclerosis. Kynurenine and tryptophan are soluble in water. 

Kynurenine does not have fluorescence. Many scientific works use for its detection the UV absorbance at 360 

or 365 nm or fluorescent derivatization. Tryptophan has native florescence at excitation 254nm and emmission 

404nm. HPLC determination of creatinine in human serum is complicated it is affected by pH and there are many 

impurities similar to creatinine in human serum. Creatinine is soluble in water and is usually detected at 235 nm. 

Urine, blood, amniotic fluid and exudate are complicated matrices containing many other compounds, which make 

an on-line analysis difficult. We have focused on finding optimal conditions for the simple and fast determination of 

all compounds using modern technologies such as different types of analytical columns (polymeric, hybrid Gemini 

Twin, monolithic, poroshell etc.) and new instrumentations for sample preparation and HPLC analysis. Supported by: 

MSM0021620822, MZO00179906, MSM0021620820, GAUK124809/2010 and Zentiva [1] Wachter H, Fuchs D, Hausen 

A, Reibnegger G, Werner ER. Neopterin as marker for activation of cellular immunity: Immunologic basis and clinical 

application. Adv Clin Chem 1989:27;81-141. 


545


P16-018 INVESTIGATIONS OF THE INTERACTIONS BETWEEN METALS AND IODINE IN PATHOLOGICAL AND HEALTHY HUMAN 

THYROID GLANDS USING ION CHROMATOGRAPHY 

Blazewicz A., Orlicz-Szczesna G., Randhawa R., Dolliver W., Sivsammye S., Deol A. 

Medical University of Lublin 

The World Health Organization estimates that more than 2 billion people are at risk of iodine deficiency in 130 

countries [1]. At the same time the level of heavy metals is rising in both the environment and thus in the human body 

[2]. The interactions between various metals and iodine in human tissues are not well understood. It is known that 

halides impede the absorption of iodine by blocking receptors in the thyroid gland. Coexisting deficiencies of certain 

metals can impair thyroid function as well [3]. Very few reports (mainly animal studies) record iron and zinc impact 

on thyroid metabolism (iron deficiency impairs thyroid hormone synthesis by reducing activity of ion-dependent 

thyroid peroxidase and zinc deficiency is associated with decreased concentrations of triiodothyronine [4, 5]). There 

is no literature pertaining to the mutual relationships between Pb2+, Fe3+, Cu2+, Ni2+, Zn2+, Co2+, Cd2+, Mn2+ 

and iodine in human thyroids (neither pathological nor healthy tissues). The aim of our study was to examine the 

correlations between the content of iodine and every above mentioned metal. Our samples included 65 nodular 

goiters and 100 healthy human thyroid tissues (50 – frozen and 50 – formalin fixed). The study group consisted of 

40 females and 25 males with an average age of 35, who had fragments of their thyroid glands (diagnosed with 

nodular goitre) removed surgically. The patients underwent thyroidectomy in three hospitals in Lublin (a town with 

a population of 380,000 in the south-eastern region of Poland). Thyroids taken from individuals killed in accidental 

events constituted the control group. All the samples were analysed using an ion chromatography method, which 

involved applying a pulsed amperometric detection (IC-PAD) for iodide analysis and spectrophotometric detection 

followed by a post column derivatization reaction – for the determination of metals. Alkaline digestion with the 

assistance of microwaves was developed and used for the comparative analysis of the two types of human thyroid 

samples. A detailed statistical analysis has been performed. Suitability of the developed IC method was supported 

by validation results. Measurement accuracy was verified using NIST standard reference material (1566a Oyster 

Tissue). [1] WHO global database on iodine deficiency Geneva, World Health Organization, 2004. [2] B. Nowak, H. 

Kozłowski Biological Trace Element Research Volume 62, Number 3, 213 1998. [3] D. Brownstein, Iodine, Why You 

Need It. Medical Alternative press, 2009. [4] Zimmermann MB, Köhrle J. Thyroid. 12(10): 867-78, 2002. [5] G. A. El- 

Sisy, A.M.A. Abdel-Razek, A.A. Ypunis, A. M. Ghallaab, M.S.S. Abdou Global Veterinaria 2 (2):46-50, 2008. 

546 



P16-019 CAPILLARY ELECTROPHORETIC DETERMINATION OF TIMOLOL MALEATE AND DORZOLAMIDE HCL IN EYE DROPS 

Caglayan M.G., Palabiyik I.M., Onur F. 


Corresponding author e-mail: caglayangokhan@gmail.com 

A capillary electrophoresis method was developed for the simultaneous determination of timolol maleate and 

dorzolamide HCl. Effect of buffer pH, buffer concentration and voltage on obtained responses were investigated. 

Separation was performed on a fused-silica capillary (50 cm total length× 50 μm I.D.) using sodium phosphate (pH 

6.00; 25 mM) as buffer, 20 kV as applied voltage at 25 oC. Detection wavelengths and limit of detections were 257 

nm and 294 nm, and 5 μg/mL and 5 μg/mL for timolol maleate and dorzolamide HCl, respectively. This method was 

successfully applied to the quantitative determination of these compounds in eye drops and was validated in terms 

of linearity, precision, accuracy and sensitivity. 


547


P16-020 INVESTIGATION OF THE INTERACTION OF MERCUROCHROME® CONSTITUENTS WITH PROTEINS USING LIQUID 


Wilken A. 

Institute for Inorganic and Analytical Chemistry 

Investigation of the Interaction of Mercurochrome® Constituents with Proteins Using Liquid Chromatography/ 

Mass Spectrometry Andrea Wilken1, Rasmus Janzen1, Michael Holtkamp1, Sascha Nowak2, Michael Sperling1,3, 

Martin Vogel1 and Uwe Karst1 1Westfälische Wilhelms-Universität Münster, Institut für Anorganische und 

Analytische Chemie, Corrensstr. 30, 48149 Münster, Germany, uk @uni-muenster.de 2Westfälische Wilhelms- 

Universität Münster, Institut für Physikalische Chemie, Corrensstr. 28-30, 48149 Münster, Germany 3European 

Virtual Institute for Speciation Analysis, Mendelstr. 11, 48149 Münster, Germany Merbromin (see fig. 1) is a 

mercury-containing compound used as pharmaceutical agent in a 2% aqueous solution and was sold under the 

trade name Mercurochrome®. Fig. 1 : Merbromin For decades, it has widely been used as topical disinfectant 

for the treatment of childhood cuts, scrapes and abrasions.[1] Such applications provide possibilities for a direct 

entry of the compound into the bloodstream and thus for reactions between blood constituents and the active 

compound. A connection between the use of Mercurochrome® and multitude of diseases were reported.[2,3] Since 

more than 80 years, the existence of toxic side products depending on the synthesis route has been known and 

discussed. Modifications of the synthesis have been considered to cause various impurities such as mercury salts. 

Therefore, the “source of origin” of the compound turned out, to be more important than the administered dose. 

[4] In contrast to current medical preparations, the exact composition of the product has never been published 

despite the many analyses that had been performed. Such lack of information motivated us to investigate the 

interaction of Mercurochrome® with free thiols in low-molecular weight peptides and in proteins by means of liquid 

chromatography (LC) and electrospray mass spectrometry (ESI-MS). β-Lactoglobulin A (β-LGA) from bovine milk 

(18.4 kDa) has been used as model protein. While the commonly expected modes of interaction between Hg species 

and thiols were observed, the heterogeneity and the stability of Mercurochrome® led to some surprising findings 

regarding the compound itself and its reaction products. It was found that, in contrast to assumptions made in 

the literature, the commercial product itself is a heterogeneous mixture of moderate chemical stability, which may 

contain precipitated Hg salts depending on storage time and conditions. Further variability results from different 

degrees of bromination of the fluorescein backbone of the compound. The formation of mercury compound-protein 

adducts was detected. The peptide sequence T13 containing a free thiol residue was identified as binding site for 

mercury species after tryptic digestion of β-lactoglobulin A. While fresh Mercurochrome® tends to the formation 

of a Hg(II)-β-LGA adducts due to excess Hg2+ in solution, investigations after precipitation of Hg salts yield 

Hg(merbromin)(β-LGA) as major product. References : [1] Risher JF, Murray E, Prince GR (2002) Toxicol Ind Health 

18:109-160 [2] Debray P, Besson-Leaud M, Lavaud J (1979) Ann Pediatrics 26:531-537 [3] Torres JLC, De Corres F 

(1985) Ann Allergy 54:230-232 [4] Burn JH, Elphick (1930) Quart J Pharm Pharmacol 3:177-186 

548 



P16-021 A QBD WITH DESIGN OF EXPERIMENTS APPROACH TO THE DEVELOPMENT OF A CHROMATOGRAPHIC METHOD FOR THE 

SEPARATION OF IMPURITIES IN VANCOMYCIN 

Plankeele J.M. 

Waters European Headquarters, UPLC 

This poster describes a novel method development approach using Quality by Design (QbD) with Design of 

Experiments to develop a UPLC® method for impurities in Vancomycin resulting in an optimally performing analytical 

method while simultaneously applying robustness limits to ensure success in final method validation and ultimately 

in method transfer. 


549


P16-022 MULTIVARIATE OPTIMIZATION OF THE EXPERIMENTAL CONDITIONS IN HIGH PERFORMANCE LIQUID 

CHROMATOGRAPHIC METHOD USING RESPONSE SURFACE METHODOLOGY 

Cigdem Aybaba, Ismail Murat Palabiyik, Mehmet Gokhan Caglayan, Feyyaz Onur 


Corresponding author e-mail: mpala@pharmacy.ankara.edu.tr 

Optimization procedures are very important in chromatographic techniques with regard to selection of the 

optimal experimental conditions and to provide relevant information depending on analyzing experimental data. 

For studying the simultaneous variation of the factors on considered responses, some designs of experiments 

techniques were suggested in optimization procedure including multivariate applications. In this study, Response 

surface methodology (RSM) was used as an optimization method for simultaneous determination of acemetacin 

and chlorzoxazone in a tablet by high performance liquid chromatographic method. An important aspect of RSM 

is design of the experiment. Central composite design was used to calculate method optimization and percentage 

of acetonitrile and ammonium acetate in mobil phase and pH of ammonium acetate solution in mobile phase were 

investigated as factors on resolution. Results were also shown graphically. This developed and optimized method 

has been proved linear, sensitive, accurate and precise to be applied in routine analysis of these drugs. 

550 



P16-023 MOLECULARLY IMPRINTED SOLID PHASE EXTRACTION COUPLED TO MICELLAR ELECTROKINETIC CHROMATOGRAPHY 

FOR THE DETERMINATION OF DIGOXIN AND DIGITOXIN IN CLINICAL SAMPLES 

Guijarro M. 1 , Paniagua G. 2 , Fernández P. 2 , Crego A.L. 1 , Marina M.L. 1 

1 


2 

National University of Distance Education 


Digoxin is a glycosylated steroid-like drug derived from the leaves and seeds of Digitalis lanata. It is one of the 

most commonly prescribed cardiac glycosides in the treatment of congestive heart failure and certain cardiac 

arrhythmias. This drug is characterized by a narrow therapeutic range of concentrations (only a few ng ml−1) and 

its use requires strict monitoring of levels to minimize toxicity [1]. Therefore, sensitive techniques are required for 

the control of this compound. On the other hand, selective methodologies are necessary to avoid interferences with 

digoxin metabolites (i.e. digoxigenin) and endogenous digoxin-like substances, which could be present in the serum 

and urine of patients. This work describes the development of a rapid and simple extraction procedure based on 

molecularly imprinted solid-phase extraction (MISPE) for cleanup of clinical samples previous to the simultaneous 

determination of digoxin and its aglycon digoxin (digoxigenin) by Micellar Electrokinetic Chromatography (MEKC). 

In addition, different in-capillary preconcentration strategies based on electrophoretic principles were studied. 

The optimization of parameters affecting the separation and preconcentration was carried out to select the best 

conditions of selectivity and sensitivity to determine these compounds at low concentration levels in human fluids. 

In order to demonstrate the suitability of the developed method several analytical characteristics (linearity, LODs, 

precision, accuracy and selectivity) were studied. The proposed MISPE-MEKC method was successfully applied to 

urine and serum samples analysis. [1] L.L. Mackstaller, J.S. Alper, Clin. Cardiol. 20 (1997) 640. 


551


P16-024 STUDY OF THE INTERACTION OF EPHEDRINE ENANTIOMERS WITH NATIVE CYCLODEXTRINS BY CAPILLARY 

ELECTROPHORESIS AND NMR 

Domínguez-Vega E. 1 , Crego A.L. 1 , Marina M.L. 1 , Salgado A. 2 , Scriba G.K.E. 3 , Chankvetadze B. 4 

1 

University of Alcalá-Spain 

2 

Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 

3 


4 

Tbilisi State University-Georgia 


Enantiomer migration order is one of the most important aspects in chiral separations especially in the case in which 

the optical purity has to be determined. Reversing the enantiomer migration order can be achieved by different ways 

being one of the most used the change of the nature of the chiral selector. In the case of native cyclodextrins (CDs) 

the difference between α-, β- and γ-CDs is the size of the cavity due to the different number of glucose units. This 

difference of size can affect to the chiral separation allowing or not the enantioseparation of the analyte or even in 

some cases reversing the migration order of the enantiomers. In this work, enantiomers of ephedrine were separated 

by capillary electrophoresis using native CDs. No chiral separation of ephedrine was achieved with γ-CD, while 

opposite migration order was observed with α- and β-CD. In order to study the mechanism of separation between 

ephedrine enantiomers with native cyclodextrins, the apparent equilibrium constants of the transient diastereomeric 

complexes were calculated using capillary electrophoresis and NMR spectroscopy as well as the stoichiometry 

and structure of the complexes were studied with the latter technique. For the reason that it showed much better 

enantiomer resolving ability towards the enantiomers of ephedrine the charged single component heptakis(2,3- 

diacetyl-6-sulfo)-β-CD was also included in this study. Significant structural differences were observed between 

diastereomeric associates of ephedrine with CDs involved in the present study. 

552 



P16-025 IDENTIFICATION OF UNKNOWN DEGRADATION PRODUCTS IN NEW PHARMACEUTICAL DOSAGE FORMS OF 

PARACETAMOL BY LIQUID CHROMATOGRAPHY–TANDEM MASS SPECTROMETRY 

Pérez I., Castro-Rubio F., Nozal L., Novella J.L., Crego A.L 



The use of sophisticated analytical techniques as high performance liquid chromatography coupled with tandem 

mass spectrometry has enabled the identification and characterization of two unknown degradation products which 

provided an unwanted coloring in new pharmaceutical dosage forms of Paracetamol, as current legislation requires. 

A new method with mass spectrometry requirements was proposed for determination of Paracetamol related 

substances in a new pharmaceutical formulation. A hybrid mass spectrometer, quadrupole-time-of-flight (QTOF), 

was used in order to increase the specificity by monitoring not only the characteristic precursor ions of the unknown 

products but also their specific product ions in MS/MS. The comparison of fragmentation patterns of unknown 

detected peaks related to degradation products with those of same chemically synthesized compounds has shown 

unequivocal identification of both. Furthermore, a new analytical methodology by LC/MS/MS was performed to 

improve the sensitivity using multiple-reaction-monitoring experiments with triple quadrupole (QqQ), where great 

quantification and detection limits were achieved. These facts make mass spectrometry a useful and essential tool 

for pharmaceutical industry during the development of new drug products. 


553


P16-026 CHARACTERIZATION OF PHENOLIC PROFILE IN FOUR ROSMARINUS OFFICINALIS EXTRACTS AND DETERMINATION OF 

THEIR ANTIOXIDANT CAPACITY 

Borrás Linares I., Herrero M., Ibánez E., Segura Carretero A., Fernández Gutiérrez A. 



Corresponding author e-mail: iborras@ugr.es 

Rosemary (Rosmarinus officinalis) is a shrub belonging to the Labiatae family that grows wild in the Mediterranean 

basin. Rosemary is one of the most appreciated sources for natural bioactive compounds. In fact, the extract of 

this plant exerts a number of pharmacological activities, such as hepatoprotective, antibacterial, antithrombotic, 

antiulcerogenic, diuretic, antidiabetic, antinociceptive, antiinflammatory and antioxidant. These activities of 

rosemary extracts are due mainly to phenolic compounds, specially phenolic diterpenes such as carnosol, carnosic 

acid, rosmadial, rosmanol, epirosmanol and rosmarinic acid among others. Furthermore antioxidant compounds 

belonging to flavonioids such as scutellarein, genkwanin and cirsimaritin have been reported. The aim of this 

research was to characterize rosemary extracts obtained by different extraction procedures. In order to obtain 

these extracts, rosemary leaves were dried in a ventilated place for 20-30 days, then they were grinding under liquid 

nitrogen and sieving to an appropriate size. Several methods to extract antioxidants from aromatic plants have also 

been reported. In this work we used supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) with 

different conditions, two environmentally friendly and selective extraction techniques. In this study, the antioxidant 

capacity was determinated using Trolox equivalent antioxidant capacity (TEAC) assay. In all extracts of rosemary 

the range were from 60 to 240 mmol Trolox equivalent / 100 g extract. A methodology for qualitative characterization 

of complex plant matrix have been developed consisting of the coupling of reversed-phase high-performance 

liquid chromatography (RP-HPLC) eqquiped with a small particle size column, with two different detection systems: 

photodiode array (DAD) and mass spectrometry with time-of-flight (TOF) analyzer. UV-visible spectrophotometry 

is a valuable tool for identifying the class of phenolic compounds, whereas MS data are useful for their structural 

characterization. The sensitivity together with mass accuracy and true isotopic pattern of TOF-MS analyzer provided 

the most probable molecular formula. In this work we have applied the described methodology to carry out the 

comprehensive characterization of these rosemary extracts. This procedure was able to determine many well-known 

phenolic compounds present in rosemary such as carnosic acid, carnosol, rosmanol, epirosmanol, scutellarein, 

genkwanin, cirsimaritin and homoplantaginin. 

554 



P16-027 CHARACTERIZATION OF PHENOLIC COMPOUNDS BY HPLC-ESI-TOF-MS IN THREE DIFFERENT PEPPER VARIETIES. 

UPDATING AND IMPROVING FOOD COMPOSITION TABLES 

Morales Soto A., Segura Carretero A., Fernández Gutiérrez A. 


Corresponding author e-mail: arms@correo.ugr.es 

A food can be regarded as functional if it is satisfactorily demonstrated to affect beneficially one or more target 

functions in the body, beyond adequate nutrition, in a way that improves health and well-being or reduces the risk 

of disease. Food Composition Tables (FCTs) are data bases of the chemical composition, energy, and nutrient 

yield of foods but major FCTs do not include information about several bioactive compounds. The pharmaceutical 

interest in these compounds has stimulated multidisciplinary research on the characterization of food and their 

inclusion in FCTs is an important contribution to the agri-food system. Pepper (Capsicum annuum L.) is a great 

importance fruit in human diet due to its high content of phenolic compounds such as phenolics acids, flavonoids, 

hydroxycinnamates and flavones. The bioactivity of the phenolic compounds could be attributed to their antioxidant 

properties. Reactive oxygen species responsible of oxidative stress are involved to the chronic diseases such as 

atherosclerosis, cancer, obesity, diabetes, and coronary diseases. Several are the evidences that the protective 

effects of these compounds against these chronic diseases are related to the antioxidant properties of phenolic 

compounds. Since the antioxidant activity as well as many other biological effects of pepper-derived phenolics 

appear to have compound-specific properties, the aim of this study was: to characterize and examine three 

different varieties called “Lamuyo Amarillo”, “California rojo” and “Italiano verde ecológico”and to include the family 

of phenolic compounds in FCTs. The phenolic compounds extraction system used for freeze dried samples was 

liquid-liquid extraction (LLE), which is based on the use of organic solvents and water in different proportions. A 

comparative study of six extraction procedures to elucidate the phenolic fraction in pepper samples were carried out 

using methanol:water (90:10, 80:20, 70:30, 50:50) and methanol-aqueos hydrochloric acid (70:30, 50:50). The best 

results were achieved using 70:30 methanol:water extraction procedure. A high-performance liquid chromatography 

(HPLC) coupled to mass spectrometry time of flight analyzer (TOF) method was used for qualitative characterization 

of pepper extracts. The chromatographic method consisted in a linear gradient using water with 0.5 % acetic acid as 

eluent A and acetonitrile as eluent B. Finally, many well-known phenolic compounds such as glicosilated species of 

flavonols (quercetin 3-O-α-L-rhamnoside) and other polar compounds such as quinic acid were identified. 


555


P16-028 COMPARISON OF DIFFERENT METHODOLOGIES FOR THE EVALUATION OF BINDING OF ANTIHISTAMINES TO HUMAN 

SERUM ALBUMIN BY CAPILLARY ELECTROPHORESIS 

Martinez Gomez M.A. 1 , Sagrado S. 1,2 ,Villanueva Camañas R.M. 1 , Medina Hernandez M.J. 1 

1 


2 


Many drugs are non-covalently bound to serum proteins or other binding agents in the circulation, feature that 

makes the determination of free/bound drug fractions and studies of their binding with serum proteins a topic of 

great interest in clinical and pharmaceutical research. 

Several methodologies have been proposed for the measurement of drug-protein binding such as equilibrium 

dialysis, ultrafiltration, nuclear magnetic resonance spectroscopy, UV-vis spectroscopy, circular dichroism and 

surface plasmon resonance. However, these methodologies present some disadvantages such as long analysis 

times, potential interferences from the sample, need for drugs with suitably high concentrations or levels of binding 

to detection and specialized equipment for the rest of methodologies. In the last years, HPLC and CE methods have 

been widely used for this purpose employing proteins as stationary phases or buffer additives, respectively. 

In this sense, our research group has used some capillary electrophoretic methodologies such as frontal analysis 

and ultrafiltration-affinity electrokinetic chromatography (AEKC) for the evaluation of affinity of racemic β-blockers, 

antihistamines, NSAIDS, phenothiazines, to HSA, AGP and all plasmatic proteins, independently of the extent of 

affinity towards proteins. In addition the enantioselective binding of chiral compounds to serum proteins has been 

also studied. In this case ultrafiltration-AEKC and different chiral selectors such as HSA or TM-β-CD have been 

used, allowing the estimation of affinity of both enantiomers of chiral drugs to plasmatic proteins. 

In this communication, comparison of frontal analysis, ultrafiltration-EKC and electrokinetic injection in CE for 

evaluating affinity of four racemic antihistamines, brompheniramine, orphenadrine, chlorpheniramine and hydoxyzine 

to HSA at near physiological conditions is proposed. In all cases, mixtures of drug and protein were incubated at 

36.5ºC for 30 min in a water bath. In frontal analysis, mixtures were hydrodynamically inyected at 50 mbar for 30 s; 

in electrokinetic injection, sample was introduced in the capillary system at 1 kV for 5 or 10 s; in ultrafiltration-EKC, 

pre-equilibrated mixtures were ultrafiltrated and free or bound drug fraction was hydrodynamically injected at 30 

mbar for 2 s after the injection of the chiral selector HSA. 

The advantage of electrokinetic injection over ultrafiltration-AEKC could be the avoidance of ultrafiltration step 

since when voltage is applied, free drug present in the mixture drug-protein would enter before than bound drug 

in the capillary system; consequently, pre-treatment of sample and cost per analysis would be reduced since no 

ultrafiltration system (ultracentrifuge and filter devices) would be required. On the other hand, the introduction of a 

chiral selector before electrokinetic injection of mixture would allow estimating affinity of both isomers of racemic 

drugs. 

Acknowledgements: The authors acknowledge the Spanish Ministry of Science and Technology (MCYT) for the 

financial support (Project SAF2008-00859). 

556 



P16-029 ENANTIOMERIC RPLC SEPARATIONS OF CHIRAL LOCAL ANESTHETICS USING POLYSACCHARIDE BASED CHIRAL 


Escuder Gilabert L., Sagrado S. 1,2 ,Villanueva Camañas R.M. 1 , Medina Hernandez M.J. 1 

1 


2 


Corresponding author e-mail: lescuder@uv.es 

Enantioselective pharmacology can occur at any site in the body where a drug interacts with an endogenous chiral 

centre. The pharmacological complication caused by drug racemates is that their component enantiomers usually 

have different pharmacodynamic effects and different pharmacokinetic properties. Therefore, there is growing 

scientific, clinical, commercial and regulatory recognition to be aware of the key issues surrounding enantiomers. To 

achieve this purpose it is necessary to develop suitable methods for the enantioseparation of chiral drugs. 

With the constant development of the methodology and applications in chromatographic enantioseparations, many 

chiral stationary phases (CSPs) have appeared for HPLC including proteins, oligosaccharides, polysaccharides, 

antibiotics, helical synthetic polymers and low-molecular-weight compounds. Among them, polysaccharide 

derivatives, such as the cellulose esters and phenylcarbamates of cellulose and amylose, exhibit a unique chiral 

recognition for a broad range of chiral compounds and have been widely used as CSPs for HPLC. 

The majority of chiral separations accomplished using polysaccharide-based CSP use normal-phase eluents 

(alkane/alcohol mixtures) although there are also some examples of useful separations with reversed-phase 

eluents (aqueous methanol or acetonitrile, or appropriate buffer/methanol or buffer/acetonitrile mixtures, or pure 

polar organic solvent). The use of alkane/alcohol solvents is possible since there is no ionic functional group in the 

polysaccharide CSPs, and since enantioselective interactions are considered to be more effective under normalphase 

conditions. Moreover, normal phase liquid chromatography is preferred for speed of method development and 

short analysis times (because of low viscosity of eluents, faster column equilibration than in RPLC and possibility 

to use higher flow rates). Nevertheless, there are a number of advantages to use reversed-phase conditions, for 

example, good solubility of polar compounds, easier preparation of aqueous samples, and use of less costly 

and toxic solvents. Reversed-phase conditions have been especially useful for assay of the enantiomeric ratio of 

pharmaceuticals in biological matrices. 

The aim of this communication is the chromatographic enantioseparation of four chiral local anesthetics 

(bupivacaine, mepivacaine, prilocaine and propanocaine) used in the clinical practice as racemates and whose 

component enantiomers have different pharmacokinetic, pharmacodinamic and toxic properties. For this purpose, 

two polysaccharide-based CSPs (phenylcarbamates of cellulose and amylose) in reversed-phase eluents are 

assayed. The effect of the mobile phase composition (nature and concentration of polar organic solvent) and pH as 

well as the separation temperature on the enantioresolution is studied. 

ACKNOWLEDGEMENTS: This work has been supported by the Spanish Ministry of Science and Innovation (MCINN, 

Project SAF2008-00859). 


557


P16-030 ELECTROKINETIC CHROMATOGRAPHY AS AN ANALYTICAL APPROACH TO EVALUATE THE ENANTIOSELECTIVE BINDING 

OF FLUOXETINE TO HUMAN SERUM ALBUMIN 



The pharmacological activity of drugs depends on some pharmacokinetic and pharmacodynamic processes. At 

the level of pharmacokinetic, the absorption, the distribution and the clearance have influence in the plasmatic 

concentration, which affects the therapeutic activity of the drug. All biological processes are affected by 

enantioselectivity, due to the chirality of biological molecules. One of the most important processes at the level of 

the distribution of the drug is the protein binding, also affected by the high enantioselectivity of the plasma proteins 

(principally human serum albumin, HSA) in their interaction with drugs. 

Psychoactive drugs are the most used in our society. Recently the industry is interested in the introduction of 

these drugs as pure enantiomers in the pharmaceutical market, due to their differences on the therapeutic activity. 

Pharmacokinetic and pharmacological properties of each enantiomer have to be studied nowadays for these drugs 

in pre-clinical phases. Fluoxetine (FLX) is a potent and selective inhibitor of the neuronal serotonin-uptake carrier 

and is a clinically effective antidepressant. FLX is used therapeutically as the racemate although a stereospecificity 

associated with its interactions with the serotonin-uptake carrier has been demonstrated. 

In this communication, the enantioselective binding of FLX to HSA has been evaluated by ultrafiltration of FLX-HSA 

mixtures and chiral analysis of unbound fractions by electrokinetic chromatography using highly-sulfated beta 

cyclodextrin (HS-beta-CD) as chiral selector. 

Protein binding (PB), affinity constants (K) and enantioselectivity (ES) were obtained for both enantiomers of FLX. 

In order to improve the consistency of the estimations, the evaluation of affinity constants of each enantiomer 

was performed using two designs, one keeping constant the total concentration of protein and varying the 

total concentration of the enantiomers, and the other in the opposite way, in both cases via an unusual shortconcentration 

interval strategy to assure model validity. Different mathematical approaches were compared and 

characterised and some of them, judged as the most consistent under the experimental conditions used, were 

selected to provide final estimates. The estimates were done with two models of binding, one independent and other 

competitive. 

Using the proposed methodology, PB estimates were 95.2% and 90.0% for the first (E1) and the second (E2) 

enantiomer eluted, in consistence with the reported in the literature for racemic FLX (94.75%). Affinity constants 

were (3.3 ± 0.3)•104 M-1 and (1.50 ± 0.07)•104 M-1 for E1 and E2, respectively, using the independent model. With 

the competitive one, these values were (6.0 ± 1.9)•104 and (2.8 ± 0.6)•104 M-1 for E1 and E2. 

Enantioselectivity values, obtained as the ratio between the affinity constants of E1 and E2, were 2.20 for the 

independent model and 2.24 for the competitive one. 

ACKNOWLEDGEMENTS This work has been supported by the Spanish Ministry of Science and Innovation (MCINN, 


558 



P16-031 ENANTIOSELECTIVITY STUDY IN BINDING OF QUINUPRAMINE TO HUMAN SERUM ALBUMIN BY ULTRAFILTRATION AND 

CAPILLARY ELECTROPHORESIS USING EXPERIMENTAL DESIGNS 

Martinez Gomez M.A. 1 , Villanueva Camañas R.M. 1 , Sagrado S. 1,2 , Medina Hernandez M.J. 1 

1 


2 


Corresponding author e-mail: maria.j.medina@uv.es 

Interactions between drugs and serum proteins are important in determining the transport, distribution, metabolism, 

excretion and activity of many pharmaceutical agents in the body. The development of methods to examine these 

interactions is of great interest. Human serum albumin (HSA) is the most abundant plasma protein and is involved in 

the transport of many drugs and small solutes. 

The extent to which binding occurs varies and depends on the affinity between drug and protein, the drug 

concentration, the concentration of protein and the presence of other substances which either compete with drug for 

binding sites or displace it through allosteric effects. While pharmacokinetics (PK) governs the relationship between 

dose and concentration, pharmacodynamic (PD) measurements may relate this concentration to effect. 

Free drug is pharmacologically active and its concentration is more closely related to efficacy and toxicity than total 

blood or plasma concentrations. Traditionally, in pharmacokinetic studies and therapeutic drug monitoring, total drug 

concentrations in plasma are measured and free concentrations are predicted from the protein-binding capabilities 

of the particular drug. However, whenever possible, free drug concentration at the receptor site should be used for 

making inferences about a drug’s pharmacological activity. 

In this communication, the evaluation of the affinity of a racemic tricyclic antidepressant, quinupramine towards HSA 

is proposed. The study was carried out by ultrafiltrating mixtures containing racemic quinupramine and HSA and 

by analyzing ultrafiltrates, that contain free fraction of both enantiomers, by affinity electrokinetic chromatography 

using HSA as chiral selector. Two experimental designs were carried out, one keeping the concentration of protein 

constant and varying the concentration of racemic drug and the other in the opposite way, in both cases via an 

unusual short-concentration interval strategy to assure model validity; in both designs 5 concentration levels were 

used and 2 independent replicates per level, totaling 18 independent mixtures. 

Protein binding and affinity constants for both enantiomers of quinupramine and enantioselectivity were calculated 

by considering both experimental designs; consequently, the variability associated to the estimations of these 

parameters by maintaining drug concentration constant and varying protein concentration or in the opposite way was 

negligible. 


The authors acknowledge the Spanish Ministry of Science and Technology (MCYT) for the financial support (Project 

SAF2008-00859). 


559


P16-032 COMPARISON BETWEEN RPLC USING POLYSACCHARIDE BASED CHIRAL STATIONARY PHASES AND CAPILLARY 

ELECTROPHORESIS WITH CYCLODEXTRINS FOR THE CHIRAL SEPARATION OF BASIC DRUGS IN AQUEOUS SAMPLES 



Drug action is the result of a large number of pharmacological and pharmacokinetic processes that take place in 

the living systems. Most of these processes present a high degree of enantioselectivity that leads to a difference 

in the pharmacological features of each drug enantiomer. It may well be that while one of them is the most active 

the other may produce side-effects or can be even toxic. Thus, the development of methods for enantioselective 

analysis became very important for drug quality control, pharmacodynamic and pharmacokinetic studies as well as 

toxicological investigations. 

Analytical methods used so far for the chiral separation include thin-layer chromatography, gas chromatography, 

high-performance liquid chromatography (HPLC), supercritical fluid chromatography and capillary electrophoresis 

(CE). Many chiral stationary phases (CSPs) have been developed for HPLC including proteins, oligosaccharides, 

polysaccharides, antibiotics, helical synthetic polymers and low-molecular-weight compounds. Among them, 

polysaccharide derivatives, such as the cellulose ester and phenylcarbamates of cellulose and amylose, exhibit a 

unique chiral recognition for a broad range of chiral compounds and have been used as CSPs for HPLC. Recently, 

the importance of capillary electrophoresis (CE) has been increased in the field of chiral analysis, due to its 

versatility, high separation efficiencies, low reagenty consumption and high speed of analysis. 

The aim of this communication is the enantioseparation of five psychoactive basic drugs (fluoxetine, bupropion, 

viloxazine, nomifensine and ethosuximide) in aqueous samples. For this purpose, the use of HPLC polysaccharide 

based stationary phases and capillary electrophoresis with cyclodextrins as chiral selectors was evaluated. In 

HPLC three Lux® Chiral Columns based on amylose tris(5-chloro-2-methylphenylcarbamate, cellulose tris(3- 

chloro-4-methylphenylcarbamate) and cellulose tris(3,5-dimethylphenylcarbamate) were used under reversed phase 

conditions. The effect on the separation of several experimental variables such as nature of stationary phase, buffer 

pH, nature and concentration of organic modifier, or temperature was studied. For the enantioresolution of basic 

drugs by capillary electrophoresis, heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TM-beta-CD) and highly sulfated 

beta-cyclodextrin (HS-beta-CD) were used as chiral selectors using the partial filling technique. In this methodology 

the capillary is partially filled with the chiral selector solution and the separations are performed with plain 

background electrolyte. The effect of CD concentration, buffer pH, temperature or applied voltage on resolution was 

studied. 

Baseline resolution of enantiomers of fluoxetine, bupropion, viloxazine and nomifensine was achieved using the 

CE methodology. For these compounds, polysaccharide based stationary phases also show enantioselectivity, but 

only in the case of bupropion and nomifensine values of resolution higher than 1.5 were achieved. In addition of 

better enantioselectivity, an advantage of CE is the speed of analysis since for all the compounds studied analysis 

times were shorter in CE than in HPLC. However, CE suffers from poor sensitivity when using UV detection, and the 

method of choice for analysis where low detection limits are required is liquid chromatography. 

ACKNOWLEDGEMENTS This work has been supported by the Spanish Ministry of Science and Innovation (MCINN, 


560 



P16-033 COMPARISON OF DART-MS AND GC-MS CAPABILITIES IN ANALYSIS OF MINT ESSENTIAL OIL SAMPLES 

Chernetsova E.S., Goryainov S.V., Ovcharov M.V., Bochkov P.O., Khomyakov Y.Y. 


Corresponding author e-mail: chern_es@mail.ru 

Direct Analysis in Real Time (DART) mass spectrometry is a new method, which is promising for fast analysis of 

different samples without a sample preparation [1,2]. DART-MS can be used for the direct analysis from thin layer 

chromatography plates [3], however, usually it does not require a chromatographic separation. Since the method 

is rather new, the number of publications on DART-MS and its comparison with other methods is very limited. 

Capabilities of DART-MS for the identification of unknown mixture compounds were not studied deeply. In the 

present work the capabilities of DART-MS as compared to GC-MS were studied for the first time with the example of 

mint essential oil samples. The special attention was paid at mass spectra composition. The respective results will 

be presented. 

This work was partially supported by the Council of the President of Russia (grant МК-594.2010.3). 

References 

1. R.B. Cody, J.A. Laramee and H. Dupont Durst // Anal. Chem., vol. 77 (2005) P. 2294. 

2. R.B. Cody // SpectroscopyEurope, vol. 18 (2006) P. S12. 

3. G. Morlock, Y. Ueda // J. Chromatogr. A., vol. 1143 (2007) P. 243-251. 


561


P16-034 STABILITY OF PHARMACOKINETIC STUDIES DATA OBTAINED USING HPLC/MS/MS SYSTEMS WITH TRIPLE QUADRUPOLE 

MASS SPECTROMETERS 

Chernetsova E.S. 1 , Ovcharov M.V. 1 , Bochkov P.O. 1 , Kovaleva A.S. 2 , Koryakova A.G. 2 

1 


2 

Chemical Diversity Research Institute 

Corresponding author e-mail: chern_es@mail.ru 

Pharmacokinetic (PK) studies of new drugs are often carried out using HPLC combined with triple quadrupole mass 

spectrometers due to their high selectivity and sensitivity in MRM (multiple reaction monitoring) mode. At that not 

less than 18 samples are used for getting one PK curve, which represent 6 time points. At each of these points blood 

or other kind of biological material (tissues, cerebrospinal fluids, etc.) from 3 laboratory animals were sampled. 

Studying liver microsomal stability of the respective potential drugs or their stability in plasma also requires analysis 

of more than ten samples. Therefore, taking into account the necessity for analysis of replicates and calibration 

samples, one analytical cycle for a PK study is very time consuming. Due to this, a high stability of the MS response 

during the whole analytical cycle is essential for getting valid resulting data. In order to increase the accuracy, 

internal standards are often used. 

In a present paper the stability of a response of 2000 QTrap mass spectrometer in MRM mode was studied for 

testosterone, midazolam and propranolol. During four days 30 ml of a model mixture, containing these compounds 

100 ng/ml each, were multiply introduced into HPLC/MS/MS system, and the respective chromatographic peak areas 

were registered. The model compounds were chosen due to the fact that testosterone and midazolam are often used 

when studying the potential drug metabolism as the specific substrates for СYP450 cytochrome, isoform 3A4. In 

some of these cases, propranolol is used as an internal standard [1]. 

The response drift was observed during a continued period (one hour and more). It was shown that testosterone 

and midazolam responses correlated with a correlation coefficient from 0.75 up to 0.95 in different days, while there 

was no any correlation of propranolol response with responses of testosterone and midazolam. In different days the 

correlation coefficient for testosterone and propranolol was from 0.02 (no correlation) up to 0.96 (linear correlation). 

In was concluded, that the response instability in time could not be compensated using an internal standard. The 

sequence for HPLC/MS/MS runs was suggested which could be suitable for PK studies without a need for an 

internal standard. 

This work was supported by the Council of the President of Russia (grant МК-594.2010.3). 

1. S.X. Peng, A.G. Barbone, D.M. Ritchie // Rapid Commun. Mass Spectrom. 2003. Vol. 17. P. 509–518. 

562 



P16-035 UTLC-MS AND TLC-MS OF SINGLE PEPTIDES OF ACE INHIBITORS 

Vovk I. 1,2 , Popovic G. 3 , Simonovska B. 2 , Agbaba D. 3 , Albreht A. 2 

1 

EN-FIST Centre of Excellence-Slovenia 

2 


3 

Faculty of Pharmacy, Belgrade-Serbia 


Angiotensin converting enzyme inhibitors (ACE) well known prils are widely used as antihypertensive drugs. The 

active forms of these drugs called prilates are polar species, most of them possess dicarboxylate structure and in 

order to be absorbed they are designed in more lipophilic ethyl ester forms. Besides that the free diacid forms i.e., 

cilazaprilat, ramiprilat and quinaprilat are required to be tested as impurities in bulk drugs and their dosage forms. 

The goal of our investigation was to compare the conventional TLC silica gel 60 plates (250 μm layer) and monolithic 

ultra-thin layer chromatographic plats (UTLC, 10 μm layer) for the separation, detection and identification of 

structurally related compounds ACE inhibitors such as, lisinopril, cilazapril, ramipril and quinapril and apart that 

their active diacid forms. For successful application of UTLC plates we had to solve several technical problems 

including the application of small volumes of analytes that was overcome by application of 0.1 μl (bandwise) or 0.02 

μl (spotwise) using automatic TLC sampler ATS 4. Due to the lack of proper development devices ascending mode 

was applied first, but it resulted in poor peak shape including tailing of all studied compounds. Since horizontal 

development chambers for UTLC plates are still not commercially available, we made a simple but very practical 

adapter for the existing horizonatal developing chamber (10 cm x 10 cm, Camag), which can also be applied for 

the 10 cm x 20 cm chambers of the same producer. Since there is no possibility for fast detection of the separated 

compounds at UV (254 nm), densitometry was performed in absorption / reflectance mode at 220 nm or the plates 

were exposed to vapors of iodine and documented by image analyzing system, which finally results in the video 

denzitograms. The obtained results showed that monolithic layer is more efficient for the separation of structurally 

similar polar compounds, such as prilates than conventional silica layers. Identification of the studied compounds 

was confirmed by UTLC-ESI(PI)-MS or TLC-ESI(PI)-MS after on-line extraction from the UTLC and TLC plates by 

means of Camag TLC-MS. 


563


P16-036 DISTRIBUTION OF METOPROLOL IN HUMAN AUTOPSY MATERIAL 

Oertel R., Pietsch J., Arenz N., Goltz L., Kirch W. 

TU Dresden 

Corresponding author e-mail: Reinhard.Oertel@tu-dresden.de 

In the legal medicine autopsy material is normally investigated to find out the cause of death. But the results of 

corresponding toxicology measurements often involve more information. With screening methods drugs were 

detected without connection to the cause of death. A liquid/liquid extraction and a LC/MS/MS method were used 

for the determination of drug concentrations. In seven cases metoprolol could be determined in different autopsy 

materials. In all cases the dosage of the drug was unknown. In cases with oral application probably the patients 

took a normal customary continuous dosage. Intoxication with metoprolol could be excluded in all cases. The 

concentrations of metoprolol in blood were all in the therapeutic range. The time between oral intake and death 

was unknown. Therefore and because of the low number of cases statistic calculations were not meaningful and 

an individual case study was necessary. In three cases the highest concentration of metoprolol was found in the 

liver. Probably, metoprolol was taken shortly before the person died. In the other cases the highest concentration of 

metoprolol was found in urine. This means the elimination process of the drug predominated at the time of death. In 

all cases the concentrations of metoprolol were similar in the compartments heart blood, venous blood and brain. 

In this study it was possible to measure the distribution of metoprolol in human directly in several compartments. 

Measurements of drug concentrations in human autopsy material deepen the knowledge of its pharmacokinetic. 

564 



P16-037 STUDY ON A METABOLIC PROFILE GENERATED BY CYTOCHROME P450 2D6 INSIDE THE SEPARATION CAPILLARY 

Zeisbergerová M., Mádr A., Glatz Z. 

Masaryk University, Faculty of Science 

Corresponding author e-mail: glatz@chemi.muni.cz 

Drug metabolism studies are essential to determine the fates of new therapeutic agents in human body. In the 

development phase, the metabolic profiles of drug candidates have to be determined definitively. Further, they are 

important for designing prodrugs and pharmacologically active metabolites. Different human-derived in vitro systems 

like human liver slices, microsomes or recombinant enzyme systems are utilized in the investigation of metabolic 

disposition of potential therapeutics. Recombinant cytochrome P450 enzymes (rCYP) are suitable for ‘frontline’ 

predictive human metabolism studies in early drug discovery. rCYP are a favorite in vitro system for their availability 

and ease employment in high throughput assays. They are a valuable tool in the CYP phenotyping, i.e. searching for 

which CYP enzyme is involved in the biotransformation of new agents. 

The cytochrome P450 2D6 (CYP2D6) isoform accounts for only a small percentage of total hepatic CYPs (


P16-038 VALIDATION OF LC–UV AND LC–MS METHODS FOR DETERMINATION OF TORASEMIDE AND ITS IMPURITIES IN TABLETS 

Jovic Z. 1 , Zivanovic Lj. 2 , Radisic M. 1 , Malesevic M. 1 

1 


2 


Corresponding author e-mail: zarko.jovic@yahoo.com 

Torasemide, 1-isopropyl-3-(4-m-toluidinopyridine-3-sulphonyl)urea is a loop diuretic. Potential impurities of 

torasemide, which may be found in the drug product, are following: 4-(3-methylphenylamino)-3-pyridinesulfonamide 

(R2), N-(ethylaminocarbonyl)-4-(3-methylphenylamino)-3-pyridinesulfonamide (R3), 3,4-dihydro-4-(3-methylphenyl)- 

2H-pyrido[4,3-e]-1,2,4-thiadiazine-1,1-dioxide (R4) and N-(butylaminocarbonyl)-4-(3-methylphenylamino)-3- 

pyridinesulfonamide (R6). The aim of this study was to develop rapid, sensitive and reliable LC–UV and LC–MS 

methods for the separation and simultaneous determination of torasemide and its impurities R2, R3, R4, and R6 for 

routine quality control of commercially available torasemide tablets. The chromatographic separation was performed 

on a Zorbax SB C18 analytical column (250 mm x 4.6 mm, 5 µm) with column temperature set at 25 °C. The mobile 

phase was an aqueous solution of ammonium formate, 10 mM, adjusted to pH 2.5 with formic acid (mobile phase A) 

and acetonitrile (mobile phase B), with gradient elution: 0 min, B 30%; 11.2 min, B 60%; 11.3 min, B 30%, hold for 10 

minutes. The flow rate was 1 mL min–1 and the injection volume was 30 µL for LC–UV analysis, and 10 µL for LC–MS 

analysis. For LC–UV analysis, detection was performed at 290 nm. For LC–MS analysis, a single quadrupole mass 

analyzer with electrospray ionization (ESI) techinique was used. The optimized parameters of the interface were: 

drying gas (N2) flow rate, 12.0 L min–1; nebulizer gas pressure, 60 psig; temperature, 350 °C; capillary voltage, 3000 

V. To quantify torasemide and its impurities, selective ion monitoring (SIM) of protonated molecular ions [M+H] + at 

m/z: 349 (torasemide), 264 (impurity R2), 276 (impurity R4), 335 (impurity R3) and 363 (impurity R6) was used. The 

both analytical methods, LC–UV and LC–MS were successfully validated according to the ICH guideline. The both 

analytical methods showed good selectivity. The concentrations of torasemide and its impurities used for linearity 

testing in LC–UV analysis were in range 70–130 µg mL–1 for torasemide and 0.1–10 µg mL–1 for impurities. For LC– 

MS analysis, the concentrations were in range 0.7–1.3 µg mL–1 for torasemide and 0.01–1.0 µg mL–1 for impurities. 

The coefficients of correlation (r) of the calibration curves were greater than 0.9982, for both methods. Accuracy 

and precision studies were performed at three different concentrations with three replicates covering the specified 

range. The obtained recovery values (95.8–104.9%) and relative standard deviations (0.12–5.56%) indicate good 

accuracy and precision of both methods. The limits of detection for all compounds were in range 0.02–0.04 µg mL–1 

for LC–UV analysis and 0.0002–0.0004 µg mL–1 for LC–MS analysis. The limits of quantification were 0.06–0.1 µg 

mL–1 and 0.0006–0.0012 µg mL–1 for LC–UV and LC–MS analyses, respectively. The proposed LC–UV and LC–MS 

methods are sensitive, specific and reproducible enough to be applied in the routine quality control of torasemide 

and its impurities in tablets. 

566 



P16-039 VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF ZOLPIDEM TARTRATE AND ITS 

DEGRADATION PRODUCTS IN TABLETS 

Jovic Z. 1 , Zivanovic Lj. 2 , Malesevic M. 1 

1 


2 


Zolpidem tartrate (bis[N,N-dimethyl-2-[6-methyl-2-(4-methylphenyl)imidazo[1,2-a]pyridin-3-yl]acetamide] 

(2R,3R)-2,3-dihydroxybutanedioate) is used as a hypnotic in the short-term management of insomnia. Potential 

degradation products of zolpidem tartrate are following: 2-[6-methyl-2-(4-methylphenyl)imidazo[1,2-a]pyridin-3-yl] 

acetic acid (zolpacid), N,N-dimethyl-2-[6-methyl-2-(4-methylphenyl)imidazo[1,2-a]pyridin-3-yl]-2-oxoacetamide 

(oxozolpidem), 6-methyl-2-(4-methylphenyl)imidazo[1,2-a]pyridin-3-carboxaldehyde (zolpaldehyde) and 5-methyl- 

2-(4-methylbenzamide)pyridine (zolpyridine). The aim of this study was to develop and validate rapid, sensitive and 

reliable reversed-phase high-performance liquid chromatographic method for the separation, identification and 

simultaneous determination of zolpidem tartrate and its four degradation products in tablets. Optimization of the 

method and method robustness evaluation were performed by usage of 32 full factorial design. Data obtained from 

the factorial design were evaluated by response surface methodology. The optimal chromatographic separation 

was obtained with a mobile phase consisting of methanol–10 mM ammonium acetate aqueous solution (70:30, 

v/v) at a flow rate of 1 mL min–1. The isocratic elution of analyzed compounds was performed on a Luna C18(2) 

analytical column (250 mm x 4.6 mm, 5 µm) with column temperature set at 35 °C. The injection volume was 10 µL 

and detection was performed at 254 nm. The total duration of analysis was about 15 min. After development and 

optimization, the analytical method was successfully validated according to the ICH guideline. The method showed 

good selectivity, because there were no interferences between analyzed substances and tablet excipients. The 

method was linear over the concentration range of 280–520 µg mL–1 for zolpidem tartrate and 0.4–40 µg mL–1 

for degradation products. The coefficients of correlation (r) of the calibration curves were greater than 0.999, for 

all analyzed compounds. Accuracy and precision studies were performed at three different concentrations with 

three replicates covering the specified range. The method showed good intra-day precision with relative standard 

deviations lower then 2.5%. The obtained recovery values (98.9–103.6%) indicate good accuracy of the method. The 

limits of detection for all compounds were in range 0.02–0.08 µg mL–1. The quantification limits were in range 

0.08–0.26 µg mL–1. The validated method was successfully applied to the analysis of commercially available 

zolpidem tartrate tablets. The proposed RP-HPLC method is sensitive, specific and reproducible enough to be 

applied in the routine quality control of zolpidem tartrate and its degradation products in tablets. 


567

P17 

LIFE SCIENCES 

AND BIOANALYSIS 

POSTER 

SESSIONS

POSTER SESSION 17 - LIFE SCIENCES AND BIOANALYSIS 

P17-001 LC-MS IDENTIFICATION OF MYCOLIC ACIDS AND THEIR METABOLITES AS BIOMARKERS FOR ANCIENT TUBERCULOSIS 

Bona A., Boros Major A., Maasz G., Jambor E., Mark L. 

University of Pecs 

Corresponding author e-mail: agnes.bona@aok.pte.hu 

It is a widely accepted view that diagnosis of tuberculosis from archaeological human skeletal remains is not an 

easy task by using classic morphological methods. A biomolecular approach to diagnosis is probably more reliable 

than gross osteological examination of archaeological skeletal remains. The structure of the recent mycobacterial 

cell envelope is well known, contains a characteristic mycoloyl arabinogalactan-peptidoglycan complex. Mycolic 

acids, as extremely hydrophobic, long chain fatty acid residues of mycobacterial cell envelope, are ideal targets 

for biomolecular detection of ancient tuberculosis. In this study, the “chemical fossilization” process of the mycolic 

acids and their derivatives have been discovered by using chemical modeling and modern analytical techniques. 

For investigations off-line HPLC MALDI TOF MS was used. In this case the fractions of the interesting ancient 

biomarkers are separated and collected by HPLC, then directly analyzed by MALDI TOF MS. On the other hand, 

nano-LC coupled with nanoelectrospray ionization ultra high resolution mass spectrometry was used for quantitative 

information and for further molecular evidence. 


569


P17-002 PROTEIN SEPARATION WITH A NEW HIGH RESOLUTION GLASS COLUMN 

Fuchs M. 

Wissenschaftliche Gerätebau Dr.Ing. H.Knauer GmbH-Germany 

Corresponding author e-mail: fuchs@knauer.net 

A new type of glass column with pressure resistant glass tubes for up to 10 MPa (100 bar) operating pressure has 

been introduced by Knauer. The pressure resistant filling funnel belonging to this column allows to fill the gel under 

pressure from the beginning while the column bed can be further compressed after the upper adjustable adapter is 

placed into the glass tube. A number of marker proteins is separated under flow and pressure conditions reaching 

the limits of the Biofox 40 SEC gel to demonstrate the power of high resolution biochromatography. It is shown that 

the operating range of this biochromatography medium is extended when using the Knauer Bioline high-resolution 

glass column. 

570 



P17-003 SEPARATION AND DETERMINATION OF ELEVEN PTERIDINES AND LUMAZINES IN HUMAN URINE BY LIQUID 

CHROMATOGRAPHY USING DIODE ARRAY AND FLUORESCENCE SERIAL DETECTION 

Cañada-Cañada F., Mancha de LLanos A., Espinosa-Mansilla A., Muñoz de la Peña A. 


Corresponding author e-mail: nuncy@unex.es 

The metabolites in study are a family of heterocyclic formed by a bicyclic pyrimidine- pyrazine that occur in a wide 

range of living systems and participate in relevant biological functions [1]. These metabolites of interest include 

pteridines (2-amino-4-hydroxy derivatives) and lumazines (2, 4-dihydroxy derivatives). In the present work a liquid 

chromatographic method, with ultraviolet and fluorimetric in serial detection, has been developed for the separation 

and determination of eleven marker pteridines and lumazines (7-hydroximethyl-lumazine; pterin-6-carboxilic acid; 

neopterin; 6-hydroximethyl-lumazine; L-monapterin; xanthopterin; isoxanthopterin; pterin; biopterin; 7-biopterin; 

6-hydroximethyl-pterin), in urine samples. The influence of mobile phase composition and buffer pH, have been 

studied. The optimized mobile phase was composed by a Tris-HCl buffer (15 mM) at pH 6.10 solution (eluent A) and 

a Tris-HCl buffer (15 mM) at pH 6.40 solution (eluent B), in gradient mode and the separation was accomplished 

in less than 25 min. The column temperature was maintained at 23 ºC. Pteridins and lumazines were determined 

by fluorimetric detection at λex = 272 nm and λem = 445, 410 and 465 nm. CREA, as a reference of metabolites 

excretion in urine, was determined by photometric detection at 230 nm. Detection limits were ranged between 0.21 

- 6.10 ng mL-1 for pteridine derivatives, and 0.22 µg mL-1 for CREA. Two different oxidation urine processes were 

optimized, Method A: alkaline iodine/iodide solution (Trehan method); Method B: neutral potassium permanganate 

solution. The urine of two groups of healthy persons was tested. The Group I was composed by persons between 

20-30 years old and the Group II by persons between 5-10 years old. Each group of urines was oxidized using both 

optimized method and the results of the pteridins/CREA and NEO/BIO ratio were compared. NEO/BIO ratio was 0.98 

and 0.86, for adults and children, respectively by mean of alkaline iodine/iodide oxidation and 0.45 and 0.57, using neutral 

permanganate oxidation. The pteridines/CREA and NEO/BIO ratio drastically depend of the chemical-physical conditions 

of the oxidation process applied. This fact must be taking account for comparative studies and for establishing 

reference values. The developed method enabled us to determine the listed pteridines and creatinine in just one 

run and, moreover, to establish a pteridines complete map for healthy persons. Acknowledgment: Finantiation from 

the Junta de Extremadura and European Social Funds (consolidation of Research Group FQM003). [1] Kappock, TJ, 

Caradonna, JP, Pterin-dependent amino acid hidroxylases, Chem. Rev., 96 (1996) 2659-2756 


571


P17-004 TRANS-RESVERATROL AND TRANS-PICEID IN HUNGARIAN WINES 

Boros Major A., Bona A., Jambor E., Montsko G., Ohmacht R., Mark L. 

University of Pecs 

Plant polyphenols are naturally occurring secondary plant metabolites, synthesized in response to environmental 

stress factors. As being anti-oxidants and free-radical scavengers they serve as essential components of the human 

diet. Among polyphenols well studied representatives are the trans-resveratrol and the trans-piceid molecules 

the latter being the glycoside of trans-resveratrol. Trans-resveratrol has been shown to modulate the metabolism of 

lipids, inhibit the oxidation of low density lipoproteins, reduce platelet aggregation is known to have anti-inflammatory, 

and has anti-tumor, cardio- and vasoprotective effects which plays a curcial role in the prevention of chronic 

cardiovascular and tumorous diseases. In the present study, forty-four Hungarian wines were analyzed using HPLC/ 

DAD detection. The wines were from Villány and Eger wine regions representing three wineries from 2003 to 2007 

vintage years. The trans-resveratrol amount in the processed wines ranged from 0.75 mg/L and 10.4 mg/L and for 

trans-piceid the results spanned from 0.1 mg/L to 3.7 mg/L. Our results show that trans-reservatrol content is mainly 

dependent on variety and vintage year. 

572 



P17-005 SEPARATION AND CHARACTERIZATION OF ALPHA 2-3 AND ALPHA 2-6 ISOMERIC SIALYLATED O-GLYCOPEPTIDES FROM 

PROTEOLYTICALLY DIGESTED CASEINOMACROPEPTIDE USING HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY 

(HILIC) - TANDEM MASS SPECTROMETRY 

Hernandez-Hernandez O. 1 , Lebrón-Aguilar R. 2 , Quintanilla-Lopez J. 2 , Sanz M.L. 1 , Moreno F.J. 3 

1 


2 

Instituto de Química-Física “Rocasolano”, Madrid 

3 

Instituto de Fermentaciones Industriales-CIAL, Madrid 

Corresponding author e-mail: j.moreno@ifi.csic.es 

Most carbohydrates can be attached to proteins by two different bonds, called N- and O- linkages. N-linked 

glycosylation requires a specific amino acidic sequence whilst O-linked glycosylation consists of attaching the 

glycans to the hydroxyl oxygen of any serine or threonine residue. Generally, O-glycans are attached to the 

protein through N-acetylgalactosamine and sialic acid (Neu5Ac) is found to be terminating branches of glycans. 

The most common linkages found in the sialic acid are to the C-3 or C-6 positions of galactose residues. Bovine 

caseinomacropeptide, a fragment derived from the action of chymosin or pepsin cleavage of the kappa-casein, has 

two previously characterized isomeric trisaccharides, containing the alpha 2-3- and the alpha 2-6- linked sialic acid, 

respectively (1). Currently, hydrophilic interaction liquid chromatography (HILIC) is gaining importance in the analysis 

of glycopeptides, but little information is found about O-glycan and O-glycopeptide analysis. Therefore, in this work 

a HILIC multi-stage mass spectrometric method (HILIC-ESI-MSn) has been developed to characterize alpha 2-3 

and alpha 2-6 sialylated trisaccharides either in their unconjugated form or linked to the bovine CMP. Proteolytically 

digested CMP and a tetrapeptide model non-enzymatically glycosylated with two different sialyl-trisaccharides were 

employed. The separation was carried out on a zwitterionic HILIC column, using a linear gradient of acetonitrile and 

water, with 0.005% v/v of formic acid, coupled to an ion trap mass spectrometer at positive and negative modes. 

CMP glycopeptides differing only in the isomeric sialylated glycan structure, i.e. linear (alpha 2-3) and branched 

(alpha 2-6) depending on the attachment of the sialic acid, could be separated by a ZIC®-HILIC column under the 

chromatographic conditions described above. Furthermore, this same behavior was also found in the tetrapeptide 

model glycosylated non-enzymatically with two isomeric sialyl-trisaccharides. Knowing the studied trisaccharide 

structures, the values of (w^w)pKa and (w^s)pKa were calculated. The branched trisaccharide was less acidic (higher 

pKa values) than the linear trisaccharide. Consequently, it could be expected that glycopeptides with the branched 

trisaccharide had a higher retention time due to a low degree of electrostatic repulsion interactions between the 

negatively charged sialic acid residue and the negatively charged terminal sulfonate group of the stationary phase 

than their counterparts with a linear isomeric trisaccharide. This behaviour was confirmed by the MS2 detection of 

the fragment Y1beta-type ion corresponding to the initial neutral loss of Gal residue, denoting the presence of this 

carbohydrate at the terminal position of the glycan structure and, therefore, revealing the presence of the branched 

trisaccharide. In conclusion, ZIC-HILIC-ESI-MS2 has shown to be a powerful technique for the separation and 

identification of alpha2-3 and alpha2-6 isomeric sialylated O-glycopeptides without any derivatization treatment. (1) 

Varki et al. (2009). Essentials of Glycobiology. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY. This 

work was financed by project PIF-SIALOBIOTIC 200870F0101 funded by CSIC. O. Hernández thanks CSIC for a JAE 

Predoc grant. 


573


P17-006 DEVELOPMENT AND VALIDATION OF AN HPLC METHOD FOR THE ANALYSIS OF A NEW NITROSYL RUTHENIUM COMPLEX, 

CIS-[RUCL(BPY)2NO](PF6)2, NITRIC OXIDE DONOR IN RAT LIVER MICROSOME 

Sesso T.A.C., Simões R.A., Santana R.S., de Oliveira A.R.M. 


Corresponding author e-mail: deoliveira@usp.br 

In the last decades, it has been established that nitric oxide (NO) plays an important role in several biological events 

including vascular tone control, and this fact has lead to the development of NO donors with therapeutic uses. One 

of the most used NO donors, sodium nitroprusside, presents some drawbacks in its use, mainly, due to release of 

cyanide ions during its metabolism. Thus, new NO donors have been synthesized to minimize these undesirable 

effects. Among these new NO donors, the ones centered in the ruthenium metal have shown interesting results, 

mainly due to the similarity of the behavior of this metal with iron in physiological medium. A particular compound 

was developed, cis-[RuCl(bpy)2NO](PF6)2, and studies have been showing that this complex induces vasorelaxation 

in aortic ring, suggesting its applicability in therapeutics. However, as one of the first steps during drug development, 

the biotransformation of the drug needs to be evaluated. Moreover, before performing a biotransformation study, 

it is necessary to validate all the methodology to guarattee the confiability of the results. In this context, the 

aim of this work was to develop and to validate an HPLC method employing rat microsomal fraction for further 

biotransformation studies employing this ruthenium complex as substrate. The method was validated employing a 

Shimadzu HPLC system composed with dual piston pump, a rheodyne injector equiped with a 20 μL loop and a diode 

array detector operating in the range of 190-800 nm. Separation was performed using a LiChrospher® 100 RP-8 (4.0 

x 125 mm) column, coupled with LiChrospher® 100 RP-8 guard column. The mobile phase employed was a mixture 

of acetonitrile : aqueous trifluoroacetic acid solution, pH 5 at a proportion of 30:70 (v/v). The flow rate employed 

was 0.5 mL min-1. For quantitative analysis the detection was set at 290 nm. The method was validated according 

to the literature guidelines. The method showed to be linear over the concentration range of 3.40 – 339.60 µM with 

correlation coefficient > 0.99. The quantification limit was 3.40 µM with RSD below 20%. Within-day and betweenday 

precision and accuracy evaluated by RSDs and relative errors, respectively, were lower than 15% for the nitrosyl 

ruthenium complex. The stability test (ANOVA) showed no degradation under 37°C during 60 minutes with p values 

higher than 0.05. A preliminary biotransformation study employing hepatic rat microsomal fraction showed that after 

30 min all the complex was consumed (compared with a control sample without NADPH cofactor), indicating that this 

complex was metabolized by rat CYP450. 

Financial support: CNPq, CAPES, FAPESP 

574 



P17-007 QUANTIFICATION OF PHENOLIC ANTIOXIDANTS IN RAT CEREBROSPINAL FLUID BY GC–MS AFTER ORAL 

ADMINISTRATION OF COMPOUNDS 

Zafra Gómez A. 1 , Luzón-Toro B. 2 , Jiménez-Díaz I. 1 , Ballesteros O. 1 , Navalon A. 1 

1 


2 


Corresponding author e-mail: azafra@ugr.es 

One of the most important factors in olive oil benefits is the presence of phenolic compounds. These compounds 

are a group of chemicals that act as strong antioxidants and radical scavengers. Olive oil is a source of a large 

amount of phenolic compounds [1]. In the last years, some authors have described that brain aging and the most 

diffused neurodegenerative diseases of the elderly proceed with oxidative damage [2]. The compounds have been 

described as potential inhibitors of amyloid aggregation and significance to Alzheimer’s disease has been suggested 

[3]. A gas chromatographic-mass spectrometric method for qualitative and subsequent quantitative analysis of 

phenolic antioxidants compounds, presents in olive oil in rat cerebrospinal fluid (CSF) after oral administration of 

compounds is proposed. The procedure involves the extraction of compounds from the samples by a traditional 

microliquid–liquid extraction method, followed by a silylation step before the GC–MS analysis. The chromatographic 

separation was performed by using a low bleed DB5-MS fusedsilica capillary column. The presence of 21 phenolic 

compounds was tested in CSF extracts and only free hydroxyphenyl ethanol (HPE), dihydroxyphenyl (DHPE) ethanol 

and ferulic acid (FA) were detected. Those compounds were then quantitatively determined. The molecular ion for 

silylated compounds appears at 370m/z for DHPW, 282m/z for HPE and 338m/z for FA respectively, while the base 

peak appears at 267m/z, 179m/z and 338m/z. α-Naphthol was used as a surrogate (216 and 201m/z). The detection 

capabilities (CCa) were 74, 92 and 79 ng•mL-1 respectively. The method was validated in term of selectivity, 

precision and accuracy, sensitivity and linearity and a recovery assay was also carried out. Recoveries found were 

from 90.0% and 110%. Finally, the method was applied to the determination of trace amounts of compounds in 

rat cerebrospinal fluid after oral administration. The animals were fed with a standard chow diet (free of phenolic 

antioxidants) in order to avoid the influence of any other component of the diet on the CSF of the animals. [1]F. 

Visioli, A. Poli, C. Galli. Med. Res. Rev. 22 (2002) 65. [2]L. Rossi, S. Mazzitelli, M. Arciello, C.R. Capo, G. Rotilio. 

Neurochem. Res. 33 (2008) 2390. [3]S. Bastianetto, S. Krantic, R. Quirion. Mini Rev. Med. Chem. 8 (2008) 429. 


575


P17-008 DETERMINATION OF BISPHENOL A AND ITS CHLORINATED DERIVATIVES IN PLACENTAL TISSUE SAMPLES BY LC-MS/MS 

Zafra Gómez A., Jiménez-Díaz I., Navea N., Ballesteros O., Navalón A., Fernández M.F., Olea N., Vilchez J.L. 

1 


Corresponding author e-mail: azafra@ugr.es 

The group of compounds commonly called endocrine disrupting chemicals (EDCs) covers a wide range of synthetic 

and natural substances able to alter the normal hormone function of wildlife and humans, consequently causing 

adverse health effects. Bisphenol A (BPA) and its chlorinated derivatives are some of these compounds. Many 

researchers hypothesize that exposure to EDCs during critical periods of development –in utero or early postnatal 

life- could cause morphologic and functional alterations in wildlife and humans affecting growth, reproduction and 

also development [1, 2]. In this work, we propose a new liquid chromatography-tandem mass spectrometry (LC-MS/ 

MS) method to determine these compounds in human placental tissue samples. The method involves an extraction 

phase of the extracts from the samples using ethyl acetate, followed by a clean-up phase by centrifugation prior to 

their quantification by LC-MS/MS using an atmospheric pressure chemical ionization (APCI) interface in the negative 

mode. Deuterated bisphenol A (BPA-d16) was used as internal standard. Found detection limits ranged from 0.2 to 

0.6 ng g-1 and quantification limits from 0.5 to 2.0 ng g-1 for bisphenol A and its chlorinated derivatives, while interand 

intra-day variability was under 8.1%. The method was validated using standard addition calibration and a spike 

recovery assay. Recovery rates for spiked samples ranged from 97% to105%. This method was satisfactorily applied 

for the determination of BPA and its derivatives in 49 placental tissue samples collected from women who live in 

the province of Granada (Spain). [1]Fernández MF, Olmos B, Granada A, López-Espinosa MJ, Molina-Molina JM, 

Fernández JM, Cruz M, Olea-Serrano F, Olea N (2007) Environ Health Perspect 115:8-14. [2]Bosquiazzo VL, Varayoud 

J, Muñoz de Toro M, Luque EH, Ramos JG (2010) Biol Reprod 82(1):86-95 

576 



P17-009 SUITABLE METHOD FOR THE DETERMINATION OF ROSIGLITAZONE AND ITS MAIN METABOLITES IN RAT LIVER 

MICROSOMAL FRACTION EMPLOYNG HOLLOW-FIBER LIQUID-PHASE MICROEXTRACTION (HF-LPME) 

Calixto L.A., Bonato P.S. 



Rosiglitazone (RSG), a thiazolidinedione antidiabetic agent, is a potent and selective activator of peroxisome 

proliferator-activated receptor γ (PPAR γ); it has been developed for the treatment of type 2 diabetes. This drug 

is extensively metabolized, with virtually no rosiglitazone being excreted unchanged; the two major routes of 

metabolism were N-demethylation and hydroxylation. The specific P450 enzymes involved in the metabolism of 

RSG are primarily cytochrome 2C8 (CYP2C8), with minor contributions from CYP2C9. A suitable method for in vitro 

metabolism study of this drug should be selective not only for the drug but also for the metabolites. Thus, the aim of 

this study was the optimization of a high-performance liquid chromatography method for the determination of RSG 

and its main metabolites (N-desmethyl rosiglitazone and ρ-hydroxy rosiglitazone) in microsomal fraction isolated 

from rat liver homogenates. The RSG and metabolites determination was carried out using an X-Terra RP-18 column, 

at 22ºC. The mobile phase was composed of water, acetonitrile and acetic acid (85:15:0.5, v/v/v) and the detection 

was performed at 245 nm. A hollow-fiber liquid-phase microextraction (HF-LPME) procedure was used for sample 

preparation. The optimization was carried out by multifactorial experiments and the following optimal condition was 

established: sample agitation at 1750 rpm, extraction for 30 min, hydrochloric acid 0.01 mol/L as acceptor phase, 

1-octanol as organic phase and donor phase pH adjustment to 8.0. The recovery rates, obtained by using 1 mL of 

microsomal preparation, were 47-70%. The method presented quantification limits (LOQ) of 50 ng/mL and it was 

linear over the concentration range of 50-6000 ng/mL for all analytes. (r ≥ 0.9961). Within-day and between-day 

precision and accuracy evaluated by RSDs and relative errors, respectively, were lower than 15% for all analytes. 

Moreover the analytes were stable under biotransformation conditions. The validated method was suitable to study 

the in vitro biotransformation of rosiglitazone employing rat liver microsomal fraction. Financial support: Capes, 

CNPq and FAPESP 


577


P17-010 VALIDATION OF A QUANTITATIVE GC METHODOLOGY FOR PHYTOSTEROLS DETERMINATION IN SERUM 

Vidal C., García-Llatas G., Lagarda M.J., Alegría A., Barberá R. 


Corresponding author e-mail: guadalupe.garcia@uv.es 

INTRODUCTION Plant sterols (Ps), due to their hipocholesterolemic effects, are used for enrichment in functional 

foods. Many methodologies, using lipid extraction, saponification and analysis by gas chromatography (GC), have 

been described to determine Ps in serum. But, some them have been validated partially and only two are described 

to have been completely validated: with total lipid extraction before saponification and solid-phase extraction 

for purification (Phillips et al., 1999), or directly saponification but using high volumes of serum (Ahmida et al., 

2006). AIM To set up and validate a relatively fast GC methodology useful to determine Ps in serum. MATERIAL 

AND METHODS Blood samples of healthy post-menopausal women were used, in order to evaluate the developed 

method. Analyses: Serum (100 µl) was saponified with 0.71 M KOH (1h, 65ºC), after internal standard and BHT 

addition. The unsaponifiable fraction was extracted with hexane and derivatized into trimethylsilylethers. Ps were 

determined by GC-FID using a capillary column (CPSil 8CB, 50m x 0.25mmID x 0.25µm). Oven was programmed from 

150ºC (3 min), then raised to 280ºC at 30ºC/min, and finally until 290ºC (10 min) (total run time = 47 min). Injector 

(splitless) and FID temperatures were 280ºC and 300ºC, respectively. RESULTS A good linearity (r > 0.99) was found 

in the assayed amount range: beta-sitosterol (0.198-1.980 µg) y = 0.601x + 0.127, and campesterol (0.054-1.080 µg) 

y = 0.518x + 0.079. Detection and quantification limits (LOD and LOQ, respectively) were determined according 

to the USP criteria by running six blanks. LOD and LOQ (ng/ml serum) were 146 and 488 (beta-sitosterol) and 16 

and 53 (campesterol), respectively. Similar results were found with the LOD also estimated as Knoll (163 for betasitosterol 

and 12 ng/ml serum for campesterol). Accuracy (%), estimated by recovery assays, was 108.9 ± 5.3 (betasitosterol) 

and 96.9 ± 4.1 (campesterol). Intraday precision expressed as the relative standard deviation, is 2.3% 

(beta-sitosterol) and 0.9% (campesterol). The method was applied to the serum samples, resulted in beta-sitosterol 

concentrations (µg/ml serum) ranging 0.780-12.112, and, for campesterol, ranged between 0.221-5.757 µg/ml serum. 

CONCLUSIONS The developed method allowed a fast (one-day analysis) and reliable quantitation of Ps in serum 

samples, as the results of linearity, accuracy and repeatability showed. Therefore, it could be used in clinical assays 

for serum Ps measurements where a high number of samples are handled. ACKNOWLEDGEMENTS Study financed 

by AGL2008-02591-C02-01 (CICYT-FEDER) and partially by Consolider Ingenio 2010 program FUN-C-FOOD 

CSD2007-063. REFERENCES Ahmida H.S.M., Bertucci P., Franzò L., Massoud R., Cortese Cl., Lala A., Federici 

G.(2006) J. Chromatogr.B. 842, 43-47. Knoll J.E. (1985) J. Chromatog. Sci. 23,422-425. Phillips K.M., Ruggio D.M., 

Bailey J.A. (1999) J. Chromatogr.B.732, 17-29. USP: The United States Pharmacopeia (USP XXIII), 1989, p. 1711. 

578 



P17-011 DETERMINATION OF 2-THIOTHIAZOLIDINE-4-CARBOXYLIC ACID IN URINE BY HPLC 

Torrado S., Rosell M.G., Garrote P., Guardino X. 


The aim of this work is to optimise and validate a reference method to determine 2-thiothiazolidine-4-carboxylic 

acid (TTCA) in urine by HPLC in order to be able to assess the exposure to carbon disulfide, as TTCA is the 

metabolite that is excreted in urine of people exposed to carbon disulfide. Among the instrumental methods found 

in the literature, a method based in a gradient with two mobile phases was chosen because previous studies had 

shown that this approach is faster and cheaper than the one based in ion-pair chromatography using one mobile 

phase. The extraction step was also optimised as different extraction solvents were found in the literature. Finally, 

we will analyse urine samples from people exposed to carbon disulfide. To extract the TTCA, 0.5 ml of 1 N HCl 

and approximately 0.3 g of NaCl are added to one ml of urine. The urine is shaken and then the TTCA is extracted 

twice. Peroxide-free ethyl ether, ethyl acetate and tert-butylmethylether will be tested. The extracts are dried with 

a nitrogen flow and the residue is regenerated with 1 ml of 50% methanol. Twenty µl of this solution are injected in 

a C-18 column (250 mm x 4.6 mm i.d., and particle size 5 µm) with a precolumn of the same stationary phase and 

detected with a PDA detector at 273 nm. The mobile phases are: 50% acetonitrile or methanol and 0.1 M acetic acid 

at pH 3 or 0.1% phosphoric acid. The flow rate was 1 ml/min. The column was held at 30ºC. Validation is performed 

with the optimised method including the following parameters: recovery, limit of detection and quantification, 

precision, accuracy and linearity or range. Linearity is required between 0.1 and 2 times the occupational exposure 

level, according the standards used for industrial hygiene. Comprehensive validation to test the suitability of the 

method as a reference method will be presented. 


579


P17-012 METABOLOMIC APPROACH TO THE NUTRACEUTICAL EFFECT OF ROSEMARY EXTRACT IN DIABETIC CHILDREN 

Balderas C. 1 , Villaseñor A. 1, García A. 1 , Rupérez F. 1 , Ibañez E. 2 , Señorans J. 3 , Guerrero-Fernández J. 4 , González I. 4 , Gracia-Bouthelier R. 4 , 

Barbas C. 1 

1 


2 

Instituto de Fermentaciones Industriales, CSIC, Madrid 

3 


4 

Hospital La Paz, Madrid 


It is generally accepted that the majority of complications associated with type I diabetes, such as cardiovascular 

disease, nephropathy or cataracts, are related to oxidative stress. A clinical trial was performed to investigate the 

potential nutraceutical antioxidant properties of a Rosmarinus officinalis extract used as food additive in diabetic 

children vs their controls. 33 type 1 diabetic children and 16 controls ranging from 6 to 11 years including both boys 

and girls were enrolled in the assay. The aim was evaluating the effect of a long term, “antioxidant diet” consisting 

on a regular diet plus the ingestion of meat products enriched with 0.02% rosemary extract and 0.3% de PUFAs 

(300 g/week). One of the strategies employed by the emergent science of metabolomics is metabolic fingerprinting; 

which involves rapid, high-throughput global analysis to discriminate between samples of different biological 

status or origin by pattern recognition. Capillary electrophoresis provides a comprehensive snapshot of multiple 

metabolites in biological samples, especially in urine because all analytes are already dissolved and most of them 

are easily separated due to its charge; despite its detection system, CE gives a general metabolic response. In 

addition, with different CE modes (CZE and MEKC, normal and reverse polarity) we can obtain the analysis of a wide 

set of compounds both charged and neutral producing an extended representation [1-3]. Punctual urine samples 

collected at time zero and after 12 months were measured in two CE modes. Method 1: normal polarity cyclodextrin 

modified micellar electrokinetic chromatography (CD-MEKC) mostly for cations and neutral compounds and method 

2: reversed polarity capillary zone electrophoresis (CZE) mainly anions. The methodology already proved to be 

successful with urine from control and diabetic rats [1,3], but humans with different diet, physiological, environment 

and genetic variation are still a challenge. CE fingerprints were first pre-treated and after that chemometric tools 

such as PCA (principal components analysis), PLS-DA (partial least squares discriminant analysis) and OPLS- 

DA (orthogonal partial least squares discriminant analysis) were applied. Regarding multivariate data analysis, a 

satisfactory result for pattern recognition was obtained for sample classification at time zero with a supervised 

analysis such as OPLS-DA using Pareto scaling (most commonly used for metabolomic research) After the treatment, 

comparing the three groups (healthy, treated and non-treated diabetic), results showed that there is a certain 

change in metabolism due to the diet. Metabolites identified so far are mainly related to Krebs cycle, amino acids 

metabolites, ketone bodies and products of catabolism. REFERENCES 1. Barbas, C.; Vallejo, M.; García, A.; Barlow, 

D.; Hanna-Brown, M., J Pharm Biomed Anal 2008, 47 (2), 388-98. 2. Garcia-Perez, I.; Couto Alves, A.; Angulo, S.; Li, 

J.; Utzinger, J.; Ebbels, T.; Legido-Quigley, C.; Nicholson, J.; Holmes, E.; Barbas, C., Anal Chem 2010, 82 (1), 203-10. 

3. Vallejo, M.; Angulo, S.; García-Martínez, D.; García, A.; Barbas, C., J Chromatogr A 2008, 1187 (1-2), 267-74. 

580 



P17-013 QUANTIFICATION OF GLUTAMATE UPTAKE IN BRAIN BY CE-LIF 

Lorenzo MªP., Valladolid I., Merino B., Ruiz-Gayo M., Fernández-Alfonso M., Cano V., Barbas C., García A. 



There is growing evidence that high-fat diets and subsequent obesity can affect brain functioning and behaviour. 

Therefore a high fat diet induces apoptosis of hypothalamic neurons, impairs memory and hippocampal morphology. 

Glutamic acid is the main excitatory transmitter in central nervous system (CNS) where glutamatergic neurons 

mediate many vital processes, like the encoding of information, the memory, spatial recognition and the maintenance 

of consciousness. An increase in the amounts of glutamate (GLU) in the extracellular fluid surrounding neurons 

can cause them to be overexcited, leading ultimately to their death. Since there is not data about the effect of 

diet-induced obesity on the physiological extracellular levels of GLU in the brain, the goal of this work was to 

evaluate changes in the ability of astrocytes to re-uptake glutamate after the consumption, during 8 weeks, of 

a high fat diet. An experimental model with mice was tested. Animals were divided into two groups with similar 

average body weight, housed six per cage, and assigned either to a low fat (LF) or high fat diet (HF). Mice were 

sacrificed by beheading and briefly, hippocampus was extracted. Hippocampal slices (300 μm thick) were obtained 

with a chopper system. Then, the slices were incubated in oxygenised ice-cold ringer buffer. Glutamate uptake of 

hippocampal slices were initiated by adding glutamate (μM) for 30 minutes. Following, slices were washed (2 x 5 

min). To perform glutamic acid release, slices were incubated in 500 μl KCl, 50 mM in Krebs-Ringer´s solution, for 1 

min and proteins amount were measured in samples. Values obtained were expressed in concentration of GLU (nM) 

per milligrams of proteins per minute (nM/mg/min). In the analytical point of view samples were a real challenge with 

ultra-trace amounts of GLU and a high concentration of salts which made difficult the use of GC-MS. CE-LIF was 

selected based on a previous method by Zhang et al [1] where GLU was detected after derivatization with fluorescein 

isothiocyanate (FITC) in human serum at nM level. The separation and the derivatization conditions with FITC were 

optimised and analytical parameters checked. Finally, GLU released to the media from brain slices previously 

incubated with increasing concentrations of GLU ranging from 0-500 μM at 5 points were measured. Results 

correlated with those obtained with an expensive assay with radioactive GLU and proved that rats taking a high 

fat diet (HF) released higher concentrations of GLU to the media than rats under a low fat diet. Those results might 

explain the impairment in spatial learning in HF mice. REFERENCES [1] J. Zhang, J. Tian, J. Liu, H. Gao, X. Chen, and 

Z. Hu. Microchim. Acta 143, (2003) 241–244. 


581


P17-014 SEPARATION OF BETA-LACTOGLOBULIN A AND BETA-LACTOGLOBULIN B BY HIGH PERFORMANCE GRADIENT 

CHROMATOFOCUSING – MASS SPECTROMETRY 

Albreht A., Vovk I., Simonovska B. 


Corresponding author e-mail: alen.albreht@ki.si 

Conventional chromatofocusing (CF) for the separation of bipolar compounds was already established in the late 

1970s by Sluyterman et al [1]. With this technique longer packed ion-exchange columns are normally used. The 

analytes are primarily bound to the ion exchanger using the starting buffer. After that an internal ascending or 

descending pH gradient is formed inside the column as the eluent buffer, usually polymeric ampholyte buffer (PAB), 

passes through the column and the analytes elute accordingly to their isoelectric points (pI). This technique excels 

in high resolution – being able to separate molecules where pI values differ by as little as 0.02 pH units. Using PABs 

has its advantages and disadvantages. They give considerably good linear pH gradients but on the downside they 

can give poor chromatographic reproducibility, are quite expensive, and they tend to associate with some proteins 

which can lead to additional complications when CF is used as a purification step. Another limitation of CF is its 

inflexibility in controlling a pH gradient slope as it can only be regulated by the elution buffer concentration. Attempts 

were made to overcome the drawbacks of conventional CF either by exchanging the PABs for mixtures of low number 

– low mass buffers [2,3] or by implementing gradient CF where the pH gradient is formed externally (i.e. outside 

the column) [4,5]. Despite the improvements the chromatographic runs are tedious and the developed methods are 

not compatible with mass spectrometry (MS), respectively. Coupling any LC system with MS requires the use of 

volatile buffers in low concentrations. Only one publication can hitherto be found where an ion-exchange column is 

directly hyphenated to the MS; in that instance 7 proteins were separated in a 70 min chromatographic run [6]. The 

aim of our study was to develop a rapid and simple high performance gradient chromatofocusing method (HPgCF), 

for the resolution of proteins, which could be coupled to a MS system. The column of choice was an analytical 

DEAE Bio-Monolith HPLC column and the test proteins subjected to HPgCF analysis were β-Lactoglobulin A and 

β-Lactoglobulin B. The proteins were resolved in less than 10 min using a simple buffering system which made this 

separation compatible with ion-spray MS detection. With this new HPgCF-MS method the chromatographic run 

time is significantly reduced and MS detection enables lower limits of detection compared to the conventional UV/ 

Vis detection. References: [1] L.A.AE. Sluyterman, O. Elgersma, J. Chromatogr. 150 (1978) 17. [2] M.S. Vakstein, P.N. 

Nesterenko, A.V. Ivanov, A.B. Tessman, J. Liq. Chromatogr. R. T. 29 (2006) 485. [3] M.S. Vakshtein, A.V. Ivanov, J. 

Anal. Chem. 62 (2007) 1040. [4] Y. Liu, D.J. Anderson, J. Chromatogr. A 762 (1997) 207. [5] L. Shan, D.J. Anderson, 

Anal. Chem. 74 (2002) 5641. [6] L. Shan, J.A.Hribar, X. Zhou, D.J. Anderson, J. Am. Soc. Mass Spectrom. 19 (2008) 

1132. 

582 



P17-015 ANALYSIS OF BIOMOLECULES BY UPLC-SEC AND UPLC-IEX 

Hong P., Fountain K., Morrison D., Bosch G. 


The complete analysis and characterization of the biopharmaceuticals is most efficient when orthogonal analytical 

techniques are applied to the samples. Each technique should be based on different physical properties of the 

protein and can include size-exclusion, ion-exchange or reversed-phase chromatography as chromatographic 

methods. In this presentation, we will demonstrate how Ultra Performance Liquid Chromatography (UPLC 

Technology) can improve chromatographic separations of biopharmaceuticals when used in conjunction with new 

ion-exchange and size-exclusion packing materials. With the advent of UPLC® Technology combining the low 

dwell volume of the chromatographic system and the higher pressure capabilities of these new packing materials, 

improved resolution, sensitivity and speed will be demonstrated over conventional low pressure, or traditional, HPLC 

methods for bioseparations. In addition, the robustness and column reproducibility of both materials will be shown. 

Size exclusion method development will include the effects of flow rate, temperature, and column length. For Ion- 

Exchange method development the effects of gradient slope, temperature and pH, will be outlined. These factors 

will be utilized to demonstrate the improved impurity detection and/or faster analysis of biomolecules achievable 

with the combination of UPLC Technology and newer packing materials available for both ion-exchange and sizeexclusion 

chromatography. 


583


P17-016 CHARACTERIZING CFEX KNOCKOUT MICE URINE BY CE-UV 

Villaseñor A. 1 , Holmes E. 2 , Barbas C. 1 , Unwin R. 3 

1 


2 

Imperial College 

3 

University College London 

The development of new and highly sophisticated tools in the analytical lab is compatible with getting the most of 

simpler, but still very interesting tools such as capillary electrophoresis (CE). Our group is interested in CE with UV 

detection because it can provide a wide picture of urine profiles. CE is a technique well suited for the analysis of 

biofluids and extracted tissue. Previous studies in our group proved that it is facile to combine data derived from a 

micellar electrokinetic chromatographic (MEKC) system separation with normal polarity, with a second and different 

CZE separation system employing reversed polarity method, to produce an extended representation of the sample. 

That coupling between two orthogonal capillary electrophoresis separation modes was successfully applied to 

classify urine samples coming from animal models [1]. This is even more interesting when small experimental animals 

are involved because the amount of sample which is available is in the microliters range and after the experiment, 

due to the low consumption, there is still a remaning volume for other measurements. Knockout mouse studies 

have demonstrated a major role of CFEX as an apical membrane Cl−/oxalate exchanger that contributes to NaCl 

reabsorption in the proximal tubule. CFEX null mice were observed to have a high incidence of calcium oxalate 

urolithiasis that was attributable to hyperoxaluria. Hyperoxaluria in CFEX null mice was found to result from a defect 

in oxalate secretion in the intestine, thereby causing increased net absorption of ingested oxalate and elevated 

plasma oxalate concentration [2]. Oxalate is not easy to be measured in low concentration in urine because it is a 

small highly polar ion, but can be easy determined in the CZE-RP method. Therefore we analysed two independent 

experiments (E1, E2), each one had 2 groups, with 6 wild and 6 null CFEX mice both including males and females. 

We performed simultaneously a target (for oxalate) and fingerprinting assay of the urine after only acidification with 

hydrochloride acid and water dilution. Knockout mice showed higher oxalate levels in urine as expected than the 

wild type animals, but in addition fingerprints permitted a clear classification of animals not only according to their 

type, but also to their sex, which was not expected. That could be related to sex differences in the expression of 

transporters responsible for oxalate secretion in the mammalian kidney. In addition to oxalate changes, looking 

at the fingerprints, important differences in other metabolites appeared which can permit a better insight into the 

metabolism of these experimental animals. REFERENCES 1. Barbas, C.; Vallejo, M.; García, A.; Barlow, D.; 

Hanna-Brown, M., J Pharm Biomed Anal 2008, 47 (2), 388-98. 2. Aronson, P. Kidney Int 2006, 70 (7), 1207-13. 

584 



P17-018 RAPID VITAMIN A AND E SAMPLE PREPARATION AND LIQUID CHROMATOGRAPHY QUANTIFICATION PROCEDURE FOR 

HUMAN BREAST MILK INVESTIGATION 

Kasparova M., Plisek J., Krcmova L., Hronek M., Zadak, Z., Solichova D. 

Charles University Medical School & Teaching Hospital-Czech Republic 

The World Health Organization recommends full breastfeeding for the first six months of life. The milk is sole 

source of liquids, nutrition, vitamins, minerals and immunity oligosaccharides during this time. Extraction and HPLC 

quantification procedure for possibility of vitamin A and E human breast milk screening were developed. 

Concentrations of breast milk constituents are influenced by several factors these include stage of lactation, 

breastfeeding routine, parity, age and others. Typically, major constituents such as fat and protein can differ by 

two- to threefold between mothers at the same stage of lactation. Moreover the concentrations of some minor 

constituents (e.g. vitamins) can be highly variable. Sample preparation procedure, performed before HPLC 

quantification, refers to human breast milk composition and has to comprehend wide range of differences. 

Procedure consists of deproteination, saponification, and liquid-liquid extraction steps carried out in one screw top 

glass tube. 

Ethanol was used for deproteination of 500 μ L breast milk (in ratio 2:1, v:v). Ascorbic acid was added before second 

step of sample preparation to avoid oxidation of vitamins. Saponification effect of potassium hydroxide (c = 10 

mol/L) was optimized by controlled conditions (30 minutes, 80 °C and protected from light). After cooling down to 

laboratory temperature vitamins were extracted by n-hexane. 1 500 μL of organic phase was evaporated and the 

residue was dissolved in methanol mobile phase. 

Quantification of the target analytes (retinol and α-tocopherol) was measured by the chromatographic system 

Prominence Shimadzu (Kyoto, Japan) composed by Rack changer 1C autosampler for microtitrate plates, Degasser 

DGU-20A5, Column Oven CTO – 20 AC, Diode array detector SPD – M20A and Communication bus module CBM-20A. 

Separation of vitamins was performed using the Chromolith Performance RP-18e, 100 mm × 4.6 mm monolithic 

column (Merck, Darmstadt, Germany). As the mobile phase 100 % methanol was used at the flow rate 2.5 ml/min. 

The detection of retinol and α-tocopherol was carried out at 325 and 295 nm, respectively by diode array detector. 

Randomised group of 120 women (age: 29 ± 4 years) was selected for vitamin A and E human breast milk screening. 

The study group implied mothers at different lactating stages (1, 3, 6 and 9 months postpartum). 

Financial support from Zentiva and projects: MZO 00179906, GAUK 124809/2010 is gratefully acknowledged. 


585


P17-020 DEVELOPMENT OF HILIC UHPLC-FD METHOD FOR DETERMINATION OF NEOPTERIN, BIOPTERIN AND ITS DERIVATES IN 

URINE USING SOLID PHASE EXTRACTION AS THE SAMPLE PREPARATION TECHNIQUE 

Vlcková H. 1 , Plíšek J. 2 , Nováková L. 1 , Solich P. 1 

1 

Charles University-Czech Republic 

2 

Teaching Hospital in Hradec Králové-Czech Republic 

HILIC (Hydrophilic interaction chromatography) has recently become a popular separation mode in modern bioanalysis. 

It is an alternative of conventional RP-HPLC (reversed-phase HPLC) or NP HPLC (normal phase HPLC) and 

is more convenient for the analysis of small polar molecules being weakly retained or eluted with dead volume in 

conventional RP-HPLC systems. HILIC is suitable for analyses of biological samples, because many biological active 

compounds possess polar structures. Under HILIC conditions, stationary phase has a polar character, including 

silica or silica modified by polar groups such as hydroxyl-ethyl-, amino groups or zwitterions. Mobile phase is 

composed of the high percentage of an organic solvent and a small percentage of water or volatile buffer. Neopterin 

is released by macrophages and it is an immunologic marker for the activation of the cell-mediated immune system 

connected with most infectious and autoimmunity diseases. Increased level of neopterin is observed in patients 

with various types of malignant diseases. Biopterin is an oxidative product of tetrahydrobiopterin and it influences 

biosynthesis of monoamine neurotransmitters. All analyzed molecules are polar basic compounds. The aim of this 

work was to develop UHPLC-FD (ultra high performance liquid chromatography with fluorescence detection) method 

using HILIC approach together with a solid phase extraction as the sample preparation step for the determination of 

neopterin, biopterin, dihydrobiopterin and dihydroneopterin in urine. During development of UHPLC-FD the influence 

of pH, buffer concentration and mobile phase composition was optimized using peak asymmetry, resolution, 

efficiency and analysis time for the evaluation. Optimized composition of mobile was acetonitrile: 50mM ammonium 

acetate buffer pH 6.8 (85: 15) at flow-rate 0.4 ml/min and isocratic elution. Acquity UPLC BEH Amide (100 x 2.1 

mm, 1.7 μm) analytical column was used for separation. The fluorescence detection was accomplished at 353 nm 

(excitation wavelength) and at 438 nm (emission wavelength) for all analytes. Total time of analyses was 7 minutes. 

The newly developed UHPLC-FD method was validated. The validation data indicated good linearity (r 0.9998), 

repeatability of retention time (RSD < 0.26%) and repeatability of peak area (RSD < 1.14%). Further, SPE method for 

the determination of biopterin, neopterin and its derivatives has been developed. Oasis MCX (mixed-mode cation 

exchange) extraction cartridges (Waters) were used. Analytes were eluted by a mixture ammonium hydroxide, 

acetonitrile and water. The SPE method was applied to urine samples. The main advantage of developed method 

was the direct connection of HILIC approach and SPE without the need for evaporation and reconstitution step. 

The elution solvent on SPE was fully compatibility with HILIC mobile phase (mostly acetonitrile). It simplifies sample 

preparation process and simultaneously decreases time requirement for whole analysis. The newly developed 

UHPLC-FD and SPE methods for the determination of neopterin, biopterin and its derivatives will be applied to urine 

samples and may be utilized to monitor these markers of immune system activation and inflammatory reactions. The 

author gratefully acknowledge: GAUK 124809/2010, MSM 0021620822 and MSM 0021620820. 

586 



P17-021 COMPREHENSIVE GCXGC, A VALUABLE TECHNIQUE FOR THE SCREENING OF BIOLOGICAL ACTIVE CHEMICAL 

COMPOUNDS IN POLYPORE SAMPLES 

Gilart-Alzuria N., Wiikinkoski E., Mantyla S., Ruiz-Jiménez J., Hartonen K., Riekkola M.L., Wiedmer S. 


Corresponding author e-mail: jose.ruiz-jimenez@helsinki.fi 

Polypores are members of the Aphyllophorales, a group of morphologically complex, terrestrial basidiomycetes. 

These funguses have a long history of medicinal use. The tinder polypore, Fomes fomentarius, was used in the 18th 

and 19th centuries in hemostatic dressings and bandages. The same polypore together with the birch polypore 

(Piptoporus betulinus) was found in the body of the famous 5300 year old “Ice Man”. According to a recent biological 

evaluation of over 200 species, more than 75% of screened polypores have shown strong antimicrobial activity. 

Antiviral, cytotoxic, antineoplastic, immunomodulatory and miscellaneous biological activities have been also found 

in polypore extracts. These activities are associated not only with small molecule secondary metabolites such as 

coumarins, polyketides, sesquiterpenes, triterpenoids, sterols, acids, polyols and phenols, but also with high molar 

mass cell wall polysaccharides. 

In this research, comprehensive two dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC- 

TOF-MS) was used for the identification and semiquantitation of biological active compounds in polypore samples. 

Because compound volatility was required, some compounds were transformed via derivatization (silylation) into 

volatile ones. The identification of the analytes was achieved by comparing the mass spectra obtained from TOF-MS, 

for a chromatographic peak, with those provided by the NIST database for TOF-MS. 

This methodology was applied to the identification of biological active compounds in six polypore varieties 

collected during the spring of 2007 in Finland. Sequential Soxhlet extraction with different polarity solvents such as, 

dichloromethane, methanol and tetrahydrofurane; was used for the extraction of the biological active compounds. 

The last step based on hydrolysis under basic conditions was used to ensure the extraction of the analytes bonded 

to the sample matrix. More than 100 compounds were identified on the samples. The differences found in the polypore 

composition can be used for the identification of different polypore species and the estimation of polypore age. 


587


P17-022 HILIC–UPLC-BASED METHOD FOR GENOMIC DNA METHYLATION MEASUREMENT 

Sotgia S., Zinellu A., Pisanu E., Murgia L., Gaspa L., Deiana L., Carru C. 

University of Sassari 

Corresponding author e-mail: carru@uniss.it 

Methylcytosine (mC) constitutes approximately 1% of the bases in mammalian genomes and it is designated 

as the fifth base of human DNA The evaluation of the extent of genomic DNA methylation may be an important 

parameter for diagnostic and biological investigation. Among the analytical approaches for measuring total DNA 

methylation, acidic hydrolysis of DNA by means of formic acid followed by quantification of the resulting free 

nucleobases, provides significant advantages in terms of speed, expense, and ease of assay. By common RP-HPLC 

methods the separation of nucleobases it is difficult since it is unsuitable for retaining or separating highly polar, 

ionic, and hydrophilic compounds such as nucleobases. To overcome these problems a hydrophilic interaction 

chromatography-based method, in combination with 1.7 μm ethylene bridged hybrid particle packed column 

(100 mm×2.1 mm I.D.) and ultraperformance liquid chromatography, was developed. Separation of cytosine and 

methylcytosine was achieved with good resolution and in fairly short times (5.5 min) by using isocratic elution with a 

mixture of 97:3 (v/v) acetonitrile/ 10 mM ammonium acetate as a mobile phase. The determination coefficients of C 

and mC were high (R2> 0.999) within the range tested. The %RSD for intraday and interday were respectively 2.2% 

and 2.5% for C and 3.5% and 3.8% for mC. The limit of detection was 0.52 μM (0.52 fmol on-column) both for C and 

mC while the limit of quantification was 1.72 μM (1.72 fmol on-column) both for C and mC. The smallest amount of 

purified DNA that yielded a measurable level of C and mC was 10 μg. 

This is the first work in which HILIC column was exploited to assess the degree of total genomic DNA methylation. 

The selectivity for polar solutes was combined with the high efficiency of 1.7 μm particle columns and the high speed 

of a UPLC system. The result is the method here described which is very fast, simple, sensitive, and easily scalable 

for high-performance liquid chromatography analysis. 

588 



P17-023 MICRO-SOLID PHASE EXTRACTION AND SELECTIVE ENRICHMENT OF GLYCOPROTEINS USING LECTIN MODIFIED GOLD 

NANO-PARTICLES ON A MONOLITHIC SUPPORT 

Alwael H., Connolly D., Clarke P., Thompson R., O’Connor B., Twamley B., Paull B. 



The selective extraction of specific proteins (non-glycosylated, glycosylated or different glycoforms) from complex 

sample matrices is of significant interest within the fields of proteomics and glycoproteomics. One approach to 

obtain selective extraction is to utilise solid phase extraction (SPE) whereby the solid phase incorporates a selective 

ligand or bio-recognition molecule for efficient trap and release of the target. Polymer monoliths have proven to be 

an excellent solid support for SPE applications in bio-analysis due to their excellent mass transfer characteristics 

for large biomolecules. Although biorecognition molecules such as lectins can be covalently immobilised directly 

onto the monolith surface using a variety of chemistries, the relatively low surface area of these monoliths results 

in a correspondingly low sample capacity. Recently, we have described the covalent attachment of 20 nm gold 

nanoparticles (AuNP) upon polymer monolith with excellent surface coverage leading to a significant increase 

in surface area. In addition, the immobilisation of proteins upon gold nanoparticles is well known and in many 

instances confers additional stability to the protein. In this work therefore we describe the in-situ preparation of an 

ethylene dimethacrylate porous polymer monolith within the confines of 20 µL polypropylene pipette tips, onto which 

methacrylate anchor sites were previously grafted to ensure intimate monolith/wall contact. Vinyl azlactone was 

photografted onto the monolith pore surface and reacted with ethylenediamine to provide amine attachment sites 

for subsequent immobilisation of AuNP. Field emission scanning electron microscopy was used to verify the high 

surface coverage of AuNP. A selective lectin (Erythrina cristagalli lectin, ECL) was immobilised upon the attached 

AuNP via a biofunctional linker (dithiobis(succinimidylpropionate). The ECL-immobilised tip was successfully applied 

for the enrichment of galactosylated protein (neuraminidase treated transferrin) versus the non- galactosylated 

protein (ribonuclease B) due to the specificity of ECL. Reversed-phase capillary HPLC was used to validate the 

efficiency and selectivity of the developed micro-extraction phase. 


589


P17-024 DETERMINATION OF CATECHOLAMINES USING MIXED-MODE REVERSED-PHASE AND CATION-EXCHANGE HIGH- 

PERFORMANCE LIQUID CHROMATOGRAPHY 

Tsunoda M. 

University of Tokyo 

Corresponding author e-mail: makotot@mol.f.u-tokyo.ac.jp 

The measurement of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) is useful for an index of dopamine 

turnover. We developed a simultaneous determination method for catecholamines and DOPAC with high-performance 

liquid chromatography-fluorescence detection. Mixed-mode reversed-phase and cation-exchage column 

which contained C18 silica particles and sulfonic acid cation-exchange particles was used for the separation 

of 3,4-dihydroxy-L-phenylalanine, norepinephrine, epinephrine, dopamine, and DOPAC. The mobile phase was 

optimized for factors such as pH and counter ion concentration. With a mobile phase of 15 mM sodium acetate 

buffer (pH 4.5), separation was achieved within 22 min. The developed method was applicable to the determination 

of dopamine and DOPAC in mouse striatum. 

590 



P17-025 URINE FINGERPRINTING WITH CE-MS REVEALS ROSMARINUS OFFICINALIS EXTRACT EFFECT ON DIABETIC RATS 

Godzien J. 1,2 , Moraes E. 1,3 , Rupérez F.J. 1 , Barbas C. 1 

1 

University San Pablo-CEU 

2 

The John Paul II Catholic University of Lublin 

3 

University of Sao Paulo 

Rosmarinus officinalis, common name rosmary, is well known for its antioxidant activity attributable to: phenolic 

diterpenes, such as carnosic acid, carnosol, rosmanol, epirosmanol, 7-methyl-epirosmanol and methyl carnosate, 

rosmadial. Senorans et al [1] isolated antioxidant extracts from rosemary with supercritical fluid extraction with 

CO2 and we tested those extracts in an animal model of oxidative stress: the rat made diabetic by streptozotocine 

injection. Metabolic fingerprinting (metabolomics) is a complex matrix profiling strategy widely adopted by many 

researchers and which can be applied to a variety of sample matrices. Urine is especially interesting as a diagnostic 

sample because it is non-invasive and easy to obtain. Many compounds in urine are ionic or highly polar and for 

them capillary electrophoresis (CE) can offer a special selectivity as compared to LC-MS. Five doses of rosemary 

extract with in vitro antioxidant properties were intragastrically administrated to adult male streptozotocin (STZ) 

diabetic rats and the corresponding controls. Urine fingerprints of control and diabetic rats, both with and without 

treatment, were obtained by CE-TOF (Agilent). Data were collected in positive ESI mode operated in full scan mode 

from 50 to 1,000 m/z. When the profiles were submitted together to pattern recognition techniques they showed the 

effects of rosemary on this acute and short term treatment animal model. After checking the analytical quality of the 

process with the corresponding QCs, a PLS-DA model was built with variables found in diabetic and control, nontreated 

groups. Afterwards, the model was used to predict treated diabetic animals and they appeared clustering 

between both, which could prove an improvement in the general status. In order to have further biochemical 

knowledge of the effect, after treatment, groups were studied by pairs and metabolites identified as responsible 

of the effect are under study. REFERENCES [1] F. J. Senorans, E. Ibanez, S. Cavero, J. Tabera, G. Reglero, J. 

Chromatogr. A 870 (2000) 491 


591


P17-026 INVESTIGATION OF THE CHROMATOGRAPHIC BEHAVIOR OF BARE TITANIA IN HYDROPHILIC INTERACTION LIQUID 

CHROMATOGRAPHY (HILIC) 

Abi jaoudé M., El Debs R., Randon J. 


Corresponding author e-mail: randon@univ-lyon1.fr 

Background 

Compared to silica, titania offers enhanced column long-term stability as well as mixed surface chemistry: (i) it can 

withstand adverse chromatographic conditions of pH between 1 to 14 and temperature up to 150 °C [1,2] and (ii) 

can also exhibit either anion- or cation- exchange at low or high pH respectively, owing to its high isoelectric point 

(between 4 and 7) [3] or even selectively adsorb Lewis hard bases such as carboxylate and phosphate through ligand 

exchange interactions [4,5]. The potential of native titanium dioxide has already been explored in normal phase (NP) 

and ion exchange (IE) chromatographic modes. More recently, hydrophilic interaction liquid chromatography (HILIC), 

established to achieve the analysis of polar compounds such as peptides, carbohydrates and basic molecules [6], 

has emerged on titania but remains at its early stages due to the complexity of the chromatographic system. First 

insights on separations carried with phosphorylated nucleotides and other carboxylate probes already revealed 

mixed ion- and ligand- exchange interactions mostly governing the retention mechanism [7,8]. 

Results 

To broaden the scope of HILIC on titania, our study focused on other families of probe molecules such as 

N-methylated xanthines and beta-blockers of highly physiological importance [9,10]. The study of the thermodynamic 

aspect of separation was performed using a commercial titanium dioxide particle-packed column (NP Sachtopore® 

Technologies, Interchim, France). Our results showed that retention and selectivity could be controlled by tuning 

several operating mobile phase parameters such as acetonitrile percentage, nature and concentration of buffering 

solution and column temperature depending on the prevalent mechanism involved. Moreover, our findings highlight 

the potential great value of titania as a high selective substrate for complex and original separations in liquid 

chromatography. 

[1] T. S. Kephart, P. K. Dasgupta, Anal. Chim. Acta, 414, 71 (2000). 

[2] J. Nawrocki, C. Dunlap, A. McCormick, P. W. Carr, J. Chromatogr. A, 1028, 1 (2004). 

[3] J. Winkler, S. Marme, J. Chromatogr. A, 888, 51 (2000). 

[4] S. Miyazaki, M. Y. Miah, K. Morisato, Y. Shintani, T. Kuroha, K. Nakanishi, J. Sep. Sci., 28, 39 (2005). 

[5] K. Tani, T. Sumizawa, M. Watanabe, M. Tachibana, H. Koizumi, T. Kiba, Chromatographia, 55, 33 (2002). 

[6] P. Hemström, K. Irgum, J. Sep. Sci., 29, 1784 (2006). 

[7] T. Zhou, C. A. Lucy, J.Chromatogr. A, 1187, 87 (2008). [8] T. Zhou, C. A. Lucy, J.Chromatogr. A, 1217, 82 (2010). 

[9] E. Kulikowska, B. Kierdaszuk, D. Shugar, Acta Biochim. Polonica, 51, 493 (2004). 

[10] M. Delamoye, C. Duverneuil, F. Paraire, P. de Mazancourta, J. C. Alvareza, Forensic Sci. Int., 141, 23 (2004). 

592 



P17-027 EVALUATION OF THE BIO-FUNCTIONALIZATION OF POROUS MONOLITHS DEDICATED TO IMMUNO-PRECONCENTRATION 

AND SYNTHESIZED IN MICROSCALE CAPILLARY FORMAT 

Chamieh J. 1 , Faye C. 2 , Vandenabeele-Trambouze O. 2 , Moreau T. 2 , Faure K. 1 , Dugas V. 1 , Demesmay C. 1 , Randon J. 1 

1 


2 

University of Montpellier II 

Trend in chemical as well as biochemical analytical methods concerns the integration of multistep processes inside 

small scale analytical systems also named “lab-on-a-chip”. Such devices for medical purpose for example would, 

ideally, treat small-volume of complex mixture and/or diluted analytes. Implementation of a sample preparation step 

is thus a prerequisite to separation and detection steps. Our interest focuses on the development of a purification 

and preconcentration step inside microchannel (microfluidic microchannel or capillary) by immunoaffinity methods. 

In such methods, the analyte (Antigen or Ag) in solution interacts “specifically” with antibodies (Ab) previously 

immobilized on a substrate. After elution of the sample solution and washing of the substrate, the analyte is 

recovered by flushing the substrate with an eluting solution. Transfer of individual steps working in macroscale 

format (mg or ml) towards miniaturized format (ng or nl) is not trivial. And some methodological tools need to be 

developed and validated to ensure correct transfer. 

Glycidyl methacrylate porous monoliths (GMA-co-EDMA) are already widely used for the immobilization of 

antibodies at macroscale format [1-3]. However, characterization of antibody immobilization inside microchannels as 

well as control of the remaining activity requires the development of dedicated methods to acquire information from 

such confined environment. 

Here, we report on the design and validation of a simple, specific and very sensitive colorimetric method for the 

analysis of immobilized Ab. This is based on recent works aiming to quantify the amine content of functionalized 

supports [4]. This method is not Ab dependant and allows working similarly on native as well as denaturated proteins 

The protocol was thus adapted and validated to the quantification of immobilized Ab onto the porous monolith 

synthesized in macroscale (static mode) and microscale “capillary” format (dynamic mode). 


593


P17-028 STUDY OF RETENTION BEHAVIOUR AND MASS SPECTROMETRY COMPATIBILITY IN ZWITTERIONIC HYDROPHILIC 

INTERACTION LIQUID CHROMATOGRAPHY FOR THE SEPARATION OF MODIFIED NUCLEOSIDES 

Rodríguez-Gonzalo E., García-Gómez D., Carabias-Martínez R. 


Corresponding author e-mail: dgg@usal.es 

In recent years the determination in urine of modified nucleosides, mainly of the hydroxylated and methylated type, 

has been confirmed to be an efficient measure for the early detection of different diseases. The analytical method 

most used for such purposes is liquid chromatography coupled to mass spectrometry (MS). Commonly, reverse 

phase (RPLC) columns have been used, although currently Hydrophilic Interaction Liquid Chromatography (HILIC) 

has been established as a highly suitable chromatographic method for the separation of polar compounds. In 

addition, HILIC has the advantage of enhanced detection sensitivity when used in conjunction with MS, owing to 

the high organic content of the mobile phase, which allows for efficient spraying and desolvation in electrospray 

MS. Here we studied the chromatographic behaviour of modified nucleosides using stationary phases of different 

polarity. Thus, we used a PentaFluoroPhenyl (PFP) reverse-phase column and two polar columns: cross-linked 

diol (Luna-HILIC) and Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) columns. As target 

analytes, six hydroxylated or methylated nucleosides were selected: 8-hydroxy-guanine, 8-hydroxy-guanosine, 

8-hydroxy-2’-deoxyguanosine, 9-methyl-guanine, 1-methyl-guanine and 7-methyl-guanine. The results obtained 

suggest that the ZIC-HILIC chromatographic column, based on a sulfobetaine residue as the stationary phase, is 

much more suitable for the separation of these modified nucleosides. We then studied the different parameters 

affecting separation with a view to elucidating theoretical aspects, including the mechanisms of retention. In this 

sense, we evaluated the content of organic solvent in the mobile phase, pH, the concentration and nature of the 

salts present in the mobile phase, and column temperature. In the study of these parameters we also took into 

account the effect of later detection via MS considering its restrictions and the different possibilities of injection. 

The experimental results suggest a mechanism of retention involving mechanisms of partition and interaction due to 

weak electrostatic forces. In the optimized method, we performed an isocratic elution with 80% ACN: 20% 2.7 mM 

formic acid. The chromatographic column allowed the injection of up to 50 uL of sample in organic medium, affording 

increased sensitivity and good compatibility with the usual extraction/preconcentration methods and allowing direct 

coupling between the separation/detection stages. Relative standard deviation (RSD) values of less than 2.5% for the 

retention times, and of less than 4% for the analytical signal were obtained. 

594 



P17-029 DETERMINATION OF TETRAHYDROFURAN AND KETONES IN URINE BY HEADSPACE TECHNIQUE 

Prado Burguete C. 1 , Marín Carrasco P. 2 , Alcaraz Rodríguez J. 1 , Periago Jiménez F. 1 

1 

Instituto de Seguridad y Salud Laboral 

2 

University of Murcia 

Corresponding author e-mail: celiaa.prado@carm.es 

Tetrahydrofuran (THF) is a compound widely used in industry as a solvent for resins and plastic material in the 

fabrication of dyes, paints, varnishes and adhesives. Also, ketones such as acetone, methyl ethyl ketone and methyl 

isobutyl ketone, are used as solvents in the fabrication of these same preparations and in some cases, appear 

together with tetrahydrofuran. The analysis of unmetabolized solvents in urine is very useful for biological monitoring 

of this kind of compounds in occupational environment [1]. So, the development of analytical methods that allow the 

determination of unmetabolized ketones and THF in urine samples is very interesting as they are biomarkers that 

have established biological limits values of occupational exposition [2]. In addition, THF can be absorbed by dermal 

via, so the measure of the environmental concentration may not be sufficient to quantify the overall exposition. The 

aim of this work has been the development of a method for the simultaneous determination of the above mentioned 

compounds. A headspace sampler (40 Turbomatrix Trap, Perkin Elmer) has been used and the analysis was 

performed using a gas chromatograph-mass spectrometer. Urine samples of 2 ml have been used. The calibration 

curves obtained for all compounds showed good linearity, with coeficient regresion values higher than 0.99 over 

the studied range (3.2-197.13 µg/ml for acetone, 0.16-40.02 µg/ml for MEK, 0.43-8.51 µg/ml for THF and 0.15-39.94 

µg/ml for MIBK). The detection limits of the developed methods are of 0.88 and 0.05 µg/ml for acetone and MEK, 

respectively, and a value of 0.06 µg/ml both for THF and MIBK. The precision of the method, based on the value 

of the relative standard deviation obtained for each concentration level, ranged from 1.5 to 7.5%. The results were 

compared with those obtained by solid phase microextraction [3], indicating a statistically significant relationship 

between the two methods. Finally, these two used methods were tested by an intercomparison programme. The 

results indicate that the proposed method is very sensitive and accurate, so very useful for the biomonitoring of 

ketones and THF in urine from occupational exposed people. [1] M. Imbriani, S. Ghittori. Int. Arch. Occup. Environ. 

Health 78 (2005) 1. [2] Límites de exposición profesional para Agentes Químicos en España. 2008. Instituto Nacional 

de Seguridad e Higiene en el Trabajo. INSHT [3] C. Prado, P. Marín, J. Alcaraz, J.F. Periago. 12a Jornadas de Análisis 

Instrumental. Barcelona. 2008. 


595


P17-030 DEVELOPMENT AND OPTIMIZATION OF THE CONDITIONS FOR THE APPLICATION OF HYDROPHOBIC INTERACTION 

CHROMATOGRAPHY (HIC) FOR THE PURIFICATION OF F(AB)’2 FRAGMENTS 

Seijo, D., Barbi L., Arathoon R., Gago-Martínez A. 

University of Vigo 

Corresponding author e-mail: anagago@uvigo.es 

Over the last years, antibodies are some of the most powerful tools in therapy and are currently one of the fastest 

growing classes of therapeutic molecules. Being the most commonly used the human immunoglobulin isotype 

G1 (IgG1). Antibody fragments (Fab and F(ab)’2) are becoming popular therapeutic alternatives to full length 

antibodies since they are smaller (around 50kDa), possess different properties that are advantageous in certain 

medical applications as rapid clearance from the circulation, and low immunogenicity because of the removal of 

the Fc fragment. Moreover these fragments can be produced more economically. Classically, F(ab)’2 fragment is 

obtained by pepsin digestion [1]. Due to the observed variations obtained when an antibody is digested with pepsin 

(depending the hoster, subclass, etc) some critical parameters (as digestion pH, E:Ab ratio and digestion time) must 

be optimized. On the other hand, the application of appropriate analytical methods is an essential requirement for 

the purification of therapeutic antibodies and their fragments. A range of analytical methods have been described in 

the literature to effectively determine the purity, identity, integrity and activity of the antibodies and their fragments. 

Recently Hydrophobic Interaction Chromatography (HIC) is emerging as an alternative for the purification of 

biomolecules, because of its particular features such as minimum sample pretreatment and the ability to be used in 

combination with other chromatographic techniques [2]. Mass Spectrometry is a powerful tool for protein analysis 

and the major technique used to study proteins in the field of proteomics. Mass spectrometry can be used to 

characterize recombinant proteins, identify proteins and characterize protein modification. In this work we describe 

the optimization of pepsin digestion to obtain a method to produce F(ab)’2 from human IgG1. HIC have been applied 

in this study to separate the fragments of human antibody (IgG1) after pepsin digestion. The identification of theses 

fragments was carried out by Western Blot and further confirmation by Mass Spectrometry. The results obtained 

are presented and discussed in this work showing the advantages and disadvantages for this particular application. 

References: [1] Inouye K., J. Biochem. Biophys. Methods, 2001, 48, 23-32. [2] Kostareva I., J. Chrom. A 2008, 1177, 

254. 

596 



P17-031 A SOLID-PHASE EXTRACTION LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY ANALYTICAL METHOD FOR 

THE DETERMINATION OF 2-HYDROXY-4-METHOXYBENZOPHENONE AND ITS METABOLITES IN BOTH HUMAN URINE AND 

SEMEN 

León Z., Chisvert A., Tarazona I., Salvador A. 


Corresponding author e-mail: alberto.chisvert@uv.es 

2-Hydroxy-4-methoxybenzophenone (HMB), also called benzophenone-3, is commonly used as a broad-band UV 

filter in sunscreen cosmetic products alone or in combination with other UV filters to protect from the deleterious 

effects of the sun. Both in vitro and in vivo studies clearly show that HMB is absorbed through the skin to a 

certain extent[1]. Besides this, studies concerning the hormonal activity showed that daily exposure to sunscreen 

formulations including HMB may have estrogenic effects in humans[2]. As an immediate consequence of this 

percutaneous penetration, a series of biotransformation reactions take place in the organism. Different toxicokinetic 

studies indicate that HMB is readily biotransformed into its three metabolites, namely 2,4-dihydroxybenzophenone 

(DHB), 2,2’-dihydroxy-4-methoxybenzophenone (DHMB) and 2,3,4-trihydroxybenzophenone (THB). In addition, 

HMB and its metabolites have been identified in both their free and conjugated forms, mainly via glucuronidation[3]. 

Thereafter, an analytical method for the sensitive determination of HMB and its three metabolites in both human 

urine and semen is presented. The described analytical method is based on a solid-phase extraction (SPE) 

procedure to clean-up and preconcentrate the target analytes from the urine and semen samples followed by liquid 

chromatography-tandem mass spectrometry (LC-MS/MS) detection. The methodology was fully validated and the 

standard addition calibration method was used to quantify the target analytes in order to correct the matrix effects 

observed. To determine the content of the analytes in their free and glucuronide-conjugated forms, urine and semen 

samples were treated with β-glucuronidase to carry out an enzymatic hydrolysis. The accuracy of the method 

was evaluated and the recoveries ranged from 98% to 115% and from 86% to 111% in urine and semen samples, 

respectively, depending on the analyte. For urine samples, the limits of detection ranged between 0.027-0.103 ng/ 

mL and the repeatability of the method, expressed as relative standard deviation, was in the range of 7.2-9.2%, 

depending on the analyte. In the case of semen samples, the limits of detection ranged between 1-3 ng/mL whereas 

the repeatability was in the range of 2.2-6.4%, depending on the analyte. The described SPE-LC-MS/MS method 

was satisfactorily applied to both urine and semen samples from a male volunteer who applied a sunscreen cosmetic 

product containing HMB. HMB and its metabolites were found and quantified in the low ng/mL range in both urine 

and semen samples, although at different extent. The presented analytical method can be applied to further in vivo 

studies concerning the pharmacokinetics of HMB, and especially of its metabolites, which might have more long 

term adverse effects than the parent compound. Furthermore, another point of interest might be the study of how do 

the estrogenic metabolites of HMB affect the variation of specific reproductive toxicity parameters by establishing 

relationships between the found amounts and semen quality. [1]Treffel P, Gabard B (1996) Pharm Res 13:770-774 

[2]Schlumpf M, Cotton B, Conscience M, Haller V, Steinmann B, Lichtensteiger W (2001) Environ Health Perspect 

109:239-244 [3]Kadry AM, Okereke CS, Abderrahman MS, Friedman MA, Davis RA (1995)J Appl Toxicol 15:97-102 


597


P17-032 DEVELOPMENT OF INFORMATION RICH URINARY SIGNATURES IN HUMANS BY LC-MS FOR POTENTIAL ANTI-DOPING 

APPLICATIONS 

Kiss A., Flament-Waton M.M., Cren-Olivé C. 

Service Central d’Analyse, Biochemistry 

Corresponding author e-mail: c.cren@sca.cnrs.fr 

Doping is a growing phenomenon generalized to all disciplines and all levels. It is nowadays recognized as a 

danger for the society and the health of the athletes. The amplitude and the evolution of this phenomenon urge 

the development of new, rapid and more reliable detection techniques. One of the main difficulties of the control 

laboratories is the continuous emergence of doping substances and methods. The new compounds have the same 

doping effect, but their structure is slightly modified and therefore they cannot be detected by traditional means. In 

order to address this challenge we chose a metabonomic approach. In contrast to the methods used at the present 

time, this approach is not focused on detecting the illicit substances, but, their effects on the metabolism of the 

athlete. The molecular prints of urine samples were obtained by UPLC-TOF in both positive and negative modes. 

Prior to analysis, the detection parameters (desolvatation and source temperatures, cone and capillary voltages) 

and the chromatographic conditions (the gradient’s steepness, temperature, and particle’s size) were optimized as 

to obtain information rich molecular prints. The effects of phase modifiers (ammonium format, ammonium acetate, 

trifluoroacetic acid and ammonia) on the efficiency of the positive ionization were equally tested. Next, four ways 

of sample preparation techniques were assessed: protein precipitation, direct infusion, centrifugation and filtration. 

Once the conditions were optimized, 45 samples of which 20 came from positive tested athletes were analyzed 

within a single run. The molecular prints were then processed by means of multivariate analysis (PCA and OPLS- 

DA) using MarkerLynx (Waters) software in order to select potential biomarkers. A system of quality control samples 

was implemented in order to track down any drift due to instruments. The results showed that a longer gradient and 

formic acid are favourable to the detection of more features Also, filtering the samples before injection is crucial 

in order to preserve the wellness of the column and the LC setup. Urinary signatures were compared showing that 

metabonomics could be a complementary tool to obtain information rich profiles. The potential biomarkers found are 

currently under study. This method could be, in the future, adapted to doping issues. 

598 



P17-033 DETECTION OF TOREMIFENE ADMINISTRATION IN ANTIDOPING CONTROL ANALYSES 

Gómez C. 1 , Pozo O.J. 1 , Vila E. 2 , Salvador J.P. 2 , Marco M.P. 2 , Segura J. 1 , Ventura R. 1 

1 

IMIM-Hospital del Mar, Barcelona 

2 

IQAC-CSIC, Applied Molecular Receptors Group, Barcelona 

Toremifene is a selective estrogen receptor modulator included in the list of prohibited substances in sport by the 

World Antidoping Agency. The aim of the present study was to investigate toremifene metabolism in humans in order 

to define the best marker to detect toremifene administration through the analysis of urine samples. 

Samples obtained after administration of toremifene (Fareston®) to healthy volunteers were studied. They were 

subjected to different preparation methods to detect free metabolites as well as metabolites conjugated with 

glucuronic acid or sulphate. Different hydrolysis procedures were also applied to study the conjugated metabolic 

fractions. Analysis of the sample extracts was performed by liquid chromatography coupled to tandem mass 

spectrometry. Metabolic experiments were performed in multiple reaction monitoring (MRM) mode by monitoring one 

transition for each potential toremifene metabolite. Transitions were selected by calculating the protonated molecular 

ion of the potential metabolite as precursor ion; according to the fragmentation pattern observed for toremifene, the 

product ions were m/z 72, or m/z 58, resulting from the cleavage of the lateral chain of the unchanged structure or 

N-desmethyl metabolites, respectively. Toremifene and 22 metabolites were detected in excretion study samples, 

excreted free or conjugated with glucuronic acid or sulphate. Proposed structures for the phase I metabolites 

include mono- and di-hydroxylated metabolites, hydroxyl-methoxymetabolites and N-desmethyl metabolites, among 

others. Some of the metabolites were identified by comparison with reference standards. Most of the metabolites 

were detected up to 82 h after administration. 


599


P17-034 METABOLITE TARGET ANALYSIS OF CELL EXTRACT OF BACTERIUM PARACOCCUS DENITRIFICANS 

Musilova J., Foltova K., Glatz Z. 


Corresponding author e-mail: glatz@chemi.muni.cz 

It is currently impossible to identify and quantify all intracellular and extracellular metabolites (i.e. metabolome 

analysis) in one single analysis. The reason is that the chemical complexity and heterogeneity of metabolites 

doesn’t allow using the analytical techniques in a reproducible and robust way. So, there are several approaches 

of metabolomics studies, e.g. the metabolite target analysis that identifies and quantifies only some of metabolites 

participating in a specific part of the metabolism. This work focuses on optimizing of separation conditions for 

selective and rapid determination of 17 energetically important metabolites (purine and pyrimidine nucleotides, 

adenine coenzymes and Acetyl-CoA) using capillary electrophoresis (CE). In order to improve the concentration 

sensitivity, the capillary zone electrophoresis was combined with the on-line preconcentration technique – field 

enhanced sample stacking. The optimal separation was reached in a bare fused silica capillary (effective length: 

84.5 cm; internal diameter: 75 µm) using separation voltage 30 kV (positive polarity), temperature of capillary 21 

°C and direct detection at 260, 280 and 340 nm. 50 mM concentration of phosphate buffer (pH 5.8) provided the 

best resolution of peaks. Metabolite samples were dissolved in deionised water and injected into the capillary 

hydrodynamically with a pressure of 50 mbar for 28 s. The method showed RSD (n = 10) in the range from 0.45 

to 2.00 % for migration time and from 2.10 to 3.15 % for relative peak area of metabolites with the exception for 

NADPH (6.15 %). Optimized method was used for analyzing of extract of bacterium Paracoccus denitrificans where 

comigrations of ATP, NADP and CTP peaks with other unknown analytes were discovered. The separation conditions 

were adjusted in order to quantify all demanded metabolites in the cell extract. Acknowledgements This work was 

supported by research project No. MSM0021622413 and research centre LC06023 both from Czech Ministry of 

Education. 

600 


P18 FORENSICS 

POSTER 

SESSIONS

POSTER SESSION 18 - FORENSICS 

P18-001 LIQUID CHOMATOGRAPHY WITH DOUBLE DETECTION AS A MONITORING TOOL OF A NEW PROTOCOL FOR THE 

ISOLATION OF NITROCELLULOSE FROM GUNPOWDERS 

López-López M. 1 , Fernández de la Ossa M.A. 1 , Sáiz Galindo J. 1 , Ferrando J.L. 1,2 , Vega A. 1 , Torre M. 1 , García-Ruiz C. 1 

1 


2 


Corresponding author e-mail: m.lopezl@uah.es 

Nitrocellulose takes part of explosive/propellant formulations such as dynamites and smokeless gunpowders. 

Nitrocellulose based gunpowders, according to their composition, are grouped in single-based gunpowders 

(containing mainly nitrocellulose), double-based gunpowders (composed of nitrocellulose and other explosive 

substances like nitroglycerin or dinitrotoluene), and triple-based gunpowders (consisting of nitrocellulose, 

nitroglycerin, and nitroguanidine). These gunpowders also contain stabilizers, plasticizers, and inert material, 

among other components. In the last decades, the need for unequivocal identification of explosives and their 

illegal use in terrorist acts requires the development of reliable analytical methods. However, there are no studies 

on the analytical determination of intact nitrocellulose and only few analytical methods were developed to quantify 

hydrolyzed nitrocellulose or for the quality control of nitrocellulose based explosives and propellants. Moreover, 

another analytical drawback is the lack of commercial standards with comparable nitrogen content to that of 

explosives (> 12.6 %, m/m). Then, the aim of this work was to develop a new protocol for the effective isolation 

of nitrocellulose from propellants, specifically single-, double-, and triple-based smokeless gunpowders, for its 

use in the identification of these materials. A multistep protocol for the rapid isolation (in less than 3 hours) of 

nitrocellulose from gunpowders was developed using sequential extractions. For single-based or double-based 

gunpowders six consecutive solvent extractions were selected: three extractions with methanol, to remove 

nitroglycerin, 2,4-dinitrotoluene, ethyl-centralite, diphenylamine, and diphenylamine derivatives; one extraction with 

dichloromethane, to remove colorants and plasticizers of organic nature; one extraction with methanol, to facilitate 

a final polar extraction; and one extraction with water, to remove ionic components. For the triple-based gunpowder 

studied, eight solvent extractions were needed due to the high concentration of the water-soluble nitroguanidine. 

In addition to the same five initial phases used for the single-based and double-based gunpowders, three water 

extraction phases at a higher temperature (75 ºC instead 35 ºC used for the other extractions) were needed. A final 

step to solubilize nitrocellulose in methyl ethyl ketone was used to remove inert components (mainly graphite). The 

unequivocally identification of the different components present in the nitrocellulose based gunpowders was made 

by High Performed Liquid Chromatography with double detection by Diode Array Detection and Mass Spectrometry 

(HPLC-DAD-MS). Nitrocellulose contained in gunpowders was effectively isolated, being the remaining components 

below the microgram mL-1 level. 

602 



P18-002 DETERMINATION OF NITROGEN CONTENT IN NITROCELULLOSE USED IN THE MANUFACTURE OF GUNPOWDERS BY 

ALKALINE HYDROLYSIS AND IONIC CHROMATOGRAPHY 

López-López M. 1 , Ramiro Alegre J.M. 1,2 , García-Ruiz C. 1 , Torre M. 1 

1 


2 



Nitrocellulose is a nitrated cellulose ester polymer whose nitrogen content determines its applicability. In fact, 

nitrocellulose containing less than 12.6 % (m/m) of nitrogen is widely used for manufacturing coatings, printing inks, 

leather finishes, films, etc; on the contrary, nitrocellulose with a nitrogen percentage above this value is employed in 

the manufacturing of explosives materials like gunpowders. The determination of the nitrogen content of gunpowders 

is of great importance not only in order to characterize these explosive materials but also as indicator of gunpowder 

ageing and stability. In Spain, the determination of the nitrogen content of gunpowders is carried out by means of a 

potentiometric titration of the nitric nitrogen obtained after the digestion of isolated nitrocellulose with concentrated 

sulfuric acid at low temperature [1]. However, this is a very time-consuming method (approximately, three days) 

and requires to acquire experience to really get good results. For these reasons, it is necessary to develop faster 

and accurate methodologies for the determination of nitrogen content in nitrocellulose. In this work, a method to 

determine the nitrogen content of nitrocellulose in gunpowders is proposed. After a nitrocellulose isolation, by a 

protocol optimized by our research group [2], a basic hydrolysis of nitrocellulose was carried out using NaOH 1 

% (m/v) at 150 ºC during 30 minutes. Then, the concentrations of nitrates and nitrites formed were determined by 

ion-chromatography with suppression and conductimetric detection. The optimized method was validated in terms 

of limits of detection and quantification, sensitivity, precision, and accuracy. Limits of detection calculated were 

0.04 mg L-1 for nitrates and 0.02 mg L-1 for nitrites and limits of quantification were 0.17 mg L-1 for nitrates and 

0.06 mg L-1 for nitrites. Precision was evaluated as repeatability (RSD < 0.6 % for nitrates and < 2.4 % for nitrites) 

and intermediate precision (RSD < 3.4 % for nitrates and < 6.7 % for nitrites). Accuracy was assessed as recovery 

percentages (~ 117 % for nitrates and ~ 95 % for nitrites). These parameters were good enough to demonstrate the 

validity of the method and its applicability to the determination of the nitrogen content in nitrocellulose contained in 

different types of gunpowders (single-, double-, and triple-based gunpowders, manufactured from 1950 to 2000). 

The nitrogen contents determined were compared with those given by the official label of the ammunition (obtained 

by the digestion/titration method) and errors, by defect, from 14.8 % to 0.6 % were obtained. The highest errors were 

obtained for the oldest gunpowders and were justified by the loss of nitrated groups in the nitrocellulose molecule 

during ageing. In addition, the total analysis time (2 hours for nitrocellulose isolation, 1.5 hours for nitrocellulose 

hydrolysis, and 0.2 hours for chromatographic separation) was about 10 times lower than in the digestion/titration 

method. [1] http://www.enac.es/web/enac/acreditados. [2] M. López-López, M. A. Fernández de la Ossa, J. Sáiz 

Galindo, J. L. Ferrando, A. Vega, M. Torre, C. García-Ruiz. Talanta 81 (2010) 1742–1749. 


603


P18-003 DIPHENYLAMINE AND ITS DERIVATIVES AS PREDICTORS OF AGEING OF SINGLE AND DOUBLE BASED GUNPOWDERS BY 

MEANS OF HPLC 

López-López M. 1 , Ortiz R. 2 , Bravo J.C. 1,2 , Ferrando J.L. 1,2 , Velasco E. 2 , García-Ruiz C. 1 , Torre M. 1 

1 


2 



Smokeless gunpowders are propellants constituted by explosive compounds that decompose under normal storage 

conditions. To stop such decomposition, some stabilizers such diphenylamine (DPA), are added. This compound 

acts through a group of reactions that lead to the formation of nitrated DPA derivatives, mainly N-nitroso-DPA and to 

a minor extent mononitro-DPAs, dinitro-DPAs, trinitro-DPAs, and nitro-N-nitroso-DPAs. From the different analytical 

techniques used for the determination of DPA and DPA derivatives, chromatographic techniques are the most 

widely employed. However, gas chromatography (GC) presents the disadvantage that N-nitroso-DPA is thermally 

decomposed into DPA during injection. Then, High Performance Liquid Chromatography (HPLC) has been the 

chromatographic technique showing the highest potential to determine DPA and its derivatives. In this work, the use 

of DPA and its derivatives as markers to follow the ageing process of nitrocellulose based gunpowders by means of 

HPLC with diode-array detection (DAD) is investigated. 

A simulated ageing of single- and double-based gunpowders (containing, in addition to nitrocellulose, nitroglycerin 

or dinitrotoluene) was carried out by heating these samples at 65º C during 120 days. Then, DPA and its derivatives 

were extracted from the gunpowders with methanol and analyzed by HPLC-DAD. The area of the chromatographic 

peaks of each compound extracted was measured and utilized to create a model to predict the ageing behavior of 

nitrocellulose based gunpowders. With this purpose, non linear univariate models as well as different multivariate 

models (multiple linear regression, multiple linear regression with either logarithmic or squared-root variable 

transformations, partial least squares regression and ridge regression) were studied. All the established models were 

validated with, approximately, 30 nitrocellulose based gunpowders, for which the manufacture year was well known. 

According to the results, lower and higher prediction errors were obtained for single-based gunpowders and doublebase 

gunpowders containing dinitrotoluene, respectively. On the other hand, multiple linear regression with squaredroot 

variable transformation and ridge regression models were those exhibiting lower error in the age prediction of 

gunpowders (< 13 years, in all cases). 

604 



P18-004 DETERMINATION OF NITROCELLULOSE CONTAINED IN SMOKELESS PROPELLANTS BY CAPILLARY ELECTROPHORESIS 

Fernández de la Ossa M., Sáiz J., Torre M., García-Ruiz C. 


Corresponding author e-mail: marianf.ossa@uah.es 

Nitrocellulose, discovered in the first half of the nineteenth century has a great interest in our society nowadays. It is 

a cellulose nitrate ester polymer obtained from the nitration of cellulose, which has its monomers linked by beta (1-4) 

bonds. The chemical formula of nitrocellulose is [C6H7O2(OH)3-x(ONO2)x]n, where x indicates the hydroxyl groups 

exchanged by nitro groups. The unique available places for nitro groups are the carbons C2, C3, and C6 and the rate 

for nitration is C6>>C2~C3. As an example, figure 1 shows the chemical structure of a nitrocellulose with a nitrogen 

content of 12.2 %. 

This macromolecule has different applications depending on their degree of nitration. Low-nitrated nitrocellulose 

is applied in paints, lacquers, varnishes, inks, etc., while high-nitrated nitrocellulose (> 12.5%) is used in almost all 

gun explosives. Nitrocellulose-containing explosives are dynamites and smokeless gunpowders. Dynamites are 

commercial explosives used with civilian proposes while smokeless gunpowders are used mostly by the international 

military community. 

The characterization and determination of nitrocellulose is a matter of great concern in the field of forensic 

chemistry. There are almost no studies on the analysis of intact nitrocellulose (without previous degradation or 

fragmentation), given the peculiarities of this macromolecule: its high chemical and structural complexity, high molar 

mass, as well as the lack of similar commercial standards. 

In this work, a capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection method is proposed, 

for the first time, for the determination of intact nitrocellulose contained in smokeless gunpowders. This method 

require a pre-capillary derivatization of nitrocellulose with 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt 

(APTS). This stage is followed with a double purpose: (i) to provide electrophoretical mobility to the derivatized 

nitrocellulose molecule (due to the three negative charges presents in the APTS molecule) and (ii) to take 

advantages of the fluorescent properties of the APTS to increase the sensitivity in the detection of nitrocellulose. 

Different derivatization conditions such as the amount of sample, the derivatization time, the temperature, and the 

propellant/APTS ratio were optimized to obtain the maximum number of signals and the better signal/noise ratio. 

CE separation conditions (nature and pH of the buffer, temperature, voltage, capillary length and injection time) 

were also investigated to achieve the better electrophoretic profiles. The optimized conditions were applied for the 

characterization of nitrocellulose-based gunpowders. 


605


P18-005 DEVELOPMENT OF A NEW METHODOLOGY FOR THE DETERMINATION OF CELLULOSE BY CAPILLARY ELECTROPHORESIS 

Fernández de la Ossa M., Torre M., García-Ruiz C. 


Corresponding author e-mail: marianf.ossa@uah.es 

Cellulose is a polysaccharide formed of hundred to over thousand D-glucose units linked by beta (1-4) bonds, with 

an empiric formula (C6H10O5)n, being n greater or equal than 200. This molecule has a fibrous structure, in which 

multiple sets of hydrogen bonds between the hydroxyl groups of different chains of glucose are juxtaposed, making 

them impenetrable to water, reason for which this is a polymer insoluble in water. 

The main component of paper is cellulose , obtained from the cellulose pulp extracted from softwoods (such as 

pine, fir or larch) and hardwood (eucalyptus or birch). Cellulose may also be extracted from recycled paper or 

cardboard to reduce the price of the manufacturing process. Paper presents a high interest in forensic chemistry 

because it has a great application in our daily lives and may be part of forensic evidences such as bank checks, 

letters, notes, etc. Nowadays, the analysis of paper in the forensic chemistry field is carried out by: (i) visual and 

microscopic examination, (ii) infrared spectroscopic techniques or thin-layer chromatography , for the analysis of 

organic components of paper, and (iii) inductively coupled plasma mass spectrometry, for the analysis of inorganic 

constituents of paper. 

In this work, a new Capillary Electrophoresis (CE) method using Laser-Induced Fluorescence (LIF) detection was 

developed for the determination of cellulose in paper. The electrophoretic determination of cellulose was carried 

out in two ways: (i) using a commercial kit for carbohydrates of Beckman-CoulterTM and (ii) applying experimental 

conditions developed in our laboratory. In both cases, cellulose was derivatized with 8-aminopyrene-1,3,6-trisulfonic 

acid trisodium salt (APTS).This molecule reacts with cellulose forming fluorescent and anionic components 

able to be analyzed by CE-LIF. Derivatization conditions (amount of sample to be derivatized, derivatization 

time, temperature, and cellulose/APTS ratio), as well as CE separation conditions (nature and pH of the buffer, 

temperature, voltage, capillary type and length, and injection time) were optimized for the in-house method 

developed in our laboratory. 

Results obtained with our in-house CE method were compared with those obtained with the commercially available 

kit (Figure 1). It is evident that with our method a better electrophoretic profile for this macromolecule was obtained 

(highest number and size of peaks), which may be essential for the characterization of cellulose in different types of 

papers, from a forensic viewpoint. 

606 



P18-006 STUDY ON LOSSES OF VOLATILE COMPOUNDS OF EXPLOSIVES AND CROSS-CONTAMINATION AMONG EXPLOSIVE 

SAMPLES STORED IN POLYETHYLENE BAGS BY CROMATOGRAPHIC TECHNIQUES 

Sáiz J. 1 , Bravo J.C. 1,2 , Atoche J.C. 2 , Ferrando J.L. 1,2 , Torre M. 1 , García-Ruiz C. 1 

1 


2 


Cross-contaminations of samples may lead to judicial mistakes in forensic cases. Contamination may be due to 

human errors as well as to improper operations previous to the analytical measurements, specially in the sample 

storage stage. A good sample storage system should maintain the integrity of samples and prevent crosscontaminations 

among samples. This is especially important when dealing with samples of forensic interest, such 

as explosives involved in terrorist attacks. In fact, explosives are composed mostly of high volatile compounds 

as ethylene glycol dinitrate (EGDN), nitroglycerine (NG), or dinitrotoluene (DNT), in addition to other non volatile 

components. In this work, the attention was focused on the polyethylene bags used by the Spanish Security Forces 

to collect, transport, and store evidence samples of explosives in forensic studies. The aim of this study was to 

demonstrate the appropriateness of these bags for the preservation of explosive samples, paying special attention 

to possible losses of those volatile compounds of explosives that may be essential for the characterization and 

identification of the explosive. A possible transfer of volatile compounds among samples stored into different bags 

and placed in a common space was also studied. 

The experimental design utilized in this work was the following: samples of two types of dynamites (one containing 

EGDN and DNT and other containing only EGDN) were prepared and placed individually inside polyethylene bags, 

and introduced into hermetically closed glass jars. The jars were stored at room temperature for several weeks. 

Losses of volatile compounds were studied by analyzing the headspaces of the jars by Gas Chromatography (GC) 

with mass spectrometry (MS) detection. In addition, the cross-contamination between samples was studied by 

analyzing, by High Performance Liquid Chromatography (HPLC) with MS detection, the extracts obtained after a 

sequential solvent extraction of the explosives with methanol and water. 

Results obtained in these studies have evidenced that polyethylene plastic bags used by Spanish Security Forces to 

transport and store explosive evidence samples allow the loss of volatile compounds because EGDN and DNT were 

detected by GC-MS in the headspaces of the jars containing the explosive samples. Moreover, cross-contamination 

among samples occurs since DNT was found in the dynamite that did not contain this compound in its formulation. 


607


P18-007 DETERMINATION OF NITROGLYCOL IN DYNAMITE-LIKE EXPLOSIVES BY LIQUID CHROMATOGRAPHY 

Sáiz J. 1 , Bravo J.C. 1,2 , Velasco E. 2 , Torre M. 1 , García-Ruiz C. 1 

1 


2 


Nitro group containing explosives constitute the most important group of explosives. Among them the nitrate ester 

ethylene glycol dinitrate (EGDN), also known as nitroglycol, is used in the formulation of dynamites. EGDN is a 

yellowish liquid explosive which replaces totally o partially nitroglycerin in dynamites due to its higher resistance 

against knocks and friction. EGDN is a good solvent for low-grade nitrocellulose, more volatile and less viscous 

than nitroglycerin. In addition, EGDN lowers the freezing point of nitroglycerin, which facilitates the employment of 

dynamite in countries with a cold climate. Most of studies on EGDN are focused on its effects on health, since this 

compound is known as a potent vasodilating agent and may provoke adverse effects on cardiac muscle, blood flow 

or mitochondrial respiration among workers who are exposed to this substance. However, as far as we know, there 

are no published works on the determination of EGDN as component of certain explosives and its use in forensic 

studies. Then, the aim of this study was to develop an extraction method of EGDN from a Goma-2-ECO dynamite 

and to validate a high performance liquid chromatography (HPLC) method for determining EGDN in this explosive. 

A sequential extraction method using water and methanol to extract EGDN from the Goma-2-ECO dynamite was 

developed. After, a HPLC method was used for the determination of EGDN in the two extractive phases. This 

chromatographic method was validated by evaluating its selectivity, sensitivity, linear range, limit of detection 

and quantitation, precision (as repeatability and intermediate precision), accuracy, and robustness. The method 

was proved to be selective for the chromatographic determination of EGDN. Sensitivity, evaluated as the slope of 

the calibration line, was close to 1.5 L/mg. Linearity was studied in the range from 3 mg/L to 400 mg/L of EGDN; 

results showed that the calibration, was linear from 10.0 to 200.0 mg/L. The limits of detection and quantitation 

were 1.7 and 5.6, respectively. Repeatability, assessed for three concentrations levels (30 mg/L, 70 mg/L, and 150 

mg/L), was lower than 6 % and intermediate precision, evaluated by analyzing a EGDN standard solution of 70 

mg/L, in different days and by different analysts, was lower than 6.6%. Accuracy was close to 100% at the three 

concentration levels studied (104% for 30 mg/L, 101% for 70 mg/L, and 106% for 150 mg/L). The robustness of the 

method was calculated through a study on the effect of the flow-rate on the stability of the peak area value of EGDN 

(ten injections of a standard solution of 3 mg/L). The results obtained were: average peak area (± 2s) of 3.3 ± 0.4 and 

an average retention time (± 2s) of 8.34 ± 0.01 min. Finally, the EGDN content of a sample of Goma-2-ECO dynamite 

was determined with this optimized HPLC method. The result found for this sample (30.29 %), is in accordance with 

the manufacturer´s specifications for this dynamite (25.7-31.4%). 

608 



P18-008 SIMULTANEOUS CE ANALYSIS OF INORGANIC ANIONS AND CATIONS IN POST-BLAST RESIDUE EXTRACTS OF ACID- 

ALUMINUM MIXTURES 

Sarazin C. 1 , Delaunay N. 2 , Varenne A. 2, Costanza C. 1 , Eudes V. 1 , Gareil P. 2 

1 

Laboratoire Central de la Préfecture de Police de Paris-France 

2 


Bursts caused by acid and aluminum mixtures are very often commited by the young delinquency in western 

countries because of its easiness of achievement. The contact between an acid (hydrochloric or nitric) and aluminum 

foils in a plastic bottle causes an important gaseous release leading to the bottle explosion and acid projections 

that can dramatically hurt citizens. A fast, simple, selective, and cost-effective method allowing the detection of 

chloride and nitrate anions and aluminum(III) was thus required. CE, admitted as an efficient technique for inorganic 

analysis, was therefore evaluated for the simultaneous analysis of these species in order to reduce the analysis 

time. Detection can be carried out using a common BGE allowing the indirect UV absorbance of chloride and nitrate 

anions and the direct UV absorbance of aluminum(III) as negatively charged chelate. 2,6-pyridinedicarboxylic acid 

(PDC) was already proposed for this purpose [1]. In aqueous solution, simulated diagrams for aluminum(III)-PDC 

systems show the presence of various species, according to pH: four hydroxylated aluminum complexes, two Al(III)- 

PDC complexes ([Al-PDC]+ and [Al-PDC2]-) and their hydroxylated forms. First, the pH of the background electrolyte 

was selected at 4.5, a value for which [Al-PDC2]- is present in a highly predominant form. Next, the study of 

selectivity for Al(III) with respect to 10 other cationic species potentially present in post-blast residues (Ca(II), Mg(II), 

Sr(II), Ba(II), Co(II), Zn(II), Cu(II), Ni(II), Fe(II), and Fe(III)) dictated the choice of 20 mM PDC concentration. The method 

validation was then carried out. The intermediate precision for Al(III) on normalized migration times and on corrected 

areas does not exceed 2% and 3.5%, respectively. LODs (calculated at S/N = 3) were 1.3 mg L-1 for chloride and 

nitrate anions and 0.3 mg L-1 for aluminum(III). Detection linearity range was checked between 2 and 30 ppm for 

Al(III) and between 4 and 30 ppm for anions. Finally, real samples, extracted in ultra-pure water from metal foil pellets 

or just diluted from an acidic yellow liquid, were analyzed in less than 15 min. This simple and fast procedure allowed 

the simultaneous determination and the quantitation of chloride and nitrate anions and aluminum(III) cation in postblast 

residue extracts, a new fold of application. [1]. Soga T., Ross G.A., J. Chromatogr. A, 1999, 834, 65-71 


609


P18-009 ANALYSIS OF INORGANIC CATIONS IN POST-BLAST RESIDUE EXTRACTS BY CAPILLARY ELECTROPHORESIS 

Sarazin C. 1 , Delaunay N. 2 , Costanza C. 1 , Eudes V. 1 , Gareil P. 2 

1 

Laboratoire Central de la Préfecture de Police de Paris 

2 


The determination of specific cations, such as ammonium, monométhylammonium, potassium, and alkalineearth 

cations, is critical for the identification of explosives and their residues. Indeed, they can be used in 

commercial explosives, such as Ammonium Nitrate-Fuel Oil (ANFO), improvised explosives, and black powder. 

Ionic Chromatography (IC) is currently used for the determination of cations in post-blast residues, but to confirm 

or not the presence of these compounds, a complementary technique to IC is mandatory. Therefore, capillary 

electrophoresis (CE) was assessed, because of its orthogonal mechanism compared to IC and its high efficiency. 

This work was focused on the development of a CE method for the analysis of 8 cations of interest (ammonium, 

monomethylammonium, potassium, sodium, calcium, barium, strontium, and magnesium), 4 metal ions potentially 

present in matrices (iron (II), iron (III), copper (II), and aluminum (III)), and lithium ion, which was used as an internal 

reference. Guanidinium cation, a non-CMR compound, was used as cationic chromophore and the conditions of 

indirect UV detections were optimized (guanidium concentration, wavelength, and bandwidth). Then, in order to 

prevent the system from EOF variations linked to potential effects of the real samples on capillary surfaces, the 

Successive Multiple Ionic-polymer Layers (SMIL) approach was implemented. Polybrene-dextran sulfate (PB-DS) 

and polybrene-polyvinylsulfonate(PB-PVS) were both evaluated. The final conditions consisted in a dynamic capillary 

coating involving first a flush (40 psi, 10 Vcap) with a solution of 1 g / 100 mL PB, followed by a second flush (20 

psi, 10 Vcap) with 0.01 g / 100 mL PVS solution. To increase resolution, temperature and 18-crown-6 ether (18-C-6) 

concentration were next optimized. The background electrolyte is composed of 15 mM guanidine acetate, 3 mM 18- 

C-6 adjusted at pH 4.0 with acetic acid and a temperature fixed at 20°C. In order to improve LODs, the performances 

of electrokinetic (EKI) and hydrodynamic (HD) injections were compared. With respect to band broadening, the best 

injection conditions were 2 kV for 20 s for EKI and 0.8 psi for 7 s for HD. With EKI (retained injection mode), LODs 

were improved by a factor 2, varying from 1 ppm for Na+ to 5 ppm for Ba2+ for a standard mixture. Finally, the main 

validation criteria were studied and real solutions extracted from post-blast residues were analyzed. 

610 



P18-010 DETERMINATION OF MESCALINE IN LOPHOPHORA WILLIAMSII PLANTS GROWN IN THE CULTURE USING CAPILLARY 

ELECTROPHORESIS WITH ELECTROSPRAY MASS SPECTROMETRY 

Svidrnoch M. 1 , Ranc V. 1 , Maier L. 2 , Sevcik J. 1 , Maier V. 1 

1 

Palacky University in Olomouc-Czech Republic 

2 



Mescaline (3,4,5-trimethoxyphenylethylamine) is a natural alkaloid from the phenylethylamine group, naturally 

occurring in several plants of the cactus family (Cactaceae), especially in plants of the genus Lophophora1-3. In 

many parts of the world, especially in the region of its presence, it is a frequently abused drug. Mescalin is an 

agonist of serotonin receptors (5-HT), and although not chemically related to LSD, its mechanism of action is 

similar3. So far many methods dealing with analysis of mescaline in biological samples, as well as in plant matter, 

have been published. The most common methods include GC-MS, LC-DAD and more recently LC-MS and CE- 

DAD analysis1-4. In this work we developed a method for determination of mescaline in samples of lophophora 

plants grown in cultural conditions in the Czech Republic using capillary electrophoresis with electrospray mass 

spectrometry (CE-ESI-MS). The presence of mescaline was confirmed using ESI-MS/MS spectra of plant extracts 

and their comparasion with ESI-MS/MS spectra of mescaline standard solution. The age of the plants was about 

8 years and their size ranged from 4 to 6 cm. Method development consisted of the study of selected extraction 

solvents (methanol, acetonitrile, ethanol and chloroform) and obtained recoveries were compared with results 

obtained by a classical Soxhlet extraction where ethanol was used as a solvent. Concerning CE-ESI-MS method, 20 

mM amonium acetate buffer with pH 3.3 was used. Mass spectrometric conditions were studied to obtain the highest 

signal of target analyte as possible. The linearity was observed in the concentration range of 0.5-50 mg/L, with 

correlation coefficient of 0.996. The limit of detection (LOD) and limit of quantification (LOQ) of developed method 

were 0.16 mg/L and 0.53 mg/L, respectively. Finally, the presented method was the first application of CE-ESI-MS 

for the determination of mescaline in lophophora plants, grown artifically out of their natural habitat. The financial 

support by the research project MSM619895216 of the Ministry of Education of the Czech Republic is gratefully 

acknowledged. References: 1. Casado R., Uriarte I., Cavero Y.R., Calvo I.M.: Chromatographia 67 (2008) 665. 2. 

Gennaro M.C., Gioannini E., Giacosa D., Siccardi D.: Anal. Letters 29 (1996) 2399. 3. Beyer J., Drummer H.O., Maurer 

H.H.: For. Sci. Int. 185 (2009) 1. 4. Helmin J.H., Brenneisen R.: J. Chrom. 593 (1992) 87. 


611

P19 INDUSTRIAL 

PROCESS 

POSTER 

SESSIONS

POSTER SESSION 19 - INDUSTRIAL PROCESS 

P19-001 PILOT SCALE RECOVERY OF PHYCOCYANIN FROM SPIRULINA PLATENSIS USING EXPANDED BED ADSORPTION 


Bermejo R., Ramos A. 

Jaén University 

Corresponding author e-mail:rbermejo@ujaen.es 

Biliproteins are a family of proteins with linked open-chain tetrapyrrole prosthetic groups (bilins) and microalgae 

are the usual source of these compounds [1]. The main applications of biliproteins are as fluorescent markers in 

biomedical research and highly sensitive fluorescent techniques. In addition, they have potential as natural colorants 

for use in foods and cosmetics. C-phycocyanin (CPC) is a major light-harvesting blue pigment of some blue-green 

microalgae. Although biliproteins have many applications, their use is limited by the high cost of extraction and 

purification. In recent years, different alternatives to the conventional purification have been developed. In this 

sense, expanded bed adsorption (EBA) chromatography is a downstream processing technique for capture of 

proteins directly from unclarified feedstocks that reduces the number of operations in purification processes by 

combining, clarification, concentration, and capture into one operation. So, EBA chromatography allows integrate 

solid-liquid separation, volume reduction by protein adsorption and partial purification in one unit operation without 

compromising on separation efficiencies, but saving considerable processing time and capital investment [2]. In 

this work, we describe the use of EBA chromatography for pilot scale recovery of CPC from the microalga Spirulina 

platensis. Biliproteins were released from the microalga cells by osmotic shock and captured by applying the 

centrifugued cell suspension to an anion exchanger (Streamline-DEAE) using EBA chromatography. Initially, EBA 

chromatography experiments were carried out by using a small column (inner diameter 15 mm) to optimize different 

parameters: degree of bed expansion (H/H0), protein loading (mg CPC/ml adsorbent) and sample viscosity. The 

adsorption capacity and the optimal mobile phase composition for EBA steps, have been selected by preliminary 

tests, obtaining the static and dynamic binding capacities of adsorbent from the breakthrough curve and the 

equilibrium adsorption isotherm. An effective expanded bed operation is achieved when H/H0 is 2.0 and the elution 

yield obtained was 76 %. The effluent from EBA (tested using SDS-PAGE and spectroscopy) corresponds to a 

protein mixture (phycocyanin rich solution). This optimized separation protocol was scaled up by using column 

diameters of 25, 40 and 60 mm respectively, maintaining the optimized parameters obtained with the small column 

and only the cross-sectional area of the bed and the volumetric flow through the column have been increased in 

linear proportion to the volume of feedstock to be processed. Finally, we have scale-up the process to pilot scale 

using a 150 mm column (Figure 1). The scale-up factor was 100, based on column area from the lab scale column 

(15 mm diameter to 150 mm diameter). These experiments demonstrate that elution yields are approximately 

constant while the column diameter increases. Thus, the EBA methodology simplifies the CPC isolation process, 

reduces processing time and utilities consumption and is easy to scale up, maintaining constant total product yield. 

Acknowledgements: The authors thank the Junta de Andalucía for financial support (Project P06-TEP-01362). [1] 

A.N. Glazer, Z.Cohen (Ed.), Chemicals from Microalgae, Chapter 11 (1999) 261. [2] H.A. Chase, Trends Biotechnol. 12 

(1994) 296-303. 


613


P19-002 ANALYSIS OF PYROLYSIS PRODUCTS FROM BIOMASS AND COAL BY COMPREHENSIVE TWO-DIMENSIONAL GAS- 


Rathsack P., Otto M. 

TU Bergakademie Freiberg 

Corresponding author e-mail: philipp.rathsack@chemie.tu-freiberg.de 

The aim of this study is the analysis of complex samples from biomass and coal pyrolysis experiments by 

two-dimensional gas-chromatography mass-spectrometry (GCxGC-TOF-MS). In a first step the intention is the 

identification of pyrolysis-derived compounds and compound-classes. Furthermore the aim is to utilise compound 

patterns or related parameters for the investigation of the influence of process parameters on compound distribution. 

At a later date the quantification will be the central point of interest. Pyrolysis oils were produced by a semi-industrial 

reactor and collected in sequenced cooling traps. Several biomasses like wood off-cuts, corn or rye and two coals 

from Germany were introduced into the reactor at varying temperatures. Different column setups for comprehensive 

two-dimensional gas-chromatography were tested and the chromatographic conditions were optimized. Pyrolysis 

products from biomasses mainly comprise of aliphatic and aromatic oxygen compounds besides aliphatic and 

aromatic hydrocarbons. Coal pyrolysis products show less oxygen compounds but some sulfur containing 

compounds in addition. Hundreds of peaks can be resolved by the superior separation power of GCxGC-TOF-MS. 

As generally known datasets from comprehensive two-dimensional mass-spectrometry are very large and dedicated 

data evaluation methods from the field of chemometrics have to be used. In this work algorithms in Matlab are used 

for the identification of compound classes, since they are of central interest for process engineering. Therefore 

different classification methods employing different subsets from the data are used. This work is funded by the 

German centre for energy resources. The German centre for energy resources is a strategic partnership between 

government, science and business to develop innovative concepts and technologies for the post-oil era. It addresses 

the issues of energy supplies security and sustainability through research into the non-energetic utilisation of coal 

and biomass, where they are not simply burnt to produce energy, but used as raw material in production processes. 

614 



P19-003 APPLICATION OF METHACRYLYC ACID-ETHYLENEGLYCOL DIMETHACRYLATE POLYMERIC SORBENT FOR THE REMOVAL 

OF ESTROGENS FROM WATER SAMPLES 

Gallego A., Bravo J.C., Garcinuño R.M., Fernández P., Durand J.S. 

Deparment of Analytical Sciences. Faculty of Sciences. National University of Distance Education, Madrid 

The presence in aquatic environment of chemicals that can cause adverse effects on human and wildlife has been 

widely reported. Some of these chemicals are capable of disrupting the endocrine system of fish and wildlife 

attracting considerable attentions worldwide. Among these endocrine disrupting chemicals (EDCs), natural and 

synthetic estrogens have been found, being of great concern because of their potential to alter the endocrine 

system of humans and animals. These estrogens are widely used in estrogen replacement therapy and as oral 

contraceptives, and often have been used as grow promoters in cattle. The main way these estrogens enter water 

environment is through sewage treatment plants receiving industrial and domestic waste waters, where human and 

animal waste products are released. The presence of estrogens in the environment could indicate that conventional 

wastewater treatment processes have limited capacity to remove these compounds. In the last years diverse 

researches have been focussed on the development of cost-effective methods for the removal of these compounds 

in water. 

In this research, a series of Methacrylic acid-Ethyleneglycol dimethacrylate polymers with different monomers 

ratio were synthesised by photochemical (UV irradiation at 365 nm) or thermal (oven at 60ºC) initiation for the 

removal of the estrogens named Estradiol (E2), Ethynylestradiol (EE2) and Dienestrol (DEN), representing natural 

steroidal estrogen, synthetic steroidal estrogen and synthetic stilbene estrogen respectively, from water samples. 

The separation and quantification of these compounds were carried out by the mean of HPLC-DAD. Batch and 

continuous flow experiments were carried out to evaluate the capacity of these polymers to adsorb E2, EE2 and 

DEN. Adsorption isotherm studies revealed that Langmuir isotherm model was fitted with a better correlation than 

Freundlich isotherm. Finally, continuous flow experiments were carried out by microcolumn studies to check the 

suitability of the polymeric sorbent for the removal of estrogens from real water samples. When continuous removal 

experiments at 8 mL/min flow rate were carried out, breakthrough adsorption capacities of 28.5, 38 and 69.7 mg/g 

for E2, EE2 and DEN respectively were achieved. 

Keywords: Estrogens, Removal, Polymer, Sorbent, Waters 


615


POSTER 

SESSIONS

POSTER SESSION 20 - ENVIRONMENT 

P20-001 BEHAVIOR OF COPLANAR AND NON-COPLANAR POLYCHLORINATED TERPHENYL (PCT) CONGENERS TOWARD 

STATIONARY PHASE OF COLUMN CHROMATOGRAPHY FOR DEVELOPMENT OF ANALYTICAL METHOD 

Wibowo A.H., Bahadir M., Vogt R., Wichmann H. 

Technical University of Braunschweig 

The clean up step as part of the method development for PCTs measurement in the environments has been 

performed. The fundamental knowledge which is developed for the purpose is the basic difference of the behavior 

of coplanar and non-coplanar form of the PCT congeners in the stationary phase. Behaviour of 16 congeners 

with different chlorinated degree and planarity orientation has been investigated in the various stationary phase of 

silica, alumina and florisil. In this study, florisil is focused to interact differently between coplanar and non-coplanar 

congeners. The rest two adsorbents used were aimed to eliminate the possible interference in the matrix before 

measurement with gas chromatography – mass spectroscopy instrument (GC-MS). The study showed that alumina 

and silica were able to be applied as stationary phase in the clean up step for the removal of interference in the 

matrix. As indicator, no lost of significant quantity of congeners occurred during the elution throughout this clean 

up step. The stationary phase of alumina was able to maintain congeners eluted in one fraction. Combination of 

neutral, acid and base silica were also able to be deployed in the clean up step for interference removal in the matrix. 

As key result of this study, Elution of congeners in the florisil column showed that good separation was obtained 

in the elution of PCT congeners in which coplanar congeners was stronger retained in the adsorbent compared to 

the non-coplanar ones. Thus, coplanar congeners were able to be eluted in the different fraction with non-coplanar 

congeners. The result indicated that extended use of the separation step. integrated in the conventional clean up in 

the analysis of coplanar congeners is widely open. A new method developed is hence based on the measurement 

of only coplanar PCT among total of 8557 theoretically calculated possible using GC-MS in the SIM mode after 

integrated clean up procedure. 


617


P20-002 DETERMINING THE ANHYDROSUGARS LEVOGLUCOSAN, MANNOSAN AND GALACTOSAN IN AEROSOLS 

Espuelas J. 1 , Kolb T. 2 , Bogenschütz G. 2 

1 

Gomensoro S.A.-Spain 

2 

Deutsche Metrohm GmbH & Co. KG 

Anhydrosugars are thermal degradation products of cellulose and hemicellulose. They form an important fraction of 

water-soluble organic carbon in atmospheric aerosols and thus have a great potential of affecting, among others, 

cloud formation and precipitation and thus the climate in general. Due to residential wood burning for heating, 

concentrations of levoglucosan and its stereoisomers mannosan and galactosan are typically high during winter, 

while increased contribution of primarily biological sugar components becomes evident during summer months. 

Levoglucosan (1,6-anhydro-beta-D-glucopyranose) is a dehydrated glucose with a ketal functional group and 

serves as a unique source-specific tracer for biomass burning. The emissions of mannosan and galactosan are 

typically less than those of levoglucosan. Biomass smoke contribution to ambient aerosol levels can be determined 

by collecting aerosols on glass fiber filters. After extraction in water, levoglucosan, galactosan and mannosan 

concentrations can be determined by ion chromatography (IC) followed by pulsed amperometric detection (PAD). 

IC-PAD is highly sensitive and provides a simple alternative to GC-MS determinations, which require intensive and 

expensive sample extraction as well as derivatization. Determination of levoglucosan, mannosan, galactosan and 

other carbohydrates as well as their separation from arabitol, mannitol and glucose is possible in a simple isocratic 

run. The limit of detection for levoglucosan and other anhydrosugars is less than 5 ng/m3 air, using a 4.7 cm 

diameter sub-filter from a 15 cm diameter filter on which aerosols from 80 m3 air were collected. 

618 



P20-003 DETERMINING TRACE LEVELS OF PERFLUORINATED COMPOUNDS IN WATER BY SUPPRESSED ION CHROMATOGRAPHY 

WITH INLINE MATRIX ELIMINATION 

Epalza A. 1 , Subramanian N.H. 2 , Manigandan P. 3 , Wille A. 4 

1 

Gomensoro S.A 

2 

Metrohm USA-USA 

3 

Metrohm India-India 

4 

Metrohm International Headquaters-Switzerland 

Commercially important perfluorinated chemicals such as perfluorooctane sulfonate (PFOS) and perfluorooctanoate 

(PFOA) are persistent, bioaccumulative and toxic. Therefore, their determination in environmental matrices is crucial. 

This poster describes a simple and sensitive method for the determination of PFOS and PFOA in water samples 

by suppressed conductivity detection. Separation was achieved by isocratic elution on a reversed-phase column 

thermostated at 35 ºC using an aqueous mobile phase containing boric acid and acetonitrile. The PFOA and PFOS 

content in the water matrix was quantified by direct injection applying a 1000 µL loop. For the concentration range 

of 2 to 250 µg/L, the linear calibration curve for both PFOA and PFOS yielded correlation coefficients (R) better than 

0.999. The relative standard deviations were smaller than 5.8%. The presence of divalent cations, such as calcium 

and magnesium, which are normally present in water matrices, impairs PFOS recovery. This drawback was overcome 

by applying Metrohm`s Inline Cation Removal. While the interfering divalent cations are exchanged for non-interfering 

sodium cations, PFOA and PFOS are directly transferred to the sample loop. After inline cation removal, PFOA 

and PFOS recovery in water samples containing 350 mg/L of calcium and magnesium improved from 90…115% to 

93…107%. The chromatographic system was validated in terms of linearity, recovery and matrix effects. 


619


P20-004 OCCURRENCE OF PERFLUORINATED COMPOUNDS IN SPANISH SEWAGE SLUDGE 

Navarro I., Sanz P., Martínez M.A. 

CIEMAT, Valencia 

Corresponding author e-mail: i.navarro@ciemat.es 

Socio-economic changes during recent decades, together with the development of the industry in different sectors 

and the exorbitant increase of human population and its consuming practices have resulted in a significant increase 

of organic waste production that, in certain situations, can generate environmental problems. 

The current waste policy the EU is based on a concept called “principle of hierarchy”, under which waste should be 

avoided, and if you can not be avoided should be reused, recycled or recovered to the extent possible (COM, 2005). 

Both Spain and the various European Union states have raised the review of the European Directive concerning 

the application of Wastewater treatment plant (WWTP) sludge (2nd Draft Biological Treatment of Biodegradable 

Waste, 2001) in soil for agricultural purposes in order to establish new limits for contaminants considered at first and 

increase the number of chemicals analyzed, mainly in regard to new emerging compounds. 

The use of the organic fraction of sewage sludge as agricultural amendment, offering benefits and acceptable risks 

to both the soil and plants and is one of the best outings environmentally sustainable wastes. However, there may be 

problems because of the danger, and bioaccumulation of these compounds along the food chain. 

Emerging Contaminants are defined as unknown or unrecognized contaminants, whose presence in the environment 

generates a growing concern regarding their potential effects on ecosystems. Thus, perfluorinated chemicals (PFCs) 

are some of the emerging pollutants currently most studied. They have been used in a wide variety of consumer and 

industrial products for more than 50 years. 

Considering the possible transfer of these chemicals from waste containing them to environmental compartments, 

the objective of this study is the determination of PFC concentration in sewage sludge coming from WWTPs of 

different sizes and geographically distributed all over Spain. 

Samples were extracted by agitation, sonication and centrifugation techniques or by pressurized fluid extraction, as 

an alternative extraction method. Subsequently, the extracts were purified by SPE sorbents based in weak anion 

exchange, and finally the analysis were performed by liquid chromatograph interfaced with a triple quadrupole mass 

spectrometer, operating in negative electrospray ionization and multiple reaction monitoring (MRM) mode. The 

criteria of isotopic dilution were used for identification and quantitation of target species. 

The evaluation of the presence of PFC in sewage sludge generates data that will be useful in developing strategies 

for management of wastewater treatment plants both locally and nationally. Additionally, innovative sustainable 

management systems could be developed and implemented for this type of organic waste. 

1. COM (2005) 666 Final. Commission of the European Communities. Taking sustainable use of resources forward: A 

thematic strategy on the prevention and recycling of waste. 

2. Draft of Biological Treatment of Biodegradable Waste (2001), 2nd edition. 

Acknowledgements - This study has been co-funded by the Spanish Ministry of Science and Innovation and 

European Regional Development Fund (Project number CTM2007-62801). 

620 



P20-005 ODOR-CAUSING ORGANIC COMPOUNDS IN WASTEWATER TREATMENT PLANTS: EVALUATION OF HS-SPME AS 

CONCENTRATION TECHNIQUE 

Godayol A., Alonso M., Besalú E., Sanchez J.M., Anticó E. 

Universitat de Girona 

Corresponding author e-mail: enriqueta.antico@udg.edu 

Odorous emissions from wastewater collection systems and treatment facilities reaching the atmosphere include 

inorganic and organic gases and vapours. Many of these compounds come from anaerobic decomposition of 

organic matter containing sulphur and nitrogen. Sewer gases contain mainly H2S, NH3, CO2, and CH4, being the 

two first strongly malodorous. Additionally, other highly malodorous compounds such as mercaptants, amines 

(e.g., indole and skatole), organic acids, aldehydes, and ketones might be present. The determination of these 

compounds presents different challenges: (i) the difficulty associated with air sampling, (ii) the different chemical 

and chromatographic behaviour of the compounds included within the group of total volatile malodorous organic 

compounds (VMOC), and (iii) the need to evaluate correlations between water concentration of target compounds, 

air concentration and human perception or olfactometric analysis. Capture on solid sorbents followed by gas 

chromatography determination is usually the technique of choice when odour emissions are investigated in air 

samples. However, little attention has been addressed to investigate the presence of odorous compounds in waste 

waters. In the present work we explore the possibility of using HS-SPME applied directly to waste water matrices 

to establish the contribution of the different compounds to odour emissions and perception. A list of odorous 

compounds belonging to different chemical families has been selected: di-methyl disulfide (DMDS), toluene, ethyl 

benzene, o-xylene, p-xylene, phenol, octanal, limonene, m-cresol, nonanal, carvone, indole, and skatole. All of them 

have previously been reported to be present in waste water emissions. Sulphides, mercaptans and ammonia have 

been discarded due to their high volatility which requires specific chromatographic conditions. Volatile fatty acids 

were also essayed using on-fiber silylation but loses of other target analytes were observed during the derivatization 

step and for this reason were not included. Several variables affecting the chromatographic behaviour of the target 

compounds were considered (e.g., injection band broadening, splitless time). Also experimental conditions affecting 

their extraction using HS-SPME (e.g., type of sorbent, time and extraction temperature) have been studied according 

to the design of experiments (DoE) methodology. Finally, application of the proposed method to real samples will be 

discussed. Acknowledgements: This study was financed by the MICINN (Spanish Ministry of Education and Science), 

projects CTM2008-06847-C02-02/TECNO and CTQ2009-09370. 


621


P20-006 DEVELOPMENT AND QUALITATIVE VALIDATION OF A WIDE SCOPE SCREENING OF EMERGING CONTAMINANTS IN 

NATURAL WATER AND WASTEWATER BY UHPLC-QTOF MS 

Ibáñez M., Díaz R., López F.J., Sancho J.V., Gracia E., Bijlsma L., Hernández F. 


Corresponding author e-mail: felix.hernandez@uji.es 

In this work, a wide-scope, multiclass, screening of organic contaminants in different types of water matrices 

has been developed and validated. The method is based on the use of ultra high pressure liquid chromatography 

coupled to hybrid quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). The work is focused on 

qualitative aspects, i.e. it pursues the reliable and sensitive identification of compounds detected in samples at 

a certain concentration level. A general procedure involving a simple solid phase extraction (SPE) with OASIS 

HLB cartridges was applied to water samples of different origin and matrix composition (surface, ground, and 

effluent urban wastewater). Samples were fortified at 1 µg/L with a standard mixture of around 150 organic 

micro-contaminants taken as a model, including mycotoxins, pharmaceuticals, drugs of abuse, pesticides, and 

several relevant metabolites. After SPE, the sample extracts were analyzed by UHPLC-QTOF in MSE mode. Full 

spectrum acquisition data, generated simultaneously at both, low and high collision energies, were processed 

using ChromaLynx XS (within MassLynx 4.1) in a targeted mode. For qualitative validation of the procedure, the 

presence of the (de)protonated molecule measured at its accurate mass (narrow-mass Extracted Ion Chromatogram, 

0.02 Da) at the expected retention time was evaluated at the 1 µg/L concentration level for the different types of 

samples. Additionally, the presence of a CID fragment (in any of the two functions acquired at low or high collision 

energy) or a characteristic isotopic peak was assessed. Most of the compounds were correctly identified in fortified 

water samples at the level tested. Around 1100 organic contaminants were investigated in different water samples. 

Compounds searched included pharmaceuticals, pesticides, drugs of abuse and mycotoxins, but also hormones, 

UV-filter agents, colorants, preservants, phenols and surfactants. When available, information about product 

fragmentation was also included in the target list to obtain a more confident identification. A notable number of 

compounds were detected and identified. Among pharmaceuticals, several antibiotics (ofloxacin, ciprofloxacin 

and clarithromycin), anti-inflammatory/analgesics drugs (ketoprofen, diclofenac, ibuprofen and salicylic acid) and 

lipid regulators (gemfibrozil and bezafibrate) were identified. Regarding drugs of abuse, cocaine and its metabolite 

benzoilecgonine were the most frequently compounds detected. Triazine herbicides and transformation products 

(simazine, terbumeton, terbuthylazine, desethyl-terbuthylazine and 2-hydroxy-terbuthylazine), and fungicides like 

thiabendazol, carbendazim and imazalil, were also identified. When the reference standard was not available in the 

laboratory, identification was initially carried out studying and evaluating CID fragments, and comparing them with 

those reported in bibliography. This was the case of angiotensin II receptor antagonists (valsartan and irbesartan) 

or the diuretic furosemide, which were finally confirmed with standards in a later stage. In these cases, empirical 

information obtained on the fragments was added to the target list in order to facilitate future screenings. In several 

cases, positive findings could be correctly identified at concentration levels below the concentration validated 

(1µg/L), which illustrates the strong potential and excellent sensitivity of the screening approach developed in the 

present work. 

622 



P20-007 ANALYSES OF ENERGETIC MOLECULES TRACES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY / MASS 

SPECTROMETRY IN AQUEOUS MATRICES 

Legendre A, Bousquet M., Pin N., Hairault L. 

Commissariat à l’Energie Atomique, DXPL/SMEO/LPC 

Corresponding author e-mail: marilyne.bousquet@cea.fr 

More and more environmental missions are set in the aim to control pollution and detect a large range of contaminant 

molecules in air, water…. Our project is to determine traces of energetic molecules in waters samples from layers to 

determine drinkability. 

US EPA gave maximum values for concentration in potable water, 400 µg/L for cyclo-tetramethylene-tetranitramine 

or octogen (HMX) and 2 µg/L for cyclo-trimethylene-trinitramine or hexogen (RDX). It’s why we have to find the best 

chromatographic method to reach the lower Limit Of Detection (LOD) and Quantification (LOQ) for traces detection. 

Protocol: 

• SPE extraction and concentration treatment with a vacuum rotary evaporator. 

• Separation on reversed mode by High Performance Liquid Chromatography (HPLC), acetic acid post-column 

addition. 

• Ultraviolet / Mass Spectrometry by ions trapping (UV/MS) detection and quantification. 

One chromatographic method is described by the US EPA 8330 Method which recommends analysing samples on 

two different kinds of columns. 

The aim of this project is to find an ingenious and good sensitive chromatographic method for detection and 

quantification of those molecules in a single run. The identification of molecules is guaranteed by a double detection 

(UV and MS). 

Our first interest is Solid Phase Extraction (SPE) samples treatment. 

This simple method will help for matrices eliminations first and for sensitivity of the detection in second part. Four 

types of SPE are potentially identified in literature for this kind of molecules. The selection will depend mainly of 

recovery results and percolation times. 

The use of a vacuum concentrator is only to get a concentration factor the higher and the more precise to decrease 

LOD and LOQ. 

The next step is to find a way to get the better detection in UV and MS. 

Add of acetic acid in mobile phase of HPLC degrades column. Retentions times of HMX and RDX are longer than 

without acetic acid in mobile phase. 

The solution found to avoid this problem is a post-column add of acetic acid to involve MS response without 

damaging column and UV signal. 

We tested add of different acetic acid concentrations to optimise the answer. 

The last step is to determine repeatability and sensitivity for the global chromatographic method (sample treatment, 

HPLC UV/MS) and then determine LOD and LOQ. 

This was realized using three methods: signal on noise, standard linear regression and weighted linear regression. 

Using the last method, all the possible weights were evaluated. The optimal method, allowing the smallest relative 

error with the best correlation coefficient was the weighted linear regression, using standard deviation of the 

intercept and weighted by the squared root of the inverse of the concentration. 


623


P20-008 ASSESSMENT OF PESTICIDE CONTAMINATION IN SOIL AND WATER SAMPLES FROM NATURAL PARK OF L´ ALBUFERA 

USING LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY 

Masiá A. 1 , Vazquez-Roig P. 1 , BlascoC. 1 , Andreu V. 2 , Picó Y. 1 

1 

Faculty of Pharmacy, University of Valencia 

2 

Centro de investigaciones sobre desertificación–CIDE, Valencia 

ASSESSMENT OF PESTICIDE CONTAMINATION IN SOIL AND WATER SAMPLES FROM NATURAL PARK OF L´ 

ALBUFERA USING LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY. 

Pesticides are an important group of agrochemicals used for food crop protection. Unfortunately, these compounds 

are easily accumulable in soils becoming a real environmental pollution risk and can contaminate surface water 

directly as spray drift or run-off, and also via drainage through the soils of treated farmland [1, 2]. For these 

reasons, there is increasing need for the development of well validated, accurate, time-saving, low cost, modern, 

multicomponents methods. 

In the light of the aforementioned, the aim of this study was to validate a sensitive multi-residue method for the 

target analysis of sixteen pesticides in soils and waters. 

The selected pesticides were: alachlor, atrazine, buprofezin, chlorfenvinphos, chlorpyriphos, diazinon, diuron, 

fenitrothion, fenthion, hexythiazox, imazalil, isoproturon, malathion, prochloraz, tolclophos-methyl and trifluralin. 

Pesticides from soil were extracted by pressurized liquid extraction (PLE), using an ASE 200 system from Dionex. 

Soil samples (10 g) were introduced into 11 mL cell and heated to 100 ºC for 7 min and extracted by ethyl acetate 

with a flush volume of 100 % in one cycle. Water was extracted by solid-phase extraction (SPE). Water samples (500 

mL) were acidified and then extracted using Oasis HLB cartridges previously conditioned with methanol and water. 

Analytes were eluted with 4 mL. 

The residue analyses were performed by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) 

multi-residue method. Separation was made with a Luna 3μm C18 (Phenomenex®) analytical column. The mobile 

phase was MeOH and H2O 5 mM ammonium formate gradient composition, used in conjunction with positive mode 

electrospray ionization tandem mass spectrometry. 

Performance characteristics of the proposed method were established. Selectivity, linearity, precision, recoveries 

and limits of detection (LODs) and quantification (LOQs) were studied. The method allowed LOQ from 0.03 μg/kg to 

0.05 μg/kg. Recoveries ranged from 75 to 95% with relative standard desviation < 18%. The results of the validation 

procedure confirmed that the method is suitable for the planned purpose. 

This method has been applied successfully to the analysis of incurred water and soil samples from Natural Park of 

L´ Albufera. The results revealed the presence of some pesticides at several concentration levels both in the soil and 

water samples. Nevertheless, they exist in a range of residual levels. 

Acknowledgements: 

This work has been supported by the Spanish Ministry of Science and 

Innovation through the project Consolider-Ingenio 2010 CSD2009-00065, 

as well as by this Ministry and the European Regional Development 

Funds (ERDF) (project GCL2007-66687-C02 01/BOS). 

References: 

[1] V. Andreu, Y. Picó, TrAC Trends Anal. Chem., 23 (2004), 772-789. 

[2] C. Blasco, Y. Picó, TrAC Trends Anal. Chem., 28 (2009), 745-757. 

624 



P20-009 DETERMINATION OF TRICLOSAN AND METHYL TRICLOSAN IN ENVIRONMENTAL SOLID SAMPLES BY MATRIX SOLID- 

PHASE DISPERSION AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

Sánchez-Brunete C., Miguel E., Albero B., Tadeo J.L. 


Corresponding author e-mail: tadeo@inia.es 

Triclosan (TCS) is an antimicrobial agent used in household and personal care products as well as in textile and 

plastic industries. TCS is transported to wastewater treatment plants (WWTPs) and may reach the terrestrial 

environment when TCS-containing sewage sludge is spread onto suitable agricultural land where it provides benefits 

as a fertilizer and soil conditioner but also could be a source of contamination for land-dwelling organisms. Methyl 

triclosan (MTCS) is a transformation product of TCS that is formed by biological methylation during the sewage 

sludge treatment process. Due to the lipophilic nature of both compounds, they tend to remain retained in sludge and 

the application of treated sewage sludge (biosolids) to agricultural soils could be a cause of concern. In this study, 

a method for the determination of triclosan (TCS) and methyl triclosan (MTCS) in soil and sewage sludge samples 

from municipal WWTPs was developed based on the extraction by MSPD. Soil (2.0 g dry weight) or lyophilized sludge 

(1.0 g dry weight) was placed in a glass mortar and mixed with C18 (2 g) and anhydrous sodium sulfate (1g). For the 

recovery studies, samples were previously spiked with a mixture of TCS and MTCS. Then, the mixture was gently 

blended with a glass pestle with circular motion to yield a homogeneous material. A 20 ml glass column with a Teflon 

frit at the end was filled with 1 g of Florisil. The blended sample was then transferred to the column and acetonitrile 

was the selected elution solvent. Afterwards, the extracts were concentrated with a gentle stream of air at ambient 

temperature in a fume hood to 1 ml. After extraction, the analytes were derivatized with N-(tert-butyldimethylsilyl)-Nmethyl-trifluoroacetamide 

(MTBSTFA) for their determination by isotope dilution gas chromatography with electron 

impact mass spectrometric detection in the selected ion monitoring mode, using 13C12 labeled compounds as 

internal standards. Recoveries of MTCS and TCS from laboratory spiked sludge samples were in the range from 95.7 

to 101.0 % and 97.4 to 101.3 % dry weight, respectively. In the case of soil samples, the recoveries of MTCS and TCS 

ranged from 98.4 to 101.0 % and 98.7 to 99.0 % dry weight, respectively. The limits of detection varied from 0.10 to 

0.12 ng/g for sewage sludge samples and from 0.05 to 0.08 ng/g for soil samples. The validated method was used to 

assess the levels of TCS and MTCS in sewage sludge collected from 19 WWTPs located in Madrid (Spain) and in soil 

samples collected from agricultural fields in the same area. Both compounds were detected in all the sludge samples 

at concentrations ranging from 54 to 2987 ng/g dry weight for TCS and from 4 to 311 ng/g dry weight for MTCS. In 

the case of soil samples, the levels encountered were much lower, 0.8 to 4.7 ng/g dry weight for TCS and 0.3 to 3.8 

ng/g dry weight for MTCS. 


625


P20-010 DISPERSIVE LIQUID-LIQUID MICROEXTRACTION COMBINED WITH NON AQUEOUS CAPILLARY ELECTROPHORESIS FOR 

THE DETERMINATION OF FLUOROQUINOLONE ANTIBIOTICS IN WATERS 

Herrera-Herrera A.V., Hernández-Borges J., Borges-Miquel T.M., Rodríguez-Delgado M.A. 



Pharmaceutical residues are among the new emerging pollutants to be monitored in different environmental 

compartments, including water or soil. The determination of quinolone residues in environmental waters, especially 

fluoroquinolones (FQs), is of great importance because of their widespread use worldwide as well as their broad 

activity spectrum and good oral intake. The literature dealing with the analysis of FQs in environmental waters is still 

low but enough to demonstrate the need of the development of suitable analytical methodologies able to determine 

these compounds in such samples at very low levels. In this sense, FQs analysis is frequently developed by HPLC, 

but during the last years capillary electrophoresis has appeared as an alternative separation technique for their 

determination. 

Dispersive liquid-liquid microextraction (DLLME) is a very simple and quick extraction procedure [1], introduced 

in 2006 by Rezaee et al. [2]. It is based on the rapid introduction of a suitable combination of an extraction and a 

disperser solvent into an aqueous sample forming a cloudy solution that is later centrifuged. Extraction equilibrium is 

quickly achieved due to the high surface contact between the sample and the extraction solvent droplets. 

In this work, dispersive liquid-liquid microextraction (DLLME) was combined for the first time with non-aqueous 

capillary electrophoresis with UV detection for the selective determination of eight fluoroquinolone antibiotics 

(lomefloxacin, levofloxacin, marbofloxacin, ciprofloxacin, sarafloxacin, enrofloxacin, danofloxacin and difloxacin) 

and pipemidic acid as internal standard (IS), in mineral and run-off waters. Field-enhanced sample injection was 

carried out in order to improve the sensitivity. The BGE that provided complete separation of the eight analytes and 

the IS, selected taking into account its possible compatibility with MS detection, was composed of acetic acid and 

ammonium acetate in a mixture methanol:ACN. Optimum DLLME conditions were achieved by means of experimental 

design methodology. Calibration curves of the whole method were obtained with correlation coefficients (R) higher 

than 0.994 in all cases. An accuracy and precision study was carried out at different levels of concentration, finding 

that there were no significant differences (Student’s t test) between real and spiked concentrations. 

References: 

[1] Herrera-Herrera, A. V., Asensio-Ramos, M., Hernández-Borges, J., Rodríguez-Delgado, M. A., Trends Anal. Chem. 

2010, in press, 10.1016/j.trac.2010.03.016 [2] Rezaee, M., Assadi, Y., Milani Hosseini, M. R., Aghaee, E., Ahmadi, F., 

Berijani, S., J. Chromatogr. A 2006, 1116, 1-9. 

626 



P20-011 IONIC LIQUID-DISPERSIVE LIQUID-LIQUID MICROEXTRACTION WITH HPLC-FD FOR THE DETERMINATION OF A GROUP OF 

PESTICIDES AND METABOLITES IN SOILS 

Asensio-Ramos M., Hernández-Borges J., Herrera-Herrera A.V., Rodríguez-Delgado M.A. 



Dispersive liquid-liquid microextraction (DLLME) was introduced by Rezaee et al. [1] in 2006 for the preconcentration 

of organic analytes in aqueous matrices. The technique is based on a ternary component solvent system, in which 

a mixture of an extraction and a disperser solvent are introduced together into an aqueous sample to form a cloudy 

solution in which small droplets are formed. Equilibrium is quickly achieved due to the huge surface area between 

the extraction solvent and the sample. After centrifugation, the extraction solvent is collected and injected into 

the chromatographic system. In this sense, room temperature ionic liquids (ILs) have been employed as extraction 

solvents in DLLME, but the number of works published is still very low and the majority of them deal with aqueous 

samples. 

In this work, an IL-DLLME procedure was applied for the extraction of a group of pesticides (carbendazim/ 

benomyl, thiabendazole, fuberidazole, carbaryl and triazophos) and some of their key metabolites in soils 

(2-aminobenzimidazole, metabolite of carbendazim and 1-naphthol, metabolite of carbaryl) from aqueous soil 

extracts. Determination of these pesticides was carried out in four soils with different physicochemical properties 

(forestal, ornamental, garden and volcanic-sandy soils) using high performance liquid chromatography (HPLC) with 

fluorescence detection (FD). The IL used was 1-hexyl-3-methylimidazolium hexafluorophosphate ([HMIm][PF6]). 

Factors affecting the DLLME procedure (sample pH, amount of ionic liquid, volume of disperser solvent and sodium 

chloride percentage) were optimized by means of an experimental design. Calibration of the whole method was 

carried out for every type of soil and accuracy and precision study was developed at two levels of concentration, 

finding that no significant differences were found between real and spiked concentrations (Student’s t test). LODs 

achieved were in the low ng/g range. 

Bibliography [1] Rezaee, M., Assadi, Y., Milani Hosseini, M. R., Aghace, E., Ahmadi, F., Berijani, S. J. Chromatogr. A 

(2006), 1116, 1-9. 


627


P20-012 EVALUATION OF A MODIFIED QUECHERS METHOD FOR THE EXTRACTION OF PESTICIDES FROM SOILS 

Asensio-Ramos M., Hernández-Borges J., Ravelo-Pérez L.M., González-Curbelo M.A., Rodríguez-Delgado M.A. 



The QuEChERS method was first introduced in 2003 by Anastassiades and coworkers for the extraction of 

pesticides from fruits and vegetables [1]. In an attempt to extend the applicability of this sample pretreatment 

procedure, some modifications have been introduced for the analysis of other matrices, or even for the extraction 

of other analytes. In the present work, a modified version of the QuEChERS method has been developed for the 

determination of a group of ten organophosphorus pesticides (i.e. ethoprofos, dimethoate, diazinon, malaoxon, 

chlorpyrifos-methyl, fenitrothion, malathion, chlorpyrifos, fenamiphos and phosmet) and one thiadiazine pesticide 

(buprofezin) in three different types of soils with diverse physicochemical properties (forestal, ornamental and 

agricultural) using gas chromatography with nitrogen-phosphorus detection [2]. Pesticides were first extracted 

from soils by means of a partitioning step using acetonitrile, MgSO4, NaCl and a mixture of citrates and later, 

the extract was cleaned up using PSA-dispersive SPE and MgSO4. The method was validated through linearity, 

recovery, precision and accuracy studies at different levels of concentration and a matrix-matched calibration 

was also carried out for the three soils owing to the existence of an evident and strong matrix effect. Acceptable 

recoveries, between 45 and 95%, were obtained for all pesticides and soils, except for malathion and malaoxon 

in forestal and ornamental soils, from which they could not be quantitatively extracted. Limits of detection of the 

whole method ranged between 0.48 and 7.78 ng/g, similar or even lower than LODs of other methodologies that 

appear in the bibliography. Finally, the method was applied to monitor the concentration of chlorpyrifos in a treated 

soil for cultivation of potatoes for a period of time of three months, clearly finding an accumulation of the pesticide 

over time. The presence of the pesticide was confirmed by GC-MS. Bibliography [1] Anastassiades, M., Lehotay, 

S.J., Štajnbaher, D.; Schenck, F.J. J. AOAC Int. (2003), 86, 412-431. [2] Asensio-Ramos, M., Hernández-Borges, J., 

Ravelo-Pérez, L.M., Rodríguez-Delgado, M.A., Anal. Bioanal. Chem. (2009), 396, 2307-2319. 

628 



P20-013 INVESTIGATION OF ORGANOPHOSPHATE ESTERS IN FRESH AND SEA WATER, BRINE AND SALT SAMPLES BY GC-TOF MS 

Nácher-Mestre J., Serrano R, Portolés T., Hernández F. 


Corresponding author e-mail: felix.hernandez@qfa.uji.es 

An advanced analytical methodology based on gas chromatography coupled to a time-of-flight mass spectrometry 

(GC-TOF MS) has been developed for screening of organophosphate esters (OPEs) in different water, brine and salt 

samples. The presence of OPEs in different environmental compartments is of rising concern, due to the relatively 

high levels reported in the aquatic environment (waters and sediments), air, or sewage treatment plants. They are 

widespread found in the environment since they have been used in different applications such as plastizers, flame 

retardants, antifouling, etc. These compounds can cause adverse effects on animal life, as they are neurotoxics and 

carcinogens, and tend to be present at high concentrations. For these reasons they are considered as “re-emerging 

contaminants”, and are subject of concern at present. In a first step, we performed a non-target screening by GC- 

TOF MS in sea water and salts from saltworks. As a result, we detected several non-targeted compounds (among 

them some OPEs), that were not included in the list of analytes in our previous target screenings. After detecting 

in several samples three of the most commonly reported OPEs, we decided to continue with the study of these 

compounds, including up to 12 selected compounds for further research in different type of samples. The analytical 

procedure applied was based on the use of SPE, using C18 cartridges, followed by GC-TOF MS analysis. In the case 

of salt samples, approximately 62.5g of salt were dissolved in 250 mL of HPLC-grade water and filtered, followed by 

SPE. A qualitative validation of the screening method developed for OPEs was performed in order to establish the 

lowest concentration for which the detection and identification of a given compound could be carried out according 

to a strict identification criterion. For this purpose, five fortified salt samples were spiked at three levels (1.6, 8.0 

and 16 ng/Kg) with 12 selected OPEs (including alkyl and chlorinated organophosphates). Additionally, two blank 

samples used in the study and two method blanks were also analyzed along the validation step. The presence of 

up to four ions measured at their accurate mass at the expected retention time was evaluated in every fortified 

sample at all levels tested. Additionally, their ion intensity ratios were compared to those from reference standards. 

The identification criterion required the presence of at least two accurate mass m/z ions with reasonable mass 

errors (typically below 2 mDa), an appropriate match in retention time, and the attainment of their Q/qi intensity 

ratio within specified tolerances. Most of investigated OPEs could be correctly identified in the validation process 

at the concentration level of 1.6 ng/Kg. The developed procedure was applied to the screening of these OPEs in 

different samples (ground water, surface water, commercial salt and brine) and allowed the detection and reliable 

identification of tris(1-chloro-2-propyl) phosphate in all samples investigated, as well as triethyl phosphate, tripropyl 

phosphate, tributyl phosphate, tris-(2-chloroethyl) phosphate, and 2-ethylhexyl diphenyl phosphate in some of the 

samples. 


629


P20-014 BEHAVIOUR OF PHARMACEUTICALS AND DRUGS OF ABUSE IN A DRINKING WATER TREATMENT PLANT USING 

CONVENTIONAL AND UF/RO TREATMENTS. 

Boleda MªR.¹, Galceran MªT.², Ventura F.¹ 

¹ AGBAR. Aigües de Barcelona, Àrea Química Orgànica 

² University of Barcelona, Departament química analítica 

Corresponding autor e-mail: mboledav@agbar.es. Telf. 933422 635 Fax. 933 422 666 

Pharmaceuticals and illicit drugs are emerging contaminants identified in the aquatic environment. The presence of 

these compounds either unaltered or as their main human metabolites in wastewater has been reported. In addition, 

it has been proved that they are often partially removed by wastewater treatment plants (WWTPs) using conventional 

treatments. This incomplete elimination leads to their release in surface receiving waters. Since these waters can 

be used for drinking water production it is important that these compounds are eliminated through drinking water 

treatment processes. 

In this context, the use of ultrafiltration (UF) and reverse osmosis (RO) in potabilization process have been gaining 

attention due to its ability to effectively remove most organic and inorganic compounds and microorganisms 

from raw water. However, most of the published studies deal with investigations made on a laboratory scale or 

demineralised water. 

This paper presents a comparison of the performances of conventional and advanced drinking water treatment 

processes to remove 83 selected pharmaceuticals (analgesics, antibiotics, lipid regulators, gastric, X-ray, 

barbiturates); illicit drugs (amphetamines, opiates, cocainics) and miscellaneous compounds (caffeine, nicotine and 

cotinine). 

The developed analytical methodology is based on preconcentration by solid-phase extraction (SPE) and 

identification by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). 

The study was carried out in a drinking water treatment plant (DWTP) that supplies a population above one 

million people. The raw water quality is affected by salt mine exploitations located in the upper course of the river 

The potabilization process consists on dioxichlorination, coagulation, flocculation, settling, sand filtration and 

groundwater dilution to improve raw water quality. Then, water is splitted in two lines following: 1) Conventional 

treatment (~70%): ozonation and GAC filtration and 2) Advanced treatment (~30%): ultrafiltration, reverse osmosis 

and remineralization. The GAC filtered water and the permeated are then blended and subjected to a final 

postchlorination. 

The behavior along the potabilization process of the 29 pharmaceuticals and 12 drugs of abuse which were identified 

at the intake of the DWTP has been studied. Removals reached levels >99% for the majority of compounds. 

Only few compounds, iopromide (up to 17.2 ng/L) and nicotine (13.65 ng/L), with removals of 98% and 94% or 

benzoylecgonine (1.90 ng/L), cotinine (3.62 ng/L), acetaminophen (15.6 ng/L) and erythromycin (1.96 ng/L) with 

elimination efficiencies of 99% were found in the final treated water. Both conventional and advanced treatment 

processes presented similar high efficiencies to eliminate both pharmaceuticals and drugs of abuse. 

630 



P20-015 OCCURRENCE OF POLYBROMINATED/CHLORINATED COPLANAR BIPHENYLS (CO-PXBS) IN HUMAN BREAST MILK FROM SPAIN 

Gomara B. 1 , Herrero L. 1 , Pacepavicius G. 2 , Alaee M. 2 , Gonzalez M.J. 1 

1 

IQOG (CSIC), Instrumental Analysis and Environmental Chemistry-Spain 

2 

Environment Canada, Water Science and Technology Directorate-Canada 

Corresponding author e-mail: mariche@iqog.csic.es 

Polybrominated/chlorinated biphenyls (PXBs) are a new and emerging persistent organic pollutant family 

with chlorine and bromine substituted in the biphenyl. The introduction of a second type of halogen into the 

polychlorinated moiety increases the number of possible congeners from 209 (in the case of polychlorinated 

biphenyls, PCBs) to 9180 (in the case of PXBs). Little is known about the origin of the presence of these compounds 

in the environment and knowledge about their behaviour is scarce. To date, only the formation of co-planar PXBs 

(Co-PXBs) during the manufacturing process of Fe3Cl has been reported [1]. The few studies involving these 

compounds suggest that their presence in the environment is probably due to incineration processes in the presence 

of brominated and chlorinated precursors such as brominated flame retardants (BFRs) and PCBs. The presence 

of five Co-PXB congeners in human samples, in some commercial food samples in Japan [2] and in fish from the 

Great Lakes in Canada [3] has captured the attention of the scientific community. The levels observed of these 

five Co-PXB congeners found in the Great Lakes were generally lower than those found in Japanese market fish 

(wild and cultured fish). These five Co-PXB congeners were also detected in human milk samples from Japan. In 

all cases, the concentration levels of total Co-PXBs (considering five congeners) were similar to that of total Co- 

PCBs (twelve congeners). These results indicate that these contaminants are bioaccumulative and persistent and 

that they have the potential of reaching high positions in the trophic food chain. As a result, information about their 

levels and behaviour in the environment are required in order to carry out a reliable risk assessment. Presently, the 

determination of PXB congeners is limited by the number of PXBs standards commercially available. We present 

here for the first time, preliminary results of the levels and accumulation profile of eight currently available congeners 

(PXB 77, 105, 118, 126A, 126B, 126C, 156 and 169, named according to IUPAC PCB nomenclature) in human breast 

milk from Spain using GC-HRMS. Detectable levels of all congeners investigated, except PXB 169, were found. The 

levels were lower than those found in human breast milk from Japan. Acknowledgements This study was supported 

by AGL2009-09733 National Project and P2009/AGR-1454 Regional Programme. References [1]. Nakano et al., 

Organohalogen Compounds 69: 2777 (2007) [2]. Otha et al., Organohalogen Compounds 69 : 2018 (2007) [3]. Alaee et 

al., Organohalogen Compounds 70: 768 (2008) 


631


P20-016 CHARACTERIZATION OF ALKYLPOLYPHOSPHONATES BY HPLC USING A POROUS GRAPHITIC CARBON STATIONARY PHASE 

Carrasco-Correa E.J., Herrero-Martínez J.M., Ramis-Ramos G. 



Alkylpolyphosphonates (APPs) are chelating agents that contain phosphonate or methylene-phosphonate groups 

attached to a short hydrocarbon skeleton which may also include amino nitrogen at the branching points. Owing 

to their excellent performance as chelating agents for calcium and heavy metals, APPs have found a wide range 

of applications including water treatment, scale and corrosion inhibition, mineral oil drilling, paper and textile 

production, agriculture, additives of cleaning products, and pharmaceuticals for bone and calcium metabolism 

diseases. The determination of APPs is necessary in the quality control of industrial products, as well as in the 

evaluation of their environmental impact. Owing to poor retention on most HPLC columns, covalent bonding 

of phosphonate groups to silica surfaces, and lack of a chromophore, analytical methods for APPs are scarce. 

Thus, APPs can be determined by ion chromatography on a cationic polymeric column using a diluted nitric acid 

solution as mobile phase. Detection is implemented by post-column addition of Fe(III) followed by UV detection of 

the phosphonate complexes; however, this separation shows a low efficiency, the Fe(III) solutions are unclean and 

prone to precipitation, and the detection limits are large. Another possibility is post-column oxidation to phosphate 

with subsequent application of the molybdenum blue method. In this work, APPs are determined by retention on a 

porous graphitic carbon (PGC) column (Hypercarb, Thermo Scientific) using isocratic elution with water/acetonitrile. 

PGC stationary phases are far more hydrophobic and stable at low and high pHs than silica-C18, showing a rather 

strong reversed-phase behaviour. In addition, hydrophilic solutes are retained on PGC due to dipole induction on 

the extense planar surfaces with large delocalized electron density. Using UV detection of the phosphonate groups 

at 210 nm, APPs showed adequate retention on PGC. The experimental conditions (mobile phase composition and 

pH, and column temperature) were optimized. Sensitivity of UV detection was enhanced by on-line post-column 

reaction of the APPs with a metallic ion. Studies to select the best reagent and working conditions for sensitivity 

enhancement, and to apply the procedure to industrial samples, are in progress. 

Acknowledgements: Project CTQ2007-61445 (MEC and FEDER funds). E. J. Carrasco-Correa thanks the University of 

Valencia for a grant. 

632 



P20-017 PARTITIONING OF PERFLUORINATED COMPOUNDS IN SEAWATER, SEDIMENT AND MUSSELS FROM THE CANTABRIC SEA 

(NORTH SPAIN) 

Gómez C., Vicente J., Porte C. Lacorte S. 



Perfluorinated compounds (PFCs) have emerged as significant global environmental pollutants with persistent, 

bioaccumulative and toxic properties. The most important characteristics of these compounds are that they are 

hydrophobic (high Kow) and very soluble in water, thus they can partition among different environmental matrices. 

PFCs are used mainly in Polytetrafluoroethylene (Teflon) production although they are widely applied in many 

industrial products such as shampoos, carpets, stain repellents, pesticides, etc (1). After use, PFCs are released 

to the marine environment from wastewater treatment plant effluents, direct discharge of waste waters and rivers 

(2), thus coastal waters become a vulnerable microecosystem with eventually high loads of contaminants. Once 

in the marine environment, PFCs can be adsorbed to sediment and accumulated in organisms. The effects PFCs 

may cause to aquatic organisms are not fully understood but in rats PFCs can produce weight loss accompanied 

by hepatoxicity, reductions of serum cholesterol and thyroid hormones (3). Moreover, PFOA can produce anorexia, 

alteration of fatty acid metabolism, bradycardia and hypothermia (4). The aim of this study was to determine the 

presence and partitioning of PFCs in various environmental matrices (port waters, seawaters from emissaries, 

sediments and transplanted mussels) in areas of high industrial impact from North of Spain. In addition, the source 

of PFCs to coastal waters was evaluated by determining PFCs in wastewaters discharging to the sea. We studied 

five PFCs of environmental importance: perfluorooctanesulfonate (PFOS), perfluorohexanesulfonate (PFHxS), 

perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorobutanesulfonate (PFBuS). Water samples 

were solid phase extracted (SPE) and sediments and mussels were ultrasonic extracted with methanol. Samples 

were analyzed by Ultra Performance Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MS/ 

MS) using an Acquity UPLC system (Waters, USA). All wastewater contained PFCs with a total concentration of 

1.10 to 10.9 ng/L, which accounted for an important source of PFCs to coastal waters. In seawater samples, total 

PFCs concentrations ranged from 0.06 to 8 ng/L and all PFCs were identified, with higher levels in port seawater. 

Adsorption to sediment was low and PFOA followed by PFOS were the major compounds present in 7 out of 

10 samples at levels of 0.06 to 0.44 ng/g dw. Accumulation in mussels was also low, and only PFOS and PFOA 

were detected at 0.007 to 0.12 ng/g ww in samples transplanted in ports and close to emissaries. PFCs were not 

accumulated in open sea mussels, and this can be attributed to the low concentration of PFCs in water and to the 

short exposure time of mussels (21 d). (1) Guo, R., Zhou, Q., Cai, Y., Jiang, G. Talanta 75 (2008), 1394-1399. (2) 

Sánchez, J., Meyer, J., Lacorte, S. Environmental Pollution, accepted. (3) Bao, J., Jin, Y., Liu, W., Ran, R., Zhang, Z. 

Chemosphere 77 (2009), 652-657. (4) Yu, J., Hu, J., Tanaka, S., Fujii, S. Water Research 43 (2009), 2399-2408. 


633


P20-018 DETERMINATION OF ANTIOXIDANTS IN NEW AND USED LUBRICANT OILS BY HEADSPACE-PROGRAMMED 

TEMPERATURE VAPORIZATION-GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

García Pinto C. 1 , del Nogal Sánchez M. 1 , Glanzer P. 2 , Pérez Pavón J.L. 1 , Moreno Cordero B. 1 

1 


2 


Corresponding author e-mail: cgp@usal.es 

Motor oils are heavy petroleum products commonly used to reduce the friction between surfaces, to prevent the 

corrosion, to remove heat and contaminants and to clean the motor. Base oil requires additives (dispersants, 

detergents, oxidation inhibitors and anti-wear agents) to satisfy the lubricating needs of an engine and to increase 

the useful lifetime of the oil. One of the most important aspects of lubricating oils is that the oxidation stability be 

maximized. Generally, engine oil compounds have a relatively high thermal/ oxidative stability in order to avoid the 

degradation of hydrocarbons which are exposed to oxygen and heat. Antioxidants [1] are the key additives that 

protect the lubricant from this degradation. In this work, a sensitive method is presented to determine antioxidants 

(2-, 3- and 4- tert-butylphenol, 2,6-di-tert-butylphenol, 3-tert-butyl-4-hydroxyanisol, 2,6-di-tert-butyl-4-methylphenol, 

1-naphthol and di-phenylamine) in new and used lubricant oil samples. Research was carried out on a GC 

device equipped with a headspace-sampler (HS), a programmed temperature vaporizer (PTV) and a MS detector 

unit. The proposed method does not require sample treatment prior to analyses, hence eliminating possible 

errors occurring in this step. Sample preparation is reduced to placing the oil sample (2.0 g) in the vial and adding 

propylacetate (20 µL) as solvent. Solvent vent injection mode permits a pre-concentration of the compounds of 

interest in the liner filled with Tenax-TA®, while venting other species present in the headspace. Thereby both the 

life of the liner and the capillary column is prolonged and unnecessary contamination of the detector unit is avoided. 

Calibration was performed by adding different concentrations of analytes to a new oil which did not contain any of 

the studied compounds. Prediction of the antioxidants in new oil samples of different viscosities (5W40, 10W40, 

15W40) was accomplished with the previous calibration and the results were highly satisfactory. To determine 

antioxidants in used engine oils standard addition method was used due to the matrix effect. The HS-PTV-fastGC- 

MS method revealed good precision and accuracy with detection limits for most of the compounds at µg/L level. It 

should be stressed that all the studied antioxidants have high boiling points which means that headspace analysis 

could not be, a priori, a suitable sampling technique for the analysis. However, the use of solvent vent (Tenax-TA® 

liner) in the PTV and SIM detection mode in the MS provided satisfactory results beside the benefits of using HS. 

634 



P20-019 HPLC-DAD-MS-MS MONITORING OF VETERINARY PHARMACEUTICALS IN WATER AFTER DEGRADATION BY FENTON 

PROCESS AND TOXICOLOGICAL EVALUATION 

Ašperger D., Djuric I., Vujevic D., Pelko S., Periša M., Babic S., Horvat A.J.M., Koprivanac N., Kaštelan-Macan M. 

Faculty of Chemical Engineering and Technology-Croatia 

Corresponding author e-mail: diva@fkit.hr 

Pharmaceuticals, both in their parent form and as metabolites, are commonly released into the environment at trace 

levels through conventional sewage treatment plants. Another source of entry of pharmaceuticals in the aquatic 

environment is a result of its veterinary use, mainly in aquaculture practices. Pharmaceuticals have been detected 

in ground, surface, drinking, tap and ocean water. Pharmaceuticals released in the environment may impose toxicity 

on any level of the biological hierarchy. In addition to toxic effects, certain classes of pharmaceuticals may cause 

long-term and irreversible change to the micro-organisms genome, making them resistant in their presence, even at 

low concentrations. It is therefore important to develop efficient treatment methodologies for limiting the presence 

of pharmaceuticals contaminants in aquatic environments. Presence of residual pharmaceuticals in the environment 

and especially in aquatic systems in particular represents a serious environmental problem as these compounds 

could be resistant to biological degradation processes and usually escape intact from conventional treatment plants, 

may impose serious toxic and other effects to humans and other living organisms. Since such compounds have been 

found to be present at minute concentrations, more sophisticated and laborious analytical tools for their accurate 

determination are required. Therefore, it is not surprising that research has recently been directed towards the 

application of non-biological processes for the destruction of pharmaceuticals in water systems with the emphasis 

on advanced oxidation processes (AOPs). [1, 2] This work has tackled degradation of fluoroquinolone antibiotic 

ciprofloxacin, glucocorticosteroid dexamethasone, anthelmintic febantel, sulfonamide antibiotic sulfamethoxazole 

and their synergist trimethoprim in water by Fenton process at lab scale. Aqueous solutions of pharmaceuticals were 

prepared by dissolving of appropriate amount of investigated compounds in double deionised water at an initial 

concentration of 1 and 10 mg/L. After 30 minutes of Fenton process samples of reaction mixture were subjected to 

TOC and HPLC-DAD analysis in order to determine mineralization i.e. removal extent of particular pharmaceutical. 

Furthermore, identification of the intermediate products generated during the process were performed using the 

liquid chromatography coupled to mass spectrometry (HPLC-ESI(+)-MS-MS). Chromatographic conditions include 

gradient separation with 0.01% formic acid in water and 0.01% formic acid in acetonitrile at 30 oC on Synergi 4 µ 

Fusion-RP 80 Å column (150x2.0 mm for HPLC-MS-MS and 150x4.6 mm for HPLC-DAD). The evaluation of toxicity by 

measuring the bioluminescence inhibition of Vibrio fischeri bioassays during the Fenton process was also performed. 

Acknowledgement This work has been supported by the Unity Through Knowledge Fund (UKF), which was 

established by the Croatian Ministry of Science, Education and Sports trough the World Bank Loan No. 7320-HR: 

Reduction of environmental risks posed by pharmaceuticals and their degradation products in process wastewaters, 

through RO/NF membrane treatment (REPHAD) and Croatian Ministry of Science, Education and Sports Projects: 

125-1253008-1350 and 125-2120898-3148. References: [1] M. Klavarioti, D. Mantzavinos, D. Kassinos, Environment 

International 35 (2009) 402-217 [2] A.G. Trovo, R.F.P. Nogueira, A. Agüera, A.R. Fernandez-Alba, C. Sirtori, S. Malato, 

Water Research 43 (2009) 3922-3931 


635


P20-020 MEASUREMENT OF VOLATILE ORGANIC COMPOUNDS IN ALGIERS URBAN AREAS USING STEREOSELECTIVE GAS 


Ladji R. 1 , Yassaa N. 2 

1 

Centre for Scientific Research and Technology in Physico-chemical analysis, Enviromental Chemistry-ALGERIA 

2 

University of Sciences and Technology Houari Boumediene-Algeria 

Corresponding author e-mail: rladji@hotmail.com 

Measurements of volatile organic compounds (VOC) including benzene, toluene, ethylbenzene, ortho-, meta- and 

para- xylene (BTEX) compounds were conducted in two urban areas located in Algiers city. Air samples were 

collected through two beds carbograph cartridges and analysis were performed by TD-GC-MSD measurement 

system. The system consists of a thermal desorber connected to a gas chromatograph equipped with a Mass 

Selective Detector. An enantiomerically selective column based on beta-cyclodextrin was employed in order 

to separate biogenic monoterpene enantiomers as well as anthropogenic BTEX isomers. The determination of 

several diagnostic ratios, e.g., benzene/toluene, ethylbenzene/(meta+para)-xylenes, ethylbenzene/meta-xylene 

and ethylbenzene/para-xylene has been possible using this chiral column and employed to study the emission 

sources and the atmospheric reactivity of VOC. Some conclusion can be drawn through the comparison of VOC 

mixing ratios observed in Algiers with other polluted cities: (i) a large variability in VOC mixing ratios has been 

observed; (ii) vehicular traffic emission is the principal source of VOC emission in Algiers and (iii) a need to perform 

more systematic measurements. Finally, the use of a chiral capillary column enabled a good separation not only of 

enantiomeric pairs of monoterpenes but also the stereoisomers of BTEX as well as meta-and para-xylenes in the 

atmospheric samples. 

636 



P20-021 DETERMINATION OF ABUSE DRUGS AND METABOLITES IN WATER SAMPLES BY IN-LINE SOLID PHASE EXTRACTION IN 

CAPILLARY ELECTROPHORESIS 

Botello I., Borrull F., Calull M., Aguilar C. 

University Rovira i Virgili, Tarragona 

Corresponding author e-mail: igor.botello@urv.cat 

Nowadays illicit drugs are extensively used. The source of contamination by residues of drugs of abuse in the 

environment is mainly from the consumers, because these drugs are excreted by the urine. The study of the levels of 

these drugs in water samples can provide information about the consumption of illicit drugs in different geographic 

areas. In the analysis of these illicit drugs in different matrices, capillary electrophoresis (CE) has been found to 

be a useful approach. The limited concentration sensitivity is one of the most important issues when capillary 

electrophoresis (CE) is applied to the analysis of environmental samples. To improve the concentration sensitivity 

of CE an interesting approach is by means of in-line solid phase extraction (SPE). In this work, in-line SPE as 

enrichment technique in CE was used for the determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpirrolidine 

(EDDP), cocaine (COC), codeine (COD) and 6-acetylmorphine (6AM) in water samples. In this communication we 

present a detailed study of the different parameters affecting in-line SPE, such as sample pH, volume of the elution 

plug and sample injection time. The SPE-CE extractor consisted on a short length of a capillary packed with Oasis ® 

HLB 60 μm near to the inlet within the separation capillary. The methodology developed resulted simple and efficient 

for the determination of the studied abuse drugs and metabolites in real water samples being a very effective 

approach to improve considerably the low sensitivity in CE. Validation for river water samples demonstrated good 

linearity, low detection limits as well as satisfactory precision in terms of repeatability and reproducibility. The in-line 

SPE-CE combination affords fully automated sample preparation in fewer sample handling steps and allows the 

whole eluate from the SPE device to be analyzed by CE. 

[1] F.J. Lara, A.M. García-Campaña, C. Neusüss, F. Alés-Barrero, J. Chromatogr. A 1216 (2009) 3372. [2] A. Macià, F. 

Borrull, M. Calull, F. Benavente, E. Hernández, V. Sanz-Nebot, J. Barbosa, C. Aguilar, J. Sep. Sci. 31 (2008) 872. [3] R. 

Ramautar, G.W. Somsen, G.J. Jong, Electrophoresis 31 (2010) 44. [4] C. Postigo, M.J. López, D. Barceló, Environment 

International 36 (2010) 74 


637


P20-022 ANALYSIS OF SHORT-CHAIN POLYCHLORINATED PARAFFINS IN BIOTA BY SELECTIVE PRESSURIZED LIQUID 

EXTRACTION AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

Olmos J.E., Santos F.J., Galceran M.T. 


Corresponding author e-mail: javier.santos@ub.edu 

Chlorinated paraffins (CPs) are commercial mixtures of polychlorinated n-alkanes (PCAs) with carbon chain lengths 

between C10 and C30 and a chlorine content from 30 to 70 % by weight. According to their carbon chain length, 

these mixtures are divided into short- (SCCPs, C10-13), medium- (MCCPs, C14-20) and long-chain chlorinated 

paraffins (LCCPs, C20-30). CPs are mainly used as additives in cutting fluids, lubricants and also in paints, plastics 

and sealants, due to their flame retardant properties.1 As a consequence of their widespread and unrestricted use, 

they are present in aquatic and terrestrial food webs. Among them, SCCPs are of particular interest because of 

their high persistence, toxicity and capability to bioaccumulate2 and the high amounts released in the environment. 

Consequently, SCCPs have been classified as possible carcinogenic to humans (Group 2B) by the International 

Agency for Research on Cancer.3 In addition, they have been identified as priority hazardous substances in the 

field of water policy (Directive 2000/60/EC). Therefore, environmental levels of PCAs should be monitored and 

reliable analytical methods and quantification procedures are required. Analysis of SCCPs is currently performed 

by gas chromatography with electron capture detector (GC-ECD) and coupled to low and high resolution mass 

spectrometry, operating in both electron ionization (EI) and negative ion chemical ionization (NICI) modes. Sample 

treatment in biological samples usually consists on an exhaustive extraction of the analytes using Soxhlet with non 

polar solvents. Alternative extraction techniques, such as pressurized liquid extraction (PLE) and matrix solidphase 

dispersion (MSPD), have also been used.4 Nevertheless, all these methods often require extensive clean-up 

procedures to remove matrix-interfering compounds which are usually time-consuming.5 

In the present work, a fast and simple selective pressurized liquid extraction (PLE) method in combination with gas 

chromatography-ion trap mass spectrometry for the analysis of SCCPs in biota samples has been developed. A onestep 

extraction and clean-up method was optimized in order to obtain extracts clean enough to be directly analyzed 

by GC and for reducing the analysis time and solvent consumption. To this end, different sorbents were evaluated 

as fat retainer and the optimal PLE conditions were established to obtain maximum sensitivity and selectivity on 

the analysis of SCCPs. The best results were achieved using silica modified with sulphuric acid (5%, w/w) inside the 

PLE extraction cell. In addition, to prevent interferences from other chlorinated compounds, such as polychlorinated 

biphenyls (PCBs), a fractionation with Florisil was performed. Recoveries higher than 90% were obtained for all the 

compounds. Quality parameters of the proposed method were established and it was used for the analysis of SCCPs 

in biota samples from the Mediterranean Sea. 

References 

1. C.A. de Wit, Chemosphere, 46 (2002) 583. 

2. WHO, Chlorinated Paraffins, Environmental Health Criteria 181, Geneva, Switzerland, 1996. 

3. IARC monograph 69, 1997. 

4. F.J. Santos, J. Parera and M.T. Galceran, Anal. Bioanal. Chem. 386 (2006) 837. 

5. S. Bayen, J.P. Obbard, G.O. Thomas, Environ. Intern. 32 (2006) 915. 

638 



P20-023 EVALUATING THE EFFICIENCY OF A NEW POLYMERIC IONIC LIQUID (POLY(VBHDIM-NTF2)) AS SORBENT COATING IN 

DIRECT IMMERSION MODE - SOLID-PHASE MICROEXTRACTION - GAS CHROMATOGRAPHY FOR THE DETERMINATION OF 

A GROUP OF ENDOCRINE DISRUPTING CHEMICALS 

López-Darias J. 1 , Pino V. 1 , Anderson J.L. 2 , Afonso A.M. 1 

1 


2 

The University of Toledo-USA 

Several environmental contaminants, termed as endocrine disrupting chemicals (EDCs), can interfere with the 

function of the endocrine system in living organisms. Chemical exposure to EDCs has been linked to neurological 

and reproductive effects on fish and wildlife, and may also affect human fertility. A high number of compounds are 

included in the increasing list of substances classified as EDCs, such as polycyclic aromatic hydrocarbons (PAHs), 

parabens, or alkylphenols (APs) such as nonylphenol (NP) and bisphenol A (BPA). Their determination in water 

matrices is therefore important, given the aforementioned health risks associated. Solid-phase microextraction 

(SPME) is a powerful solventless technique which integrates extraction, preconcentration, and sample introduction 

in one step. The largest disadvantage associated with SPME is arguably the limited number of stationary phases 

commercially available. The most common coating materials are polydimethylsyloxane (PDMS) and polyacrylate 

(PA), which are adequate for non-polar and polar analytes, respectively. There has been an increasing interest 

in developing new coating materials in SPME in order to achieve better sensitivity and selectivity. Ionic liquids 

(ILs) as well as their polymeric analogues (PILs) can be cited among the new materials candidate for coating 

materials in SPME. ILs are non-molecular solvents that have recently gained significant attention as a newer class 

of designer solvents. These ionic media result from the combination of organic cations and various anions, with 

the asymmetrically substituted nitrogen-containing cations being the most common in IL structures. ILs typically 

possess negligible vapor pressure, high thermal stability, and unique catalytic properties compared to conventional 

molecular solvents. The work evaluates the efficiency of a new coating material for SPME based on the PIL: 

poly(1-(4-vinylbenzyl)-3-hexadecylimidazolium bis(trifluoromethylsulfonyl)imide) (poly(VBHDIm-NTf2)), in comparison 

with the commercial coating PDMS (30 µm). It must be highlighted that this material has specifically been designed 

to possess high hydrophobic properties. This material has proven to be stable and quite reproducible. The 

performance of this PIL fiber has been applied in direct immersion mode SPME-GC-FID for the determination of a 

group of 6 APs (including NP and BPA), 6 PAHs and 2 parabens in different waters with increasing salty content. 


639


P20-024 APPLICATION OF PY-GC/MS TO STUDY CHANGES IN THE ORGANIC MATTER OF MACRO- AND MICROAGGREGATES OF A 

MEDITERRANEAN SOIL UPON HEATING 

Campo J. 1 , Cammeraat E. 2 , Andreu A. 1 , Rubio J.L. 1 

1 

Centro de Investigaciones sobre Desertificacion, Degradacion y conservacion de suelos 

2 

Universiteit van Amsterdam 

Corresponding author e-mail: julian.campo@uv.es 

The study of the changes in soil organic matter (SOM) composition has made great progress due to the use 

of techniques such as solid-state nuclear magnetic resonance (NMR), pyrolysis gas chromatography/mass 

spectrometry (Py-GC/MS), thermally assisted hydrolysis and methylation (THM), etc. Field and laboratory studies 

applying 13C and 15N NMR indicate that the thermal modifications of organic matter during charring include 

dehydration, aromatisation, loss of methoxylic and carboxylic functional groups, condensation of carbohydrates into 

furan-like structures and enrichment of heterocyclic aromatic N-compounds. In terms of spectroscopic properties, 

the effect of heating on SOM leads to the loss of the O-alkyl and di-O-alkyl structures that dominate wood and 

a large increase in aromatic C. The Py-GC/MS is an alternative approach that involves thermal degradation of 

organic molecules into small fragments that are analyzed by GC/MS, providing information related to their structure. 

According to some researchers, pyrolysis products released by unburned soils include a wide variety of molecules 

arising from carbohydrates, lignin, lipids and proteins. On the contrary, in soils affected by wildfires, most of the 

pyrolysis products present in unburned soils are absent and the dominance of charred ‘‘non-pyrolysable’’ refractory 

carbonaceous material is evident. The composition of a Mediterranean soil organic matter was studied in two 

different fractions (macro- and microaggregates) and in two environments (soil under canopy of Quercus coccifera 

and bare soil between plants). Samples were heated under laboratory conditions at different temperatures (220, 380 

and 500º C) to establish their effects on the SOM quality by comparison with unheated or control samples (25º C). The 

organic matter extracted with NaOH had a similar composition, as analysed by Py-GC/MS, in macro- and microaggregates 

of control samples, both under canopy and on bare soil. Obtained pyrolysates were characterized by 

the presence of polyphenol and other aromatic pyrolysis products (lipids and some polysaccharides derivatives, 

proteins and lignin). Some of these products, together with the presence of black carbon (BC) markers, confirmed 

the occurrence of past wildfires in the study zone. The composition of the organic matter, extracted from the soils 

heated at 220º C, was quite similar to that obtained from unheated soils. Changes in SOM characterization started 

at 380º C when the products derived from polysaccharides and lignin, and some coming from polyphenols, were not 

detected in the pyrolysates suggesting that labile organic matter was likely destroyed by the high temperatures. The 

lower proportion of pyrolysed material detected at 380 and 500º C could be partly due to selective enrichment of 

heat-resistant aromatic components. 

640 



P20-025 ANALYSIS OF SILOXANES IN WATER BY HEADSPACE-SOLID PHASE MICROEXTRACTION-GAS CHROMATOGRAPHY-MASS 

SPECTROMETRY 

Companioni E.Y., Santos F.J., Galceran M.T. 


ANALYSIS OF SILOXANES IN WATER BY HEADSPACE-SOLID PHASE MICROEXTRACTION-GAS 

CHROMATOGRAPHY-MASS SPECTROMETRY. E. Y. Companioni, F. J. Santos, M. T. Galceran. Department of 

Analytical Chemistry. University of Barcelona. Martí i Franquès 1 – 11, 08028-Barcelona, Spain. Volatile siloxanes 

constitute a new group of emerging environmental pollutants that have recently attracted a growing interest 

because some of them cause harmful effects in animals and are potentially carcinogenic compounds.1 Due to their 

widespread use in consumer products, such as cosmetics, body lotions, hair styling products, creams, deodorants, 

polished, and in other industrial applications, they have been detected in environment and biota samples, such 

water, sludge, soil, sediment, air and fish,2,3 and are bioaccumulated through the food chain. As a consequence, 

several European Environmental Agencies have launched monitoring programs to collect data about their occurrence 

and distribution in the environment. Due to their high volatility, siloxanes are analyzed by purge and trap and 

headspace techniques, although the methods reported in the literature are very scarce and are only applied to few 

cyclic siloxanes.4 Therefore, there is a great interest to dispose rapid, selective and sensitive analytical methods for the 

reliable determination of these compounds in environmental samples. In the present work, a headspace solid-phase 

microextraction method (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) has been 

developed for the determination of linear and cyclic siloxanes in water at low concentration levels (ng L-1). To this 

end, different fibres were evaluated and the divinylbenzene-polydimethylsiloxane (DVB-SPME) provided the best 

results. Parameters affecting the efficiency of the extraction and desorption of siloxanes were optimized. In addition, 

to diminish the instrumental and environmental contributions of siloxanes, a clean room with laminar flow for sample 

preparation and a free silicone Merlyn Microseal System for GC injection were used. The developed HS-SPME-GC-MS 

method provided good linearity (0.005-19.8 ng L-1 for linear and 15-213 ng L-1 for cyclic siloxanes), low limits of 

quantification (0.0053-0.1094 ng L-1 for linear and 7.2-50.2 ng L-1 for cyclic siloxanes) and an adequate precision 

(RSD%


P20-026 A SIMPLIFIED QUECHERS APPROACH FOR THE DETERMINATION OF THMS AND BTEX IN SOIL MATRICES BY FAST GAS 

CHROMATOGRAPHY WITH MASS SPECTROMETRY DETECTION 

Herrero Martín S., García Pinto C., Pérez Pavón J.L., Moreno Cordero B. 


Corresponding author e-mail: sara_hm@usal.es 

The analysis of volatile organic compounds (VOCs) from soil samples is a challenging task due to the diversity and 

complexity of the samples and to the low concentrations and high volatility of the compounds. The most critical 

step of the whole method is compound extraction. During the last years the new trend in analytical chemistry is 

the development of “environmentally friendly” sample pre-treatment techniques, which are solvent-free or solventminimised. 

Within these techniques the most widely employed for the analysis of VOCs in soils are the different 

modalities of headspace: static headspace (S-HS), purge-and-trap (P&T) and HS-solid-phase microextraction 

(HS-SPME). Despite the advantages of these methods, some of them (especially two-step procedures) employ 

extraction times of the order of 30 min. With a view to finding an alternative method able to provide satisfactory 

results without the need for complex and expensive instruments, and maintaining the requirements of minimum 

sample pre-treatment and low solvent consumption, our research group has explored the possibilities of the 

QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction procedure for the analysis of VOCs in 

soil samples. A method based on simplified QuEChERS extraction followed by large-volume injection-fast gas 

chromatography and mass spectrometry detection has been developed for the determination of THMs (chloroform, 

bromodichloromethane, dibromochloromethane and bromoform) and BTEX (benzene, toluene, ethylbenzene and 

xylenes) in soil samples. The simplified version of QuEChERS used meets the requirements of the “green chemistry” 

and provides reliable results with high sample throughput, low solvent consumption, little labour and the use of 

materials commonly employed in laboratories. The GC device used is equipped with a programmed temperature 

vaporizer (PTV), with a liner packed with Tenax-TA®. Using the solvent vent mode, this injector allows the injection of 

large volumes of sample, affording an improvement in the sensitivity of the method. The chromatographic conditions 

used here allowed the separation of the compounds in less than 5.50 min. Good linearity was obtained for all the 

target compounds, with highly satisfactory repeatability and reproducibility values. The limits of detection were in 

the 0.2 to 15 µg/kg range. The method was validated by the analysis of two certified reference materials (CRMs). 

642 



P20-027 ANALYSIS OF ANTICOAGULANT RODENTICIDES IN WATER, SOIL AND MICROTUS ARVALIS TISSUES BY LIQUID 


Nozal M.J., Bernal J.L., Toribio L., Hernandez A., Martin M.T. 



In this work, it is presented a rapid and selective analytical method to determine four rodenticides (chlorophacinone, 

bromadiolone, brodifacoum and difenacoum) in animal tissues, water and soil by HPLC with several detectors: 

fluorescence (FLD), diode array (DAD) and electrospray ionisation -mass spectrometry ESI-MS. Since the early 

1950s, anticoagulant rodenticides have been worldwide used for controlling commensal rodents. They are also used 

in field treatment against crop pests, as it was done in Castilla y León Region (Spain) during 2007 and 2008 when a 

very high density of Microtus arvalis impacted drastically on grass production. Field control was firstly carried out 

with chlorofacinone afterwards with bromadiolone, and nowadays it is starting a study for searching other possible 

rodenticides. The collateral effects associated to the use of those compounds on the environment, mainly on 

non-target fauna that can eat dead rodents, must be always taken into account. For this reason, it is required to 

develop analytical methodologies that allow the determination of rodenticides residues in the environment (water and 

soil) or in animals (muscle or viscera of rodents). The most used anticoagulant rodenticides are 4-hydroxycoumarins 

and indandiones. The different chemical structures present an interesting task for their high-performance liquid 

chromatography (HPLC) determination when it is used a fluorescence detector, as indandiones do not have 

fluorescence. For this reason, we have also employed other LC detectors (DAD and MS) as well FLD, to determine 

traces of the studied rodenticides in three matrices., Rodenticides in soil and animal tissues were extracted with 

different volumes of methanol, meanwhile the water samples were subjected to a solid -phase extraction procedure 

to extract the selected compounds. After the LC optimization study, it was selected a Gemini 5 µ C18 column and 

a mobile phase constituted by a mixture of ammonium formate 30mM in water and methanol (73:27, v/v).. High 

recoveries were obtained for all the spiked samples in a wide range of concentration (0.040-5 ppm) by using the 

proposed methodology. The LOD for the FLD and DAD were between 5 and 30 ppb and lower limits were obtained 

for the ESI-MS (between 0.2 and 4 pbb). Acknowledgements Authors thank ITACyL (Spain) for the financial support. 

Mª.T.M. would like to thank the Spanish Ministry of Ciencia e Innovación for her “Ramón y Cajal” contract. 


643


P20-028 NEW METHOD FOR PREPARATIVE LIQUID CHROMATOGRAPHY FRACTIONATION OF CRUDE OIL SAMPLES USING A 

FILTRATION SYSTEM BY GRADIENT OF SILICA GEL 

Vicente M.A. 1 , Vicente A.R. 1 , Camacho C.F.B. 2 , Tamanqueira J.B. 1 , Lange R. 1 , Sad C.M.S. 1 , Neto R.R. 1 , Castro E.V.R. 1 , Medeiros E.F. 1 , Dias J.C.M. 3 

1 

Universidade Federal do Espirito Santo 

2 

Fundação Gorceix , CENPES P&D de Produção-Brazil 

3 

Petrobras, CENPES-Brasil 

Corresponding author e-mail: maristelavicente@gmail.com 

Comprehensive analysis of crude oil is usually unattainable due to the large amount of compounds present on 

it. Rather, its common practice to rely on fractioning of classes of compounds based on chemical properties 

for simplifying analysis. Saturate, aromatic and polar components analysis of petroleum is commonly used in 

geochemistry and environmental forensics. Separation of these components into individual fractions is often 

required because compounds of interest, such as biomarkers are present in trace quantities compared to the bulk 

of the sample and must be enriched before they can be accurately analysed. Open column liquid chromatography 

methods are frequently used as a less costly alternative to HPLC methods. Traditionally, open column methods use 

adsorbents such as silica, alumina or their mixtures, as a solid phase. Silica is the most widely used adsorbent for 

the separation of petroleum into compound classes. Saturate, aromatic and polar fractions are collected by the 

successive elution with solvents of increasing polarity. Unfortunately, separation between saturated hydrocarbons 

and aromatic components using open column liquid chromatography is often not complete. Several of such methods 

have been published, however they usually require long time for sample preparation and do not produce enough 

sample for complementary methods studies. Herein, we propose a new preparative liquid chromatography method 

which is simple, fast and efficiently fractionate samples of oil through a vacuum microfiltration system using a 

gradient of silica gel and alumina developed to achieve compound class separation. It is based on compound class 

solubility in different solvents and their weak interaction with an adsorbent (silica-gel) with different granulometry. 

In order to evaluate this new method, we used crude oil samples (28.3 and 35.8 °API), derived from the southeastern 

Brazilian continental margin, with water limits of less than 1%. Fractions obtained from this process were subjected 

to high temperature gas chromatography, gas chromatography-mass spectrometry and gravimetric analysis for 

evaluating recovery and separation efficiency. Results showed a considerable fraction recovery with no crosscontamination 

(different classes of compounds on the same fraction). The procedure resulted in 48% saturate, 

23% aromatic and 7.5% polar fractions, with 0.0003 variance for the medium crude oil; and 54.9% saturate, 12.2% 

aromatic and 4.2 % polar fractions with 0.0001 variance for the light crude oil. An excellent reproducibility has been 

attained allowing oil sample fractionation in quantities adequate for further studies. 

644 



P20-029 DETERMINATION OF VOLATILE HALOGENATED ORGANIC COMPOUNDS AND CHLOROBENZENES USING MODIFIED 

QUECHERS EXTRACTION AND GC-µECD ANALYSIS 

Moreno Cordero B., Casas Ferreira A.M., Fernández Laespada M.E., Pérez Pavón J.L. 


Corresponding author e-mail: bmc@usal.es 

Currently, soils are subject to intensive contamination with chemical compounds. Soil is a complex matrix 

whose composition is highly heterogeneous. Following their arrival in the soil, contaminants enter into various 

physicochemical interactions with the mineral and organic components of the matrix. Volatile organohalogen 

compounds (VHOCs) and chlorobenzenes have received special attention as soil pollutants. They have mainly been 

used as solvents, cleaning and degreasing agents, blowing agents, polymerization modifiers, and heat-exchange 

fluids. These groups of chemicals have been included in the EPA Soil Screening Guidance to help standardize and 

accelerate the evaluation and cleanup of contaminated soils at sites on the National Priorities List (NPL) for future 

residential land use. A sensitive method based on a modified version of the Quick Easy Cheap Effective Rugged 

and Safe (QuEChERS) sample preparation method and gas chromatography with µ-ECD detection for the analysis 

of volatile halogenated hydrocarbons and chlorobenzene compounds in soils has been developed. A programmed 

temperature vaporizer (PTV) was used in the solvent-vent mode. Different extraction solvents were evaluated. Ethyl 

acetate was chosen, after comparing it with acetonitrile, owing to its better chromatographic behaviour. Clean soil 

extracts were obtained without a clean-up step, thereby reducing sample handling and the errors associated with 

this. Upon comparing the slopes obtained on preparing the calibrations in two different types of soil, the absence 

of a matrix effect was seen for most of the compounds, with slight differences in the case of carbon tetrachloride 

and 1,3-dichloropropylene, which ranged between 16.8 and 9.5 %, respectively. The method proposed here is highly 

sensitive, with detection limits ranging from 25 ng/Kg to 2.61 µg/Kg. The linearity, reproducibility and accuracy of the 

method were analyzed in certified reference materials and proved to be satisfactory. 


645


P20-030 COMPARATIVE STUDY OF THE ADSORPTION PERFORMANCE OF A MULTI-SORBENT BED (CARBOTRAP, CARBOPACK X, 

CARBOXEN 569) AND A RADIELLO DIFFUSIVE SAMPLER FOR THE ANALYSIS OF VOCS 

Gallego E. 1 , Roca F.X. 1 , Perales J.F. 1 , Guardino X. 2 , Rosell M.G. 2 

1 

LCMA-Technical University of of Catalonia 

2 


A comparison between two types of adsorbent tubes and methodologies, an active multi-sorbent bed (Carbotrap, 

Carbopack X, Carboxen 569) tube developed in our laboratory, and the Radiello diffusive sampler indicated for 

thermal desorption (filled with Carbograph 4), has been done to evaluate their usefulness in the analysis of VOCs in 

ambient air. Daily duplicate samples of multi-sorbent bed tubes were taken during a period of 14 days. On the other 

hand, during the same period of time, quadruplicate samples of Radiello tubes were taken in 3 days, 4 days, 7 days 

and 14 days samples. The sampling was done indoors during the months of February and March 2010 in Tarragona 

city. The analysis was performed by automatic thermal desorption (ATD) coupled with capillary gas chromatography 

(GC)/mass spectrometry detector (MSD). This methodology had been used in previous studies to identify and 

determine a wide range of VOCs that cause odour nuisance and affect outdoor air quality. Generally, only a few 

of the studied compounds do not show significant differences in the concentrations observed between the two 

different sampling methodologies, being higher the concentrations obtained with the Radiello samplers. Daily 

variability of VOCs concentrations was observed through the multi-sorbent bed samples; hence, as Radiello passive 

samplers represent the average concentration during a period of time, the daily variability may not be showed 

properly. On the other hand, the effect of sampling exposure time to Radiello samplers was evaluated. The results 

showed that, for several compounds, the sum of the amount of VOCs collected at two shorter sampling occasions 

was significantly different than the amount collected at a single longer sampling period (3 days + 4 days vs. 7 days, 

and 7 days + 7 days vs. 14 days). For short sampling periods, the total amount of VOCs in a tube exposed during a 

longer period (7 days) is generally significantly higher than the sum of the amount of VOCs collected in two shorter 

periods (3 days + 4 days). However, for longer sampling periods, generally, lower amounts of VOCs are significantly 

observed in the longer period samples (14 days vs. 7 + 7 days of exposure). 

646 



P20-031 EVALUATION OF WORK-RELATED VOLATILE ORGANIC COMPOUNDS (VOCS) EXPOSITION IN AUTOMOTIVE PAINTING 

CABINS INDUSTRIAL CLEANING 

Rosell M.G. 1 , Carrasco A. 2 , Gallego E. 3 

1 


2 

ACCIONA Facility Services, Departamento de Seguridad y Salud de las Personas 

3 

LCMA-Technical University of Catalonia 

Automotive painting cabins are cleaned with several solvents, being great part of them mixtures of volatile organic 

compounds (VOCs), where the three xylene isomers are the most important constituents. To evaluate the workrelated 

exposition of the cleaners that use these mixtures of solvents, xylenes have been evaluated in the working 

ambient air as well as its metabolite, o-m-p-methyl hipuric acid, has been evaluated in urine to determine the dermic 

and inhalatory exposition. 

A total of 12 painting cabins of two different companies were studied. Personal air samples and urine air samples 

were taken. In addition to that, a query form was filled by each employee to determine personal aspects that 

could interfere in the study (e.g. smoking habits). Gas phase VOCs were dynamically sampled connecting custom 

packed glass multi-sorbent cartridge tubes (Carbotap, Carbopack X and Carboxen-569) to an air collector pump 

sampler SKC to determine the compounds present in ambient air qualitatively. Air was sampled at a velocity of 100 

ml min-1.The analysis of VOCs was performed by automatic thermal desorption (ATD) coupled with capillary gas 

chromatography (GC)/mass spectrometry detector (MSD). ATD-GC/MS was chosen as analytical method due to its 

possibility of determination of a wide range of volatility and polarity VOCs in air samples. On the other hand, ORSA 

5 passive samples were used to evaluate the workers’ diary exposition. Passive samplers were analyzed by GC-FID. 

Finally, urine samples were collected at the end of each working shift and were analyzed through HPLC-UV. 


647


P20-032 SOLID-PHASE EXTRACTION ON-LINE COUPLED TO HYDROPHIIC INTERATION CHROMATOGRAPHY- MASS SPECTROMETRY 

TO DETERMINE POLAR DRUGS FROM WATER SAMPLES 

Fontanals N., Marcé R.M., Borrull F. 


Corresponding author e-mail: nuria.fontanals@urv.cat 

Hydrophilic interaction liquid chromatography (HILIC) is becoming used in recent years for separation and 

determination of polar analytes [1]. One of the features of the HILIC is the highly organic mobile phases which 

provide low column back pressure, increased ionisation efficiency for mass spectrometry (MS) detection [2], 

eventually, might solve the on-line SPE elution problems encountered with the conventional reversed-phase liquid 

chromatography (RPLC) separation systems. 

This study presented for the first time the on-line solid-phase extraction (SPE) coupled to HILIC – (electrospray (ESI)) 

mass spectrometry (MS) to determine a group of polar drugs, that include illicit drugs (such as cocaine, morphine, 

codeine and metabolites), and pharmaceuticals in environmental water samples. 

The on-line SPE was performed using a previously in-house synthesised sorbent (HXLPP) [3] that owns an 

hypercrosslinked structure, which showed very high retention for polar compounds. The HILIC separation was 

optimised and the initial high organic content of the chromatographic mobile phase, which is really influent in the 

HILIC separation, was also suitable for the proper elution of the analytes retained in the SPE column as well as the 

enhancement of the ESI ionisation efficiency. 

The developed method allows the loading of up to 250 ml of ultrapure water or 10 ml of environmental water sample 

(after a step with aqueous solution due to the presence of higher ionic strength matrices) spiked at low ng l-1 levels 

of the studied analytes with recoveries higher than 92% for all of them. The method was validated with environmental 

water and the linear range, the quantification and detection limits, repeatability and reproducibility were satisfactory. 

[1] Y. Wang, X. Lu, G. Xu, J. Sep. Sci. 31 (2008) 1564. 

[2] D.V. McCalley, J. Chromatogr. A 1217 (2010) 858. 

[3] N. Fontanals, P. Manesiotis, D.C. Sherrington, P.A.G. Cormack, Adv. Mater. 20 (2008) 1298. 

648 



P20-033 DEVELOPMENT OF A MULTI-RESIDUE METHODOLOGY FOR THE ANALYSIS OF HORMONAL STEROIDS AND VETERINARY 

COMPOUNDS TRACES IN SOIL 

SALVIA M.V., Vulliet E., Wiest L., Baudot R., Cren-Olive C. 

Service Central d’Analyse (CNRS)-France 


Chemical products are more and more used for agriculture and domestic activities and are responsible for the 

spread of several substances in the environment which are harmful for humans. Among these products, hormonal 

steroids and pharmaceutical compounds are of growing concern [1]. If several analytical methods are available to 

determine the content of hormonal steroids or veterinary substances in aquatic environment [2, 3], few methods 

were described to allow their analysis in solid matrices. Only few data concerning the content of hormonal steroids 

in soil are available and these ones reveal contaminations which can reach hundreds of ng/kg [4]. Consequently, the 

aim of this study was to develop an analytical method for the analysis of traces of hormonal steroids or veterinary 

substances in soil. Thus, 47 products were selected including 25 veterinary products, 11 hormonal steroids and 11 

other well-known compounds as human contaminants and used in this study as pollution tag. An analytical method 

both selective and sensible based on liquid chromatography – tandem mass spectrometry was developed. The 

optimization allowed the determination of the best conditions for the separation by chromatography (choice of the 

column, the gradient…) and for the detection by mass spectrometry (ionization mode, MRM transitions). The analysis 

of complex matrices such as soil needed a rigorous sample preparation to obtain a repeatable and enough sensible 

analysis to achieve the detection limits required. For this purpose, an extraction step by PLE (Pressurized Liquid 

Extraction) was developed. As the 47 studied molecules have characteristics and physical/chemical properties 

totally different, this stage was really delicate to optimize. In order to obtain the best parameters for this extraction 

(pressure, temperature, static time, cycle number and the choice of the solvent) and to find the best compromise 

to extract well each compound, a chemiometric approach by an experimental design was done. The analytical 

procedure allows the determination of the target analytes in the lower ng/g range. [1] Kuldip Kumar, Satish C. Gupta, 

Yogesh Chander, and Ashok K. Singh, Advances in Agronomy, 87, 1 (2005). [2] Marıa J. Lopez de Aldaa, Silvia 

Dıaz-Cruzb, Mira Petrovica, Damia Barcelo, Journal of Chromatography A, 1000, 503 (2003). [3] Emmanuelle Vulliet, 

Laure Wiest, Robert Baudot, Marie-Florence Grenier-Loustalot, Journal of Chromatography A, 1210, 84 (2008). [4] O. 

Finlay-Moore, P. G. Hartel and M. L. Cabrera, Journal of Environmental Quality, 29, 1604 (2000). 


649


P20-034 DETERMINATION OF 52 ORGANIC COMPOUNDS IN THE WHOLE WATER SAMPLE BY SPE-GC-MS CONSIDERING THE 

WATER FRAMEWORK DIRECTIVE 

Erger C. 1 , Balsaa P. 1 , Werres F. 1 , Schmidt T.C. 2 

1 

IWW Water Centre 

2 


Corresponding author e-mail: c.erger@iww-online.de 

Polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (BDEs), a range of volatile halogenated 

hydrocarbons and some organochlorine pesticides (OCPs) are ubiquitous environmental contaminants due to their 

widespread current or past use. Therefore, the Water Framework Directive of the European Parliament and the 

Council (WFD, Directive 2000/60/EC) requests an extensive monitoring on surface water of these compounds. Many 

of these substances are more or less non-polar. They can be almost totally sorbed on suspended particulate matter 

(SPM) and sediment. For this reason, the WFD requires the determination of the whole water sample including the 

SPM. Usually, SPM disturbs the commonly used analytical sample preparation methods for example by incomplete 

release of particulate bonded analytes by use of liquid extraction methods. Therefore, the SPM is often separated 

from water samples, by, e.g., filtration and analyzed separately. However, commonly used sample preparation 

procedures which analyze separately the solid and liquid phases are frequently characterized by low efficiency and 

reproducibility. A superior alternative for samples containing SPM is the use of solid phase extraction disks (SPE 

disks). Due to the enhanced diameter (diameter: 47-50 mm) a lower risk for plugging is given in comparison to usual 

solid phase extraction catridges (diameter: 0.6-13 mm). Thus, the whole water sample can be enriched on the SPE-disk 

without plugging and without prior filtration of the sample. Following this, the procedure of sample preparation 

includes a “one-step” extraction of the analytes, from the SPM and the sorbents. Additionally, using the SPE-disk 

offers the reduction of organic solvent volumes and process duration time in comparison to generally used methods 

such as liquid liquid extraction or Soxhlet extraction. Based on those facts a new SPE-disk method was developed 

by IWW for the determination of 52 organic xenobiotics in SPM containing water samples. In the first step, the 

SPE-disk is conditioned with an organic solvent (e.g. acetone) followed by water. Subsequently, 1 liter of water 

sample containing SPM in amounts up to 1000 mg/l is loaded in 20 minutes, without separation of the SPM. 

Afterwards the SPE-disk must be dried for some minutes to reduce interferences of the analytical method caused by 

residual water. For this reason the drying step was intensively investigated during the method development. Then, 

the fractional extraction and elution of analytes are carried out by using acetone. A volumetric standard is added 

before the combined eluates are concentrated to improve limits of detection. The eluate is analyzed by GC-EI/ 

MS. To increase the detection sensitivity, the selected ion monitoring (SIM) mode is used. All 52 target analytes are 

separated within 30 min in one GC-MS run. The overall processing time is about two hours, including the GC-MS run 

time. Next the validation of the total analytical method will be finished. 

650 



P20-035 VALIDATION OF FAST AUTOMATED SPE AND ISOTOPE DILUTION-GC/MS ANALYSIS OF PRIORITY PESTICIDES IN WATER 

Planas C., Caixach J. 

IDÆA (CSIC)-Spain 

Corresponding author e-mail: cppeco@cid.csic.es 

A method based on fast automated solid-phase extraction (SPE) and isotope dilution gas chromatography/mass 

spectrometry (GC/MS) was developed for the analysis of priority pesticides in water samples. The method combines 

the versatility and flexibility of a new automated SPE (Horizon Technology) using different types of SPE disks with the 

selectivity and sensitivity of GC/MS analysis. 

The purpose of this study was to apply USEPA methods, such us USEPA 525.2 (semi volatile organics in drinking 

water) and USEPA 625 (base/neutral and acid compounds in water) to the analysis of priority pesticides (lists from 

USEPA method 525.2 and Directive 2008/105/CE) using fast automated SPE with extraction disks. 

After optimisation according to the type of SPE disk, concentration of analytes, sample volume, drying time of the 

SPE disk before elution and elution solvent, the method was validated by extracting samples of mineral water spiked 

with different concentration levels of priority pesticides and by participating in an interlaboratory exercise. 

The validated method (based on SPE with extraction disks) is a rapid, accurate and sensitive procedure and it 

has several advantages compared to other extraction methods (SPE with extraction cartridges and liquid-liquid 

extraction (LLE)): 

- Lower total extraction time (30min). Two hours and 1h are needed when using SPE with cartridges and LLE, 

respectively. 

- Lower amounts of solvent used (about 20mL). The same amount is required when using SPE with cartridges and 

100-300mL are needed with LLE. 

- Suitable as a multiresidue method (high extraction recoveries obtained for different types of analytes), while SPE 

methods with cartridges are usually optimised for a specific family of analytes. 

- Higher reproducibility of the results. The automated procedure, as well as the use of isotope dilution with the 

addition of labeled standards before the extraction minimize and compensate errors due to the manual handling of 

samples. 


651


P20-036 EVALUATING THE CONTENT OF ALKYL- AND METHOXY-PHENOLIC COMPOUNDS IN BIOMASS SMOKE BY 

HEADSPACE-SINGLE-DROP-MICROEXTRACTION (HS-SDME) IN COMBINATION WITH HIGH-PERFORMANCE LIQUID 

CHROMATOGRAPHY (HPLC) 

Rincón A.A., Pino V., Ayala J.H., Afonso A.M.., González V. 


Several biomass materials are commonly used in the Canary Islands to smoke cheeses, such as almonds skin, 

pine needles, prickly pear, and rock rose. Cheeses smoking is accomplished not only to give cheeses a particular 

organoleptic profile but also to inactivate the actions of enzymes and microorganisms. Many organic compounds 

can be found in biomass smoke. Among them, alkyl- and methoxy-phenols are components of great importance for 

the smoke flavor, preservation of smoked cheeses, and antioxidant effects. The analytes studied in this work were 

vanillin, 3-metoxyphenol, 2,6-dimetoxyphenol, 2-ethylphenol, 3-ethylphenol, phenol, o-cresol, m-cresol, p-cresol, 

and eugenol. The sampling and extraction of volatile compounds present in biomass smoke have traditionally been 

carried out using conventional methods, which are known for being time-consuming. The conventional methods are 

also hazardous to human health as they often use large amounts of organic solvents, which are known for being 

hazardous, flammable, and damaging to the environment. These disadvantages have been the basis of a trend to 

develop analytical methods aimed at minimizing the solvent consumption. Headspace single-drop microextraction 

(HS-SDME) is a relatively novel technique which presents several advantages: low solvent consumption (from 1 to 5 

microliters of an organic solvent), low sample requirements (usually


P20-037 DETERMINATION OF VOLATILE ORGANIC COMPOUNDS IN WATER BY HEAD SPACE-SPME GC-MS/MS WITH TRIPLE 

QUADRUPOLE ANALYZER 

Cervera M.I., Beltrán J., Hernández F. 


Corresponding author e-mail: hernandf@qfa.uji.es 

DETERMINATION OF VOLATILE ORGANIC COMPOUNDS IN WATER BY HEAD SPACE-SPME GC-MS/MS WITH 

TRIPLE QUADRUPOLE ANALYZER Cervera M I, Beltrán J, Hernández F Research Institute for Pesticides and 

Water (IUPA), University Jaume I,12071 Castellón, Spain, Tel. +34 964 387334, E-mail: cerveram@qfa.uji.es Volatile 

organic compounds (VOC’s) include many different contaminants, like chlorinated solvents and fuel components. 

They can move easily through the environment and eventually end up in ground or surface water. Therefore, it 

is necessary to develop advanced analytical methodology that allows a reliable quantification and identification 

of VOC’s in environmental water. In this work, a quantitative method for the determination of selected volatile 

organic compounds in different water samples (surface, groundwater and urban wastewater) has been developed 

and validated. The procedure is based on solid phase microextraction from the sample headspace followed by 

determination by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) using triple quadrupole 

analyzer (QqQ) in electron ionization mode. The best conditions for the extraction were obtained using a factorial 

design, taking into account the interaction between different parameters and not only individual effects of variables. 

Extraction was carried out using 4 mL of water sample in a 10 mL vial, adding 10% of NaCl, with magnetic stirring, 

and a Carboxen/Polydimethylsiloxane 75 um fiber for 30 minutes after a pre-heating time of five minutes necessary 

to equilibrate the sample to 50ºC. Chromatographic determination was carried out by GC-MS/MS under Selected 

Reaction Monitoring (SRM) mode. Two transitions were acquired for most analytes, although low mass of some 

compounds made sometimes difficult to find characteristic fragments. In these cases, a pseudo Selected Ion 

Monitoring (pseudo-SIM) was used instead, with at least three ions being selected as precursors in the first 

quadrupole, applying collision energy zero and isolating the same ion as product ion in the second quadrupole. 

Thus, to ensure a satisfactory identification, two MS/MS transitions or three characteristic SIM ions were selected for 

each compound and their intensity ratios were used as a confirmatory parameter. Several approaches were tested 

for quantification of the selected compounds in different water samples (considering different matrix complexity). 

Thus, the external calibration, the use of isotopically labelled internal standard, the standard additions method, or 

the multiple headspace (MHS) approach were applied and compared for different sample matrices. The optimised 

method was validated at three concentrations at the sub-ppb level in several water matrices. Accuracy and precision 

were evaluated by means of recovery experiments (n=6 at each level). Most recoveries were between 70-120% and 

relative standard deviations (RSD) were lower than 20%. The linearity was also tested showing satisfactory values 

with coefficients of correlation higher than 0.99. The developed procedure was finally applied to the analysis of 

samples of three types: groundwater, surface water and wastewater samples obtained from a wastewater treatment 

plant (influent and effluent samples). 


653


P20-038 PESTICIDES IN WATER INTENDED FOR HUMAN CONSUMPTION BY UPLC-MS/MS AND GC-MS 

Gonzalez C., Castillo M., Carbonell E., Perez J.A. 

Centro Superior de Investigación en Salud Pública, Valencia 

Corresponding author e-mail: gonzalez_carmac@gva.es 

In view of the importance of the quality of water intended for human consumption for human health, it is necessary 

to lay down at Community and national level the essential quality standards with which water intended for that 

purpose must comply (RD 140/2003). The parametric value (PV) applied to ‘pesticides-total’ is 0.5 μg/l and for each 

individual pesticide is 0.1 μg/l. In the case of aldrin, dieldrin, heptachlor and heptachlor epoxide, the parametric 

value is 0.03 μg/l. The performance characteristics apply to each individual pesticide and will depend on the 

pesticide concerned. For pesticides the specified performance characteristics should be those that allowed the 

method of analysis used, be capable of measuring concentrations equal to the parametric value with a trueness 

of 25%, precision of 25% and a 25% limit of detection. The limit of detection will be 0.01 μg/l for aldrin, dieldrin, 

heptachlor and heptachlor epoxide and 0.025 μg/l for other substances. 

In order to detect these low levels is necessary to use sample concentration techniques. The most used are the 

liquid-liquid extraction, solid phase extraction (SPE) and solid phase microextraction (SPME). The methods of 

analysis for the determination of pesticide residues are performed by gas and liquid chromatography depending 

on the type of substance. Among the latter advances in liquid chromatography, is the Ultra Performance Liquid 

Chromatography (UPLC) improves the resolution and speed of analysis in comparasion with conventional 

chromatography, offering certain advantages in the methods of analysis multiresidues. Mass-spectrometric 

thechiques allows the confirmation of the different substances simultaneously with detection. 

In this paper, we describe a procedure based on EPA method 525-2 for determining 89 pesticides in water intended 

for human consumption. The concentration of these is by solid phase extraction using Bond Elut columns plexus 

(Varian) and Lichrolut EN (Merck). 

The process consists of the following phases: 

- Sample preparation: Removal chlorine with 50 mg / l of sodium sulfite. Separate one aliquot for GC-MS and two 

aliquot UPLC-MS/MS. Adjust the pH of each aliquot and adds the surrogate. 

- Concentration and elution of the aliquots in an automated solid phase extraction (Autotrace Caliper). 

- Evaporation and redisolution the extract with a suitable solvent for each technique. 

- Analysis by GC-MS and UPLC-MS/MS. 

The guidance in Document No SANCO/10684/2009 is intended for laboratories control or in the monitoring of 

pesticide residues in food involved in official and feed in the EU. The document describes the method validation and 

analytical quality control requirements to support the validity of data used for checking compliance with maximum 

residue limits (MRLs), enforcement actions, or assessment of consumer exposure to pesticides. This document 

provides guidelines which are also useful for the analysis of water for human consumption. 

With the procedure developed, it is possible with only one liter of sample the screening of a large number from 

pesticides of different families in a large number of samples. 

654 



P20-039 OPTIMITATION OF SOME STEPS FOR THE ANALYSIS OF PER- AND POLYFLUORINATED ALKYL SUBSTANCES (PFAS) IN 

FINE AIRBONE PARTICULATE MATTER (PM 2.5) BY LC-MS/MS 

Beser M.I. 1 , Pardo O. 1 , Yusa V. 1 , Beltrán J. 2 

1 

Public Health Research Center (CSISP), Valencia 

2 


Corresponding author e-mail: yusa_vic@gva.es 

In recent years, long chain perfluorinated carboxylates (PFCA) and sulfonates (PFSA) such as perfluorooctanoate 

(PFOA) and perfluorooctane sulfonate (PFOS) have been found to be persistent, bioaccumulative, and entailing 

toxic properties [1,2]. Furthermore, global distribution to remote regions caused by extensive industrial application 

and consumer use has been demonstrated for these kinds of compounds [3] and the European Union Directive 

2006/122/EC lays down restrictions on the marketing and the use of PFOS for new products [4]. PFAS are used in 

numerous industrial and consumer applications, ranging from water repellents for leather, paper and textiles, coating 

formulations, metal plating and cleaning, fire-fighting foams, and as polymerisation aids [1, 5, 6]. Some steps of a 

ESI(-)-LC-MS/MS multirresidue method to determinate the 13 most abundant ionic PFAS in atmospherical particulate 

matter (PFBS, PFHxS, PFHpS, PFOS, PFDS, 6:2 FTS, PFBA, PFPeA, PFPHxA, PFHpA, PFOA, PFNA, PFDA) have 

been optimized. PFAS have been extracted using microwave assisted extraction (MAE) at 50 ºC. Different cleanup 

methodologies (centrifugation, C18 dispersive phase and C graphitized dispersive phase) have been tested and 

matrix effect has been evaluated. Several mobile phases (methanol:water and acetonitrile:water) at various pH 

and using different gradients have been tested. SRM mode has been compared to a pseudo-SRM methodology in 

which ions are fragmented on the ionization source and ion source settings have been optimized automatically with 

the equipment software. The final method selected consists of extraction using MAE followed by a centrifugation. 

Separation has been accomplished on a Luna C18 analytical column of 150mm×2mm and 5 µm particle diameter 

from Phenomenex. Methanol:water with ammonium acetate at 5mM as mobile phase has been selected, reaching 

the 90% of methanol from the initial conditions to the minute 23, remaining 1 minute and decreasing to 35% until 

the minute 27 at 300 µl/min. References [1] A. Dreyer, R. Ebinghaus. Atmospheric Environment 43 (2009) 1527–1535. 

[2] A. Jahnke et al. Journal of Chromatography A. 1164 (2007) 1-9. [3] A. Jahnke et al. Environ. Sci. Technol. 41 

(2007) 745-752. [4] Directive 2006/122/ECOF of the European Parliament and the Council of 12 December 2006. [5] 

P. de Voogt, M. Saez. Trends Anal. Chem. 25 (4) (2006) 326. [6] Jonathan L. Barber et al. J. Environ. Monit. 9 (2007) 

530–541. 


655


P20-040 MULTI RESIDUE ANALYSIS OF ENDOCRINE DISRUPTOR COMPOUNDS IN ENVIRONMENTAL SAMPLES USING LIQUID 


Camilleri J., Vulliet E., Wiest L., Baudot R., Cren-Olivé C. 

Service Central d’Analyse du CNRS 


Many man-made or natural compounds are known or supposed to interfere with the endocrine, causing behaviour 

disorders [1], decreased fertility [2] and birth malformations [3]. Most of those compounds are listed as emerging 

contaminants by the European Union [4], [5] and need development of specific and highly sensitive methods to 

enable their monitoring in the environment. Endocrine disruptor compounds (EDCs) display a wide range of structural 

diversity and chemical properties and they occur in the aquatic environment at trace levels in complex matrices. 

Almost all available analytical methods only target one or two families of EDCs, such as steroids, pesticides or 

alkylphénols [6], [7], [8]. In consequence, the development of a multi-residue methodology to surface water and river 

sediment analysis is necessary, in order to monitor as many EDCs as possible. Moreover, such a method should 

involve limited sample preparation steps with sufficiently selectivity and sensitivity. 

In this context, the objective of this study was to quantify traces of 29 selected EDCs from various families 

in a single analysis using on line SPE coupled to liquid chromatography tandem mass spectrometry (LC/MS- 

MS) for surface water and pressurized liquid extraction (PLE) LC/MS-MS for river sediments. Molecules of 

interest have been chosen using lists of priority pollutants [4], [5] and compounds known or supposed to be 

EDCs : 6 hormones (estrone, 17β-estradiol, megestrol acetate, progesterone, testosterone and tamoxifen), 16 

pesticides (2,4-dichlorophenoxyacetique acid, acetochlor, alachlor, atrazine, carbendazime, diuron, iprodion, 

linuron, prochloraz, thiram and ziram), 1 UV-filter (4-methylbenzyliden camphor), alkylphenols (tert-butylphenol, 

n-octylphenol, tert-octylphenol, n-nonylphenol and 4-para-nonylphenol), 3 phenolic compounds (2,4-dichlorophenol, 

bisphenol A and resorcine) and 3 pharmaceuticals used as pollution indicators (3,4-dichloroaniline, carbamazepine 

and diclofenac). Several on line SPE cartridges have been tested to compare concentration capacities for aqueous 

samples. PLE extraction was developed for sediments using a design of experiment to optimize extraction solvent, 

pressure, temperature, static time and number of cycles. The excellent selectivity and sensitivity allowed us 

satisfactory quantification and confirmation at the lower ng/L and ng/g range. 

[1]. S. S. Madsen, Søren Skovbølling, Christian Nielsen and Bodil Korsgaard, Aquatic Toxicol, 68, 109 (2004) 

[2]. A. Giwercman, L. Rylander and Y Lindberg Giwercman, Reprod Biomed Online, 15, 633 (2007) 

[3]. D. M. Schreinemachers, Environmental health perspectives A, 111, 1259 (2003) 

[4]. Communication de la commission au conseil et au parlement européen sur la mise en œuvre de la stratégie 

communautaire concernant les perturbateurs endocriniens COM (2001) 262 final 

[5]. European commission DG ENV , Towards the establishment of a priority list of substances for further évaluation of 

their role in endocrine disruption, bkh-annex 15 

[6]. J. B. Baugros, B. Giroud, G. Dessalces, M. F. Grenier-Loustalot and C. Cren-Olivé, Analytica Chimica Acta, 607, 

191 (2008) 

[7]. S. Barrek, C. Cren-Olivé, L. Wiest, R. Baudot, C. Arnaudguilhem and M. F. Grenier-Loustalot, Talanta, 79, 712 (2009) 

[8]. J. B. Baugros, C. Cren-Olivé, B. Giroud, J. Y. Gauvrit and M. F. Grenier-Loustalot, J. Chromatogr. A, 1216, 4941 (2009) 

656 



P20-041 DEVELOPMENT OF A RELIABLE AND SENSITIVE QUANTIFICATION METHOD FOR THE DETERMINATION OF POLAR 

PESTICIDES METABOLITES IN SURFACE WATER, GROUND WATER AND DRINKING WATER 

Kowal S. 1 , Balsaa P. 1 , Werres F. 1 , Schmidt T.C. 2 

1 

IWW Water Centre-Germany 

2 

University of Duisburg Essen 

Corresponding author e-mail: s.kowal@iww-online.de 

Numerous pesticides continue to be applied several times a year over large areas with the risk – especially when 

used incorrectly – that they may enter ground water and surface water by leaching and drainage processes. Polar 

and more easily degradable pesticides have been replacing non-polar and more persistent ones. Formed metabolites 

are as well polar. That means, those components often cannot be effectively removed during water treatment. 

Additional risks may occur when these substances are chemically modified by oxidative reaction of ozone within 

the process, forming products of toxicological concern. A set of pesticides metabolites known from lysimeter tests 

possesses a high potential to reach the groundwater. Therefore, it is important to monitor the aquatic environment 

using reliable and sensitive analytical methods, to respond immediately if changes occur in the water quality. 

Objective of this study was therefore to develop a secure and sensitive analytical method with a prior purification 

and/or enrichment step for the determination of selected metabolites (table 1) in drinking water, groundwater and 

surface water. The quantification of the metabolites by using direct injection HPLC-ESI-MS/MS leads sometimes 

problems in quantification because of the acidic or ionic nature of some metabolites. This is caused by a disruptive 

influence on the ionisation process during ESI-MS measurement by cations within the original water sample. 

Therefore a sample preparation including a solid-phase extraction (SPE) was introduced to achieve reliable data and 

high sensitivity for the detection of all polar substances under investigation. The measurement was by a UPLC-MS/MS 

system. A weakly basic ion exchange SPE phase was found best in comparison to other SPE phases to interact 

with the acidic character of metabolites. An important advantage of this phase is its ability of removing all cations 

from the original sample, which leads to a partly or complete suppression of all interfering matrix effects and a 

significantly improved sensitivity. The validation work included various optimization steps concerning sample 

preparation, chromatography and spectroscopy. Methanol containing ammonia was used as SPE eluent. The eluate 

was evaporated to dryness by a gentle steam of nitrogen. The residue was dissolved again by adding of pure water. 

The chromatographic separation was performed using a 3 x 50 mm (2.6 µm) Phenomenx Kinetex C18-column. The 

ionic and acidic properties of the target compounds require attention in terms of choosing a suitable mobile phase in 

order to achieve reproducible retention times. The influence of the buffer system within the used solvent (formic acid 

and ammonium) was investigated. A very good compromise between requirements coming from ionization within 

the ESI-MS process and a stable chromatography could be found. Excellent results of reproducibility, linearity and 

accuracy could be derived from comprehensive measurements. The sensitivity of the procedure was in all cases at 

least 1 ng/L showing a S/N-ratio of at least 3:1. The robustness of the procedure when analyzing spiked samples of 

different matrices was found high. 


657


P20-042 DEVELOPMENT OF A CAPILLARY CHELATION ION CHROMATOGRAPHY SYSTEM FOR APPLICATION WITHIN 

ENVIRONMENTAL MONITORING/TRANSITION METAL ANALYSIS 

Moyna A., Connolly D., Currivan S., Macka M., Paull B. 

Irish Separation Science Cluster, Dublin City University 


The determination of trace metals in complex samples such as seawater is problematic for ion exchange 

chromatography due to the high salt concentrations which swamp the ion exchange sites. Chelation ion 

chromatography (CIC) utilises chelating stationary phases to provide the enhanced selectivity to help overcome 

this issue. Previous studies have utilised iminodiacetic acid (IDA) immobilised resins for the pre-concentration and 

separation of metals using a single column.1 To improve sensitivity in CIC the use of post column reaction detection 

is common practice. The use of post column mixing presents considerable difficulty when used at the capillary 

scale, as any extra-column band broadening in the system leads to a substantial loss in efficiency. An in-house 

capillary liquid chromatography (capLC) system was constructed consisting of two analytical pumps, a 20 nL injector 

valve, a polymer monolith (laurylmethacrylate-co-ethylene dimethacrylate (LaMA-co-EDMA) functionalised with 

vinyl azlactone followed by immobilisation with IDA, nano-fluidic mixer and capillary absorbance detection. In this 

work, the selectivity of the IDA functionalised polymer monolith was evaluated for the analysis of transition metals 

(e.g. copper, cobalt and nickel) utilising 4-(2-pyridylazo)resorcinol (PAR) as a post column reagent. The effect of 

temperature on both retention and detector sensitivity was monitored using in-house constructed capillary and 

post-column mixer micro-heaters. Examples of transition metal separations on the new IDA functionalised polymer 

monolith will be shown, and method performance data presented. 

(1) Nesterenko, J. Chromatgr. A, 770, (1997), 129-135 

658 



P20-043 RAPID ANALYSIS OF 52 PERSISTENT ORGANIC POLLUTANTS IN SOLIDS BY MEANS OF ULTRASONIC SOLVENT 

EXTRACTION AND GAS CHROMATOGRAPHY MASS/MASS SPECTROMETRY 

Martinez E., Llorca J. 

LABAQUA, Murcia 

Corresponding author e-mail: julio.llorca@labaqua.com 

Rapid analysis of 52 persistent organic pollutants in solids by means of ultrasonic solvent extraction and gas 

chromatography mass/mass spectrometry 

Esther Martínez , Julio Llorca* and I. Valor 

LABAQUA S.A. c/ del Dracma, Parcelas 16-18 03114-Alicante, Spain 

* Julio.llorca@labaqua.com 

A novel rapid method based on ultrasonic solvent extraction for the analysis of 52 persistent organic pollutants 

including polychloronaphthalenes (PCNs), polychlorinated biphenyls (PCBs), polychlorinated biphenyls Dioxin.like 

(PCBs Dioxin.like), polycyclic aromatic hydrocarbons (PAHs), and polybrominated diphenylethers (PBDEs) in solid 

samples was developed. The different parameters that affect the extraction of analytes from the solid samples, 

solvent volume, mass of solid, extraction time and clean-up agent were studied. The final selected conditions 

consisted of the extraction of 1 g of solid with 15 mL hexane: acetone (1:1) by sonication for 5 min. After cleaning 

the hexane: acetone extract with PSA Bonded Silica, 50 µL of this extract was injected by large volume injection and 

analyzed by chromatography mass/mass spectrometry. Different matrixes were studied using three kind of solid with 

very different physicochemical properties like soils, sediments and sludges. The effects of the matrix on the recovery 

of the various pollutants under the developed method were avoided by using a calibration in a matrix similar to that 

of the samples and that had been subjected to the same conditions of extraction as samples. Method sensitivity, 

linearity, repeatability, and reproducibility were also studied. Validation and accuracy of the method were conducted 

by analyzing one commercial certified reference materials. The main advantage of this rapid method resides in 

that a small amount of toxic solvents (hexane and acetone) is needed for the extraction of only 1 g of solid sample 

allowing limits of detection ranging from 0.01 to 2.0 µgkg-1 for PBDEs, PCBs and PCNs and 0.5 to 2.0 µgKg-1 for 

PAHs. Repeatability and reproducibility variation were lower than 40% for all investigated compounds. Results of the 

certified reference materials verify the high accuracy of this method. 


659


P20-044 DETERMINATION OF ALKYL SULFATES IN MARINE SEDIMENTS BY LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY 

Fernández-Ramos C. 1 , Blanc R. 1 , Ballesteros O. 1 , Zafra-Gomez A. 1 , Navalón A. 1 , Crovetto G. 1 , Vílchez J.L. 1 , Verge C. 2 , Lopez I. 2 , De Ferrer J. 2 

1 


2 

Cepsa Química, Centro de Investigacion 

Corresponding author e-mail: oballest@ugr.es 

Coastal ecosystems receive large quantities of a wide range of organic contaminants from urban wastewater which 

are discharged either treated or untreated. Among these contaminants, surfactants constitute the main proportion of 

the total, in quantities above 15 million tonnes per year [1]. 

When they reach the environment they undergo a rapid degradation in water, and the fate of the remaining quantities 

are incorporated in the sediments due to their high affinity for the organic carbon in the particulate phase. We 

therefore focused our studies on sediments because there are relatively few published papers about Alkyl Sulphates 

(AS) in marine sediments [2]. 

AS are a group of anionic surfactant used mainly in the formulation of detergents and other cleaning products and 

their European production is approximately 65.602 tons/year (CESIO 2003) [3]. 

A new pressurized liquid extraction followed by liquid chromatography-mass spectrometric analytical method (LC- 

MS) with electrospray ionization (ESI) operating in negative mode was developed, which was aimed at facilitating the 

determination of AS in complex matrices, like marine sediments. 

This new analytical procedure was satisfactorily applied for the determination of these chemical compounds in 

three different marine matrices, which are located in three different geographical areas of Spain, Almería coast, 

Mediterranean coast and Tenerife coast. 

Quality parameters were determined and satisfactory results were obtained. 

References: 

[1] D.R. Karsa, Chem. Ind., 9, 685 (1998). 

[2] Lara–Martín Pablo, Gómez-Parra Abelardo, González-Mazo Eduardo, Presence of surfactants and their 

degradation intermediates in sediment cores and grabs from the Cadiz Bay area, Environmental Pollution 144 (2), 

483-491, 2006. 

[3] HERA Alkyl Sulfates March 2002. 

660 



P20-045 CHEMICAL CHARACTERIZATION OF THE TARSAL SECRETION OF THE LOCUST S. GREGARIA 

Gradl M., Nicholson T., Schmitt C., Betz O., Albert K. 


To allow adhesion to diverse surfaces insects have developed various kinds of adhesive devices. Previous studies 

showed that the attachment is supported by a fluid secreted by the tarsal glands of these insects. [1,2,3] Further 

research indicates that the liquid is a two-phasic emlusion consisting of hydrophilic and hydrophobic components. 

[4,5] 

Our interest is focused on the identification and characterization of the secretion of smooth attachment pads. 

Therefore we obtained secretion samples of the desert locust (Schistocerca gregaria) by solvent extraction and 

solid-phase microextraction (SPME) of this insect’s feet. 

First chromatographic results were achieved by hyphenation of gas-chromatography to mass-spectrometry (GC-MS) 

because this sensitive method enables the characterization of small sample amounts in the nano- and microgram 

range. This approach indicates the presence of a large amount of hydrocarbons in the hydrophobic solvent 

extraction as well as in the SPME sample. The hydrophilic solvent extraction on the other hand contains amino acids 

as well as few carbohydrates. Cap-NMR spectroscopic studies confirm the presence of sugars in the aqueous part 

of the secretion. Further information is provided by HPLC separation of the mixture. 

[1] S. N. Gorb, Attachment devices of insect cuticle, Kluwer Academic Publishers, 2001. 

[2] S. Ishii, Appl. Entomol. Zool, 1987, 22, 222. 

[3] O. Betz, Arthropod Struct. Dev, 2004, 33, 3. 

[4] W. Federle, Integr. Comp. Biol, 2002, 42, 1100. 

[5] W. Vötsch, Insect Biochem. and Molec. Biol, 2002, 32, 1605. 


661


P20-046 ENANTIOMER DETERMINATION OF M- AND P-IMAZAMETHABENZ METHYL ISOMERS IN POTATO SAMPLES BY TWO- 

DIMENSIONAL CHIRAL HPLC USING A PROTEIN BASED COLUMN 

Santos-Delgado M.J., Polo-Díez L.M. 


Corresponding author e-mail: mjsantos@quim.ucm.es 

The importance of herbicide enantiomer analysis is increasing due to their different activity and toxicity; this is the 

case of Imazamethabenz-methyl (IMBM) chiral herbicide which belongs to the Imidazolinone family (IMIs) containing 

a stereogenic centre in the imidazolinone ring. IMBM is used in modern agriculture and it is applied both to foliage 

and through the soil, being an inhibitor of ramified chain amino acids. Enantioselectivity of IMI enantiomers has been 

recognized, R-enantiomers having a greater inhibiting action on acetohydroxy acid synthase than S-enantiomers. 

IMBM persistence in agriculture soils is high, affecting crop rotations. The lack of chiral universal phases for direct 

chiral HPLC makes critical the choice of a suitable stationary phase; even then, the mobile phase must be optimized 

carefully. Moreover, since only very clean samples must be injected in the chiral column, two-dimensional HPLC 

using a first column in reversed mode to isolate the racemic and a second chiral column to separate the enantiomers 

is particulary suitable. In this work, we have studied the simultaneous enantiomer separation of the p- and m- IMBM 

isomer herbicides by HPLC bidimensional chiral using UV detection at 247 nm and their determination in potato 

samples. The enantiomer identification was made by HPLC-CD. Elution strength of the mobile phase for the first C18 

column was ACN/HAc/H2O (25/10/65) and it was compatibilized with the mobile phase required for the second chiral 

column, which was ACN/HAc-NH4Ac (3%; 40 mM at pH 4.00). The whole process was optimized using factorial 

experimental design. The two-dimensional HPLC method allows an easy and fast determination of the IMBM 

herbicide enantiomers. The analytical characteristics for IMBM standard solutions were studied and the proposed 

method was applied for p- and m- IMBM determination and their ratio in a potato sample. The usefulness of the 

method is due to differences in the biological activity of IMBM enantiomers and their implications in agriculture and 

in the environment. 

662 



P20-047 SYNTHESIS OF STRONG ANION-EXCHANGE MONOLITHIC CAPILLARY COLUMN FOR THE MINIATURIZED ANALYSIS OF 

RADIOACTIVE SAMPLES 

Bruchet A. 1 , Dugas V. 1 , Goutelard F. 2 , Randon J. 1 

1 

University Claude Bernard-Lyon 

2 

CEA-Saclay 

Corresponding author e-mail: anthony.bruchet@hotmail.com 

Since its introduction Ion Chromatography (IC) had been widely used for inorganic cations and anions but also 

for small organic acids. Nowadays this chromatographic mode encounters a renewal of interest concerning new 

developments as hyphenated IC systems, bioanalysis and miniaturization [1]. This is in part due to the emergence 

of monolithic stationary phases. Indeed, since their introduction in the early 1990s by Svec and Fréchet [2], 

polymethacrylate monoliths have encountered growing interest due to their intrinsec enhanced convective mass 

transfer able to produce high separations efficiency at high mobile phase velocities and their easy preparation 

directly in capillary or in-microsystems (in-situ synthesis). Miniaturizing ion separations presents a great interest with 

respect to the analysis of substances hazardous to health like toxic biohazard or radioactive samples. Miniaturization 

aims to i) minimize human exposition to contaminant, ii) reduce the quantity of contaminated wastes (liquid and 

solid), and iii) allow automation of the entire analytical process. 

Specifications with respect to the developement of micro-total analysis systems (µ-TAS) for the ions analysis 

involve the use of polymeric monolithic columns for ions’ separation. For instance, acrylate-based monoliths 

are characterized by a relatively high chemical resistance, versatile surface fonctionalization and ease of in-situ 

preparation. Ethylene DiMéthacrylate–co–Glycidyl MethAcrylate (EDMA-co-GMA) monoliths as described by Ueki et 

al. [3] are widely used in ion chromatography. Their surface modification with DiEthylAmine (DEA) is typically used in 

order to create Weak Anion Exchange (WAX) support, but there is no consensus about optimal reaction conditions. 

Moreover direct grafting of ternary amines like TriEthylAmine (TEA) for the direct preparation of Strong Anion 

Exchanger has not been described yet. The present work aims to fulfill the lack of information regarding the extent 

of reaction of binary and ternary amine on GMA-co-EDMA monolith for the elaboration of anion-exchange monolithic 

columns. Kinetics and thermodynamics studies were carried out for both amines allowing comparing their reactivity. 

A chemometric approach has been used determine the optimal conditions for TEA grafting (time, temperature, extent 

of reaction≥90%) allowing to reach ion-exchange capacity of 300µmol/g. 

Transfer of the reaction to 100µm ID capillary has been successfully carried-out allowing to prepare high efficiency 

columns (H up to about 20µm for common anions), with relatively high retention in µ-IC. Use of these micro-IC 

columns to the sequential separation of radioactive elements (e.g. Uranium and Plutonium) where high concentration 

of hydrochloric acid is required imposes to test the chemical resistance of the developed monolithic stationary 

phase. The chemical resistance to these hard conditions was hence confirmed through the stability of the retention 

and affinities parameters of selected anions before and after acidic exposure proving the reliability of the developed 

support. 

The next step of the project is the design of a µ-TAS coupling treatment and collection of mono-elementary fraction 

with off-line High Resolution – Inductive Coupled Plasma – Mass Spectrometry isotopic measurements. 

1. PR Haddad, PN Nesterenko,W Buchberger, J. Chrom. A. 1184 (2008) 456-473. 

2. F Svec,JMJ Fréchet, J. Chrom. A. 702 (1995) 89-95. 

3. Y Ueki, T Umemura, J Li, T Odake,K-i Tsunoda, Anal. Chem. 76 (2004) 7007-7012. 


663


P20-048 DETERMINATION OF PARABENS IN HOUSE DUST BY PRESSURISED HOT WATER EXTRACTION FOLLOWED BY STIR BAR 

SORBENT EXTRACTION AND THERMAL DESORPTION - GAS CHROMATOGRAPHY - MASS SPECTROMETRY ANALYSIS 

Ramírez N., Borrull F., Marcé R.M. 


Corresponding author e-mail: francesc.borrull@urv.cat 

p-Hydroxybenzoic esters (parabens) are the most common preservatives and antimicrobial agents used in personal 

care, pharmaceutical and food products. Their widespread use contributes to the discharge of these compounds 

in the environment. Although there is no clear evidence of their bioaccumulation, parabens have been detected in 

human tissue, including breast tumours [1], and have endocrine activity [2]; therefore they are considered endocrine 

disrupting chemicals (EDCs). The main route of the human exposure to these compounds is their direct application 

to the skin through the use of consumer care products, such as creams or soaps. However, the inhalation and oral 

ingestion of parabens should also been considered. In this respect, recent studies have demonstrated the presence 

of parabens in air [3] and in house dust [4]. 

Due to the high complexity of the solid matrices, the analytical methods for determining semi-volatile compounds, 

such as parabens, generally comprise an extraction step with an organic solvent and time-consuming clean-up step 

previous to the analysis. The aim of this study is the development of a new method for the determination of parabens 

in house dust based on pressurised hot water extraction (PHWE) followed by the in situ derivatisation of the extracts, 

the stir bar sorbent extraction (SBSE) and thermal desorption - gas chromatography – mass spectrometry analysis. 

The method developed, is environmentally friendly, because uses water as solvent extraction, and showed high 

sensibility due to the SBSE preconcentration in combination with the thermal desorption. Method validation showed 

good linearity, repetitivity and repeatability, and detection limits at ng g-1 levels for all the target parabens. 

The applicability of the technique to real samples was tested in different house dust samples. In general, the most 

abundant parabens were methyl and propyl paraben, which are used together due to their synergic effects. 

[1] P.D. Darbre, A. Aljarrah, W.R. Miller, N.G. Coldham, M.J. Sauer, G.S. Pope, J. Appl. Toxicol. 24 (2004) 5. 

[2] E.J. Routledge, J. Parker, J. Odum, J. Ashby, J.P. Sumpter, Toxicol. Appl. Pharmacol. 153 (1998) 12. 

[3] N. Ramírez, R.M. Marcé, F. Borrull, J. Chromatogr. A (2010, in press), doi: 10.1016/j.chroma. 2010.04.049. 

[4] P. Canosa, D.Pérez-Palacios, A. Garrido-López, M.T. Tena, I. Rodríguez, E. Rubí, R. Cela, J. Chromatogr. A, 1161 

(2007) 105-112. 

664 



P20-049 DETERMINATION OF ACIDIC DRUGS IN RIVER WATER SAMPLES BY IN-LINE-SOLID-PHASE EXTRACTION-CAPILLARY 

ELECTROPHORESIS 

Maijó I., Borrull F., Aguilar C., Calull M. 


Corresponding author e-mail: irene.maijó@urv.cat 

Pharmaceutical compounds can enter into the aquatic environment. The need for their monitoring in natural waters 

is essential to verify the quality of the water. In this sense, different analytical methods have been developed to 

analyze them in wastewater and surface water at low concentration levels. Capillary electrophoresis (CE) combined 

with a chromatographic preconcentration such as SPE before the electrophoretic separation has proven to be a 

good alternative to the analysis of these drugs owing to its advantageous characteristics of high efficiency and fast 

analysis [1-6]. A solid-phase extraction (SPE) coupled in-line to CE with UV–vis detection has been developed for 

the determination of some acidic drugs: benzafibrate, indomethacin, piroxicam, diclofenac, naproxen and clofibric 

acid in water samples. In-line SPE has the following advantages over off-line SPE: (i) lower volumes of samples 

are required; (ii) few microliters of organic solvent are consumed making this approach environmentally friendly; 

(iii) the in-line SPE process is entirely carried out in the CE instrument; (iv) the small quantity of sorbent material 

needed together with the lower consumption of organic solvent makes in-line SPE cheaper than off-line. In this 

study, an in-line frit-free SPE preconcentration was prepared. A microcartridge containing an OASIS HLB sorbent 

was placed near the inlet within the separation capillary column. The frit-free configuration avoids the backpressure 

problems and the peak broadening than are observed in the frit configuration [3]. Different parameters affecting the 

preconcentartion, such us pH and volume of the sample, and composition and injection time of the elution plug were 

studied. Under the optimum conditions, the recovery of this procedure was higher than 80% for all the compounds 

and the acidic drugs could be detected at a concentration around 5 µg/L in the case of standard samples. Finally, 

the method was validated for the determination of the studied drugs in river water samples. The main advantages 

of this methodology is that it can be easily automated in fewer sample handling steps and that it allows the whole 

eluate from the SPE device to be analyzed by CE. [1] Macià, A., Borrull, F., Calull, M., Aguilar, C., Trends Anal. Chem. 

2007, 26, 133-153. [2] Benavente, F., Vescina, M., Hernández, E., Sanz-Nebot, V., Barbosa, J., Guzman, N.A., J. 

Chromatogr. A. 2007, 1140, 205-212. [3] Saavedra, L., Maeso, N., Cifuentes, A., Barbas, C., J. Pharm. Biomed. Anal. 

2007, 44, 471-476. [4] Macià, A., Borrull, F., Calull, M., Benavente, F., Hernández, E., Sanz-Nebot, V., Barbosa, J., 

Aguilar, C., J. Sep. Sci. 2008, 31, 872-880. [5] Lara, F. J., García-Campaña, A. M., Alés-Barrero, F., Bosque-Sendra, 

J. M., Electrophoresis 2008, 29, 2117-2125. [6] Lara, F. J., García-Campaña, A. M., Neusüss, C., Alés-Barrero, F., J. 

Chromatogr. A. 2009, 1216, 3372-3379. 


665


P20-050 ON-LINE ANALYSIS OF CARBONYL COMPOUNDS WITH DERIVATIZATION BY IN-TUBE SOLID-PHASE MICROEXTRACTION 

COUPLED TO CAPILLARY LIQUID CHROMATOGRAPHY 

Prieto-Blanco M.C. 1 , López-Mahía P. 1 , Campíns Falcó P. 2 

1 

University of Coruña 

2 


Corresponding author e-mail: pilar.campins@uv.es 

The use of miniaturized techniques both in the sample preparation and separation provides an increase of the 

selectivity and the sensitivity and a decrease of solvent volume1. The carbonyl compounds, mainly formed by 

oxidative processes, are found in numerous matrices such as atmosphere2 (air and particulate matter), treated 

water samples, in biological fluids (urine, plasma, serum...), in foods or in emission of plants. In this work, 

the determination of carbonyl compounds in particulate matter (PM10) by means a capillary microextraction 

technique such as in- tube solid-phase microextraction (IT-SPME) was optimized. The derivatization using 

2,4-dinitrophenylhydrazine was tested using different options (derivatization in solution or supported in ITSPME). 

In addition, several approaches (ITSPME coupled to conventional LC or coupled to capillary LC) were evaluated. 

The influence of sample volume, composition and volume of washing solvent on wide range of carbonyl derivatives 

with different functionalities was examined. Different behaviour was observed depending of types of carbonyl 

derivative. The best results were obtained for aliphatic aldehydes with longer alkyl chain because the capillary 

stationary phase of IT-SPME has a low polarity. Simultaneous derivatization/ITSPME was tested on-line coupled to 

the chromatographic system in order to improve the repeatability, the cost and the analysis time. A capillary column 

with an internal diameter of 500 µm and flow rates up 25 µL min-1 were used. A new procedure for the simultaneous 

derivatization/ITSPME option which is based derivatization-extraction in multiple steps is proposed. When this 

option was applied to PM10 samples the main carbonyl compounds were detected. Nevertheless a lower number 

of compounds at low concentration are detected with this procedure than with the option of solution derivatization/ 

ITSPME. Limits of detection (LODs) ranged from 30 to 198 ng L-1 were obtained using the option solution 

derivatization and IT-SPME coupled to capillary LC. These values improve the most LODs existing in the literature 

which are at ng mL-1 level. [1] H. Kataoka, A. Ishizaki, Y. Nonaka, K. Saito, Analytica Chimica Acta 655 (2009) 8. 

[2] M.C. Prieto-Blanco, M. Piñeiro Iglesias, P. López-Mahía, S. Muniategui Lorenzo, D. Prada Rodríguez, Talanta 80 

(2010) 2083. 

666 



P20-051 COMPARISON OF BIOMARKER ASSEMBLAGE WITH POTENTIAL FOR PALEOCLIMATE EVOLUTION IN COASTAL 

PEATLANDS FROM ASTURIAS (NORTH SPAIN) 

López Días V., Blanco C.G., Borrego A.G. 

Instituto Nacional del Carbón, Oviedo 

Corresponding author e-mail: Veneld@incar.csic.es 

The North of Spain is one of the southern limits in which peatlands as relics of the last deglaciation occur. They 

keep the record of environmental transformations having occurred in the area during different time spans over 

the last 10,000 years. This study covers the comparison of three peat bogs developed over a siliceous substrate 

close to the coast at heights of 125 m and 250 m and with thickness ranging between 50 and 300 cm. For each 

peat locality, a core has been obtained using a manual probe and has been split into 1 cm thickness samples for 

organic geochemical analysis. Freeze-dried samples were extracted with Dichloromethane/methanol (3/1) and 

the extracts separated into neutral (n-hexane) and polar fractions (dichloromethane/metha-nol 2:1). The polar 

compounds predominated over neutral compounds in all the samples with ratio being peat-dependant. The main 

components identified with palaeoenviron-mental potential were n-alkane, aliphatic ketone and different saturated 

and functionalised triterpenoids with potential for source input. Significant differences were observed in the relative 

proportion of these compounds in the various localities indicating different status of organic matter degradation 

and different organic matter input. The Ratio n C23/n C29 has been used to find out about dry and humid periods 

with higher plants vs. Sphagnum predominance in Huelga de Bayas (López Dias et al., J. Chromatogr. A, 2010, 1227: 

3538); Las Dueñas (López Dias et al., Int. J. Coal Geol in press) and Roñanzas (Ortiz et al., Org. Geochem. 2010, 41: 

454). The results indicate various humid episodes which can be correlated at regional scale. Acknowledgements: 

Financial support of the Principality of Asturias (PCTI IB08-072 C2) and the Spanish Ministry for Science and 

Innovation (CGL2009-13990-C02-01) is gratefully acknowledged. 


667


P20-052 IDENTIFICATION OF ORGANIC COMPOUNDS FROM ARCHAEOLOGICAL SAMPLES BY MEANS OF IN-SITU THERMALLY 

ASSISTED PYROLYSIS-SILYLATION GC-MS 

Doménech-Carbó M.T. 1 , Osete-Cortina L. 1 , Ahmadi H. 2 

1 


2 

Art University of Esfahan-Iran 


A number of natural products, among them, blood, egg, fats, plant gums, terpenoid resins and drying oils have been 

used as components of protective finishes and binding media of art objects since ancient times as they are organic 

products recognized as film-forming materials. Thus, a number of specialists in ancient cultures and rock art have 

conjectured on the use of these products as components of adhesives of all kind of objects and binding media 

of petroglyphs and rock paintings in earlier times. In addition to these specialised studies, organic compounds, 

in general, have attracted attention of scientists in the fields of art and conservation of heritage. Studies dealing 

with characterization and photochemical and thermal degradation of organic compounds, used as binding media 

and varnishes in art objects, have been performed by means of a variety of instrumental techniques. The present 

contribution presents the results obtained in the analysis of several archaeological and rock art samples in which 

was suspected that some organic compound was used as binding medium or adhesive. In-situ thermally assisted 

pyrolysis-silylation GC-MS using hexamethyldisilazane as derivatisation reagent has been used for this purpose. 

This technique has the analytical advantage of having high sensibility and reducing considerably the size of the 

samples tested. For this reason, it is especially suitable for detecting the presence of organic compounds in 

objects highly deteriorated such as rock art and buried archaeological remains. The study has been focused on 

the identification of the binding medium present in several rock paintings found in shelters of the Peruaçu valley 

(Minas Gerais, Brazil) as well as in the characterization of several glazed tiles from the Takht-e Soleyman palaces 

(Iran) (13th-15th centuries, Ilkhanian period), where it was suspected that gold sheets were fixed by means of an 

adhesive consisting of drying oil and sandarac resin (Kaman oil). This recipe is nowadays used by craftsmen in this 

region. A series of trials were performed at different temperatures ranging from 500ºC to 800ºC and pyrolysis time 

in order to determine the most suitable conditions for the analysis of the samples. A second series of analysis were 

performed with reference products reproducing the composition of the traditional Kaman oil that were naturally 

aged. The results obtained in the analysis of the series of samples from Peruaçu rock paintings evidenced that a 

fatty compound from animal origin was used as binding medium of these paintings. On the other hand, linseed oil 

was identified in the series of samples excised of the glazed ceramics from Takht-e Soleyman. Interestingly, this 

material has been preserved in an extraordinary low degree of oxidation due to the protective effect of the metallic 

layer. Acknowledgments Financial support is gratefully acknowledged from the Spanish MICINN project CTQ2008- 

06727-C03-01/BQU supported by ERDEF funds and the “Generalitat Valenciana” I+D project ACOMP/2009/171. The 

authors would like to acknowledge also Dr Helena David that kindly supplied samples from rock paintings of Peruaçu 

Valley. 

668 



P20-053 IDENTIFICATION OF MATERIALS EXTRACTED FROM CONTEMPORARY PAINTS AFTER SOLVENT CLEANING BY MEANS OF 

IN-SITU THERMALLY ASSISTED PYROLYSIS-SILYLATION GC-MS 

Silva M.F., Osete-Cortina L., Doménech-Carbó M.T. 



The study of the effects of solvent cleaning in the physical and chemical properties of contemporary paints, in 

particular, modern materials such as acrylic media whose use is nowadays widely extended (B Ormsby, T Learner, 

2009). is a subject that has been increasingly attracting the attention of the researchers in the field of science 

of conservation. Loss of extractable matter present in the paint as binding medium or additives (surfactants, 

thickeners, preservative, flame retardants, etc...) is one of the more evident changes that can be recognized in the 

composition of the paint film after a cleaning process carried out in current conservation practice. This loss of 

materials results in a noticeable change in the composition of the film as well as in its morphology and mechanical 

properties. In this context, it has been proposed a new analytical method for identifying additives present in 

commercial acrylic brands using in-situ thermally assisted pyrolysis-silylation GC-MS. This technique, that has been 

previously applied by the authors for the identification of acrylic and poly(vinyl acetate) paints, has the analytical 

advantage of having high sensibility and reduces considerably the size of the samples tested. Simple tests have 

been performed consisting of subjecting acrylic paint specimens of a number of commercial brands to immersion 

and swabbing in water and a selection of organic solvents. The proposed method consists of analysing solid 

dry extracts using hexamethyldisilazane as derivatisation agent. This method improves the conventional direct 

pyrolysis as it is possible the identification of the trimethylsilyl derivatives of acrylic and methacrylic acid that could 

be released from the polymeric binder in more or less extent depending on the ageing conditions of the paint. A 

series of trials were performed at different temperatures ranging from 500ºC to 800ºC in order to determine the 

most suitable temperature conditions for the analysis of the extracts. Thus, characteristic fragmentation pattern 

of polyethoxylate type compounds (PEO) were identified in the pyrograms of solid aqueous extracts from both 

immersion and swabbing tests. In contrast to this result, characteristic fragmentation patterns of acrylic polymers 

were dominating the pyrograms of the solid extracts obtained when the cleaning tests were performed using 

organic solvents with lower polarity such as ethyl acetate or acetone. The results obtained lead to conclude that 

the composition of extractable materials from acrylic paints is mainly depending on the solvent used for performing 

the cleaning of the film and, consequently, it influences the short, medium and long term behaviour of the paints. 

References Ormsby B., Learner T. 2009. The effects of wet surface cleaning treatments on acrylic emulsion artists’ 

paints, Reviews in Conservation 10: 29-42. Acknowledgments Financial support is acknowledged from the Spanish 

“I+D+I MEC” project CTQ2008-06727-C03-01/BQU supported by ERDEF funds, the “Generalitat Valenciana” I+D 

project ACOMP/2009/171 and the AP2006-3223 project ascribed to the program of predoctoral stages of universitary 

professors and researchers in Spanish universities and research centers from the Ministerio de Ciencia e Innovación 

(MICINN). 


669


P20-054 ON-LINE COLUMN-SWITCHING FAST LC-MS/MS FOR THE ANALYSIS OF PERFLUORINATED ORGANIC COMPOUNDS IN 

WATER SAMPLES 

Esparza X., Núñez O., Moyano E., Galceran M.T. 


Due to the capability of lowering the surface tension, perfluorinated organic compounds (PFCs) have been used 

since early 1980s in a variety of industrial and commercial applications (surfactants, paints, food packaging, and 

flame retardant foams), so they can enter in the environment from different stages. The persistence and ubiquity 

of perfluorocarboxylic acids (PFCAs) and perfluorosulfonic acids (PFSAs) in combination with their toxicological 

behaviour and global distribution has resulted in voluntary and regulatory actions by many manufacturers in United 

States to phase them out. Nevertheless, these compounds have been found in appreciable concentrations in food 

and environmental samples such as, sediments biota and surface water. The state of the art analysis of PFCs in 

environmental sample involves extraction generally followed by LC-MS/MS. Moreover when analyzing water samples 

at low levels of perfluorinated compounds (low ppt) a high concentration factor during sample preconcentration 

is required. For this purpose off-line solid phase extraction is currently used. However blank concentration must 

be avoided in order to obtain reliable data. In this work, in order to reduce sample manipulation an on-line SPE 

column-switching fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has been developed 

for the analysis of 13 PFCs in water. Water sample was loaded on a C18 reversed phase column without any 

sample treatment except filtration and the PFCs were backflush eluted and introduced in a LC-MS/MS system. The 

chromatographic separation was performed on a Fused-Core C18 reversed phase column (peak width < 20 s) in less 

than 5 min using a methanol:ammonium acetate (2 mM) mobile phase. Two small C18 columns sited at two different 

places between mobile phase reservoir and injection port have been used to eliminate contamination from mobile 

phase. Furthermore, to reduce interferences and increase selectivity and sensitivity, a triple hyperbolic quadrupole 

operating at 0.1 m/z unit FWHM in highly-selective selected reaction monitoring (H-SRM) was used. Method 

performance was evaluated and good linearity and precision (RSD < 20%) were obtained providing quantitation 

limits down to ng L-1 level. 1 E.I. du Pont de Nemours and Company. 2005. DuPont global PFOA strategy 

Comprehensive source reduction. U.S. EPA public docket AR226-1914. U.S. Environmental Protection Agency, 

Washington, DC. 2 van Leeuwen S.P.J., de Boer J. J. Chrom. A 1153 (2007) 172–185. 3 Kuklenyik, Z.; Needham, L. 

L.; Calafat, A.M. Anal. Chem. 2005, 77, 6085-6091. 4 Takino, M.; Daishima, S.; Nakahara, T. Rapid Comm. Mass 

Spectrom. 2003, 17, 383-390. 

670 



P20-055 LC-MS/MS FOR THE ANALYSIS OF PERFLUORINATED PHOSPONIC ACIDS IN WATER AND SEDIMENT SAMPLES 

Esparza X. 1 , Moyano E. 1 , Galceran M.T. 1 , van Leeuwen S.P.J. 2 

1 


2 

VU University 

Corresponding author e-mail: xaviesparza@gmail.com 

Since the early 1980s the use of perfluorinated organic compounds (PFCs) increased due to their surface tension 

lowering properties for a variety of industrial and commercial applications including surfactants, paints, food 

packaging, and fire-retarding foams. Recently, a new class of PFCs has emerged with a phosphonic acid as 

hydrophilic group1. The perfluorooctyl phosphonic acid (PFOPA) was listed as a high production volume chemical 

in the early of this century with an annual production between 4.500 to 230 tones2. The lack of available hazard 

data and potential concerns about persistence and toxicity were contributing factors in the U.S. EPA decision to 

no longer permit the use of these chemicals in food-use pesticides3. Perfluorinated phosphonic acids have no 

precursor compounds known. Migration from the emission sources will take place in the aqueous phase and they 

are also expected in soils and sediments. In this work, several columns, for basic pHs, perfluorooctyl column and 

conventional C18, have been tested to obtain the best peak shape for these compounds as well as the solvent 

injection (mobile phase, pH 9 and ion-pair). Both water and sediment sample needed a preconcentration and cleanup 

step and for that purpose Oasis WAX cartridges were evaluated. For sediment samples different extraction 

solvents have been studied. Finally an LC-MS/MS method for the determination of PFPAs and perfluorooctane 

sulfonate in environmental samples has been developed using a ZORBAX Rapid Resolution High Throughput column 

(30 x 2.1 mm, 1.8 µm). Acetonitrile:2mM ammonium acetate was used as mobile phase at a flow rate of 300 μL min-1 

with gradient elution. MMI mode working in soft APCI condition was used as ionisation source. 0.5L were passed 

through WAX cartridges, eluted, evaporated and reconstituted in MeOH:water (1:1) with a 25mM tetrabutylammonium 

(TBA) concentration. Sediment samples were extracted with a mixture of THF:water and then loaded into the 

cartridges and followed the same procedure than the water samples. Before injecting 5 μL in the LC-MS/MS system 

extracts are filtered through 0.22 μm GHP filters. The method showed limits of detection at ng L-1 level and provided 

good linearity and precision (RSD < 20%). 1 D’eon J., Crozier P. W., Furdui V. I., Reiner E. J., Libelo E. L., and Mabury 

S. A. Environmental Toxicology and Chemistry, 28, 2101-2107, 2009. 2 Howard P.H., Meylan W. 2007. EPA Great 

Lakes study for identification of PBTs to develop analytical methods: Selection of additional PBTs—Interim Report. 

EPA Contract EP-W-04-019. U.S. Environmental Protection Agency, Syracus, NY. 3 U.S. Environmental Protection 

Agency. 2006. Inert ingredient; revocation of the tolerance exemption for mono- and bis-(1H,1H,2H,2H-perfluoroalkyl) 

phosphates where the alkyl group is even numbered and in the C6–C12 range. U.S. EPA Public Docket OPP-2006- 

0253. Washington, DC. 


671


P20-056 A NEW IN-VIAL CONFIGURATION FOR MEMBRANE ASSISTED LIQUID-LIQUID MICRO-EXTRACTION. APPLICATION TO THE 

DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN SEAWATER AND MARINE SEDIMENTS BY GC-MS 

March J.G. 1 , Moukhchan F. 2 , Cerdà V. 1 , Simonet Suau, B.M. 3 

1 

University of Balearic Islands 

2 

University Abdelmalek Essaâdi-Marroco 

3 


Corresponding author e-mail: joan.march@uib.es 

A novel arrangement for microporous membrane liquid-liquid extraction is presented. The device consisted of a 

stoppered glass micro-vial containing the organic solvent where the septum of the screw stopper was replaced 

by a sized piece of membrane (PTFE bonded to a high density polyethylene support 150 micro-m wall thickness, 

porosity 85%, 0.22 micro-m pore size) which is hermetically assembled to the volumetric flask containing the 

aqueous donor solution. The placement of the membrane in alternative contact with the solutions was achieved 

by orbital agitation. The organic extract can be analysed by GC-MS without any further handling, being the small 

quantity of organic solvent used, the achieved sample cleanup, and the minimal risk of cross contamination 

significant operational advantages of the proposed arrangement. As a preliminary work, the extraction of seven 

polycyclic aromatic hydrocarbons, namely: naphthalene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene 

and benzo(a)anthracene from spiked aqueous solutions were studied. Main variables that affected the extraction 

efficiency were the organic solvent, the presence of organic solvents miscible with water in the donor solution, 

the ionic strength of the donor solution and the extraction time. Working with 150 micro-L of decane, as organic 

solvent, after a three hours extraction time, the extraction efficiency was of the order of 20 % (depending on the 

ratio of volume of phases). Alternative configurations, as the complete immersion of the micro-vial into a large 

volume of sample (of the order of 1 L), and the irradiation with ultrasound are really under study. Polycyclic aromatic 

hydrocarbons are frequently detected in marine environments being its possible origin anthropogenic (pyrolytic and 

petrogenic) and also natural. Being the origin anthropogenic the most important, the distribution is very dependent 

on the geographic area. Due to the adverse health effects on marine organisms and humans, its determination has 

attracted the attention of the scientific community. In fact several of such compounds are included in the priority 

list of the European Union’s Water Framework Directive. Due to the hydrophobic character of such compounds, in 

seawater the occurrence is at very low concentration (< 1 ng L-1) due to the tendency to absorb onto particulate 

matter so that they are transported to and accumulate in sediments. The analytical methods typically involved 

extraction with considerable amounts of organic solvents (Soxhlet and ultrasonic or microwave assisted), followed 

by cleanup and chromatographic analysis. For mentioned reasons, (real) marine sediments and spiked seawater 

were selected to asses the usefulness of the in-vial membrane liquid-liquid extraction with the aim of improve the 

procedure for sample preparation. Acknowledgements This work was supported by the Spanish “Ministerio de 

Ciencia e Innovación” (project CTQ2006-01851). F. Moukhchan acknowledges the fellowship from the Averroes 

program. 

672 



P20-057 DEVELOPMENT OF DATA PROCESSING WORKFLOWS FOR THE AUTOMATED DETECTION OF TRANSFORMATION 

PRODUCTS OF EMERGING CONTAMINANTS IN THE ENVIRONMENT) 

García-Reyes J.F. 1 , Pérez-Parada A. 2 , Gómez-Ramos M.M. 2 , Fernández-Alba A.R. 2 , Robles-Molina J. 1 , Domínguez-Romero J.C. 1 , Gilbert- 

López B. 1 , Molina-Díaz A. 1 , Agüera A. 2 

1 


2 


Corresponding author e-mail: jfgreyes@ujaen.es 

Current research efforts in environmental analytical chemistry include the development of advanced instrumentation 

and the development of analytical methodologies with the ability of screening and detect hundreds of contaminants 

at ultratrace levels. This performance should not be limited to known species (target analysis approach) but also 

to unknown or unexpected species (non-target analysis approach). A typical example of the later is the analytical 

studies conducted for the evaluation of advanced oxidation technologies, which involves the detection of the target 

species subject to degradation and the formation of different transformation products. These studies are usually 

accomplished manually after a comprehensive visual inspection of the files that may take days or even weeks. In this 

work, we propose the use of different approaches in order to automate the procedure of identifying transformation 

products of emerging contaminants in samples of environmental interest. The proposed strategies are based on 

the use of advanced LC-MS data processing (molecular feature extraction) software combined with user-created 

libraries of target species of interest (including molecules and characteristic fragment ions common to a chemicalclass, 

so called “diagnostic ions”). A tentative 2-step approach would consist of a target database search using 

an AMRT database (accurate mass of parent ions + retention times), followed by a non-target screening step using 

the diagnostic ions of the detected species during the first step. Besides, alternatively an in-silico approach based 

on the theoretical calculation of tentative transformation products of the targeted parent species (based on typical 

metabolization reactions (neutral losses of functional groups, (de)methylation, oxidation, (de)chlorination, etc)) is also 

being evaluated as a complementary tool. The different proposed approaches are being tested with degradation 

studies of emerging contaminants using advanced oxidation technologies. The authors acknowledge funding from 

the Spanish Ministry of Education and Science (Project CSD2006-00044 CONSOLIDER INGENIO 2010 “TRAGUA 

project” entitled Treatment and Reuse of Wastewaters for Sustainable Management). 


673


P20-058 DETERMINATION OF PENICILLINS AND ITS MAIN DEGRADATION PRODUCTS IN INFLUENT AND EFFLUENT FROM A 

WASTEWATER TREATMENT PLANT BY LC-Q-TOF 

Pérez-Parada A. 1 , Heinzen H. 2 , Gómez-Ramos M.M. 1 , García-Reyes J.F. 3 , Agüera A. 1 , Fernández-Alba A.R. 1 , 

1 


2 

Universidad de la República, Pharmacognosy & Natural Products-Uruguay 

3 


Corresponding author e-mail: amadeo@ual.es 

Penicillins are a sub class of β-Lactam antibiotics that have been the most widely used as antimicrobial for more 

than 80 years and still constitute the most important group of antibiotics to treat or prevent bacterial infections in 

humans and animals. Antibiotic contamination in the aquatic environment has paid special concern as it may include 

the appearance of resistance. Penicillins are not usually thought to be a serious threat to environment because of 

the poor stability of the β-lactam ring but the continuous and high level introduction of these compounds in aquatic 

systems needs to be monitored. Despite that, scarce information is available of their residues in water as well as 

metabolites and degradation products. Concerning analysis, penicillins are known for their instability at the common 

conditions used in multi-residue determination that involves the use of organic solvents like methanol usually applied 

in extraction and clean-up steps. On the other hand, degradation due to pH conditions also occurs. In this sense, 

the determination of penicillins has to involve proper analytical methods in sample preparation protocols, as well as 

enhanced quality control of standards and increased capabilities of used instrumental techniques. To investigate the 

fate of penicillins in wastewater from a treatment plant, an analytical method was developed for the simultaneous 

determination of six penicillins (amoxicillin, cloxacillin, dicloxacillin, oxacillin, penicillin-G and penicillin-V), and 

the screening of its main pH dependant degradation products (penilloic, penicilloic, penillic, isopenillic acids and 

penicilloaldehyde intermediates) and degradation products during analysis (penicillin methyl esters) using off-line 

solid phase extraction (SPE) followed by liquid chromatography – quadrupole-time of flight mass spectrometry 

(LC-QTOF-MS/MS). We focus the determination of penicillin residues but also the unequivocal detection of 

degradation products in environmental (mainly hydrolysis degradation products) and analytical conditions 

(methanolysis degradation products) by tandem (MS/MS) accurate mass spectrometric measurements. We found 

that residues of penicillins were absents in real influent and effluent wastewater samples whereas amoxicillin 

2’,5’diketopiperazine, amoxicillin penamaldic acid and penicillin-G penillic acid and their penicilloaldehydes were 

present in both. We suggest that there are two simultaneous routes of degradation of penicillins in wastewater 

through hydrolysis products and internal rearrangement of the molecule and that these compounds are more stable 

in the aquatic environment than penicillins itself, even through wastewater treatment. 

674 



P20-059 LEVELS OF DRUGS OF ABUSE IN SUPERFICIAL WATERS OF THE OLIVA-PEGO MARSH (VALENCIA, SPAIN) 


1 


2 


Corresponding author e-mail: pablovroig@gmail.com 

The use of drugs of abuse is increasing worldwide and causes not only a well-known serious social problem but also 

concern as environmental emerging contaminants. Data provided by the European Monitoring Centre for Drugs and 

Drug Addiction (EMCDDA) estimate that, in the last year, 22.5 million Europeans (between 15 and 64 years) smoked 

cannabis (22% of European adults), 12 million sniffed cocaine, 11 million took amphetamines and 9.5 millions took 

ecstasy. Regarding opiates, the report did not show a decline in the epidemic problems linked to heroin. Drugs 

of abuse are excreted unmetabolized or as metabolites in urine and feces, in fact, most consumed ones have 

been determined in the sewage systems and in natural waters [1,2]. However, due to the limited extent of research 

undertaken in this field, there is limited data and minimal understanding of the environmental occurrence, transport, 

fate and exposure for these compounds. One of the most important environments in the Mediterranean areas of 

Spain are saltmarshes. 

The aim of the present work is to expand the range of Spanish wetlands investigated to the Oliva-Pego marsh. This 

area, with an approx. area of 1,290 ha, is found in the extreme south of the Gulf of Valencia, bounded to the north by 

the Mustalla mountains and the Bullent river, bounded to the east by the alluvial delta of the Pego plain and to the 

west by the Mediterranean Sea. In order to check the levels of drugs of abuse, twenty-three samples were taken in 

this wetland, on April 2009. Samples were taken from the lagoon and from the most important channels that flow in 

itself. 

A simple and robust method using solid-phase extraction (SPE) and liquid chromatography tandem mass 

spectrometry (LC-MS/MS) for the simultaneous determination of 14 drugs of abuse and their metabolites (cocainics, 

amphetamine-like compounds, cannabinoids, and opiates) in surface waters was developed. The highest recoveries, 

as well as the simplest protocol, were obtained for Oasis HLB cartridges (6 mL/200 mg) using 250 mL of water. 

The proposed method was linear in a concentration range from 0.03–6 ng/L to 300–60,000 ng/L depending on 

the compound, with correlation coefficients higher than 0.998. Matrix effects have been studied in surface water 

samples, and several isotope-labelled internal standards have been evaluated as a way to compensate the signal 

suppression observed. Limits of quantification (LOQs) ranged from 0.03 to 5.13 ng/L, respectively. Recoveries were 

71–102% at the LOQ level and 77–104 at 50 ng/L. 

Many of the tested substances were found at concentrations in the range of ng/L. The results of these samples 

confirmed that the method is suitable for screening these compounds in the environment, highlighting the 

convenience to carry out a better treatment of residual waters before its discharge in the environment. 

References: 

[1] C. Postigo, M. J. Lopez de Alda, D. Barceló, TrAC-Trends Anal. Chem. 27 (2008), 1053-1069. 

[2] P. Vazquez-Roig, V. Andreu, C. Blasco, Y. Picó, Anal. Bioanal. Chem., In press 


675


P20-060 POLLUTION BY PHARMACEUTICALS IN A TYPICAL MEDITERRANEAN WETLAND ECOSYSTEM 


1 


2 


Corresponding author e-mail: pablovroig@gmail.com 

In this study, 17 pharmaceuticals belonging to different and most common therapeutical classes were analyzed in 

samples of water, soil and sediment into the natural park of L´Albufera (Valencia, Spain). These compounds occurred 

in the environment through undepurated sewage waters from urban origin, together with those from wastewater 

treatment plants [1,2]. 

Since pharmaceutical products can have long half-lives, they accumulate, reaching detectable and biologically 

active amounts. The environmental persistence of several medicinal drugs, such as Clofibric acid, the main 

metabolite of clofibrate, has an estimated persistence in the environment of 21 years. The most known problem with 

pharmaceuticals is the possibility of producing resistance genes in bacteria that can render particular antibiotics 

useless. Among other antibacterials, resistance genes of tetracycline have been reported in lagoons and the 

groundwater underlying two swine-production facilities [3]. 

In April 2008 and October 2008 a total of 70 samples of water, soil and sediment were collected, corresponding to 

different sampling points previously designed, and covering the most important channels that flow in to the lake. 

Pharmaceuticals were isolated and concentrated from water samples by solid phase extraction (SPE) through an 

Oasis HLB cartridge eluting with methanol. From sediment and soil samples, these compounds were extracted 

with water at 70 ºC using pressure liquid extraction (PLE) prior to SPE All extracts were analyzed by liquid 

chromatography–tandem mass spectrometry (LC-MS/MS), using both, positive and negative ionization with an ESI 

interface. Except for ibuprofen, two transitions were selected for each analyte to obtain unambiguous confirmation. 

Ibuprofen-d3 for negative ion mode, and carbamazepine-d10 and acetaminophen-d3 for positive ion mode were 

utilized as internal standards. 

Recoveries ranged from 74% to 101% in waters, and from 62 to 119% in soil and sediments. The LOQ ranged from 

0.03-6 ng/L in waters and 0.06-46 ng/g in soils and sediments. 

All water samples analyzed were contaminanted by pharmaceuticals. Carbamazepine was the substance more 

frequently detected followed by ibuprofen, acetaminophen and sulfamethoxazole. Tetracycline, oxytetracycline and 

fenofibrate were not present in water samples but they were in soil or sediment samples and norfloxacin was not 

detected in any of the samples. The highest concentrations correspond to acetaminophen (17.70 µg/L) and ibuprofen 

(3.91 µg/L) both of them in the same sample. 

The 70% of the samples of sediments taken contain pharmaceuticals. Carbamazepine was the substance more 

frequently detected (9 samples) followed by codeine and acetaminophen. Oxytetracycline and fenofibrate were 

present in some samples. The highest concentrations correspond to ibuprofen (35.83 µg/kg). 

In soils, the concentration of pharmaceuticals was lower than in sediment samples. Carbamazepine was the 

substance more frequently detected (9 samples). The highest concentrations correspond to acetaminophen (16.05 

µg/kg). 

Literature: 

[1] Y. Picó, C. Blasco, TrAC Trends Anal. Chem. 28 (2009) 745-757 

[2] P. Vazquez-Roig, R. Segarra, C. Blasco, V, Andreu, Y. Picó. J. Chromatogr. A 1217 (2010) 2471-2483 

[3] S.C. Kim, K. Carlson. TrAC Trends Anal. Chem. 24 (2005) 635-644. 

676 



P20-061 HYPHENATED TECHNIQUES FOR THE ANALYSIS OF LINEAR ALKYLBENZENESULFONATES IN TECHNICAL FORMULATIONS 

Piechotta C. 1 , Schmidt S. 1, Nehls I. 1 , Godejohann M. 2 , Mügge C. 3 

1 

Federal Institute for Materials and Testing (BAM)-Germany 

2 

Bruker BioSpin GmbH 

3 

Humboldt-University Berlin 

Commercially available linear alkylbenzenesulfonates (LASs) are a mixture of various homologues and isomers, 

leading to 20 major species. In this work we investigated the technical LASs formulations by liquid chromatographysolid 

phase extraction-nuclear magnetic resonance spectroscopy-mass spectrometry (LC-SPE-NMR/MS). The 

commercial product was separated into 17 fractions by liquid chromatography (LC). After chromatographic 

separation, 5% of the flow was split to a mass spectrometer (MS) while 95% was send to post-column solid phase 

extraction cartridges for enrichment of the analytes (LC-SPE). After elution from the SPE-cartridges a NMRspectrometer 

equipped with a cryo-probe was used for the characterisation of the different LASs species. For the 

first time 1H-1D and H-H-COSY spectra for 14 LASs species out of 20 major isomers are presented, whereas the 6 

remaining species are detected as mixtures in 3 1H-1D and H-H-COSY spectra. These data were used to correlate 

the chromatographic retention of the LASs isomers to the substitution pattern of the alkyl chain [1]. 

[1] S. Schmidt, C. Piechotta, M. Godejohann, T. Win, I. Nehls, C. Mugge, Characterisation of commercially available 

linear alkylbenzenesulfonates by LC-SPE-NMR/MS (liquid chromatography-solid phase extraction-nuclear magnetic 

resonance spectroscopy-mass spectroscopy), Talanta, Volume 82, Issue 1, 30 June 2010, Pages 143-150 


677


P20-062 DETERMINATION OF NITROMUSKS IN WATER SAMPLES BY DISPERSIVE LIQUID-LIQUID MICROEXTRACTION FOLLOWED 

BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

Chisvert A., Lopez-Nogueroles M., Salvador A., Carretero A. 


Corresponding author e-mail: alberto.chisvert@uv.es 

Musk compounds have been used as fragrances in many consumer products as they are valuable compounds not 

only for their unique odour but also for their fixative properties. Natural musk was already used in ancient times 

[1]. The term musk is also used to name other compounds, with totally different chemical structure but possessing 

musk-like odour properties. These are commonly named synthetic musks and appeared due to economical and 

ethical reasons. Artificial musks have been generally divided in three subgroups: nitromusks, polycyclic musks and 

macrocyclic musks. Nitromusks are basically formed by musk xylene (MX), musk ketone (MK), musk ambrette (MA), 

musk tibetene (MT) and musk moskene (MM). Nitromusks are believed to be persistent pollutants due to their strong 

tendency to bioaccumulate and health risky agents as they are shown to be related with different types of dermatitis, 

carcinogenic effects and endocrine dysfunction. In fact, the use in cosmetic products of MA, MT and MM is banned 

in the European Union while the use of MX and MK is restricted [2]. The removal of nitromusks in municipal sewage 

treatment plants has been estimated to be between 40 and 60% [3]. Thus, they appear in the aquatic environment 

and it is important to evaluate its potential for bioaccumulation on aquatic ecosystems. The purpose of this work 

is to develop a dispersive liquid-liquid microextraction (DLLME) gas chromatography-mass spectrometry (GC- 

MS) method for the simultaneous determination of the five above-mentioned nitromusks in natural water samples. 

DLLME is a very simple and rapid extraction technique developed by Reazee et al. [4]. This technique is based on 

a ternary component solvent system in which the non-miscible extraction solvent is dispersed into the aqueous 

sample with the aid of a disperser solvent (which is miscible in both extraction solvent and sample). The surface area 

between the extraction solvent and the aqueous sample is infinitely large, so equilibrium state is achieved quickly. 

The variables involved in the DLLME process, such as the ionic strength and the pH of the sample and the type and 

volume of both extraction and disperser solvents, were optimized. Under optimized conditions, 1000 μL of ethanol 

(disperser solvent) containing 50 μL of carbon tetrachloride (extraction solvent) were injected into 5 mL of aqueous 

sample adjusted to pH 4. Then, 1 μL of the sedimented phase was injected to the GC-MS system. The linearity 

studied reached 20 μg/L in all cases and the limit of detection was in the ng/L range. [1] A. Chisvert, A. Salvador, 

Perfumes in Cosmetics. Analytical Methods in: A. Salvador, A. Chisvert (Eds.), Analysis of Cosmetic Products, 

Elsevier, Amsterdam,2007. [2] Regulation (EC) Nº 1223/2009 of the European Parliament and of the Council of 30 

November 2009 on cosmetic products [3] D. R. Dietrich, B. C. Hitzfeld, Bioaccumulation and Ecotoxicity of Synthetic 

Musks in the Aquatic Environment in: G.G. Rimkus (Ed), The Handbook of environmental Chemistry 3 (2004) 233 [4] 

M. Rezaee, Y. Assadi, M.R. Milani Hosseini, E. Agahaee, F. Ahmadi, S. Berijani, J.Chromatogr. A, 1116 (2006) 1 

678 



P20-063 MOLECULARLY IMPRINTED POLYMERIC FIBERS (MONOLITHS) FOR SOLID-PHASE MICROEXTRACTION FOR THE 

DETERMINATION OF TRIAZINES COUPLED WITH GAS CHROMATOGRAPHY 

Díaz-Álvarez M., Paniagua E., Turiel E., Martín-Esteban A. 


Corresponding author e-mail: mdalvarez@inia.es 

Solid-phase microextraction (SPME) is widely used in analytical laboratories for the analysis of organic compounds, 

thanks to its simplicity and versatility. However, the current commercially available fibers are based on nonselective 

sorbents, making difficult in some cases the final determination of target compounds by chromatographic 

techniques. In the present work, a simple polymerization of molecularly imprinted polymeric fibers to be used in 

SPME is proposed. The polymerization strategy is based on the direct synthesis of molecularly imprinted polymeric 

fibers (monoliths) using silica capillaries as molds, with silica being etched away after polymerization. The system 

propazine: methacrylic acid was used as a model for the preparation of molecularly imprinted fibers, and its ability 

to selectively rebind triazines was evaluated. Imprinted fibers were thermally stable up to 300 ºC which permitted 

thermal desorption of target analytes at the injection port of the gas chroamtograph. The effect of extraction time, 

desorption time and temperature on extraction efficiency of the analytes were investigated. Besides, the use of 

prometryn as internal standard, bound to the fiber prior sample extraction, was evaluated. Acknowledgements: 

Authors wish to thank Spanish Ministry of Science and Innovation for financial support (CTQ2008-02607) 


679


P20-064 MULTI-RESIDUE METHOD FOR THE ANALYSIS OF SYNTHETIC SURFACTANTS AND THEIR DEGRADATION METABOLITES 

IN AQUATIC SYSTEMS BY LIQUID CHROMATOGRAPHY – TIME-OF-FLIGHT – MASS SPECTROMETRY 

Lara-Martín P.A. 1 , Traverso-Soto J.M. 1 , Brownawell B.J. 2 , González-Mazo E. 1 

1 

University of Cádiz 

2 

Stony Brook University 

Corresponding author e-mail: pablo.lara@uca.es 

Synthetic surfactants are economically important chemicals, as they are widely used in household cleaning 

detergents, textiles, paints, polymers, personal care products… In this work we have focused our attention on 

developing a method capable of the simultaneous analysis of some of the most widely used surfactants (linear 

alkylbenzene sulfonates, LAS, nonylphenol ethoxylates, NPEO, and alcohol ethoxylates, AEO) in aqueous (surface 

water, pore water) and solid (sediments, suspended solids) matrices. First, analytes were extracted by ultrasonic 

extraction from sediments and suspended solids, using methanol at 50ºC as solvent and 3 cycles (30 min per 

cycle). Clean-up and pre-concentration of the extracts and water samples were carried out by means of solid-phase 

extraction (SPE), using Oasis HLB cartridges and methanol and dichloromethane as eluting solvents. Recoveries 

were generally about 80% for most compounds, decreasing for those homologues having highest hydrophobicity 

(e.g. C13LAS or C18AEO). Identification and quantification of target compounds were performed by liquid 

chromatography - time-of-flight - mass spectrometry (LC-ToF-MS), using full-scan and electrospray ionization (ESI) 

in both positive and negative mode. Confirmation of the presence of degradation metabolites such as sulfophenyl 

carboxylic acids (SPC), nonylphenol ethoxycarboxylates (NPECs) and polyethylene glycols (PEG) in environmental 

samples was also carried out not only by accurate mass measurement, but also using several ions (Na+ and NH4+ 

adducts) and 13C isotopes, which, in addition, were useful to improve the quantification of the target compounds 

despite the relatively limited dynamic range of ToF detectors. Finally, this methodology was validated by measuring 

the concentration of analytes in several marine sediment and seawater samples collected at Long Island Sound (NY, 

USA). 

680 



P20-065 MULTI-RESIDUE ROUTINE ANALYSIS OF 57 PESTICIDES BY GAS CHROMATOGRAPHY COUPLED WITH TIME OF FLIGHT 

MASS SPECTROMETRY IN HONEYBEE’S POLLENS 

Wiest L., Arnaudguilhem C., Giroud B., Buleté A., Fratta C. 

Service central d’analyse CNRS 

Corresponding author e-mail: l.wiest@sca.cnrs.fr 

Bee’s mortality has never been so high all over the world and it is very important for beekeepers to understand 

relationships between pesticides treatment and bees’ mortality. The aim of this study is, at the same time, to develop 

a fast, cheap and sensitive tool to analyse pesticides and to obtain a global view of contaminants presence in 

honeybees’ environment. The increasing number of launched pesticides induces the development of methods which 

allow the extraction of pesticides of very different physico-chemical properties, like QuEChERS, and the use of 

analytical techniques which provide information on untargeted contaminants, such as, gas chromatography coupled 

with Time of Flight mass spectrometry. 

A multi residue analysis was developed for quantification of 57 pesticides, belonging to different chemical classes, in 

pollens. It consists in one single extraction, based on QuEChERS method, followed by gas chromatography coupled 

with Time of Flight mass spectrometry. QuEChERS method, which involves a salting-out liquid-liquid extraction with 

acetonitrile followed by a dispersive-SPE clean up, was adjusted to pollen, an high fat matrix, by taking out lipids, 

which interfered with mass spectrometry analysis, with a small fraction of hexane in acetonitrile. 

This method, combined with high resolution detection, allowed quantification and confirmation at levels as low as 10 

ng/g with recoveries between 70 and 120 %. This methodology will be extended to liquid chromatography coupled 

with tandem-mass spectrometry in order to quantify 30 other pesticides and adjusted to honeys and honeybees. 

Application to 40 samples of each matrix is planned for a global view of pesticide presence in honeybees’ 

environment. 


681


P20-066 ANALYSIS OF THE OCCURRENCE AND RISK ASSESSMENT OF PESTICIDES IN THE LLOBREGAT RIVER (NE, SPAIN) BY 

ONLINE SPE-LC(ESI)-MS/MS 

Köck-Schulmeyer M., Osorio V., Pérez S., de Alda M.L., Ginebreda A., Barceló D. 

CSIC, IDÆA, Barcelona 

The Llobregat River basin (NE part of Spain) covers a catchment area of about 4.948 km2 and during the last 

decades it has been highly polluted by industrial and urban wastewaters, and by surface runoff from agricultural 

areas. The river has two main tributaries, the Cardener River and the Anoia River, and all three rivers receive the 

input of various sewage treatment plants effluents. With the aim of investigating the occurrence and impact of 16 

polar pesticides coming from these sources, a multi-residue method based on isotope dilution on-line solid phase 

extraction-liquid chromatography-electrospray-tandem mass spectrometry (on-line SPE-LC-ESI-MS/MS), was 

applied to the analysis in triplicate of 22 surface water samples collected from two sites of the Llobregat River: site 

SJD (Sant Joan Despì), which is located close to the mouth, and site MT (Minas Públicas Aigües Terrasa), which is 

located some 30 Km upstream, before the confluence of the Anoia tributary. Samples were collected at intervals 

of two-to-eight days during the period October 13-November 18, 2009. Chemical analysis showed the presence of 

simazine, tertbuthylazine, diuron, and diazinon in more than 90% of the samples, and the absence of cyanazine, 

alachlor, and molinate. The maximum individual concentration was observed for diazinon (130 ng/L in SJD the last 

sampling date). Total pesticides concentrations varied between 161 and 4 ng/L, and increased with rainfall and 

river discharge (flow-rate). Comparatively, site SJD presented, as expected, higher concentrations than site MT, 

due to the contribution of two important tributaries. In terms of ecotoxicological risk, the Short-Term Pesticide Risk 

Index for the Surface Water System (PRISW-1)(ref1) calculated for each sample considering the pesticides toxicity 

against three aquatic organisms (algae, Daphnia, and fish), indicated low risk in site MT with Daphnia being the 

most sensitive organism, and medium risk in site SJD, specially for algae. Acknowledgement: This work has been 

supported by the Catalan Water Agency and the Spanish Ministry of Science and Innovation through the projects 

SCARCE (Consolider-Ingenio 2010 CSD2009-00065) and CEMAGUA (CGL2007-64551/HID). Merck is acknowledged 

for the gift of LC columns. Marianne Köck acknowledges the European Social Fund and AGAUR (Generalitat de 

Catalunya, Spain) for their economical support through the FI pre-doctoral grant. ref1: A. Finizio, M. Calliera and M. 

Vighi, Ecotoxicology and Environmental Safety, 2001, 49, 262-274. 

682 



P20-067 ANALYSIS OF 18 PERFLUORINATED COMPOUNDS IN SOILS AND BIOTA FROM TIERRA DEL FUEGO AND ANTARCTIC BY 

LC-MS/MS 

Llorca M. 1 , Farré M. 1 , Alonso B. 2 , Barceló D. 1 

1 


2 

Área Táctica-Spain 

Corresponding author e-mail: mfuqam@cid.csic.es 

Perfluorinated compounds (PFCs) are persistent, bioaccumulable and biomagnified due to their physicochemical 

properties, and they are dispersed globally. Different studies have reported the presence of these com pounds 

in Arctic fauna: Polar Bears [1] and marine food web [2]. However much less studies reported results from the 

Antartic continent [3-5]. This study reports the results of different samples from the Antarctic continent and Tierra 

de Fuego. The sampling campaign was carried out under the frame of “Premios Antárticos de Ciencia, Tecnología 

y Medio Ambiente 2010”, along February and March of 2010, and the main aim of this work was to assess PFCs in 

fauna, biota and soils from these areas. The presence of 18 PFCs including perfluorobutanoic, perfluoropentanoic, 

perfluorohexanoic, perfluoroheptanoic, perfluorooctanoic, perfluorononanoic, perfluorodecanoic, 

perfluoroundecanoic, perfluorododecanoic, perfluorotridecanoic, perfluorotetradecanoic, perfluorohexadecanoic 

and perfluorooctadecanoic acids, perfluorobutanesulfonate, perfluorohexasulfonate, perfluorooctanesulfonate and 

perfluorodecanesulfonate; and perfluorooctanesulfonamide. A total number of 35 samples and blanks including 

fish (trout), superficial soils, guano, algae (Macrocystis Pyrifera), dung and tissues of Papua penguin samples were 

included. The samples were pre-treated in a central laboratory located in Ushuaia (Argentina) prior to their shipment 

to Spain. Sample pre-treatment were based on ultrasonic extraction of soils, and alkaline digestion of biota samples 

followed by solid phase extraction (SPE), in both cases. Extract were analyzed using liquid chromatography coupled 

to a quadrupole linear ion trap mass spectrometry (LC-QLIT-MS/MS). 44 % of the total PFCs were quantifiable in 

analyzed samples with concentrations ranging 0.12-200 ng/g and 100 % of samples presented PFCs, however most 

of the analyzed PFCs were below method limit of quantification, or detection in some cases. Comparing results from 

Tierra de Fuego and Antarctic, highest values were obtained in Tierra del Fuego as was expected. However, should 

be pointed out the presence of PFCs and quantifiable levels in all samples. PFCs acids presented, in general, the 

highest levels and PFBA, PFPA, PFHxA, PFOA, PFOS and PFNA have been found more frequent. References: [1] 

R.Dietz, R.Bossi, F.F.Rigét, C.Sonne, E.W.Born. Environmental Science and Technology, 2008, 42, 2701– 2707. [2] 

G.Tomy, W. Budakowski, T.Hallordson, P.Helm, G.Stern, K.Friesen, K.Pepper, S.Tittlemier, A.Fisk. Environmental 

Science and Technology, 2004, 38, 6475-6481. [3] A.Schiavone, S.Corsolini, K.Kannan, L.Taob, W.Trivelpiece, 

D.Torres Jr., S.Focardi. Science of the Total Environment. 407 (2009) 3899–3904. [4] L.Ahrens, Z.Xie, R.Ebinghauset. 

Chemosphere, 78 (2010) 1011–1016. [5] A.Dreyer, I.Weinberg, C.Temme, R.Ebinghaus. Environmental Science and 

Technology, 2009, 43, 6507–6514. 


683


P20-068 GEOGRAPHIC AND TROPHIC PATTERNS OF OCS IN PELAGIC SEABIRDS FROM THE NORTHEAST ATLANTIC: 

A MULTI-SPECIES/MULTI-LOCALITY APPROACH 

Roscales J.L. 1 , Muñoz-Arnanz J. 1 , González-Solís J. 2 , Jiménez B. 1 

1 

Institute of Organic Chemistry, CSIC, Madrid 

2 


Corresponding author e-mail: jlroscales@gmail.com 

Trophic ecology and geographic location have been shown to be crucial factors explaining OC levels in marine 

vertebrates, but these factors are often difficult to disentangle (1). To examine their relative influence and thus, 

evaluate the suitability of pelagic seabirds as indicators of organochlorine contamination from open waters in 

northeast Atlantic, PCBs and DDTs as well as stable-nitrogen isotope signatures (δ15N) were analyzed in the blood 

of several species across multiple breeding localities from the northeast Atlantic Macaronesian archipelagos (i.e. 

Azores, Madeira, Canary Is. and Cape Verde). High-Resolution Gas Chromatography with Micro-Electron Capture 

Detection was the chromatographic technique used for the identification and quantification of Organochlorine 

compounds. Since δ15N can be used to delineate the trophic position of marine consumers (2) and seabird species 

were sampled across several localities, this approach offers an excellent opportunity to understand the relevance 

of breeding locality and trophic ecology shaping the contaminant burdens of marine predators. Overall, our results 

show that both breeding locality and species have a marked influence on seabird organochlorine contamination. 

Large scale geographic patterns emerged due to the greater OC levels found in northern (Azores and Madeira) 

compared to southern Macaronesian archipelagos (Canary Is. and Cape Verde). This pattern is probably associated 

with long-range pollutant transport from North American coasts over mid-north Atlantic regions. In addition, our 

results suggest recent inputs of DDTs in the case of southern Macaronesian archipelagos, which could be related to 

the use of this pesticide in the Sub-Saharan African countries. Trophic ecology also was a major factor explaining 

OC levels. We found consistent differences among species contaminant burdens within each breeding locality 

but δ15N showed a strong overlap for most of them. That is, seabirds feeding on mesopelagic (200-1000 m depth) 

preys showed significant greater OC levels than those feeding on epipelagic preys (0-200 m depth), resulting in a 

lack of correlation between seabird OC levels and trophic position as shown by δ15N. This indicates that different 

seabird species may show similar trophic positions but, due to vertical migrations of some fish and squid species, 

may feed on prey from different levels of the water column, resulting in different OC burdens and obscuring the 

biomagnification processes. In sum, our findings suggests that multispecific approaches could be used to monitor 

the vertical dynamics of OCs in the marine environment, thereby providing a more comprehensive evaluation of 

marine pollution. Moreover, this study illustrate that multispecies/multi-localities approaches can better help us to 

understand the relative contribution of geography and feeding ecology to the OC burdens than a single speciesbased 

strategy. (1) Fisk, A. T. et al. Environmental Science & Technology. 2001. 35, 732-738. (2) Kelly, J. F. Canadian 

journal of Zoology. 2000. 78, 1-27. 

684 



P20-069 ENANTIORESOLUTION OF SOME TRIAZOLE AND RELATED CHIRAL PESTICIDES BY CAPILLARY ELECTROPHORESIS- 

PARTIAL FILLING TECHNIQUE USING HIGHLY SULFATED β-CYCLODEXTRIN AS CHIRAL SELECTOR 

Martin-Biosca Y. 1 , Villanueva-Camañas R.M. 1 , Moreno L. 1 , Sagrado S. 1,2 , Medina-Hernandez M.J. 1 

1 


2 


Corresponding author e-mail: martiny@uv.es 

Separation of enantiomers has become one of the most important tasks of analytical chemistry especially in the 

field of pharmaceutical, clinical and agrochemical sciences since the stereochemistry has a significant influence 

on the biological activity. It is well known that a pair of enantiomers may exhibit quite different properties with 

respect to uptake, distribution, receptor binding, metabolism, excretion, etc. As a consequence of these differences, 

enantiomers generally tend to have unequal biological activities and fates in their courses of application. 

Today, up to 25% of pesticides are chiral molecules; however, almost all of them are manufactures and applied as 

racemic mixtures. The agrochemical industry and government regulators are beginning to take enantioselectivity 

into account to make a more accurate risk assessment of chiral pesticides. To obtain information on the respective 

properties of individual enantiomers, e.g., physiological, toxicological and metabolic behaviors, methods for 

enantiomeric separation become indispensable. 

Capillary electrophoresis had experienced an explosive growth the last decade because of its high separation 

efficiencies, small sample size requirements, low reagent consumption and high speed of analysis. Thus, it 

is suitable for chiral separations because of the usual high economic cost of the chiral selectors used in the 

enantiomeric separation. A wide range of chiral selectors has been used in chiral capillary electrophoresis separation 

such as cyclodextrins (CDs), oligo-sacharides, chiral surfactants, antibiotics and proteins. A general drawback of 

these conventional chiral separations are the relative high cost of analysis since chiral selector solution should be 

replaced after few runs because they become electrolyzed during them. A useful approach to overcome this problem 

is the partial filling technique. In this methodology the capillary is partially filled with the chiral selector solution and 

the separations are performed with plain background electrolyte. In such conditions the same chiral selector solution 

can be reused several times thus reducing the cost per analysis. 

This communication deals with the application of the partial filling technique to the separation of four triazole 

pesticides (hexaconazole, penconazole, tebuconazole, myclobutanil) and the related imazalil and benalaxyl by 

capillary electrophoresis using the highly sulfated β-cyclodextrin (HS-βCD) as chiral selector. The effect on the 

separation of several experimental variables such as HS-βCD concentration, temperature and chiral selector plug 

length is evaluated. Complete enantioresolution was achieved for all the studied compounds using 50 mM phosphate 

buffer at pH 7.0 as background electrolyte and different concentrations of HS-βCD in the chiral selector plug. 


The authors acknowledge the Spanish Ministry of Science and Technology (MCYT) for the financial support (Project 

SAF2008-00859). 


685


P20-070 NANO-LIQUID CHROMATOGRAPHY - NANOSPRAY - MASS SPECTROMETRY FOR THE DETECTION AND QUANTIFICATION 

OF TRACES OF ENDOCRINE DISRUPTORS IN BENTHIC INVERTEBRATES 

Bulete A., Baudot R., Wiest L., Cren-Olive C. 

USR 059 CNRS, Service central d’analyse-France 


Introduction 

Due to industrialization and use of chemical products in everyday life, different kinds of drugs or pesticides are 

present in our environment, causing negative impacts and threats on humans. Consequences of these pollutions are 

gradually highlighted and one of the challenges of environmental science is to evaluate risks. 

The objective of this study is to develop analytical tools for extraction and analysis of traces of endocrine disruptors 

in biotic environmental matrices like water’s benthic invertebrates. These gastropods, weighing just a few milligrams, 

are used to evaluate ecosystems state. 

With such a small sample size, extraction step and analysis are more difficult. So, advanced technologies are 

required to seek drugs traces in complex matrices like gastropods: nano-chromatography coupled with mass 

spectrometry (nano-LC-nano-ESI MS/MS) increases sensitivity, reduces the required initial sample amount and is a 

good tool to answer this ecotoxicological issue. 

Keywords: Nano-liquid chromatography, gastropods, small molecules, extraction, validation 

Experimentation 

In this context, we developed a new methodology using nano-LC-nano-ESI MS/MS to quantify traces of two 

substances (a neuropharmaceutical, fluoxetine and an anti convulsant, carbamazepine) in two gastropods (10 to 

20mg): Potamopyrgus antipodarum and Valvata piscinalis prosobranch gastropods. An easy and quick extraction 

method was developed. The procedure involves an extraction of about 10 milligrams of matrix by 500µL of a mixture 

of acetonitrile:water:hexane (50/20/30) and 100mg of buffer salt. Recoveries were 86% for carbamazepine and 87% 

for fluoxetine. 

Nano-LC-nano-ESI MS/MS analysis was performed with a nano Ultimate3000 (Dionex®) coupled with a Qtrap3200 

detector (AB Sciex®). Separation was performed on a Pepmap C18 column (Dionex®) with an injection volum of 

1µL. MS/MS detection was performed in the Multiple Reaction Monitoring (MRM) mode using a NanoSpray ® II (AB 

Sciex®) in the positive mode. Numerous experiments using solutions of the individual analytes were performed to 

determine the optimal MRM transitions, collision energies and declustering potentials for the two compounds. 

Validation 

The results show that the developed method presents a good robustness and obtained limits of detection and 

quantification are enough low to detect contaminants in the environment: for carbamazepine and fluoxetine, LOD 

was respectively of 4ng/g and 30ng/g with only 10 milligrams of matrix. The validation of the method was carried out 

over three days: within-day precision, inter-day variation and recoveries were determined by analysing three extracts 

of three different concentrations of analytes (low, intermediate and high concentration level of the linearity range). 

Conclusion and perspectives 

In order to handle small volumes, to work with a limited matrix quantity and to keep analysis sensitivity, an analytical 

method was developed using nano-LC-nano-ESI MS/MS. This quantification method was applied to gastropods 

exposed to fluoxetine in Cemagref ecotoxicological laboratory. After a short exposure (14 days) at different doping 

concentrations, we studied bioaccumulation of fluoxetine in the two gastropods. As this method was carried out on 

one gastropod, a metabonomics approach could be possible to characterize biological responses. 


Thanks to the CNRS which funded this work (PEPS-EDD 2009) and J. GARRIC and M. GUST from Cemagref 

laboratory. 

686 



P20-071 ANALYSIS OF METHANOL AND ETHANOL IN AUTOMOTIVE EXHAUST 

Cirera-Domènech E., Ribas Font C., Gotor Navarra G., Comellas Riera L., Broto-Puig F. 

Ramon Llull University Barcelona 

Corresponding author e-mail: francesc.broto@iqs.edu 

Alcohols are oxygenated VOCs that are involved in the photochemical production of tropospheric ozone. The use of 

ethanol as a fuel for explosion motor vehicles can represent an important source of alcohols emissions, essentially 

ethanol and methanol. Based on SOP No.101 Rev.2.1. (Air Resources Board- CEPA, California Environmental 

Protection Agency), a method for the analysis of methanol and ethanol in automotive exhaust has been developed 

(HRGC-FID). The method has been optimized and validated. However, in the analysis of some samples it has been 

observed the presence of a substance that interferes in methanol determination. It has been checked that the 

presence of this interference is associated with the appearance in the chromatogram of another signal to a smaller 

retention time. By HRGC-MS the interfering substance has been identified as ATB (tert-butyl alcohol) and the other 

signal as ETBE (ethyl tert-butyl ether). Both compounds are used as gasoline additives and ATB can also be used 

as ethanol denaturant. Due to the presence of this interferent, a new chromatographic method has been developed 

employing a lower polarity column (dimethylpolysiloxane - 6% cyanopropylphenyl instead of polyethylene glycol 

column used in the original method). This method allows a good separation of methanol, ethanol, ATB and other 

possible interferent compounds. 


687


P20-072 VOLATILE ORGANIC COMPOUNDS VARIATIONS IN URBAN AND INDUSTRIAL AREAS BY CONTINUOUS MONITORING 

Barrero M.A., Goikoetxea M., Cantón L. 


Non-methane hydrocarbons (NMHC) play a main role in air quality, being involved in the formation of tropospheric 

ozone. In addition, some of them are hazardous to human health (e.g. benzene, 1,3-butadiene). Therefore, knowing 

their atmospheric concentrations is a matter of interest, which was pointed out by the European Directive 2002/3/ 

CE, which gives a list of 31 compounds to be monitored in urban and suburban ambients. 

Monitoring of NMHC provides useful information about the evolution of both levels and composition. This information 

can be used to assess the behaviour of emission sources in a certain area. The aim of this work was to characterise 

NMHC variations in various environments in the surroundings of Donostia-San Sebastián, in the Basque Country, 

and relate them with the main sources in the area. We carried several sampling campaigns in two urban, an industrial 

and a suburban site with an automated thermal desorption-gas chromatograph. 

The automated NMHC analyzer system used for the determination of hourly concentrations consisted of a sampling 

pump, a thermal desorption unit (TDU), a gas chromatograph and a data processing unit. Hourly, an air sample was 

drawn by the pump at a flow rate of 25 mL/min, set up by a mass flow controller, through a sorbent trap in the TDU, 

held at -30ºC by a Peltier device. After sampling, the trap was heated up to 325ºC and desorbed with a stream of 

helium, which was transferred to the gas chromatograph through a heated transfer line. The gas chromatograph was 

equipped with two capillary columns, a BP-1 and a PLOT. The desorbed stream entered the BP-1 first and, after a 

certain time, a Deans’ switch changed the disposition from serial to parallel, which made the lighter hydrocarbons 

(C2-C5) elute from the PLOT column and the heavier ones (C6-C10) from the BP-1. At the end of each column a flame 

ionization detector (FID) detected the eluted compounds. 

In addition, major pollutants concentrations (CO, NO, NO2, O3, SO2) and meteorological variables corresponding to 

the study periods were measured in situ at the two urban sites by automatic stations of the Basque Government’s Air 

Quality Monitoring Network. 

A total of 53 compounds were identified in the four sites. Concentrations of the regulated NMHC (benzene) were 

below the EC limit value for the annual average (5 μg/m3). 

The hourly temporal resolution of the measurements allowed to obtain the diurnal evolution of the hydrocarbon 

concentrations. Four types of diurnal-weekly behaviour were identified and associated with different sources of 

emission: natural gas and domestic, traffic related, natural and industrial emissions. An example of a traffic related 

compound variation is shown in Figure 1, using carbon monoxide as traffic marker. 

688 



P20-074 OCCURRENCE AND ELIMINATION OF EMERGING CONTAMINANTS IN SEWAGE TREATMENT PLANTS. USE OF POLAR 

ORGANIC CHEMICAL INTEGRATIVE SAMPLERS (POCIS) VS GRAB SAMPLING METHODOLOGIES 

Dávila T.J.,); Céspedes R., Valle M.C., De la Cal A., Ventura F., Martin J. 


Passive sampling techniques have become an alternative sampling methodology to study the occurrence and 

behaviour of organic and inorganic pollutants in the environment. Applicability of this technology in different 

environmental compartments, matrixes, and to diverse families of contaminants has been under investigation during 

the last years, and the most relevant advantages and drawbacks when compared to grab sampling methods have 

been reported in these studies. The holistic interpretation of data from passive sampling techniques has showed 

to be as one of the most relevant advantages, whereas the need of specific calibration experiments previous to 

sampling campaigns is generally considered an important drawback. 

In the present work we go into the use and application of passive sampling techniques versus traditional methods of 

sampling in depth. Thus, an assessment of the contamination profile for the two different tertiary treatments applied 

in two wastewater treatment plants in the east cost of Spain (STP1 and STP2) is presented. Levels of different 

pharmaceuticals, pesticides, and alkylphenolic compounds were measured by using passive sampling techniques 

and compared with those obtained from spot water samples. With a markedly polar character for the majority of 

compounds in study (0.16 ≤ Log Kow ≤ 5.16), the Polar Organic Chemical Integrative Sampler POCIS was selected in 

order to test the group of pollutants. The two commercially available configurations (i.e. resins prepared specifically 

for “pharmaceuticals” and “pesticides”) of this device were used and deployed in several treatment stages of 

the wastewater facilities. Furthermore, grab samples of wastewater were simultaneously collected to the passive 

sampling devices in each one of the stages. 

. In STP 1, an UV disinfection open channel was applied as the previous to final distribution step, whereas a reverse 

osmosis treatment was applied to the final effluent in STP 2. The UV disinfection system did not work under normal 

operational conditions during the sampling period, and strong differences for the estimated concentrations of 

most of the studied compounds between STP1 and STP2 were found in the final treated water, both in grab and 

passive sampling devices samples. An optimal efficiency in the elimination for STP2 was observed for the most of 

compounds, in comparison with the UV tertiary treatment applied in STP1. The most ubiquitous pollutants were 

ibuprofen, diclofenac, carbamazepine and diuron in both studied STPs. 

In conclusion, a complete study on the suitability of using passive sampling techniques versus classical sampling 

methodologies was done for a wide range of medium-high hydrophilic contaminants in two wastewater treatment 

plants. The results showed that the concentrations obtained from POCIS in wastewater properly explain the 

behaviour for most of compounds during the treatment, and emphasize the importance of determining the time 

weighted average (TWA) concentrations for monitoring contaminants in the environment. 


689


P20-075 EVALUATION OF THE SPATIAL VARIABILITY OF ATMOSPHERIC VOLATILE ORGANIC COMPOUNDS 

Goikoetxea M., Barrero M.A., Cantón L. 

University of the Basque Counts 

In urban areas, vehicular exhaust emissions are usually the most significant source of Volatile Organic Compounds 

(VOCs). However, some specific industrial contributions may not be negligible. Identification of the major sources 

of airborne pollutants and their contributions are a matter of interest in developing effective pollution control 

and mitigation strategies. The evaluation of VOCs concentrations in several points in a region can yield relevant 

information about which are the main processes having influence on pollutants levels. This work focuses on the 

variations of VOCs in six urban locations with different anthropogenic inputs placed in the surroundings of Donostia 

- San Sebastián, in the Basque Country. Air samples were collected weekly over a 24 hour period simultaneously 

at all locations (Andoain, Astigarraga, Hernani, Lasarte, Urnieta, Usurbil) in two consecutive winter sampling 

campaigns. VOCs were adsorbed on charcoal tubes attached to personal sampling pumps (SKC) operating at a 

flow rate of 1 L/min. Once collected, samples were desorbed with carbon disulphide and extracts analysed by gas 

chromatography (GC-FID and GC/MS) equipped with an HP-1701 capillary column. In addition, major pollutants 

concentrations (CO, NO, NO2, O3, PM10, SO2) and meteorological variables corresponding to the study period were 

measured in each site by automatic stations of the Basque Government’s Air Quality Monitoring Network. A total of 

30 VOCs were identified and quantified, ranging from aliphatics C7 to C3-benzenes (Fig. 1). They were classified into 

five groups: aromatic and aliphatic hydrocarbons, and oxygenated, biogenic and chlorinated compounds. Figure 

1. VOC chromatographic profile obtained from the atmosphere of Andoain. The average VOC composition varied 

from site to site: BTEX accounted for 17% to 61%; aliphatic hydrocarbons contribution ranged from 19% to 59%; the 

third main group, composed by oxygenated compounds, contributed between 5% and 19% to the total VOCs. Three 

sites exhibited composition profiles similar to those found in the nearby urban site of San Sebastián, while the other 

three had clearly distinct profiles. Some compounds (e.g. ethyl acetate, methyl isobutyl ketone) were found to be 

characteristic of each sampling site. Several VOCs showed significant, high correlations between sites (e.g. benzene, 

b-pinene, Fig. 2), pointing at common sources of emission, while others did not correlate at all (e.g. ethyl acetate, 

methyl isobutyl ketone), revealing the incidence of local, specific sources. Figure 2. Correlation matrix between ethyl 

acetate, benzene, MIBK (methyl isobutyl ketone) and β-pinene concentrations in the different sampling locations. 

AND=Andoain, AST=Astigarraga, HER=Hernani, LAS=Lasarte, URN=Urnieta, USU=Usurbil. 

690 



P20-076 INTEGRATED ASSESSMENT OF EMERGING COMPOUNDS BY THE COMBINATION OF POCIS AND UPLC-QQLIT-MS. 

APPLICATION IN MEDITERRANEAN SEWAGE TREATMENT PLANTS 

Céspedes-Sánchez R., Dávila T.J., De la Cal A., Martin-Alonso J., Ventura F. 


Corresponding author e-mail: rcespedes@agbar.es 

Occurrence of pharmaceuticals and other polar compounds in the environment and their possible effects in human 

health are of increasing concern. Urban wastewaters are the major pathway for aquatic contamination by these 

compounds, and advanced analytical methodologies are needed for their determination. Passive sampling offers a 

promising alternative, highly complimentary to traditional sampling methods in environmental analysis, which provide 

more representative measure of the pollution levels. In this work, the development of an analytical procedure which 

makes use of passive sampling techniques and of the LC–QqLIT-MS system is presented. Both pharmaceutical and 

pesticide configuration of Polar Organic Chemical Integrative Samplers (POCIS) were examined. Laboratory-based 

calibration experiments were carried out in order to assess the uptake of the selected compounds by exposing 

them for different time periods. In addition, an analytical procedure of POCIS extraction and Solid Phase Extraction 

of waters followed by Ultra Performance Liquid Chromatography–Quadrupole-Linear Ion Trap Mass Spectrometry 

(UPLC–QqLIT-MS) was developed and validated for the determination of the target compounds at low ng/L levels. 

The recoveries obtained and optimized analysis parameters are reported. The applicability of POCIS and the 

developed method to estimate the concentrations of pharmaceuticals, polar pesticides and alkylphenols was tested 

in two Sewage Treatment Plants (STP) in the Mediterranean coast. On-site passive and spot sampling campaigns 

were carried out in raw water and effluent of secondary and tertiary treatments. The results obtained in the POCIS 

deployed at two exposition times and those from water analyzed show the most ubiquitous compounds (diclofenac, 

ibuprofen, carbamazepine and diuron) in the studied STPs and the different efficiency of the tertiary treatments 

evaluated. Thus, whereas some pesticides showed similar levels between both STPs, lower levels of pharmaceuticals 

were detected in the Reverse Osmosi treatment. In addition, the use of POCIS permitted the detection of the 

influence of the heavy and intermittent rainfalls produced after a severe drought, missed by conventional grab 

sampling. Thus, lower concentrations derived from POCIS exposed longer time could be obtained due to the dilution 

produced by the rains during the last days of the sampling campaign. In conclusion, POCIS provides time weighted 

average concentrations (TWA) and therefore more representative water quality monitoring levels with a reduction of 

the detection limits, and relevance to ecological risk assessments not easily obtainable with traditional methods. In 

addition, with the overall analytical procedure developed by using the passive samplers POCIS and UPLC-QqLIT-MS 

system, the method sensitivity increases, providing reliable and more representative water quality monitoring at trace 

concentration levels data across EU member states. Key words: Polar Organic Integrative Sampler, pharmaceuticals, 

polar pesticides, sewage treatment plants, Liquid Chromatography–Quadrupole-Linear Ion Trap Mass Spectrometry. 

Acknowledgements: This work leaded by Aguas de Barcelona has been financed by the SOSTAQUA Project through 

the CDTI (Ingenio 2010 Programme under the CENIT call) and by the Alliance Project HE0605. 


691


P20-077 ANALYTICAL CHARACTERISATION OF MANNOSYLERYTHRITOL LIPID BIO SURFACTANTS PRODUCED BY BIOSYNTHESIS 

BASED ON FEEDSTOCK SOURCES FROM AGRO-FOOD INDUSTRY 

Onghena M. 1 , Wijnants M. 1 , Pico Y. 2 , Covaci A. 3 , Lemiere F. 3 

1 

Karel de Grote University College-Spain 

2 


3 


A fermentation based on feedstock sources from agro-food industry for the production of mannosylerythritol 

lipid (MEL) biosurfactants (BIOMEL project; M. Wijnants) yields in a secretion of a complex blend of biomolecules 

with promising properties in food-, pharmaceutical, cosmetic and domestic housecare applications. The goal of 

this project is to define detailed protocols of standard procedures for isolation, purification, quantification and 

characterisation of current and novel MELs from a complete fermentation broth. All the MELs that are known up 

to now (A,B, C and D) consist of a hydrophilic part with 4-O-β-D-mannopyranosyl-D-erythritol and a hydrophobic 

part with two fatty acid acyl chains. Each homologue possesses one or two acetylgroups on the C-4’ and/or on the 

C-6’ mannose moiety. Literature concerning the isolation and the characterisation of mannosylerythritol lipids out 

of crude broths (matrices) is very rare. On the other hand, the downstream processing of the fermentation broth 

obtained after the production from the studied carbon feedstocks is clearly described. Those protocols serve as 

an analytical starting platform for the separation and the isolation out of the crude blend. In order to quantify the 

isolated product, the first step was obtaining analytical standards because there are no commercial MEL standards 

available on the market. For this purpose, purification and separation into the different kinds of MELs was developed 

using a glass column filled with silica beads and applying a gradient elution with an organic solvent system. Once 

standards are obtained, LC-methods using organic solvent systems are described in literature for the further 

quantification of the MELs. Because of the wide variety of MELs that can be produced, depending on the used 

feedstock and yeast strain, MS was used for the characterization. Due to the inability of NP chromatography systems 

to work with MS, a new RP chromatography approach with specific parameters was applied using HILIC, fluoro- and 

(modified) C18-stationary phases. Using this new chromatography approach, an appropriate LC-MS method was 

developed to verify the identity of the produced MELs and to detect new types of homologues. 

692 



P20-078 EVALUATION OF SEQUENTIAL INJECTION CHROMATOGRAPHY FOR THE ANALYSIS OF ANTICOCCIDIAL AGENTS IN 

AQUEOUS MATRICES 

Maya F. 1 , Björklund E. 2 , Estela J.M. 1 , Cerdà V. 1 

1 


2 

University of Copenhagen 

Corresponding author e-mail: fernando-maya@hotmail.com 

This paper explores the possibility of applying sequential injection chromatography (SIC) as a means to perform direct 

injection and analysis of the two anticoccidial agents Lasalocid and Toltrazuril in aqueous matrices. Reversed-phase 

sequential injection chromatography was performed by connecting a 25x 4.6mm monolithic C18 column to a 2m long 

pathlength capillary flow cell, where the usage of a flow cell lower the detection limit compared to a conventional 

short-distance flow cell, providing a simple detection system for these two compounds which are initially poorly UV 

absorbents. The obtained method provides a high injection throughput of 12 analysis per hour. A limit of detection of 

0.019 and 0.010 mg/L for Toltrazuril and Lasalocid, respectively. The repeatibilities obtained (n=10) were lower than 

2% and 4% for Toltrazuril and Lasalocid, respectively. The usefulness of the proposed method has been evaluated 

for groundwater samples as well as in the quality control of Toltrazuril contained in oral suspensions for veterinary 

use. Figure 1. Schematic depiction of the SIC manifold used to separate the two anticoccidial agents in reversedphase 

liquid chromatography. MP = mobile phase; SP = syringe pump; E = two-way connector; SV = solenoid valve; 

HC = holding coil; MV = 8-port multi-position valve; GC = guard column; MC = monolithic column; UV = ultra violet 

light source; DET = spectrophotometer; OF = optical fibre. All filled lines represent Teflon tubing (0.8mm i.d.), while 

all dotted lines represent electronic communication with the personal computer (PC). 


693


P20-079 STUDY OF THE VOLATILE ORGANIC COMPOUNDS (VOCS) EMISSION FROM AN URBAN WASTE LANDFILL IN MALLORCA 

(SPAIN) BY TD-GC-MS. 

Rodriguez-navas Gonzalez C., Forteza R., Cerdà V. 


Corresponding author e-mail: victor.cerda@uib.es 

Thermal desorption coupled to gas chromatography-mass spectrometry (TD-GC-MS) is a common technique 

used to quantify volatile organic compounds in environmental samples. This study is the first data ever report on 

the occurrence of volatile organic compounds (VOCs) emitted by a landfill in the island of Mallorca (Mediterranean 

sea, Spain) where 200.000 tones of urban solid wastes are dumped every year. Samples were collected from three 

different sampling sites, depending on the waste aging, in august 2008 during the main tourist season and the 

hottest weather conditions. The standard European method EN 13725 was followed to collect the samples into 

Nalophan® bags. Afterwards, VOCs were adsorbed into Carboxen 1000® and Tenax TA® prior to the thermal 

desorption and the analysis by GC-MS. In this work, 43 VOCs have been analyzed, using external standard, from 

which 37 have a positive identification. This data was also examined with active olfactometry to determine a 

statistical linear correlation between odour and VOC concentrations. Detected VOCs were monoaromatics (83.2 

- 105.6 ug•m-3), alcohols (35.5 – 67.4 ug•m-3), aldehydes (55.1 – 96.0 ug•m-3), ketones (78.4 – 151.6 ug•m-3), 

esters (25.4 – 32.7 ug•m-3), halogenated compounds (16.3 – 39.4 ug•m-3) or terpens (1.4 – 2.4 ug•m-3), showing 

different patterns each one of the three sites. Benzene-to-toluene ratio (B: T) also shows characteristic values within 

0.13 – 0.2 for landfill emissions. In spite of the olfactometric data for the three different sites (152 – 242 OUE•m-3), 

it has not been possible to make a significant linear correlation between odour and VOC concentration, due to 

the high complexity of the samples, having more than 200 peaks, what illustrates the necessity of new and more 

comprehensive studies. 

694 



P20-080 COMPARATIVE STUDY USING LARGE-VOLUME INJECTION COUPLED-COLUMN REVERSED PHASE LIQUID 

CHROMATOGRAPHY WITH FLUORESCENCE DETECTION AND LIQUID CHROMATOGRAPHY-TIME-OF-FLIGHT MASS 

SPECTROMETRY IN THE DETERMINATION OF BETA-BLOCKERS IN GROUNDWATER 

Parrilla M.M., Martínez Galera M., Parrilla P. 


Corresponding author e-mail: parrilla@ual.es 

The need to reduce the overall sample preparation time, as well as the quantities of organic solvents needed for the 

extraction of organic pollutants from environmental samples has led to the development of several new extraction 

approaches. Coupled chromatographic techniques are an advantageous alternative available for the tracelevel 

determination of pollutants in environmental samples. In particular, coupled-column reversed-phase liquid 

chromatography combined with large volume injections (LVI), is a powerful and appropriate technique for the rapid, 

sensitive and selective determination of polar pollutants in environmental samples, which allows automated sample 

processing using the separation power of a first column (C-1) and analysis in an analytical column C-2. Besides the 

opportunity of enlarging the sample injection volume to improve sensitivity, it offers the possibility of removing a 

large excess of early-eluting polar interferences encountered in environmental analysis. This latter capability is the 

key to success for enhancing selectivity in the analysis of polar compounds. A simple multidimensional system for 

direct injection of large volumes has been developed for the determination of five beta-blockers (atenolol, nadolol, 

metoprolol, bisoprolol and betaxolol) in groundwater using fluorescence detection. An AQUASIL C18 50 x 4.6 mm id 

column coupled to a Discovery RP Amide C16 150 x 4.6 mm id column for sample clean-up and determination were 

used, respectively. The capability of a first column for eliminating large interfering molecules, combined with an 

optimised, coupled-column separation procedure (LC-LC), large volume injection (LVI) and fluorescence detection 

(FD), gave excellent sensitivity and selectivity for target analytes. The LVI-LC-LC-FD method combines analyte 

isolation, preconcentration and determination into a single step. Detection limits obtained in groundwater matrix 

were lower than, or equal to, 6.4 ng L-1. Average recoveries ranged from 82 to 107% (n=3) and relative standard 

deviation (RSD) values ranged from 0.8 to 9%. The LVI-LC-LC-FD method was applied for determining the target 

analytes in groundwater samples from Almería (Spain). Low enough LOQ values were obtained to permit analysis of 

the beta-blockers at ng L-1 levels. The method compares favourably with others previously developed in terms of 

sensitivity, material and time required. The lesser sample handing and the short run time made possible the analysis 

of at least 30 samples per day. This methodology does not require manipulation of the groundwater samples before 

injection. The automation of the system is subject to a minimum of human errors and contamination. Significant 

reductions in costs for sample pre-treatment (solvent and solid phases for clean-up) and method development 

times are also achieved. A method using solid-phase extraction (SPE) and liquid chromatography-time-of-flight mass 

spectrometry (LC-TOF-MS) (ESI positive mode) has been developed for the confirmatory analysis of the five beta-blockers 

in groundwater samples. Extraction of the beta-blockers was carried out by an Oasis MCX cartridge with a strong 

cation-exchange mixed-mode polymeric sorbent. Recoveries ranged between 81-102% (n=3) and relative standard 

deviation (RSD) values were on average less than 10%. The method limits of quantification ranged from 0.5 to 5 ng 

L-1. 


695


P20-081 MATRIX EFFECTS IN PESTICIDE RESIDUE ANALYSIS: THE BATTLE IS NOT YET OVER 

Fernandez-Alba A.R. 


Matrix effects in pesticide residue analysis: The battle is not yet over. Amadeo R. Fernandez-Alba Pesticide Residue 

Research Group, University of Almeria. EU-CRL for Pesticide Residues in Fruits and Vegetables. 04120-Almería 

(Spain). amadeo@ual.es Matrix effects in food analysis remain as the single greatest operational drawback for GC 

and LC-MS techniques. In LC-MS, high concentration of co-eluting matrix components compete with the analytes 

in the ionization mechanism, which causes reduced ionization efficiency for the analytes and a reduced signal 

compared to the same conditions when matrix is absent. Signal enhancement is also known to sometimes occur 

in API due to indirect effects on the ionization process in the presence of matrix components. In GC-MS methods, 

the matrix-induced chromatographic response enhancement effect often occurs for relatively polar analytes 

when matrix components fill active sites in the injection liner, column, and ion source, thereby reducing losses 

of susceptible analytes that interact more greatly with those sites in the absence of matrix. Furthermore, matrix 

effects can cause false positive or negative results as a consequence of the presence of isobaric components and 

masking compounds of the matrix. One very promising approach is to reduce the amount of matrix injected in the 

chromatographic system by, for example, dilution of the final extracts. The appearance of new generation analytical 

systems in the market make possible this attempt, as highly sensitive instruments are available for these purposes. 

In this way, matrix effect can be avoided for many fruit and vegetable matrices. In order to evaluate matrix effect 

behaviour in fruit juice, direct injection of diluted matrix matched standards was performed by LC-MS/MS. Orange, 

pineapple and peach juice were tested, and only around 8 % of the pesticides studied showed appreciable matrix 

effect with a 10-fold dilution. Another approach in the case of GC is considering its feasibility to work in GCXGC, 

that allows a much greater separation of the analytes from the matrix. In our study we focused in the analysis 

of chlorinated pesticides in red and black pepper and in tea by GC×GC TOF-MS, obtaining two-dimensional 

separation. Typically, 25-50 ng kg-1 concentrations were the LODs for chlorinated (including organochlorine) 

pesticides in solvent and also in red pepper. Another approach is the use of mass accuracy LC-TOF-MS that allows 

a greater discrimination from the matrix compounds based on exact mass detections. That advantages together an 

increase of the resolution up to values of 15000 or higher can solve the majority of the interferences presented in 

food analysis. This promising approach is recently reported as a very effective and robust tool to overcome matrix 

or isobaric interferences. But in general quantification processes are not well resolved by using the currently sample 

extraction procedures applied Generally speaking in all cases studied we can find situations where matrix effects 

remain as a difficult battle not enough solved for the analysts. 

696 



P20-082 EFFECTIVENESS OF GC-MS/MS AND GC×GC TOFMS TECHNIQUES ON THE DETERMINATION OF CHLORINATED 

PESTICIDES FROM DRY MATERIALS 

Kemellár B. 1 , Gómez M.J. 2 , Martínez Uroz M.A. 3 , Fernández-Alba A.R. 3 

1 

Corvinus University of Budapest-Hungary 

2 


3 


Effectiveness of GC-MS/MS and GC×GC TOFMS techniques on the determination of chlorinated pesticides from dry 

materials Kmellár B1,2, Gomez Ramos M J1, Martínez Uroz M A1 and Fernández-Alba A R1 1 University of Almería, 

Carretera de Sacramento s/n 04120 La Cañada de San Urbano, Almería, SPAIN 2 Corvinus University of Budapest, 

Villanyi str. 29-43, H-1118, Budapest, HUNGARY; e-mail: bela.kmellar@uni-corvinus.hu The majority of the published 

pesticide multiresidue methods (e.g.>50 pesticides) were focusing on high water (like fruits and vegetables) and 

high fat (like olives and olive oil) content commodities and on crops in recent years. Whereas the analysis of dry 

materials like spices and tea has been wind-mindedness, but their control is also necessary being a potential source 

of contamination. Our aim was to compare the matrix-induced effects on overlapping peaks, limits of detection, 

quantification, recovery and robustness of the system by measuring six selected GC-amenable pesticides in spices 

and tea using GC-MS/MS and GC×GC-TOFMS instruments and a very simple sample preparation technique. Matrix 

effect plays an important role even with 10-time dilution that results many overlapping matrix peaks in 1D-GC. That 

induces the application of 2D-GC, where the complete pesticide-matrix separation was achieved in red pepper and 

in tea. The recoveries were acceptable, except for chlorothalonil in red pepper and lindane in black tea. Endosulfans 

(including endosulfan α and β isomers and endosulfan sulfate) have to be measured together for the reason of MRL 

definition. Whereas this was not feasible using 1-D separation: endosulfan β is insensitive for the co-eluting matrix, 

this problem was solved by 2-D GC thanks to the complete separation. Tea seems simpler matrix and all selected 

pesticides can be determined by 1-D GC. With the lack of clean-up, high matrix-induced signal enhancement was 

observed at several pesticide-matrix pairs, but matrix-matched calibration is fit for quantitative purposes. Limits 

of detection by 2-D GC meet the criteria settled by the current MRLs in all cases studied, unlike not all pesticide/ 

commodity pairs by 1-D GC. Moreover TOFMS serve to detect non-target pesticides that prove a remarkable benefit 

of GC×GC TOFMS technique over GC-MS/MS for determination of pesticides in dirty spice extracts. References 

[1] Banerjee K., Patil S.H., Dasgupta S. D., Oulkar D. P., Patil S. B., Savant R.,Adsule P. G., J Chrom. A 1190 (2008) 

350–357 [2] Ferrer Amate C., Unterluggauer H., Fischer R. J., Fernández-Alba A. R., Masselter S., Anal Boianal 

Chem (2010) DOI 10.1007/s00216-010-3526-x [3] Manirakiza P., Covaci A., Schepens P., Chrom 52 (2000) 787- 

790 ACKNOWLEDGEMENTS Béla Kmellár would like to thank the financial support of the Hungarian State Eötvös 

Scholarship. 


697


P20-083 ELECTROCHEMICALLY SYNTHESIZED MOLECULARLY IMPRINTED POLYMER FILMS: NOVEL SORBENTS FOR SPME 

Mazzotta E. 1 , Malitesta C. 1 , Díaz-Álvarez M. 2 , Martin-Esteban A. 2 

1 

Università del Salento-Italy 

2 


Corresponding author e-mail: mdalvarez@inia.es 

Due to the importance of sample preparation, Solid-phase microextraction (SPME) has been widely used in 

analytical laboratories as it provides simultaneous extraction and pre-concentration of analytes from sample matrix. 

However, recent years have seen an increase in interest in the preparation of tailor-made sorbents with the aim of 

increasing selectivity of the extraction process. Molecularly imprinted polymers (MIPs) are stable polymers with 

selective molecular recognition abilities providing a powerful l tool in the development of highly selective analytical 

methods. In this work, preparation of a molecularly imprinted polymer (MIP) film and its recognition properties 

for levofloxacin (a fluoroquinolone antibiotic) were investigated. For the first time, electrosynthesis is proposed 

as a viable way to directly interface MIP to SPME conducting fiber. The proposed approach aims to combine MIP 

selectivity with advantages of electropolymerization, including the possibility to deposit an uniform layer with a 

controlled thickness. The imprinted film was prepared by deposition of polypyrrole in the experimental conditions 

selected with a template molecule (levofloxacin) and then electrosynthesized films have been characterized by X-Ray 

Photoelectron Spectroscopy (XPS) to verify the template entrapment and its removal after washing steps. Also some 

rebinding experiments have been carried out and results will be described. Acknowledgements: Authors wish to 

thank Spanish Ministry of Science and Innovation for financial support (CTQ2008-02607 and HI2008-0101) 

698 


AUTHOR 

INDEX

INDEX - AUTHORS 

122, 173 

135 

135 

122 

592 

196, 538 

508 

458, 639, 652 

563 

113, 233 

531 

109 

278, 279 

172, 509, 673, 674 

174, 637, 665 

112 

177 

135 

516 

668 

233 

631 

266 

625 

91, 158, 421, 438, 661 

81 

522, 523, 563, 582 

105, 208 

595 

165 

114 

321 

507 

257, 286, 287, 475, 578 

257 

354 

683 

86, 147 

190 

323 

104 

381, 589 

453, 454, 465, 470 

639 

168 

124 

293 

640 

624, 675, 676 

137, 527 

340 

503 

399 

86, 147, 444, 621 

429 

Abad, E. 

Abad-Fuentes, A. 

Abad-Somovilla, A. 

Ábalos, M. 

Abi Jaoudé, M. 

Adam, O. 

Afonso Morales, D. 

Afonso, A. M. 

Agbaba, D. 

Aghakhani, A. 

Agocs, A. 

Agrafiotou, P. 

Agrawal, N. 

Agüera, A. 

Aguilar, C 

Aguilera-Herrador, E. 

Aguilera-Luiz, M. M. 

Agulló, C. 

Agut, M. 

Ahmadi, H. 

Akbari 

Alaee, M. 

Alañón, E. 

Albero, B. 

Albert, K. 

Albertí, J. 

Albreht, A. 

Albuquerque, F. C. 

Alcaraz Rodríguez, J. 

Alcón, M. J. 

Alcudia-León, M. C. 

Alden, P. G. 

Alechaga, E. 

Alegria, A. 

Alemany-Costa, L. 

Allwood, W. 

Alonso, B. 

Alonso, M. 

Alonso-Villegas, R. 

Altmann, F. 

Álvarez, J. 

Alwael, H. 

Amigo-Benavent, M. 

Anderson, J. L. 

Andersson, J. T. 

Andrási, N. 

Andrés, A. 

Andreu, A. 

Andreu, V. 

Andujar-Ortiz, L. 

Anres, P. 

Anta- Saiz, L. 

Antal, P. 

Anticó, E. 

Antunes Lopes, F. 

173 

95, 361, 362 

399 

596 

564 

378 

301, 482, 486 

252 

164, 348 

681 

526 

558, 560 

627, 628 

91 

635 

204 

607 

497, 498 

305, 306, 367 

180 

458, 652 

113, 232, 233 

430 

635 

242, 243, 244 

255 

113, 232, 233 

617 

224 

580 

542, 575, 576, 660 

650, 657 

484 

444 

373 

426 

155, 259, 534 

300, 472 

143, 228, 235, 536, 580, 581 

584, 591 

134 

257, 286, 287, 475, 578 

596 

468 

104, 123, 221, 318, 682, 683 

265 

146 

688, 690 

400 

225 

539 

221 

486 

Antunes, P. 

Aragon, A. 

Aranyi, A. 

Arathoon, R. 

Arenz, N. 

Arias Abrodo, P. 

Aristoy, M. C. 

Armstrong, D. W. 

Arnaiz, E. 

Arnaudguilhem, C. 

Arroyo-Manzanares, N. 

Asensi-Bernardi, L. 

Asensio-Ramos, M. 

Ashu-Arrah, B. A. 

Ašperger, D. 

Ates, H. 

Atoche, J.C. 

Aum, H. S. 

Aurand, C. 

Avakyan, A.P. 

Ayala, J. H. 

Ayazi, Z. 

Ayuso, M.C. 

Babic, S. 

Baeza-Baeza, J. J. 

Báez-Aglio, M. I. 

Bagheri, H. 

Bahadir, M. 

Baier, H. U. 

Balderas, C. 

Ballesteros, O. 

Balsaa, P. 

Banic Simicic, J. 

Bañeras, L. 

Baños-Pérez, C. 

Baras, M. C. 

Barattini, V. 

Barba, F. J. 

Barbas, C. 

Barber, E. 

Barberá, R. 

Barbi, L. 

Barbosa, J. 

Barceló, D. 

Barco Bonilla, N. 

Barend van Drooge, L. 

Barrero, M.A. 

Barth, T. 

Bartolomé, B. 

Basmakci, G. 

Batalla, R. 

Batlle, N. 

700 



649, 656, 686 

80 

168 

305, 306, 367 

263 

525, 653, 655 

283 

468 

452 

85, 296, 326, 390 

391 

199 

613 

333, 335 

164, 329, 348,466 

164, 344, 348, 466, 643 

430 

318 

252 

161, 173 

86, 621 

525, 655 

426 

357 

661 

168 

622 

693 

660 

378, 412 

510, 624, 675, 676 

221 

546 

460, 469 

561, 562 

130 

194 

618 

496 

358, 392 

74 

392 

430 

554, 545 

630 

269, 569, 572 

400, 401, 402, 577 

358 

626 

569, 572 

554 

667 

159, 174, 637, 648, 664, 665 

109, 247, 293, 537 

132, 540, 583 

Baudot, R. 

Baumann, A. 

Belder, D. 

Bell, D. S. 

Belotti, E. 

Beltrán, J. 

Beltran-Martinavarro, B. 

Benavente, F. 

Bendini, A. 

Beneito-Cambra, M. 

Benito-Peña, E. 

Bergna, M. 

Bermejo, R. 

Bernabé-Zafón, V. 

Bernal, J. 

Bernal, J. L. 

Bernalte, M.J. 

Berrojalbiz, N. 

Berthod, A. 

Bertolero, A. 

Besalú, E. 

Beser, M. I. 

Bet, Z. F. C. 

Bettermann, H. 

Betz, O. 

Beyreiss, R. 

Bijlsma, L. 

Björklund, E. 

Blanc, R. 

Blanco Gomis, D. 

Blasco, C. 

Blasco, J. 

Blazewicz, A. 

Bobalova, J. 

Bochkov, P. O. 

Boehm, G. 

Boesten, J. M. M. 

Bogenschütz, G. 

Bognar, A. 

Bogolitsyna, A. 

Buszewski, B. 

Böhmdorfer, S. 

Bohoyo-Gil, D. 

Bohuslav, M.. 

Boleda, Mª. R. 

Bona, A. 

Bonato, P.S. 

Borgards , A. 

Borges-Miquel, T. M. 

Boros Major, A. 

Borrás Linares, I. 

Borrego, A.G. 

Borrull, F. 

Bosch, E. 

Bosch, G. 

278, 279, 282, 283 

459 

154 

174, 637 

373 

623 

288 

92, 193 

299 

141 

305, 306 

159 

118 

604, 607, 608, 615 

454 

282 

451 

687 

680 

663 

262 

96 

305 

681, 686 

378 

314, 315, 316, 317 

667 

318 

537 

547 

517, 651 

400, 577 

174, 637, 665 

266 

644 

656 

640 

184, 666 

640 

387 

490 

581 

688, 690 

571 

428 

199, 386 

386 

594 

476, 654 

240, 241, 529, 245, 252, 277 

278, 279, 280, 281, 282, 283 

319, 518, 519, 520 

112, 114 

Bose, D. 

Bosque-Sendra, J. M. 

Bosserhoff, A. K. 

Botello, I. 

Bourdin, D. 

Bousquet, M. 

Bozal, B. 

Brabazon, D. 

Brabcová, I. 

Brack, W. 

Brandes, H. K. 

Bratkoswka, D. 

Brauckmann, C. 

Bravo, J. C. 

Bravo, L. 

Briñón, M. C. 

Brokl, M. 

Broto-Puig, F. 

Brownawell. B. J. 

Bruchet, A. 

Buchanan, M. 

Buchberger, W. 

Buckendahl, K. 

Buleté, A. 

Busto, E. 

Butchart, K. 

C.G. Blanco 

Caballero, G. 

Cabot, J.M. 

Caglayan, M. G. 

Caixach, J. 

Calixto, L. A. 

Calull, M. 

Calvo, E. 

Camacho, C. F. B. 

Camilleri, J. 

Cammeraat, E. 

Campins-Falcó, P. 

Campo, J. 

Canals Hernández, A. 

Canela, R. 

Cano, V. 

Cantón, L. 

Cañada-Cañada, F. 

Cao, X. L. 

Cappiello, A. 

Capriotti, F. 

Carabias-Martínez, R. 

Carbonell, E. 

Carda-Broch, S. 

Cardelle-Cobas, A. 

Cárdenas, S. 


701


184 

647 

390, 632 

267, 492, 493 

533 

678 

186, 229 

588 

128, 645 

182 

109 

256, 476, 479, 654 

644 

404 

512, 553 

266, 491 

468 

250 

110 

416, 672, 693, 694 

522 

452, 455 

653 

210 

211, 689, 691 

593 

395 

552 

90, 176, 535 

427 

514 

561, 562 

455 

279 

387, 597, 678 

407 

83 

143 

72, 329, 341, 436 

550 

248, 310 

687 

589 

127 

306 

434 

460 

215 

280, 283 

517 

92, 193, 380 

516, 687 

641 

450 

221 

Carracedo-Marzo, R. 

Carrasco , A. 

Carrasco-Correa, E. J. 

Carrasco-Pancorbo, A. 

Carrera, F. 

Carretero, A. 

Carrillo-Carrión, C. 

Carru, C. 

Casas Ferreira, A.M. 

Castaño-Álvarez, M. 

Castells, C. 

Castillo, M. 

Castro, E. V. R. 

Castro-Puyana, M. 

Castro-Rubio, F. 

Castro-Vázquez, L. 

Català-Clariana, S. 

Çelik, H. 

Centrich, F. 

Cerdà, V. 

Cernelic, K. 

Cerretani, L. 

Cervera, M. I. 

Cesio, V. 

Céspedes, R. 

Chamieh, J. 

Chankvetadze, B. 

Chankvetadze, B. 

Chapuis-Hughon, F. 

Checa, A. 

Cheon, S. H. 

Chernetsova, E. S. 

Chiavaro, E. 

Chin-Chen, M. L. 

Chisvert, A. 

Chrastina, J. 

Corcoran, C. 

Ciborowski, M. 

Cifuentes, A. 

Aybaba, C. 

Cimpan, G. 

Cirera-Domènech, E. 

Clarke, P. 

Claude, B. 

Claus, J. E. 

Clemente, A. 

Cmelik ,R. 

Cochran, J. 

Collado-Sánchez, M. A. 

Collgrós, F. 

Collins, D. 

Comellas Riera, L. 

Companioni, E. Y. 

Concha-Herrera, V. 

Conde, C. 

92, 379, 381, 589, 658 

429 

159 

95, 361, 362 

319, 518, 519, 520 

463 

256 

227, 609, 610 

538 

347, 692 

158 

500, 501, 505, 506, 512, 551 

552, 553 

598, 649, 656, 686 

236 

660 

504 

112 

516 

225 

379, 658 

154 

376, 415 

373 

206 

318 

126, 198, 200, 201, 383 

497, 498 

538 

380 

412 

166 

211, 689, 691 

198, 201 

682 

660 

235, 237, 342 

365 

215, 349, 353, 354, 355 

448, 449 

211, 689, 691 

97, 181 

134 

236 

574 

213 

267 

588 

453, 454, 465, 470 

236 

260, 634 

137, 225, 527 

440, 443 

Connolly, D. 

Cordeiro Maia Saglioni, E. 

Cormack, P. A. G. 

Cortes, J. M. 

Corzo, N. 

Corzo-Martinez, M. 

Coscollà, C. 

Costanza, C. 

Cotte, J. F. 

Covaci, A. 

Crean, C. 

Crego, A. L. 

Cren Olive, C. 

Crevillén, A. G. 

Crovetto, G. 

Cruces-Blanco, C. 

Cruz-Vera, M. 

Cuadrench-Tripiana, A 

Cueva, C. 

Currivan, S. 

Czech, B. 

D’Arrigo, M. 

D’Orazio, G. 

D’Orlyé, F. 

Dachs, J. 

Dadson, A. 

Dae-Hwan, K. 

Dalençon, F. 

Danilevics, U. 

Dapena de la Fuente, E. 

Davankov, V.A. 

Dávila, T.J. 

Davis, R. 

de Alda, M. L. 

De Ferrer, J. 

de Frutos, M. 

de Kanter, F. 

de Koning, S. 

de la Cal, A. 

De La Guardia, M. 

de la Iglesia, P. 

de la Puerta, A. 

de Oliveira, A. R. M. 

De Wit, A. 

Deelder, A. M. 

Deiana, L. 

del Castillo, M. D. 

del Mar Barrios Romero, M. 

del Nogal Sánchez, M. 

Del Pozo-Bayón, M. A. 

del Río, C. 

702 



227, 340, 609, 610 

593 

250 

546 

154 

644 

412 

378, 508 

416 

253 

622 

679, 698 

266 

164, 344 

235, 236, 237, 342 

134 

220, 431 

131 

635 

288 

546 

393, 668, 669 

437 

673 

430 

505, 506, 552 

416 

538 

194 

593, 663 

98 

98 

322 

533 

615 

278 

374 

533 

249 

219, 295 

332 

144 

143, 235 

75, 331 

307, 308 

139, 261, 488 

592 

273 

87, 218 

221 

329 

389 

619 

Delaunay, N. 

Demesmay, C. 

Denizli, A. 

Deol, A. 

Dettmer, K. 

Dias, J. C. M. 

Díaz Llorente, D. 

Díaz Romero, C. 

Diaz, G. 

Díaz, P. 

Díaz, R. 

Díaz-Álvarez, M. 

Diaz-Maroto, Mª. C. 

Diego, J. C. 

Diez-Masa, J. C. 

Diogène, J. 

Dirinck, P. 

Divan, K. 

Djuric, I. 

Dogan-Topal, B. 

Dolliver, W. 

Doménech-Carbó, M. T. 

Domingues, V. 

Domínguez-Romero, J. C. 

Domínguez-Valhondo, D. 

Domínguez-Vega, E. 

Duarte, C. M. 

Dubayle, J. 

Duchateau, A. L. L. 

Dugas, V. 

Dugo, G. 

Dugo, P. 

Dullnig, V. 

Dulsat, J. F. 

Durand, J. S. 

Durgbanshi, A. 

Dyatchkov, I.A. 

Ebri, I. 

Eckhardt, A. 

Edge, A. 

Edwards, K 

Eggink, M. 

Egido, J. 

Ehala, S. 

Eisele, T. 

Eke, Z. 

El Debs, R. 

Elian, M. 

Elmashni, D. 

Elosegi, A. 

Elvira, C. 

Emmenegger, C. 

Epalza, A. 

353 

650 

539 

335, 461 

557 

670, 671 

271 

389 

571 

371 

618 

271 

693 

440, 443 

300, 472 

277, 278, 279, 280, 281 

282, 283 

135, 181 

227, 609, 610 

195 

199, 386 

168 

77, 373 

420 

104, 551, 683 

481 

259, 377, 534 

593 

593 

413 

418 

150 

532 

319 

602, 605, 606 

267, 492, 493, 554, 555 

128, 645 

404 

542, 576 

615 

76, 100, 172, 189, 210, 509 

673, 674, 696, 697 

581 

536 

265 

246 

660 

182 

341 

602, 604, 607 

262, 263, 305, 306, 367 

105 

189 

Erban, A. 

Erger, C. 

Ertas, N. 

Escrig-Doménech, A. 

Escuder-Gilabert, L. 

Esparza, X. 

Esperatti, M. 

Espinosa, M. 

Espinosa-Mansilla, A. 

Espuelas, C. 

Espuelas, J. 

Esquinas, C. 

Estela, J. M. 

Esteve, C. 

Esteve, M. J. 

Esteve-Romero, J. 

Esteve-Turrillas, F. A. 

Eudes, V. 

Evers, D. 

Famiglini, G. 

Fan, Y. 

Fanali, S. 

Fanjul, P. 

Farré, M. 

Farré, R. 

Faulkner, W. 

Faure, K. 

Faye, C. 

Fedorkova, A. 

Fehrenbach, T. 

Felinger, A. 

Fenclova, Z. 

Fernandes, J. C. 

Fernández de la Ossa, M. A. 

Fernández Gutiérrez, A. 

Fernández Laespada, M.E. 

Fernández, M. A. 

Fernández, M. F. 

Fernández, P. 

Fernandez-Alba, A. R. 

Fernández-Alfonso, M. 

Fernández-Fidalgo, C. 

Fernández-Moreno, J. L. 

Fernández-Navarro, J. J. 

Fernández-Ramos, C. 

Fernánedez-la-Villa, A. 

Ferragut, J. A. 

Ferrando, J. L. 

Ferrari, R. 

Ferreira, C. G. 

Ferrer, C. 


703


471, 483, 485 

127 

471 

357 

307, 308 

598 

460, 469 

381 

600 

159, 648 

156 

694 

132, 540, 583 

324 

221 

480 

681 

300, 472 

570 

537 

94 

399 

180 

596 

209, 363, 364, 369, 414, 507 

630, 638, 641, 670, 671 

289, 292, 463 

363, 364 

615 

646, 647 

276 

515 

437 

459, 504, 526 

634, 642 

267 

281, 536, 580 

397, 398 

440, 443, 503, 505, 506 

240, 241, 242, 243, 244, 245 

246, 372, 529 

519, 520 

459, 504, 526 

72, 436 

594 

376, 415 

257, 475, 578 

430 

673,674 

441, 442, 500, 602, 603, 604 

605, 606, 607, 608 

106, 267 

615 

227, 340, 609, 610 

Fiechter, G. 

Filaquier, J. P. 

Fischer E. 

Fischer, B. 

Fischer, L. 

Flament-Waton, M. M. 

Flodrova, D. 

Floris, P. 

Foltova, K. 

Fontanals, N. 

Foret, F. 

Forteza, R. 

Fountain, K. 

Fox D. 

Francés, F. 

Franquet, H. 

Fratta, C. 

Frigola, A. 

Fuchs, M. 

Fuguet, E. 

Fukuhara, K. 

Fülöp, F. 

Gabrielyan, E.S. 

Gago-Martínez, A. 

Galceran, MªT. 

Galindo-Iranzo, P. 

Gallart-Ayala, H. 

Gallego, A. 

Gallego, E. 

Galushko, S. 

Gamboa-Santos, J. 

Gameiro, P. 

Gámiz-Gracia, L. 

García Pinto, C. 

García- Villalba, R. 

García, A. 

García, M. A. 

García, M. C. 

García-Álvarez-Coque, M. C. 

García-Bermejo, A. B. 

García-Campaña, A. M. 

García-Cañas, V. 

García-Gómez, D. 

García-Lafuente, A. 

García-Llatas, G. 

García-Parra, J. 

García-Reyes, J. F. 

García-Ruiz, C. 

García-Villalba, R. 

Garcinuño, R. M. 

Gareil, P. 

177, 265, 313, 368 

237 

579 

377 

588 

134 

458 

227 

165 

144, 365 

587 

673 

294 

468 

494, 495 

682 

167, 339, 406, 407 

681 

634 

565, 600 

523 

83, 91, 158 

86, 146, 444, 621 

677 

591 

539 

688, 690 

564 

289, 292, 404, 631 

313 

341, 342 

599, 633 

100, 697 

420 

673, 674 

378 

479, 654 

580 

404 

436 

652 

445, 628 

430 

257 

680 

446, 447, 464 

513 

684 

354 

561 

378 

687 

663 

622 

580 

Garrido-Frenich, A. 

Garrido-Medina, R. 

Garrote, P. 

Gartland, J. 

Gaspa, L. 

Gemma Giménez, G. 

Germán-Hernández, M. 

Ghasemi, M. 

Gibert, J. M. 

Giera, M. 

Gilart-Alzuria, N. 

Gilbert-López, B. 

Gil-Ramírez, A. 

Giménez, E. 

Gimeno-Adelantado, J. V. 

Ginebreda, A. 

Ginterova, P. 

Giroud, B. 

Glanzer, P. 

Glatz, Z. 

Glavnik, V. 

Glennon, J. D. 

Godayol, A. 

Godejohann, M. 

Godzien, J. 

Goger, N. 

Goikoetxea, M. 

Goltz, L. 

Gómara, B. 

Gómez Perez, M. L. 

Gomez, A. 

Gómez, C. 

Gómez, M. J. 

Gómez-Cordovés, C. 

Gómez-Ramos, M. M. 

González Álvarez, J. 

Gonzalez, C. 

González, I. 

Gonzalez, M. J. 

Gonzalez, R. 

González, V. 

González-Curbelo, M. A. 

González-Gómez, D. 

González-Larena, M. 

González-Mazo, E. 

González-Peñas, E. 

González-Porto, A. V. 

González-Solís, J. 

Goodacre, R. 

Goryainov, S. V. 

Gotor Fernández, V. 

Gotor Navarra, G. 

Goutelard, F. 

Gracia, E. 

Gracia-Bouthelie, R. 

704 



438. 661 

323 

153 

155 

187 

213 

433 

385 

517 

579, 646 

87, 88, 130, 218 

541 

271 

580 

87, 88, 218 

195 

551 

376, 415 

73, 303 

396 

70 

270, 288, 304 

378, 412 

305, 306, 367 

538 

514 

623 

102 

322 

258 

254 

298 

371 

348, 587 

346 

382 

213 

210, 674 

124 

154 

395 

392 

90 

327, 328, 427 

508 

643 

420 

254, 275, 622, 629, 653 

430 

480 

445, 626, 627, 628 

301 

434, 435, 451, 491, 573 

Gradl, M. 

Grass J. 

Greibrokk, T. 

Griffiths, J. 

Grimalt J.O. 

Grivel, C. 

Grulichova, H. 

Grunwaldova, V. 

Guadayol, M. 

Guardino, X. 

Guazzotti, S. 

Guermouche, S. 

Guerrero, L. 

Guerrero-Fernández, J. 

Guifeng Jiang 

Guignard, C. 

Guijarro, M. 

Guillamón, E. 

Guillarme, D. 

Guillén-Casla, V. 

Guiochon, G 

Gumustas, M. 

Gutiérrez Álvarez, M. D. 

Gutierrez, P. 

Haensler, J. 

Hahm, J. H. 

Hairault, L. 

Hajji, A. A. 

Hallermayer, K. 

Hamilton, S. 

Hancock, P. 

Hara, T. 

Hartmann, T. 

Hartonen, K. 

Haun, J. 

He, X. 

Heinisch, S. 

Heinzen, H. 

Helenkár, A. 

Hellerbrand, C. 

Hendrickx, A. 

Henniges, U. 

Hennion, M. C. 

Hernández Cassou, S. 

Hernández Rodríguez, L. 

Hernandez, A. 

Hernandez, D. 

Hernández, F. 

Hernández, M. T. 

Hernández, S. 

Hernández-Borges, J. 

Hernández-Cázares, A. 

Hernández-Hernández, O. 

504 

172 

184 

100 

626, 627 

642 

631 

72, 329, 470, 473, 489, 554 

85, 296, 326, 333, 335, 336 

390, 450, 452, 456, 457, 461 

462, 632 

298 

420 

96 

429 

195 

349 

432, 433 

584 

82 

583 

330 

416 

635 

531 

151 

448 

585 

322 

209 

219 

198, 200 

492, 493 

274 

531 

72, 341 

72, 294, 470, 473, 489, 554 

580 

662 

446, 447 

531 

524 

399 

285 

365, 144 

402 

334, 338 

80 

269, 569, 572 

285 

215, 352, 448, 449 

248 

Hernández-Mesa, M. 

Hernando, M. D. 

Herráez-Hernández, R. 

Herrera, S. 

Herrera-Herrera, A. V. 

Herrero Martín, S. 

Herrero, L. 

Herrero, M. 

Herrero-Martínez, J. M. 

Heuser, S. 

Hevia, D. 

Himmelsbach, M. 

Hoffmann Kowalski, C. 

Hoffmann, L. 

Hofmeister, S. 

Hohnova, B. 

Holmes, E. 

Holopainen, J. M. 

Hong, P. 

Horká, M. 

Horstkotte, B. 

Horvat, A. J. M. 

Horvath, A. 

Hoyos, M 

Hrnĉiřík K. 

Hronek, M. 

Huber, C. G. 

Huerta-Fontela, M. 

Hui, Y. 

Hung, D. 

Hurtado-Fernández, E. 

Hüser, T. 

Huszar, M. 

Ibáñez, C. 

Ibáñez, E. 

Ibáñez, M. 

Ibáñez-Vea, M. 

Idei, M. 

Igualada, C. 

Ilisz, I. 

Imramovsky, A. 

Irth, H. 

Jabor, V. A. P. 

Jac, P. 

Jahn, S. 

Jambor, E. 

Jampilek, J. 

Janssen, H. G. 

Japertas, P. 


705


123 

126, 198, 200, 201, 383 

684 

542, 576, 575 

542, 575, 576 

497, 498 

337 

148, 178, 357 

320 

359 

303 

566, 567 

533 

497, 498 

483 

311 

156 

532 

94 

497, 498 

339 

198 

250 

432, 433 

80, 116, 119 

75, 331, 337 

544, 545, 585 

635 

284 

353, 354, 355 

355 

350, 697 

367 

531 

180 

259 

561 

497, 498 

514 

514 

295 

564 

154 

284 

598 

96 

366 

403, 406 

322 

682 

618 

248 

424 

Jelic, A. 

Jensen, D. 

Jiménez, B. 

Jiménez, M. 

Jiménez-Díaz, I. 

Jin, S. N. 

Jiracek, J. 

Jochmann, M. A. 

Johnsen, E. 

Jones, M.D. 

Josephine, R. 

Jovic, Z. 

Julià, M. 

Jung-Boem Kim 

Jungmayr, A. 

Jurica, J. 

Juskova, P. 

Kacer, P. 

Kakiuchi, T. 

Kang, S. H. 

Kaniansky, D. 

Kanyal, S. S. 

Karakoç, V. 

Karasek, P. 

Karst, U. 

Kasicka, V. 

Kasparova, M. 

Kaštelan-Macan, M. 

Kawabe, T. 

Kay, L. 

Keck, M. 

Kemellár, B. 

Kennedy, J. H. 

Keri, G. 

Khachvankyan, G. Y. 

Khan, A. 

Khomyakov, Y. Y. 

Kim, K. C. 

Kim, K. S. 

Kim, S. H. 

King, B. 

Kirch, W. 

Kirovski, G. 

Kishi, N. 

Kiss, A. 

Klampfl, C. W. 

Klejdus, B. 

Knob, R. 

Kobold, U. 

Köck-Schulmeyer, M. 

Kolb, T. 

Kolovanov, E. 

Kominkova, J. 

365 

353 

423, 424 

373 

635 

562 

102 

117 

392 

505 

562 

657 

385 

261 

307, 308 

544, 545, 585 

144 

348 

330 

136 

178, 357 

97 

119 

160 

532 

148 

249 

286 

140, 161, 173, 663 

636 

257, 286, 287, 475, 481, 578 

296 

644 

410 

680 

221 

367 

532 

289, 292, 463, 573 

514 

497, 498 

195 

195 

623 

692 

186 

436 

524, 597 

597 

323 

386 

255, 396 

Kool, J. 

Kopka, J. 

Koplik, R. 

Kopperi, M. 

Koprivanac, N. 

Koryakova, A. G. 

Koseoglu, O. R. 

Köster, J. 

Kostic, M. 

Kotkowska, O. 

Kovaleva, A. S. 

Kowal, S. 

Kral, V. 

Kramarics, Á. 

Kranz, B. 

Krcmova, L. 

Kretschmer, A 

Kronholm, J 

Kubesová, A. 

Kuebler, K. 

Kujawinski, D. M. 

Kuligowski, J. 

Künnemeyer, J. 

Kusuma, I. W. 

Kuzma, M. 

Laaks, J. 

Lacinova, K. 

Lacomba, R. 

Lacorte, S. 

Ladji, R. 

Lagarda, M. J. 

Lämmerhofer, M. 

Lange, R. 

Laniewski, K. 

Lara-Martín, P. A. 

La-Roca, F. 

Laughlin, B. 

Lebedova, J. 

Lebrón-Aguilar, R. 

Lee, H. J. 

Lee, J. B. 

Lefèvre, I. 

Legay, S. 

Legendre, A. 

Lemiere, F. 

Lendl, B. 

Leon, C. 

León, N. 

León. Z. 

Léonard R. 

Leonardis, I. 

Leon-González, M. E. 

706 



336, 450, 452, 455, 456, 457 

461, 462 

360 

139 

271 

271 

219 

274, 296 

126, 200, 201, 383 

144, 365 

131 

446, 447, 464 

456, 457 

185, 659 

104, 683 

71 

80 

366 

82 

504 

429 

667 

127 

275, 622 

660 

123 

639 

602, 603, 604 

666 

678 

104 

531 

581 

203 

499 

189, 509 

362 

369 

112, 114 

320 

83 

271 

455 

575 

334 

181 

365 

269, 569 

92, 380, 658 

565 

90 

396 

611 

Lerma-García, M. J. 

Letzel, T. 

Lezsak, G. 

Li Bassi, G. 

Liapikou, M. 

Liddicoat, T. 

Lindner, W. 

Linford, M. R. 

Lingeman, H. 

Liu, Y. 

Lizarraga, E. 

Lliberia, J. L. 

Llorca, J. 

Llorca, M. 

Lobinski, R 

Lohmann, W. 

Lojkova, L. 

Lokajová, J. 

Lombardo-Agüí, M. 

Lopes Modro, A. 

López Días, V. 

Lopez, C. 

López, F. J. 

Lopez, I. 

Lopez, R. 

López-Darias, J. 

López-López, M. 

López-Mahía, P. 

Lopez-Nogueroles, M. 

López-Teijón, M. 

Lorand, T. 

Lorenzo, MªP. 

Lough, W. J. 

Lozada-Castro, J. J. 

Lozano, A. 

Lozano, Y. 

Lucci, P. 

Lucena, R. 

Lundanes, E. 

Luong, J. H. T. 

Luque, N. 

Lusardi, R. 

Luzón-Toro, B. 

Luzova, V. 

Ly-Verdú, S. 

Maada, I. 

Maasz, G. 

Macka, M. 

Mádr, A. 

Madru, B. 

Magro-Moral, J. 

Maier, L. 

167, 339, 403, 405, 406, 407 

408, 611 

174, 665 

331 

509 

566, 567 

698 

325 

476 

571 

376, 415 

412 

204, 395 

619 

437 

199 

587 

164 

339 

391 

159, 648, 664 

672 

599 

283 

595 

294 

397, 398, 440, 441, 442, 443 

500, 501, 503, 505, 506, 512 

551, 552, 

269, 569, 572 

484, 496 

81 

365 

538 

689 

213 

344, 418, 466, 467, 647 

691 

137, 225, 235 

558, 560, 685 

679, 698 

695 

697 

177, 265, 313, 368 

659 

376, 415 

620 

447 

536 

172, 189, 210 

264 

253, 435, 439, 491, 502 

556, 559 

271 

465 

414 

Maier, V. 

Maijó, I. 

Makrlik, E. 

Malato, O. 

Malesevic, M. 

Malitesta, C. 

Mallon P. 

Mancebo, M. T. 

Mancha de LLanos, A. 

Manchón, N. 

Mangas Alonso, J. J. 

Mangelings, D. 

Manigandan, P. 

Mansilha, C. 

Mantegazza, A. 

Mantyla, S. 

Manzano, P. 

Marak, J. 

Marazuela, M. D. 

Marcé, R. M. 

March, J. G. 

Marco, M. P. 

Marco-Peiró, S. 

Marín Carrasco, P. 

Marín, F.R. 

Marina, Mª. L. 

Mark, L. 

Marošanovic, Biljana 

Marque, H.Z. 

Marques, L. A. 

Martial, F. 

Martin, J. 

Martin, M 

Martin, Mª. T. 

Martin-Alonso, J. 

Martín-Álvarez, P.J. 

Martin-Biosca, Y. 

Martín-Esteban, A. 

Martínez Galera, M. 

Martínez Uroz, M.A. 

Martínez Vidal, J. L. 

Martinez, E. 

Martínez, J. A. 

Martínez, M. A. 

Martínez, R. 

Martínez-Alcázar, M. P. 

Martínez-Bueno, M. J. 

Martínez-Cañas, M. A. 

Martínez-Castro, I. 

Martinez-Gomez, M. A. 

Martínez-Olondris, P. 

Martínez-Rodríguez, A. 

Martínez-Villalba, A. 


707


363, 364 

143, 235 

187 

122 

511 

298 

510, 624 

337 

287 

495 

494 

127 

416, 693 

267 

223, 471, 483, 485 

698 

248 

303 

351 

418 

644 

462 

556, 557, 558, 559, 560, 685 

223 

143 

80 

456, 457 

294 

106 

263 

135 

581 

477 

423, 424 

438 

140, 173 

161 

509 

143 

625 

249 

377, 534 

465 

205 

479 

473 

213 

273 

673 

184, 229 

184 

124 

467 

98 

441, 442 

Martins, C. P. B. 

Martín-Ventura, J. L. 

Martrat, B. 

Martrat, M. G. 

Masa, A. 

Mascotto, S. 

Masia, A. 

Masova, A. 

Matencio, E. 

Mateo, E. M. 

Mateo, R. 

Max, J. P. 

Maya, F. 

Mayboroda, O. A. 

Mayer, H. K. 

Mazzotta, E. 

McBrien, M. 

McCalley, D. 

McGillicuddy, N. 

McLeod, F. 

Medeiros, E. F. 

Medina-Escrivà, L. 

Medina-Hernandez, M. J. 

Meier, J. 

Meilhac, O. 

Melles, D. 

Méndez, A. 

Mendiola, J. A. 

Menéndez, J. A. 

Meni, L. 

Mercader, J. V. 

Merino, B. 

Messina, G. A. 

Mestek, O. 

Metzger, M. 

Meyer, J. 

Meyer, J. I. 

Mezcua, M. 

Michel, J. B. 

Miguel, E. 

Miksik, I. 

Milton, D. 

Mingo, E. 

Minguillón, C. 

Miralles, A. 

Miron, T. L. 

Mishra, M. 

Mokhtar, H. 

Molina-Díaz, A. 

Moliner-Martinez, Y 

Molins-Legua, C. 

Molnár-Perl, I. 

Moncadas,Mª. J. 

Mondello, L. 

Montealegre, C. 

290 

434, 453, 515, 518, 519 

572 

321 

301, 482 

228, 591 

524 

555 

173 

235 

409 

593 

128, 260, 634, 642, 645 

434, 463, 573 

685 

122 

137, 225, 527 

391 

459 

510 

127 

132, 540, 583 

672 

363, 364, 369, 414, 507, 670 

671 

658 

123 

102 

677 

571 

110 

221 

147 

684 

137, 225 

588 

600 

629 

94 

488 

516 

542, 575, 576, 620 

660 

221 

336 

255, 391 

576 

677 

92, 193, 231, 350, 380, 351 

163 

644 

254 

438 

Montesdeoca-Esponda, S. 

Montilla, A. 

Montsko, G. 

Moore, D. 

Mora, L. 

Moraes, E. 

Moragues, F. 

Morales Soto, A. 

Morales, L. 

Morales-Cid, G. 

Morante-Zarcero, S. 

Moreau, T. 

Moreno Cordero, B. 

Moreno, F. J. 

Moreno, L. 

Moreno, V. 

Moreno-Arribas, M. V. 

Moreno-Bondi, M. C. 

Moreno-González, D. 

Morillas, F. 

Morin, P. 

Morrison, D. 

Moukhchan, F. 

Moyano, E. 

Moyna, A. 

Mozeto, A. A. 

Mueller, H. 

Mügge, C. 

Muñoz de la Peña, A. 

Muñoz, G. 

Muñoz, I. 

Muñoz, S. 

Muñoz-Arnanz, J. 

Muñoz-González, C. 

Murgia, L. 

Musilova, J. 

Nácher-Mestre, J. 

Nakajima, Y. 

Nász, S. 

Navajas, H. 

Navalón, A. 

Navarro, I. 

Navarro-Ortega, A. 

Navarro-Pascual-Ahuir, M. 

Navarro-Villoslada, F. 

Navea Noemí 

Nehls, I. 

Nesterenko, E. 

Nesterenko, P. N. 

Neto, R. R. 

Newton, A. 

Nicholson, T. 

708 



365 

121 

168 

586 

512, 553 

512, 553 

344, 466, 467, 643 

363, 364, 369, 480, 670 

278 

191, 208 

325 

320 

154 

564 

360 

168 

269, 572 

94 

400 

285, 385 

434, 518, 519, 520 

542, 576 

429 

327, 328, 427 

101 

173, 638 

200, 383 

91, 158 

692 

547, 550 

413 

413 

546 

604 

393, 668, 669 

682 

156 

614 

561, 562 

270, 288, 304 

323 

267 

631 

368 

136 

547, 550 

156 

247 

199, 386 

122 

102 

Niessen, W. M. A. 

Nocun, M. 

Nolte, T. 

Nováková, L. 

Novella, J. L. 

Nozal, L. 

Nozal, Mª. J. 

Núñez, O. 

Ochoa-Aranda, E. 

Ochs, S. M. 

O’Connor, B. 

Odsbu, I. 

Oefner, P. J. 

Oertel, R. 

Oeste, K. 

Ohla, S. 

Ohmacht, R. 

Okamoto, H. 

Okano, L. T. 

Oktabec, Z. 

Olano, A. 

Olea, N. 

Olivares, I. R. B. 

Oliver, R. 

Olivier, B. C. 

Olmos, J. 

Olsen, R. 

Omamogho, J. O. 

Onghena, M. 

Onur, F. 

Orinak, A. 

Orinakova, R. 

Orlicz- Szczesna, G. 

Ortiz, R. 

Osete-Cortina, L. 

Osorio, V. 

Ostatna, V. 

Otto, M. 

Ovcharov, M. V. 

Ozkan, S. A. 

Pabst M. 

Pacchiarotta, T. 

Pacepavicius, G. 

Padilla-Sánchez, J. A. 

Paetzold, R. 

Palabiyik, I. M. 

Palecek, E. 

Pallicer, J. M. 

Palma, P. 

Palomera, E. 

Panda, S. K. 

679 

551 

525, 655 

210 

309 

122 

497, 498 

695 

695 

272, 530 

181, 256 

399 

249 

269 

92, 192, 231, 350, 351, 379 

381, 382, 589, 658 

285 

532 

635 

347 

127 

646 

101, 105, 191, 208 

155, 219, 259, 295, 377, 534 

203 

128, 260, 634, 642, 645 

205 

205 

553 

654 

682 

255, 396 

146 

266 

397, 398 

673, 674 

595 

635 

279,281,283 

215 

309 

167, 403, 404, 405, 406, 407 

123 

334, 338 

90, 176, 535 

104, 221, 510, 624, 675, 676 

692 

677 

564 

623 

110 

437 

458, 639, 652 

319 

271 

Paniagua, E. 

Paniagua, G. 

Pardo, O. 

Pareja, L. 

Parenica, J. 

Parera, J. 

Park, Y. B. 

Parrilla, M. M. 

Parrilla, P. 

Pashkova, E. B. 

Pastor, A. 

Pataj, Z. 

Pataridis, S. 

Patko, B. 

Paull, B. 

Pejchal, V. 

Pelclova, D. 

Pelko, S. 

Pena-Abaurrea, M. 

Penna, R. 

Perales, J. F. 

Pereira Netto, A. D. 

Pereira, L. 

Perera, R. W. H. 

Pérez Pavón, J. L. 

Pérez, A. M. 

Pérez, E. 

Pérez, I. 

Perez, J. A. 

Pérez, S. 

Pérez-Arribas, L. V. 

Pérez-Ballesta, P`. 

Pérez-Coello, Mª. S. 

Pérez-Fernández, V. 

Pérez-Parada, A. 

Periago Jiménez, F. 

Periša, M. 

Peris-Vicente, J. 

Peroni, D. 

Pes, O. 

Petr, J. 

Petrovic, M. 

Petru, K. 

Pichon, V. 

Picó, Y. 

Piechotta, C. 

Pietsch J. 

Pin, N. 

Pineda, L. 

Pinho, C. 

Pino, V. 

Pintado, M. 

Piñer, R. 


709


272, 374, 530 

258 

588 

275 

651 

549 

366, 470, 473, 489 

177, 265, 368 

544, 545, 585, 586 

334, 338 

396, 499, 662 

208 

310 

563 

633 

360 

254, 629 

358, 392 

321 

242, 243, 372 

599 

182 

595 

444 

130 

87, 218 

666 

500 

235, 342 

480 

82 

400 

98 

289, 292, 573 

97 

223, 483, 485 

566 

109, 247, 537 

187 

277, 278, 279, 280, 281, 282 

283 

664 

603 

85, 296, 326, 336, 390, 455 

632 

613 

347, 439 

167, 403, 405, 406, 407, 

408, 611 

546 

592,593,663,196 

Pirogov, A.V. 

Pirzada, Z. 

Pisanu, E. 

Pitarch, E. 

Planas, C. 

Plankeele, J .M. 

Plaza, M. 

Plaza-Bolaños, P. 

Plíšek, J. 

Polasek, M. 

Polo-Díez, L. M. 

Pontes Massa, M. C. G. 

Ponzio, T. 

Popovic, G. 

Porte, C. 

Portner, C. 

Portolés, T. 

Potthast, A. 

Potts, W. 

Pous-Torres, S. 

Pozo, O. J. 

Pozo-Ayuso, D.F. 

Prado Burguete, C. 

Prat, C. 

Preiswerk, T. 

Preston, K. 

Prieto-Blanco, M. C. 

Puchalska, P. 

Puerta, A. 

Puignou, L. 

Pukkila, J. 

Pupo, M. T. 

Purcaro, G. 

Quintanilla-López, J. E. 

Quintás, G. 

Raba, B. 

Radisic, M. 

Ràfols, C. 

Rama, O. 

Rambla-Alegre, M. 

Ramírez, N. 

Ramiro Alegre, J. M. 

Ramis-Ramos, G. 

Ramos, A. 

Ramos, L. 

Ranc, V. 

Randhawa, R. 

Randon, J. 

151 

331 

614 

445, 628 

278, 279, 282 

437 

342 

301, 478, 482, 486 

332 

206 

464 

385 

687 

127 

332, 348, 373, 587 

271 

139, 261 

652 

378 

275 

85 

122 

490 

673 

646 

476, 524 

341 

437 

508 

391 

137, 225, 527 

445, 626, 627, 628 

594 

694 

253, 439, 502 

514 

387 

177, 265, 313, 368 

255, 396 

684 

579, 646, 647 

358 

109, 247, 293, 537 

376, 415 

640 

205 

73, 303 

263 

513 

435, 439 

240, 241, 245, 246, 252, 372 

529 

581 

348, 373, 587 

491, 518, 519, 520 

Rarier, C 

Rathore, R. 

Rathsack, P. 

Ravelo-Pérez, L. M. 

Raviolo, M. A. 

Rebelo, H. 

Regueiro-Vilar O. 

Reig, M. 

Reijmar, K. 

Reiller, P. E. 

Remiro, R. 

Rezacova, A. 

Ribas Font, C. 

Ribet, J. P. 

Riekkola, M-L. 

Rigol, M. 

Rikker, T. 

Rincón, A. A. 

Ríos Lombardía, N. 

Ripollés, C. 

Ripoll-Seguer, L. 

Rivera, J. 

Rivera, S. 

Robles-Molina, J. 

Roca, F. X. 

Roca, M. 

Rocamora, L. 

Rocha, S. 

Rodríguez Rodríguez, E. 

Rodriguez, E. 

Rodríguez-Bencomo, J. J. 

Rodríguez-Delgado, M. A. 

Rodríguez-Gonzalo, E. 

Rodriguez-Navas Gonzalez, C. 

Rodríguez-Sánchez, S. 

Roh, J. S. 

Román Falcó, I.P. 

Romero-Gonzalez, R. 

Rosales-Conrado, N. 

Roscales, J. L. 

Rosell, M. G. 

Rosenau, T. 

Rosés, M. 

Rostagno, M. A. 

Rubio, J. L. 

Rubio, N. 

Rudaz, S. 

Ruggeri, M. 

Ruiz, L. V. 

Ruiz-Aceituno, L. 

Ruiz-Ángel, M. J. 

Ruiz-Gayo, M. 

Ruiz-Jiménez, J. 

Ruiz-Matute, A. I. 

710 



228, 536, 580, 591 

371 

330 

221 

644 

556, 557, 558, 559, 560, 685 

602, 605,607,608 

373 

286, 287 

247 

552 

460, 469 

81 

387, 597, 678 

599 

649 

513 

416 

86, 147, 621 

140 

625 

500, 501, 512 

221 

318 

254, 275, 622 

350 

270 

270 

574 

290, 291 

191 

638, 641 

173 

499, 662 

423 

253, 502 

253, 434, 435, 439, 451, 491 

502, 573 

620 

468 

227, 609, 610 

165 

539 

299 

314 

81, 327, 328, 427 

480 

75, 337 

258 

148 

337 

223 

418 

Rupérez, F. J. 

Ruth, K. 

Ružicka, F. 

Sabater, S. 

Sad, C. M. S. 

Sagrado, S. 

Sáiz, J. 

Sakeye, M. 

Salcedo, J. 

Sales, J. 

Salgado, A. 

Salplachta, J. 

Salvà, M 

Salvador, A. 

Salvador, J. P. 

Salvia, M. V. 

San Andrés, M. P. 

Sanchez, A. T. 

Sanchez, J. M. 

Sánchez-Avila, J. 

Sánchez-Brunete, C. 

Sánchez-Hernández, L. 

Sanchez-Vila, X. 

Sanchis, J. 

Sancho, J. V. 

Sandron, S. 

Sanli, N. 

Sanli, S. 

Santana, R. S. 

Santana-Rodríguez, J. J. 

Santos, A. M. S. 

Santos, F. J. 

Santos, J. 

Santos-Delgado, Mª. J. 

Santrucek, J. 

Sanz, J. 

Sanz, M. L. 

Sanz, P. 

Sanz-Nebot, V. 

Sarazin, C. 

Sarrión, N. 

Satana, H. E. 

Šatínsky, D. 

Saunders, K. 

Saurina, J. 

Saurina, X. 

Sazelova, P. 

Schafer, W. 

Schilling, B. 

Schimperkova, T. 

Schmid, A. 

Schmidt, C. 

677 

108, 148, 178, 346, 650, 657 

661 

214, 216, 417 

485 

221 

274 

96 

141 

168, 552 

249 

267, 489, 554, 555 

599 

596 

81 

580 

533 

110 

629 

122 

574 

167, 339, 403, 405, 406, 407 

408, 611 

295 

367 

272, 297, 374, 530 

262 

409 

123 

393, 477, 669 

72 

341 

85, 333, 335, 336, 450, 452 

455, 456, 457, 461, 462 

400, 401, 574 

186, 229, 672 

672 

521, 522, 523, 563, 582 

310 

201 

334, 338 

199 

546 

546 

413 

543 

330 

298 

373 

469 

374 

155 

297 

521 

366 

Schmidt, S. 

Schmidt, T. C. 

Schmitt, C. 

Schoenmakers, P. J. 

Schreiner, M. 

Schuhmacher, M. 

Schuster, G. 

Schwarzinger, C. 

Schymanski, E. 

Scriba, G. K. E. 

Sedlakova, P. 

Segura Carretero, A. 

Segura, J. 

Seijo, D. 

Sentellas, S. 

Señorans, J. 

Serra, C. 

Serrahima, E. 

Serrano, R. 

Serra-Prat, M. 

Sesso, T. A. C. 

Sevcik, J. 

Shick, L. 

Shirey, R. 

Shpigun, O.A. 

Sidisky, L. M. 

Sierra, I. 

Silva, B. F. 

Silva, M. F. 

Simo C 

Simó, S. 

Simó-Alfonso, E. F. 

Simões, R. A. 

Simonet Suau, B.M. 

Simonet, B. M. 

Simonovska, B. 

Sinclair, T. 

Singh, S. 

Siroka, J. 

Sistani, H. 

Siviero, A. 

Sivsammye, S. 

Skantarova, L. 

Sklenarova, H. 

Šlais, K. 

Smarsly, B. 

Smått, J. H. 

Smetalova, D. 

Smirnov, K.N. 

Smith, D. 

Smolenkov, A. D. 

Smrke, S. 

Snoblova, M. 


711


544, 545 

100 

299, 543, 544, 545, 586 

544, 545, 585 

75, 337 

477 

201 

538 

451, 502, 515 

290, 291 

588 

466, 467 

271 

158 

484, 496 

339 

432, 433 

449 

477 

418 

262 

154 

307, 308 

619 

408, 611 

367 

532 

399 

261 

Sobotka, L. 

Solé, D. 

Solich, P 

Solichova, D. 

Solinova, V. 

Sombra, L. L. 

Song, J. 

Sonnery, S. 

Soria, A. C. 

Sosa-Ferrera, Z. 

Sotgia, S. 

Soto, E. 

Soy, D. 

Stack, E. M. 

Stankov, V. 

Stanova, A. 

Stavikova, L. 

Steenbergen, H. 

Stege, P. W. 

Steiner, F. 

Stenerson, K. 

Stevens, A. P. 

Stressler, T. 

Subramanian, N. H. 

Svidrnoch, M. 

Sweeney, B. 

Syslova, K. 

Szatmári, I. 

Szekeres, Z. 

344, 348, 467, 643 

139, 261 

579 

602, 603, 604, 605, 606, 607 

608 

271 

241, 242, 243, 244, 372 

284 

98 

680 

444 

590 

166 

143, 235 

679 

250 

589 

359 

481 

172 

417 

141 

584 

342 

288 

250 

Toribio, L. 

Torkos, K. 

Torrado, S. 

Torre, M. 

Torres, A. 

Torres-Lapasió, J. R. 

Toyo’oka, T. 

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Trias, R. 

Tsunoda, M. 

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Ubillús, F. 

Uclés, S. 

Uliyanchenko, E. 

Ulrich, N. 

Unwin, R. 

Uriel C. 

Uslu, B. 

Uzun, L. 

160 

625 

474 

426 

284 

538 

413 

644 

597 

119 

293 

374 

489 

333, 335 

108, 346, 357, 360 

90, 176, 535 

194 

589 

301,478, 482, 486 

95, 361, 362 

331 

309, 311 

284 

402 

Tachibana, S. 

Tadeo, J. L. 

Tahrani A. 

Taka, T. 

Takemura, A. 

Talaga, P. 

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Thompson, R. 

Toldrá, F. 

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Toman, P. 

Tomandl, J. 

Tomitsuka, T. 

Tonon, M. A. 

126, 198, 200, 201, 383 

332 

112, 114, 186, 229 

366 

581 

689 

494, 495 

220, 431 

214, 417 

215 

671 

216 

593 

204, 395 

198, 201 

531 

227, 609 

531 

286, 475 

124 

248 

95, 361, 362 

193 

624, 675, 676 

602 

Vail, M. A. 

Vainikka, K. 

Valcárcel, M. 

Valentova, E. 

Valladolid, I. 

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Valle-Algarra, F. M. 

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Vasanits-Zsigrai, A. 

Vazhentsev, A. 

Vázquez, A. 

Vazquez, M. 

Vazquez-Roig, P. 

Vega, A. 

712 



291 

542 

329 

604, 608 

517 

209, 211, 630, 689, 691 

599 

513 

184 

462 

660 

432, 433 

73, 303 

161, 173 

644 

633 

644 

161 

578 

255 

80 

599 

511 

490 

542, 576, 660 

463, 515 

556, 557, 559, 685 

376, 415 

580, 584 

95, 361, 362 

517 

214, 216, 352 

366 

586 

532 

96 

276 

617 

117 

521, 522, 523, 563, 582 

635 

649, 656 

Vega-Morales, T. 

Vela, F. 

Velasco, D. 

Velasco, E. 

Vendrell, E. 

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Vo, T. D. T. 

Vogel-da Silva, F. 

Vogt, R. 

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Vovk, I. 

Vujevic, D. 

Vulliet, E. 

587 

692 

144 

548 

371, 389, 619 

69 

320 

367 

314, 315, 316, 317 

514 

94 

539 

636 

231 

231 

250 

275 

91, 158, 421 

132, 540 

514 

497, 498 

321 

256, 252,655 

264 

585 

542, 575, 576, 660 

543 

511 

124 

297 

565 

448 

311 

178, 357 

83, 158 

588 

566, 567 

249 

167, 405, 406, 407 

Wiikinkoski, E. 

Wijnants, M. 

Wijtmans, M 

Wilken A. 

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Wilson, I D 

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Yazawa I. 

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Zendulka, O. 

Zhang, L. 

Zhou, L. 

Zinellu, A. 

Zivanovic, L. 

Zmatlikova, Z. 

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380 

117 

298 

258 

650, 657 

617 

617 

82, 373, 477, 587 

108, 357 

198, 649, 656, 681, 686 

126, 200, 383 

Walsh, Z. 

Weber, G. 

Weidmann, C. 

Welch, C. J. 

Werres, F. 

Wibowo, A. H. 

Wichmann, H. 

Wiedmer, S. 

Wiese, S. 

Wiest, L. 

Wiest, L. A. 


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