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A high‐throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography–tandem mass spectrometry
Author(s) -
Qin ChongZhen,
Ren Xian,
Tan ZhiRong,
Chen Yao,
Yin JiYe,
Yu Jing,
Qu Jian,
Zhou HongHao,
Liu ZhaoQian
Publication year - 2014
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.3003
Subject(s) - chromatography , chemistry , mass spectrometry , tandem mass spectrometry , liquid chromatography–mass spectrometry , high performance liquid chromatography
ABSTRACT A sensitive and high‐throughput inhibition screening liquid chromatography–mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7‐hydroxycoumarin, CYP2A6; 4‐hydroxytolbutamide, CYP2C9; 4′‐hydroxymephenytoin, CYP2C19; α ‐hydroxymetoprolol, CYP2D6; and 1‐hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C 18 column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC 50 values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13–103.37%), and inter‐day (RSD < 6.20%) and intra‐day (RSD < 6.13%) precision. Also, the incubation extracts of the sample were stable at room temperature (20 °C) for 48 h and for 96 h in the autosampler (4 °C). The presented method is the first HPLC‐MS/MS method of this combination for simultaneous detection of the five metabolites 7‐hydroxycoumarin, 4‐hydroxytolbutamide, 4′‐hydroxymephenytoin, α ‐hydroxymetoprolol and 1‐hydroxymidazolam in a single‐run process. It is possible that the high‐quality and ‐throughput cocktail provides suitable information in drug discovery and screening for new drug entities. Copyright © 2013 John Wiley & Sons, Ltd.