High-sensitivity liquid chromatography–tandem mass spectrometry for the simultaneous determination of five drugs and their cytochrome P450-specific probe metabolites in human plasma

► We develop simultaneous quantitation tool of 5 CYP probe drugs and their metabolites. ► High recovery of all analytes was achieved by dual solvent extraction from plasma. ► LC/MS/MS method afforded high sensitivity to detect pg level of the drugs. ► This method is suitable for mini-dose cocktail clinical trials for drug interactions. A sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method with electrospray ionization was developed for the simultaneous quantitation of five probe drugs and their metabolites in human plasma for assessing the in vivo activities of cytochrome P450 (CYP). CYP isoform specific substrates and their metabolites of CYP1A2 (caffeine), CYP2C9 (losartan), CYP2C19 (omeprazole), CYP2D6 (dextromethorphan) and CYP3A (midazolam) were all simultaneously analyzed using LC–MS/MS after administration of a mixture of five drugs (i.e., a “cocktail approach”) to healthy volunteers. The assay uses propranolol as an internal standard; dual liquid extraction; a Xbridge MS C18 (100mm×2.1mm, 3.5μm) column; a gradient mobile phase of 0.1% formic acid/acetonitrile (7/3→3/7); mass spectrometric detection in positive ion mode. The method was validated from 5 to 500ng/mL for caffeine and paraxanthine, 0.1–40ng/mL for losartan and EXP3174, 0.05–20ng/mL for omeprazole and 5-hydroxyomeprazole, 0.008–0.8ng/mL for dextromethorphan and dextrorphan, 0.01–1.0ng/mL for midazolam, and 0.04–4ng/mL for 1′-hydroxymidazolam. The intra- and inter-day precision over the concentration ranges for all analytes were lower than 12.5% and 13.8% (relative standard deviation, %RSD), and accuracy was between 86.5% and 108.4% and between 87.0% and 107.0%, respectively. This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10–100 times lower than typical therapeutic doses.