Development and validation of a fast and sensitive UPLC–MS/MS method for the quantification of six probe metabolites for the in vitro determination of cytochrome P450 activity

A fast and sensitive UPLC–MS/MS method was developed and validated for the simultaneous quantification of six probe metabolites for the in vitro cytochrome P450 activity determination in hepatic microsomes from patients with hepatic impairment. The metabolites acetaminophen (CYP1A2), 4′-hydroxy-mephenytoin (CYP2C19), 4-hydroxy-tolbutamide (CYP2C9), dextrorphan (CYP2D6), 6-hydroxy-chlorzoxazone (CYP2E1) and 1-hydroxy-midazolam (CYP3A4), together with the internal standard chlorpropamide, were separated on a Waters Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm particle size) with VanGuard pre-column (5 mm × 2.1 mm, 1.7 μm particle size). A short gradient elution (total run time of 5.25 min), using water with 0.1% formic acid (eluent A) and acetonitrile with 0.1% formic acid (eluent B) at a flow rate of 400 μl/min, was used. The metabolites were detected with a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Two runs, one in the positive ionization mode and one in the negative mode, were necessary for the detection of all metabolites. The method was selective and showed good accuracy (84.59–109.83%) and between-day (RSD% < 5.13%) and within-day (RSD% < 9.60%) precision. The LOQ was in full accordance with the intended application, and no relative matrix effects were observed. Also, the sample incubation extracts were stable after three freeze–thaw cycles. The usability of the method was demonstrated by the incubation of pediatric microsomes with subsequent quantification of the formed metabolites and CYP activity calculation.