A sensitive and high-throughput LC-MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates.

Abstract

A sensitive and high-throughput LC-MS/MS method was established and validated for the simultaneous quantification of seven probe substrate-derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy-bupropion (CYP2B6), n-desethyl-amodiaquine (CYP2C8), 4'-hydroxy-diclofenac (CYP2C9), 4'-hydroxy-mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1'-hydroxy-midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well-known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC50 values of these inhibitors determined on this cocktail assay were highly correlated (R(2) > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89-113.35%) and between-day (RSD <13.95%) and within-day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze-thaw cycles. This seven-CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds' potential inhibition of the seven major CYPs in drug development settings.