title: A highly sensitive liquid chromatography tandem mass spectrometry method for simultaneous quantification of midazolam, 1'-hydroxymidazolam and 4-hydroxymidazolam in human plasma.
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.
authors.affiliation: Laboratory of Nephrology, University Hospitals Leuven, K.U.Leuven, Belgium.
authors.forename: Henriette
authors.initials: H
authors.lastname: de Loor
authors.forename: Hylke
authors.initials: H
authors.lastname: de Jonge
authors.forename: Kristin
authors.initials: K
authors.lastname: Verbeke
authors.forename: Yves
authors.initials: Y
authors.lastname: Vanrenterghem
authors.forename: Dirk R
authors.initials: DR
authors.lastname: Kuypers
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D016030:Kidney Transplantation; D008874:Midazolam / Q000031:analogs & derivatives* / Q000097:blood* / Q000493:pharmacokinetics; D017711:Nonlinear Dynamics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C042940:4-hydroxymidazolam; C040081:1-hydroxymethylmidazolam; D008874:Midazolam