Rapid determination of five probe drugs and their metabolites in human plasma and urine by liquid chromatography/tandem mass spectrometry: application to cytochrome P450 phenotyping studies

A liquid chromatography/mass spectrometry method, for rapid determination of five cytochrome P450 (CYP) probe drugs and their relevant metabolites in human plasma and urine, is described. The five specific probe substrates/metabolites, caffeine/paraxanthine (CYP1A2), tolbutamide/4‐hydroxytolbutamide/carboxytolbutamide (CYP2C9), omeprazole/5‐hydroxyomeprazole (CYP2C19), debrisoquine/5‐hydroxydebrisoquine (CYP2D6) and midazolam/1′‐hydroxymidazolam (CYP3A), together with the internal standards (phenacetin and paracetamol), in plasma and urine, were extracted using solid‐phase extraction. The chromatography was performed using a C 18 column with an isocratic mobile phase consisting of acetonitrile and 0.1% formic acid in water (70:30). The triple‐quadrupole mass spectrometer was operated in both positive and negative modes, and multiple reaction monitoring was used for quantification. The method was validated over the concentration ranges 0.05–5 μg/mL for caffeine and paraxanthine, 0.02–2 μg/mL for tolbutamide, 0.1–20 μg/mL for 4‐hydroxytolbutamide, carboxytolbutamide, debrisoquine and 5‐hydroxydebrisoquine, 5–2500 ng/mL for omeprazole and 5‐hydroxyomeprazole, and 1–100 ng/mL for midazolam and 1′‐hydroxymidazolam. The intra‐ and inter‐day precision were 0.3–13.7% and 1.9–14.3%, respectively, and the accuracy ranged from 93.5–107.2%. The lower limit of quantification varied between 1 and 100 ng/mL. The present method provides a robust, fast and sensitive analytical tool for the five‐probe drug cocktail, and has been successfully applied to a clinical phenotyping study in 16 subjects. Copyright © 2004 John Wiley & Sons, Ltd.