A highly sensitive liquid chromatography tandem mass spectrometry method for simultaneous quantification of midazolam, 1'-hydroxymidazolam and 4-hydroxymidazolam in human plasma.
Biomed Chromatogr, 2011/10;25(10):1091-8.
de Loor H[1], de Jonge H, Verbeke K, Vanrenterghem Y, Kuypers DR
Affiliations
PMID: 21204116DOI: 10.1002/bmc.1576
Impact factor: 1.911
Abstract
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.
MeSH terms
Chromatography, High Pressure Liquid; Drug Stability; Humans; Kidney Transplantation; Midazolam; Nonlinear Dynamics; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry
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