A liquid chromatography-tandem mass spectrometry method was developed for the simultaneous quantification of 26 benzodiazepines and metabolites, zolpidem and zopiclone, in blood, urine, and hair. Drugs were extracted from all matrices by liquid-liquid extraction with 1-chlorobutane. Chromatography was achieved using a XTerra MS C18 column eluted with a mixture of methanol and formate buffer. Data were acquired using positive electrospray ionization and multiple reaction monitoring using one precursor ion/product ion transition per compound. Quantification was performed using 13 deuterated analogues. Further confirmation of the identity of the compounds was achieved through a second injection of positive samples, monitoring two transitions per compound. The limits of quantification for all benzodiazepines ranged from 1 to 2 ng/mL in blood, 10 to 25 ng/mL in urine, and 0.5 to 10 pg/mg in hair. Linearity was observed from the limit of quantification of each compound to 200 ng/mL, 1000 ng/mL, and 1000 pg/mg for blood, urine, and hair, respectively (r2 > 0.99). Precision for quality control samples, spiked at three concentrations, was calculated (CV < 20% in most cases). Extraction recoveries for the three matrices ranged from 25.1 to 103.8%, except for one compound (cloxazolam in urine). Ion suppression was studied for all matrices. The validated assay was applied to authentic blood, urine, and hair samples from forensic cases.

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