Simultaneous Determination of the Activities of Five Major Cytochrome P450 Enzymes by an in Vitro Cocktail Method Coupled with LC-MS/MS Assay

Abstract

Abstract: Objective To develop an in vitro Cocktail method coupled with liquid chromatography/tandem mass spectrometry ( LC -MS/ MS ) assay for simultaneous determination of the activities of five major cytochrome P450 (CYP450) isoenzymes by monitoring the metabolites of five probe substances including omeprazole(CYP2C19), dex-tromethorphan (CYP2D6), testosterone (CYP3A4), chlorzoxazone (CYP2E1), tolbutamide (CYP2C9). Methods The five probes were incubated with human microsome CYP450 isoenzymes at 37 ℃ in the presence of NATPH. The concentrations of produced metabolites in the reaction solution were determined by LC-MS/MS method, and compared for evaluating the activities of isoenzymes. Results The various conditions for enzyme reactions and LC -MS/MS method have been established for monitoring the activities of five CYPP450 isoenzymes. The data showed that the method possesseda good linearity and sensitivity, as well as the excellent accuracy and repeatability, and can be used for the rapid evaluation of drug -drug interactions. Conclusion A sensitive and validated Cocktail Method coupled with LC -MS/MS assay has been developed and can be applied to rapidly evaluate the CYP450 enzyme activities for predicting the potential drug-drug interactions.

Key words: P450, Cocktail, LC-MS/MS, drug-drug interactions, in vitro

CLC Number:

R969

YE Lin -Hu ,KONG Ling –Ti, XIAO Bing -Xin ,WANG Qian, HE Xiao –Xi, CAO Fang -Rui ,CHANG Qi. Simultaneous Determination of the Activities of Five Major Cytochrome P450 Enzymes by an in Vitro Cocktail Method Coupled with LC-MS/MS Assay [J]. Chinese Journal of Pharmacovigilance, 2013, 10(5): 263-268.

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