Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1′-hydroxymidazolam, and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry

Mrdazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity A rapid, sensitive. and selective LC-MS/MS method was developed for simultaneous quantum ion of midazolam and its metabolites (1'-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D-5) analog of midazolam was utilized as an internal standard Sample preparation either from human plasma (100 mu L) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 mu L) with an average recovery of >90% for all compounds The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2 1 mm x 100 mm, 3 5 mu m) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B) The flow rate was 0 25 mL/min and total run time was 55 min Calibration curves were lineal over the concentration range of 0 100-250 ng/mL The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85-115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam (C) 2010 Elsevier B.V All rights reserved