Determination of CYP1A2 and CYP2D1 activities in rat liver microsomes and kinetic studies using ultra performance liquid chromatography tandem mass spectrometry / 中国药学杂志

ABSTRACT

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 μmol·L-1, respectively. The kinetic studies showed that the Michaelis constant (Km) for CYP1A2 and CYP2D1 were (28.4±2.7) and (13.9±1.3) μmol·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 μmol·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.