Quantitation of Benzodiazepines in Urine, Serum, Plasma, and Meconium by LC-MS-MS

Abstract

A single method for confirmation and quantitation of a panel of commonly prescribed benzodiazepines and metabolites, α-hydroxyalprazolam, α-hydroxyethylflurazepam, α-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam, oxazepam, temazepam, clonazepam, and 7-aminoclonazepam, was developed for three specimen types, urine, serum/plasma, and meconium. Quantitation was by liquid chromatography tandem-mass spectrometry (LC-MS-MS) using a Waters Alliance-Quattro Micro system. The instrument was operated in multiple reaction monitoring mode with an electrospray ionization source in positive ionization mode. The method was evaluated for recovery, imprecision, linearity, analytical measurement range, specificity, and carryover. Average recovery and imprecision (within-run, between-run, and total % CV) were within ± 15% of the target concentrations for urine (10 to 5000 ng/mL) and serum/plasma (10 to 2500 ng/mL) and within ± 20% for meconium (10 to 5000 ng/g). In all, 205 patient specimens were analyzed, and the results compared to a previous in-house gas chromatography-MS method or LC-MS-MS results from an outside laboratory. Oxazepam glucuronide was evaluated as a hydrolysis control for the urine and meconium specimens.