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.

Development and validation of a sensitive assay for analysis of midazolam, free and conjugated 1-hydroxymidazolam and 4-hydroxymidazolam in pediatric plasma: Application to Pediatric Pharmacokinetic Study

“…This data showed acceptable stability for all three compounds during the analytical runs and under the storage conditions. These results are also consistent with previously reported results for midazolam and 1-hydroxymidazolam [6, 8] and 4-hydroxymidazolam [16]. …”

“…There are several LC-MS/MS methods reported for the analysis of midazolam and 1-hydroxymidazolam in human plasma and other biological samples [6–26]. However, there are few LC-MS/MS methods reported for the analysis of 4-hydroxymidazolam, which is an acid sensitive metabolite [8, 9, 16, 23]. Three of the reported methods [8, 16, 23] require a larger volume of plasma samples (0.5 to 1.0 mL), which is not feasible in pediatric studies where blood draws are limited to smaller volume to minimize risk.…”

“…However, there are few LC-MS/MS methods reported for the analysis of 4-hydroxymidazolam, which is an acid sensitive metabolite [8, 9, 16, 23]. Three of the reported methods [8, 16, 23] require a larger volume of plasma samples (0.5 to 1.0 mL), which is not feasible in pediatric studies where blood draws are limited to smaller volume to minimize risk. A recent study utilized 1-hydroxymidazolam glucuronide that was isolated from human urine as a standard for quantitation [6].…”

Direct urine analysis for the identification and quantification of selected benzodiazepines for toxicology screening

“…These studies use either immunological methods [4][5][6] or chromatographic techniques [7][8][9][10][11][12][13]. Enzyme immunoassay assays (EIAs) are known to provide rapid results for preliminary screening in urine for the presence of benzodiazepines with usual cut-off level of 100 ng/mL; however, specificity remains a significant disadvantage of such methods [14] where this class of drugs appears to cross react with other substances in a number of cases [15][16][17].…”

In vivo CYP3A4 activity does not predict the magnitude of interaction between itraconazole and tacrolimus from an extended release formulation

“…Plasma concentrations of MDZ were measured using a high‐performance LC‐MS/MS method, as previously described . Whole blood concentrations of tacrolimus were measured using an ultraperformance LC‐MS/MS method, adapted from a previously described method …”