A highly sensitive liquid chromatography tandem mass spectrometry method for simultaneous quantification of midazolam, 1′‐hydroxymidazolam and 4‐hydroxymidazolam in human plasma

A highly sensitive liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of midazolam and its major metabolites 1′‐hydroxymidazolam and 4‐hydroxymidazolam in human plasma was developed and validated. Stable isotope‐labeled midazolam‐D 4 and 1′‐hydroxymidazolam‐D 4 were used as internal standards. Compounds were extracted from 0.5 mL plasma by liquid–liquid extraction with ethyl acetate–heptane (1:4). Chromatography was achieved using a Sunfire C 18 column. The mobile phase was a gradient with 10 m m formic acid in Milli‐Q water and methanol at a flow rate of 0.3 mL/min. Total run time was 10 min. Detection was performed using a tandem mass spectrometer with positive electrospray ionization. Calibration curves were linear over the range of 0.10–50.0 ng/mL for midazolam and 0.025–25.0 ng/mL for both metabolites. For all compounds the lower limit of quantification was 0.10 ng/mL. Imprecision was assessed according to the NCCLS EP5‐T guideline and was below 10% for all compounds. Mean recoveries were between 94 and 109% for midazolam and its metabolites. The validated method was successfully applied in a pharmacokinetic study investigating in vivo CYP3A‐activity in a large cohort of renal allograft recipients using sub‐therapeutic doses of midazolam as a drug‐probe. Copyright © 2010 John Wiley & Sons, Ltd.