Validation of higher-throughput high-performance liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in humanliver microsomes

In the early stage of drug discovery, thousands of new chemical entities (N CEs) may be screened before a single drug candidate can be identified for d evelopment. In order to accelerate the drug discovery process, we have deve loped higher-throughput enzyme assays to evaluate the inhibition of cytochr ome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes, The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was r educed from similar to 20 minutes for the conventional HPLC assay to 30 sec onds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- a nd intraday validations were satisfactory. Since the implementation of the LC/ MS/MS assays, our sample throughput has increased by over 40-fold. Copy right (C) 2000 John Wiley & Sons, Ltd.