Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies
An automated in-capillary assay requiring very small quantities of reagents was developed for performing in vitro cytochrome P450 (CYP450) drug metabolism studies. The approach is based on the following: (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sand...
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| Veröffentlicht in: | Analytical and bioanalytical chemistry 2010-11, Vol.398 (5), p.2163-2171 |
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| creator | Curcio, R Nicoli, R Rudaz, S Veuthey, J.-L |
| description | An automated in-capillary assay requiring very small quantities of reagents was developed for performing in vitro cytochrome P450 (CYP450) drug metabolism studies. The approach is based on the following: (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sandwich mode, within a capillary; (ii) mixing the reagents by diffusion across the interfaces between the injected solutions; (iii) collection of the capillary content at the end of the in-capillary assay; and (iv) off-line analysis of the incubation mixture by ultrahigh pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After optimizing the injection sequence of the reagents, the in-capillary approach was applied to the quantitative determination of the kinetics of drug metabolism reactions catalyzed by three CYP450 isozymes involved in human drug metabolism: CYP1A2, CYP2D6, and CYP3A4. It was demonstrated that this in-capillary method was able to provide similar kinetic parameters for CYP450 activity (e.g., Michaelis constants and turnover values) as the classical in vitro method, with a drastic reduction of reagent consumption. [graphic removed] |
| doi_str_mv | 10.1007/s00216-010-4175-9 |
| format | Article |
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The approach is based on the following: (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sandwich mode, within a capillary; (ii) mixing the reagents by diffusion across the interfaces between the injected solutions; (iii) collection of the capillary content at the end of the in-capillary assay; and (iv) off-line analysis of the incubation mixture by ultrahigh pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After optimizing the injection sequence of the reagents, the in-capillary approach was applied to the quantitative determination of the kinetics of drug metabolism reactions catalyzed by three CYP450 isozymes involved in human drug metabolism: CYP1A2, CYP2D6, and CYP3A4. It was demonstrated that this in-capillary method was able to provide similar kinetic parameters for CYP450 activity (e.g., Michaelis constants and turnover values) as the classical in vitro method, with a drastic reduction of reagent consumption. 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The approach is based on the following: (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sandwich mode, within a capillary; (ii) mixing the reagents by diffusion across the interfaces between the injected solutions; (iii) collection of the capillary content at the end of the in-capillary assay; and (iv) off-line analysis of the incubation mixture by ultrahigh pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After optimizing the injection sequence of the reagents, the in-capillary approach was applied to the quantitative determination of the kinetics of drug metabolism reactions catalyzed by three CYP450 isozymes involved in human drug metabolism: CYP1A2, CYP2D6, and CYP3A4. It was demonstrated that this in-capillary method was able to provide similar kinetic parameters for CYP450 activity (e.g., Michaelis constants and turnover values) as the classical in vitro method, with a drastic reduction of reagent consumption. [graphic removed]</description><subject>Analysis</subject><subject>Analytical Chemistry</subject><subject>Assaying</subject><subject>Biochemistry</subject><subject>Biological Assay - methods</subject><subject>Capillarity</subject><subject>Capillary pressure</subject><subject>Characterization and Evaluation of Materials</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Chromatographic methods and physical methods associated with chromatography</subject><subject>Chromatography, Liquid - methods</subject><subject>CYP450</subject><subject>CYP450 kinetics</subject><subject>Cytochrome P-450</subject><subject>Cytochrome P-450 Enzyme System - analysis</subject><subject>Cytochrome P-450 Enzyme System - metabolism</subject><subject>Cytochromes P450</subject><subject>Drug metabolism</subject><subject>Drug-drug interactions</subject><subject>Drugs</subject><subject>Exact sciences and technology</subject><subject>Food Science</subject><subject>Humans</subject><subject>In vitro testing</subject><subject>In-capillary enzymatic assays</subject><subject>Isoenzymes</subject><subject>Kinetics</subject><subject>Laboratory Medicine</subject><subject>Liquid chromatography</subject><subject>Mass Spectrometry</subject><subject>Metabolism</subject><subject>Methods</subject><subject>Monitoring/Environmental Analysis</subject><subject>Nanostructure</subject><subject>Original Paper</subject><subject>Other chromatographic methods</subject><subject>Physiological aspects</subject><subject>Reaction kinetics</subject><subject>Spectrometric and optical methods</subject><issn>1618-2642</issn><issn>1618-2650</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1v1DAQhiMEoqXwA7iALwguKePPxMeqKh9SJZCgZ2vWdrauEntrJ5X23-NVlnIrsqwZjZ93PPbbNG8pnFOA7nMBYFS1QKEVtJOtftacUkX7likJzx9zwU6aV6XcAVDZU_WyOWHQc9krcdrYqwccF5xDiiQNBCMJsbW4C-OIeU9wt8sJ7S0ZUiY7n2uYQtyS-wXjHOaqe_DE7udkb3OaPPkpJBC0tRzmPSnz4oIvr5sXA47FvznGs-bmy9Xvy2_t9Y-v3y8vrlsrlZ5bzjQ4Cs47B1YCdRQ7Bai944Ownd5oKTSTFDYDdh1DQMeZRUTtaOfEhp81H9e-deb7xZfZTKFYX18SfVqK6ZWWXQdU_ZfspJaMCUEr-elJknZC1JZ1V_R8Rbc4ehPikOaMti7np2BT9EOo9Quuey44iIOArgKbUynZD2aXw1S_3VAwB4PNarCpBpuDwUZXzbvjPMtm8u5R8dfRCnw4AlgsjkPGaEP5x3GuVM955djKlXoUtz6bu7TkWP158vb3q2jAZHCba-ObXwwoB6qBQ8_4H0AzxlU</recordid><startdate>20101101</startdate><enddate>20101101</enddate><creator>Curcio, R</creator><creator>Nicoli, R</creator><creator>Rudaz, S</creator><creator>Veuthey, J.-L</creator><general>Berlin/Heidelberg : Springer-Verlag</general><general>Springer-Verlag</general><general>Springer</general><scope>FBQ</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7SR</scope><scope>7U5</scope><scope>8BQ</scope><scope>8FD</scope><scope>JG9</scope><scope>L7M</scope><scope>7X8</scope><scope>7QH</scope><scope>7UA</scope><scope>C1K</scope></search><sort><creationdate>20101101</creationdate><title>Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies</title><author>Curcio, R ; 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| subjects | Analysis Analytical Chemistry Assaying Biochemistry Biological Assay - methods Capillarity Capillary pressure Characterization and Evaluation of Materials Chemistry Chemistry and Materials Science Chromatographic methods and physical methods associated with chromatography Chromatography, Liquid - methods CYP450 CYP450 kinetics Cytochrome P-450 Cytochrome P-450 Enzyme System - analysis Cytochrome P-450 Enzyme System - metabolism Cytochromes P450 Drug metabolism Drug-drug interactions Drugs Exact sciences and technology Food Science Humans In vitro testing In-capillary enzymatic assays Isoenzymes Kinetics Laboratory Medicine Liquid chromatography Mass Spectrometry Metabolism Methods Monitoring/Environmental Analysis Nanostructure Original Paper Other chromatographic methods Physiological aspects Reaction kinetics Spectrometric and optical methods |
| title | Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies |
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