Detection of Benzodiazepines and z-Drugs in Hair Using an UHPLC-MS/MS Validated Method: Application to Workplace Drug Testing.

Abstract

A sensitive and reproducible Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry method has been developed for the simultaneous quantification of the 29 commonly prescribed benzodiazepines and z-drugs in hair. The method was validated according to international guidelines. After decontamination (with dichloromethane and water), compounds were extracted from 20 mg of pulverized hair samples using methanol at 45°C and sonication for 2 hours. The drugs were recovered by liquid-liquid extraction using 1-chlorobutane, evaporated to dryness, and reconstituted with 100 μL of methanol before injection in the UPLC-MS/MS. The applied gradient ensured the elution of all the compounds within 7 minutes using 0.1% formic acid in water and methanol as mobile phase. The lower limit of quantification values ranged from 0.5 to 5 pg/mg of hair. Calibration curves were linear for almost all the compounds and ranged from the limit of quantification to 620 pg/mg hair. The bias and relative standard deviation of the intraday and interday imprecision were lower than 15% in 3 fortified "in-house" quality control samples, 1 external quality control sample, and 1 authentic hair sample (from a diazepam user). No significant matrix effects were observed for most of the compounds, and the extraction efficiency of the sample cleanup procedure ranged from 19% to 82% with a relative standard deviation <15% [except for clobazam (16%), loprazolam (20%), brotizolam (18%), and 7-aminoclonazepam (20%)]. The method was then successfully applied to the analysis of 40 hair samples from the workplace drug testing, containing alprazolam, estazolam, clonazepam, diazepam, zolpidem, and desalkylflurazepam (and metabolites). The method was completely validated and can be of interest to clinical and forensic laboratories.