A high‐throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography–tandem mass spectrometry

• DOI: 10.1002/bmc.3003
• Publication Date: 2013-08-15T14:51:51Z
• Authors: Chong‐Zhen Qin; Xian Ren; Zhi‐Rong Tan; Yao Chen; Ji‐Ye Yin; Jing Yu; Jian Qu; Hong‐Hao Zhou; Zhao‐Qian Liu
• Keywords: Reproducibility of Results; Sensitivity and Specificity; Recombinant Proteins; High-Throughput Screening Assays; Inhibitory Concentration 50; 03 medical and health sciences; 0302 clinical medicine; Cytochrome P-450 Enzyme System; Tandem Mass Spectrometry; Microsomes, Liver; Cytochrome P-450 Enzyme Inhibitors; Humans; Drug Interactions; Enzyme Inhibitors; Chromatography, Liquid; Enzyme Assays

ABSTRACT A sensitive and high‐throughput inhibition screening liquid chromatography–mass spectrometry (LC‐MS/MS) method was developed validated for the simultaneous quantification of five probe metabolites (7‐hydroxycoumarin, CYP2A6; 4‐hydroxytolbutamide, CYP2C9; 4′‐hydroxymephenytoin, CYP2C19; α ‐hydroxymetoprolol, CYP2D6; 1‐hydroxymidazolam, CYP3A4) in vitro cytochrome P450 activity determination human liver microsome recombinant. All internal standard, tramadol, were separated on a Waters 2695 series chromatograph with Phenomenex Luna C 18 column (150 × 2.0 mm, 5 µm). Quality control samples positive CYP inhibitor included method. The IC 50 values determined typical inhibitors reproducible agreement literature. selective showed good accuracy (99.13–103.37%), inter‐day (RSD < 6.20%) intra‐day 6.13%) precision. Also, incubation extracts sample stable at room temperature (20 °C) 48 h 96 autosampler (4 °C). presented is first HPLC‐MS/MS this combination detection 7‐hydroxycoumarin, ‐hydroxymetoprolol 1‐hydroxymidazolam single‐run process. It possible that high‐quality ‐throughput cocktail provides suitable information drug discovery new entities. Copyright © 2013 John Wiley & Sons, Ltd.