LC-MS/MS同时测定5种探针底物代谢产物和快速评价细胞色素P450同工酶的活性

目的建立同时测定奥美拉唑（CYP2C19）、右美沙芬（CYP2D6）、睾酮（CYP3A4）、氯唑沙宗（CYP2E1）和甲苯磺丁脲（CYP2C9）5种探针底物代谢产物的Cocktail体外方法,快速评价药物对人肝微粒体细胞色素P450同工酶活性的影响。方法通过人肝微粒体与5种探针底物在还原系统NADPH的作用下,于37℃水浴条件下共同孵育,采用液质联用（LC-MS/MS）分析方法,测定探针底物的代谢产物生成量来评价各P450同工酶的活性。结果建立了同时测定5种P450同工酶活性的酶反应条件和LC-MS/MS分析方法,数据显示该方法具有良好的线性和分析灵敏度,以及具有良好的准确度、精密度、重现性,可用于药物间相互作用的体外快速评价。结论可用于体外快速评价药物对相应的CYP450亚型酶活性的影响,以预测潜在的药物相互作用。

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

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）,dextromethorphan（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 possessed a 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.