A high-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry.
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 C18 column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC50 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.
Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University Xiangya School of Medicine, Changsha, Hunan, 410078, People's Republic of China.
Chong-Zhen
CZ
Qin
Xian
X
Ren
Zhi-Rong
ZR
Tan
Yao
Y
Chen
Ji-Ye
JY
Yin
Jing
J
Yu
Jian
J
Qu
Hong-Hao
HH
Zhou
Zhao-Qian
ZQ
Liu
D002853:Chromatography, Liquid / Q000379:methods*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004347:Drug Interactions; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D008862:Microsomes, Liver / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System
cocktail; cytochrome P450; drug-drug interactions; high-throughput; liquid chromatography-mass spectrometry