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

Background: 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.

Methods: 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.

Results: 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).

Conclusions: The method was completely validated and can be of interest to clinical and forensic laboratories.