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Determination of CYP1A2 and CYP2D1 Activities in Rat Liver Microsomes and Kinetic Studies Using Ultra Performance Liquid Chromatography Tandem Mass Spectrometry

??OBJECTIVE To establish a robust, fast and convenient method for in vitro assay of rat liver CYP1A2 and CYP2D1, and explore their kinetic features.METHODS Two selective substrates including phenacetin and dextromethorphan, which are probes of CYP1A2 and CYP2D1, were chosen for liver microsomes incubation, respectively; the corresponding ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS) methods were developed for kinetic studies.RESULTS The fast and convenient UPLC-MS methods with high resolution and short running time(4~5 min) were established and validated for two assays of CYP1A2 and CYP2D1 activities;both methods showed good accuracy and precision, and the values of LOQ for CYP1A2 and CYP2D1 assays could reach 0.267 and 0.007 ??mol??L^-1, respectively. The kinetic studies showed that the Michaelis constant(K_m) for CYP1A2 and CYP2D1 were (28.4??2.7) and (13.9??1.3) ??mol??L^-1, respectively. Their activities were determined to be (1.47??0.12) and (3.98??0.09) nmol??mg^-1, respectively,when the substrate concentration was 10 ??mol??L^-1.CONCLUSION UPLC Tandem MS technique is proved to be a rapid, convenient and efficient approach with high sensitivity and selectivity for the assays of CYP1A2 and CYP2D1 in drug metabolism.