CN101963600A - Method for measuring cotinine in body fluid through molecular-imprinting solid-phase extraction-liquid chromatography - Google Patents
Method for measuring cotinine in body fluid through molecular-imprinting solid-phase extraction-liquid chromatography Download PDFInfo
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- CN101963600A CN101963600A CN 201010182271 CN201010182271A CN101963600A CN 101963600 A CN101963600 A CN 101963600A CN 201010182271 CN201010182271 CN 201010182271 CN 201010182271 A CN201010182271 A CN 201010182271A CN 101963600 A CN101963600 A CN 101963600A
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- phase extraction
- tianning
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Abstract
The invention relates to a method for measuring the cotinine in a body fluid, in particular to a method for measuring the cotinine in the body fluid through molecular-imprinting solid-phase extraction-liquid chromatography. The method sequentially comprises the following steps of: (1) synthesizing a cotinine molecular-imprinting polymer by using the cotinine as a template; (2) preparing a solid-phase extraction small column by using the synthesized polymer as a solid-phase extraction adsorbent; (3) sequentially leaching the solid-phase extraction small column by adopting an acetonitrile solution containing organic acid and water, alcohol and a buffer solution; (4) concentrating an eluant to dryness, and adding an acetate buffer solution to obtain a detected liquid; and (5) measuring the cotinine in the detected liquid by utilizing high-efficiency liquid chromatography. In the invention, the cotinine in the body fluid is firstly completely extracted and then eluted through solid-phase extraction, thereby realizing the rapid and accurate quantitative analysis of the cotinine thereof.
Description
Technical field
The present invention relates to the assay method of Tianning in the body fluid, particularly a kind of method of utilizing the method for Tianning in the synthetic Tianning molecularly imprinted polymer mensuration body fluid.
Background technology
Along with the continuous propelling of global Tobacco Control campaign, the public is for smoking and health problem growing interest.Tianning is the primary product of nicotine at body metabolism, advantage such as have long half time, can accurately measure from biological fluid.Urine, blood and blood plasma, tell the Tianning in the liquid, be considered to a kind of special efficacy, highly sensitive, the most believable biological marker, can reflect that human body takes in the size of nicotine dosage by smoking or diet.Smoking population and non-smokers are exposed to that there is good positive correlation in detected Tianning concentration in the degree of smoke of tobacco and its body fluid.Therefore Tianning is the biomarker that suitable evaluation human body exposes main flow flue gas in tobacco or environmental tobacco smoke.
The analytical approach of measuring at present Tianning in the human body fluid comprises spectrophotometric method, gas chromatography-nitrogen phosphorus detection method, ion trap mass spectrometry method or ionization source mass spectroscopy, high performance liquid chromatography (HPLC) uv detection method, HPLC atomic force chemical ionization source mass spectrometry, pre-column derivatization HPLC analytic approach,
125I radio-immunity detection method and monoclonal antibody enzyme immunodetection etc.In order to purify or the pre-concentration sample, nearly all analytical approach all adopts liquid-liquid extraction (LLE), the Solid-Phase Extraction sample pre-treatments technology such as (SPE) of comprising.The LLE process is loaded down with trivial details, time-consuming, consume a large amount of organic solvents, healthy and environment caused adverse effect to the operator.And at the complex system sample, classical SPE adsorbent lacks selectivity again, can't carry out quantitative test to the Tianning in the human body fluid fast and accurately.
Summary of the invention
Purpose of the present invention provides the method for Tianning in a kind of molecular imprinting Solid-Phase Extraction-liquid chromatogram measuring body fluid just at the deficiencies in the prior art part.The Tianning molecularly imprinted polymer that this method at first prepares high selectivity extracts Tianning in the body fluid fully, passes through the Solid-Phase Extraction wash-out then, thereby realizes wherein quick, the accurately quantitative test of Tianning.
The objective of the invention is to be achieved through the following technical solutions:
The method of Tianning in a kind of molecular imprinting Solid-Phase Extraction-liquid chromatogram measuring body fluid may further comprise the steps successively:
(1) will seal behind Tianning, monomer, crosslinking chemical and initiating agent, pore generating agent ultrasonic degas, the logical nitrogen, polymerization under water-bath, then polymkeric substance is ground, sieves, collect the polymkeric substance of 25-36 μ m, obtain required particle behind the sedimentation removal fine particle repeatedly with acetone.Required particle is packed in the glass chromatography column, use organic acid soln and the drip washing of 100mL-300mL alcohol of 100mL-400mL respectively, the template molecule Tianning in the wash-out imprinted polymer.Polymer beads behind the wash-out places baking oven dry, the Tianning molecularly imprinted polymer that must synthesize.
(2) with synthetic polymkeric substance as solid phase extraction adsorbents, take by weighing 200mg-300mg fill in 3mL SPE polypropylene void column (Supelco, Bellefonte, USA) in, the preparation solid phase extraction column.
(3) adopt the acetonitrile solution contain organic acid, water, alcohol, buffer solution drip washing solid phase extraction column successively, go up sample after adopting buffer solution to regulate the body fluid sample pH value, acetonitrile solution drip washing pillar with buffer solution and organic amine, remove salt and other matrix compounds in the humoral sample, adopt the acetonitrile solution that contains organic acid, water that pillar is carried out wash-out.
(4) collect respectively step (3) drip washing, flow out the solution of link, be concentrated near doing, add acetate buffer solution, must detect liquid.
(5) utilize high-performance liquid chromatogram determination step (4) gained to detect Tianning in the liquid.
Chromatographic condition: chromatographic column: Zorbax SB-C18 (250 * 4.6mm, 5 μ m, Agilent company); Moving phase: A is buffer salt solution mutually, and B is acetonitrile mutually, isocratic elution, wash-out ratio: A: B=88%: 12%; Comprising concentration in the moving phase is that 0.02mol/L heptane base sodium sulfonate is made ion-pairing agent; Flow velocity: 1.0mL/min; Detecting device: diode array detector; The detection wavelength is 260nm; Sample size: 20 μ L.
Among the present invention, described body fluid can be urine, blood, blood plasma or tells liquid.
Monomer is the olefin(e) acid that comprises one, two or three carbon atom substituted alkyl in the described step (1).Crosslinking chemical is olefin(e) acid monobasic alcohol ester or the polyol ester that comprises one, two or three carbon atom substituted alkyl.Pore generating agent is that one of one, two or three carbon atom replaces or polysubstituted chlorohydrocarbon; Initiating agent is that one of one, two or three alkyl replaces or polysubstituted azonitrile.
The degassing time is 5-20min in the described step (1); The logical nitrogen time is 5-20min; Bath temperature is controlled at 40 ℃-80 ℃; The water-bath time is 12h-36h.
Organic acid is the monobasic organic acid or the multicomponent organic acid of one, two or three carbon atom in the leacheate of described step (1).Alcohol is the monohydroxy alcohol or the polyvalent alcohol of one, two or three carbon atom.The polymer beads baking temperature is controlled at 100 ℃-150 ℃.Be controlled at 12h-36h drying time.
Organic acid is that one of one, two or three carbon atom replaces or polysubstituted fluoro organic acid in the acetonitrile solution that contains organic acid, water of described step (3); The organic acid ratio is 1%-5%, and the water ratio is 1%-5%.
The alcohol of the adjusting solid phase extraction column of described step (3) is the monohydroxy alcohol or the polyvalent alcohol of one, two or three carbon atom.
Adjusting, drip washing solid phase extraction column in the described step (3), the buffer salt of regulating humoral sample are monoacid ammonium or the polyprotonic acid ammonium and the ammonia spirit composition of one, two or three carbon atom.The pH scope of regulating humoral sample is 8-11.Organic amine is one, two or three alkyl replacement ammoniums in the organic amine acetonitrile solution of drip washing solid phase extraction column.
The process of doing that is concentrated in the step (4) is carried out under inert gas shielding, and temperature is 30-50 ℃.Acetate buffer solution pH scope: 3-5, amount is controlled at 100-500 μ L.
Outstanding advantage of the present invention is: the present invention is masterplate with the Tianning, adopt non-covalent synthetic method to prepare the Tianning molecularly imprinted polymer, the Tianning molecularly imprinted polymer of high selectivity can extract Tianning in the body fluid effectively fully, pass through effective solid-phase extraction column wash-out then, thereby realize wherein quick, the accurately quantitative test of Tianning.Method of the present invention not only can be measured such as Tianning content in the such complex system of body fluid, and selectivity is strong, highly sensitive, accuracy is good, easy and simple to handle fast.
Embodiment
The present invention is further described below with reference to embodiment, but does not limit the present invention.
Embodiment
A, the non-covalent synthetic method of employing prepare the Tianning molecularly imprinted polymer.With the Tianning of 1mmol, the methacrylic acid of 4mmol and the methylene chloride of 5.6mL join in the ampere bottle of 20mL, after fully mixing, add the ethylene glycol dimethacrylate of 20mmol and the azoisobutyronitrile of 0.24mmol again.Ultrasonic degas 10 minutes, logical nitrogen 5 minutes, sealing was inserted in 60 ℃ of water-baths polymerization 24 hours.Broken ampere bottle takes out bulk polymer, grinds, screening, collects the polymkeric substance of 25-36 μ m particle diameter, with acetone repeatedly sedimentation remove fine particle.Get gained particle 1g, in the glass chromatography column of packing into, methyl alcohol/acetate (90: 10) of using 300mL respectively (v/v) and the drip washing of 100mL methyl alcohol, the template molecule in the wash-out imprinted polymer.Polymer beads placed 105 ℃ of baking oven dried 24 hours, stored for future use.
B, the 200mg polymer beads filled in preparation SPE pillar in the SPE polypropylene void column of 3mL.
C, extraction humoral sample.Before last sample, use acetonitrile-H of 50mmol/L respectively
2The O-trifluoroacetic acid (95: 2.5: 2.5, v/v) solution 5mL, methyl alcohol 3mL and CH
3COONH
4-NH
3 (aq)Buffer solution (pH=9.0) 2mL regulates the SPE pillar.Use CH
3COONH
4-NH
3 (aq)Buffer solution (pH=9.0) is regulated the pH value of urine sample and with the water system membrane filtration of 0.45 μ m, last sample volume is 1.0mL.Use 1.0mLCH respectively
3COONH
4-NH
3 (aq)(99: 1, v/v) drip washing SPE post was to remove salt and other matrix compounds in the urine sample for buffer solution and 1mL triethylamine acetonitrile solution.Elute soln is acetonitrile-H
2The O-trifluoroacetic acid (95: 2.5: 2.5, v/v) solution 2.0mL.
All solution of d, collection drip washing, outflow link, 40 ℃ of N
2Blow near and do, add the acetate buffer solution of 200 μ LpH 3.1 again.
E, the gained sample is carried out liquid chromatography-diode array detector analysis.
Claims (9)
1. the method for Tianning in molecular imprinting Solid-Phase Extraction-liquid chromatogram measuring body fluid is characterized in that: may further comprise the steps successively:
(1) will seal behind Tianning, monomer, crosslinking chemical and initiating agent, pore generating agent ultrasonic degas, the logical nitrogen, polymerization under water-bath, then polymkeric substance is ground, sieves, collect the polymkeric substance of 25-36 μ m, obtain required particle behind the sedimentation removal fine particle repeatedly with acetone, required particle is packed in the glass chromatography column, use organic acid soln and the drip washing of 100mL-300mL alcohol of 100mL-400mL respectively, template molecule Tianning in the wash-out imprinted polymer, polymer beads behind the wash-out places baking oven dry, the Tianning molecularly imprinted polymer that must synthesize;
(2) with synthetic polymkeric substance as solid phase extraction adsorbents, take by weighing 200mg-300mg and fill in the SPE polypropylene void column of 3mL, the preparation solid phase extraction column;
(3) adopt the acetonitrile solution contain organic acid, water, alcohol, buffer solution drip washing solid phase extraction column successively, go up sample after adopting buffer solution to regulate the body fluid sample pH value, acetonitrile solution drip washing pillar with buffer solution and organic amine, remove salt and other matrix compounds in the humoral sample, adopt the acetonitrile solution that contains organic acid, water that pillar is carried out wash-out;
(4) collect respectively step (3) drip washing, flow out the solution of link, be concentrated near doing, add acetate buffer solution, must detect liquid;
(5) utilize high-performance liquid chromatogram determination step (4) gained to detect Tianning in the liquid:
Chromatographic condition: chromatographic column: Zorbax SB-C18 (250 * 4.6mm, 5 μ m, Agilent company); Moving phase: A is buffer salt solution mutually, and B is acetonitrile mutually, isocratic elution, wash-out ratio: A: B=88%: 12%; Comprising concentration in the moving phase is that 0.02mol/L heptane base sodium sulfonate is made ion-pairing agent; Flow velocity: 1.0mL/min; Detecting device: diode array detector; The detection wavelength is 260nm; Sample size: 20 μ L.
2. the method for Tianning in a kind of molecular imprinting Solid-Phase Extraction according to claim 1-liquid chromatogram measuring body fluid, it is characterized in that: described body fluid is urine, blood, blood plasma or tells liquid.
3. the method for Tianning in a kind of molecular imprinting Solid-Phase Extraction according to claim 1-liquid chromatogram measuring body fluid is characterized in that: monomer is the olefin(e) acid that comprises one, two or three carbon atom substituted alkyl in the described step (1); Crosslinking chemical is olefin(e) acid monobasic alcohol ester or the polyol ester that comprises one, two or three carbon atom substituted alkyl; Pore generating agent is that one of one, two or three carbon atom replaces or polysubstituted chlorohydrocarbon; Initiating agent is that one of one, two or three alkyl replaces or polysubstituted azonitrile.
4. the method for Tianning in a kind of molecular imprinting Solid-Phase Extraction according to claim 1-liquid chromatogram measuring body fluid is characterized in that: the degassing time is 5-20min in the described step (1); The logical nitrogen time is 5-20min; Bath temperature is controlled at 40 ℃-80 ℃; The water-bath time is 12h-36h.
5. the method for Tianning in a kind of molecular imprinting Solid-Phase Extraction according to claim 1-liquid chromatogram measuring body fluid, it is characterized in that: organic acid is the monobasic organic acid or the multicomponent organic acid of one, two or three carbon atom in the leacheate of described step (1); Alcohol is the monohydroxy alcohol or the polyvalent alcohol of one, two or three carbon atom; The polymer beads baking temperature is controlled at 100 ℃-150 ℃; Be controlled at 12h-36h drying time.
6. the method for Tianning in a kind of molecular imprinting Solid-Phase Extraction according to claim 1-liquid chromatogram measuring body fluid is characterized in that: organic acid is that one of one, two or three carbon atom replaces or polysubstituted fluoro organic acid in the acetonitrile solution that contains organic acid, water of described step (3); The organic acid ratio is 1%-5%, and the water ratio is 1%-5%.
7. the method for Tianning in a kind of molecular imprinting Solid-Phase Extraction according to claim 1-liquid chromatogram measuring body fluid, it is characterized in that: the alcohol of the adjusting solid phase extraction column of described step (3) is the monohydroxy alcohol or the polyvalent alcohol of one, two or three carbon atom.
8. the method for Tianning in a kind of molecular imprinting Solid-Phase Extraction according to claim 1-liquid chromatogram measuring body fluid, it is characterized in that: adjusting, drip washing solid phase extraction column in the described step (3), the buffer salt of regulating humoral sample are monoacid ammonium or the polyprotonic acid ammonium and the ammonia spirit composition of one, two or three carbon atom; The pH scope of regulating humoral sample is 8-11; Organic amine is one, two or three alkyl replacement ammoniums in the organic amine acetonitrile solution of drip washing solid phase extraction column.
9. the method for Tianning in a kind of molecular imprinting Solid-Phase Extraction according to claim 1-liquid chromatogram measuring body fluid is characterized in that: the process of doing that is concentrated in the step (4) is carried out under inert gas shielding, and temperature is 30-50 ℃.Acetate buffer solution pH scope: 3-5, amount is controlled at 100-500 μ L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200082983A (en) * | 2018-12-31 | 2020-07-08 | 중앙대학교 산학협력단 | Nicotine metabolites extraction from hair and nail using QuEChERS sample preparation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6699722B2 (en) * | 2000-04-14 | 2004-03-02 | A-Fem Medical Corporation | Positive detection lateral-flow apparatus and method for small and large analytes |
| CN101477090A (en) * | 2009-01-19 | 2009-07-08 | 陈枢青 | Method for simultaneously detecting cotinine, phenyl hydroxyacetic acid and phenylglyoxalic acid in human urine based on derivation method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6699722B2 (en) * | 2000-04-14 | 2004-03-02 | A-Fem Medical Corporation | Positive detection lateral-flow apparatus and method for small and large analytes |
| CN101477090A (en) * | 2009-01-19 | 2009-07-08 | 陈枢青 | Method for simultaneously detecting cotinine, phenyl hydroxyacetic acid and phenylglyoxalic acid in human urine based on derivation method |
Non-Patent Citations (3)
| Title |
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| 《中国科学技术大学学位论文》 20070531 杨俊 可天宁和尼古丁分子印迹新材料及其固相萃取分析研究 1-120 1-9 , * |
| 《光谱学与光谱分析》 20070630 杨俊 等 可天宁印迹聚合物分子识别特性的光谱与XPS研究 1152-1155 1-9 第27卷, 第6期 * |
| 《分析测试学报》 20060331 胡雁 等 反相离子对高效液相色谱法同时测定头发中的尼古丁和可天宁 77～80 1-9 第25卷, 第2期 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200082983A (en) * | 2018-12-31 | 2020-07-08 | 중앙대학교 산학협력단 | Nicotine metabolites extraction from hair and nail using QuEChERS sample preparation |
| KR102211890B1 (en) | 2018-12-31 | 2021-02-03 | 중앙대학교 산학협력단 | Nicotine metabolites extraction from hair and nail using QuEChERS sample preparation |
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Application publication date: 20110202 |