Our aim was to develop and validate a fast, sensitive, and specific method determining 17 designer benzodiazepines (adinazolam, clobazam, clonazolam, delorazepam, deschloroetizolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam, flunitrazolam, N-desmethylclobazam, nifoxipam, nitrazolam, meclonazepam, pyrazolam and zolazepam) in hair by liquid chromatography tandem mass spectrometry (LC-MS/MS).

In recent years, identification and analysis of designer benzodiazepines have become a challenge in forensic toxicology. These substances are analogues of the classic benzodiazepines, but their pharmacology is not well known, and many of them have been associated with overdoses and deaths. As a result, there has been a surge in efforts to develop analytical methods to determine these compounds in different biological samples. Most publications have been focused on blood and urine; however, publications concerning alternative matrices, such as hair, are scarce.

Hair samples were decontaminated, pulverized, and a 20mg aliquot was incubated in methanol in an ultrasonic bath (1h, 25°C). The supernatant was evaporated and reconstituted in 200μL of mobile phase composed of 0.1% formic acid and acetonitrile (95:5). The extracts were filtered before injection into the LC-MS/MS. Chromatographic separation was performed in reversed phase using a C18 column in gradient mode. All analytes eluted from the chromatographic column in 8min. Two multiple-reaction monitoring transitions in positive mode were used to identify each compound. The method was validated based on the AAFS Standards Board (ASB) Standard Practices for Method Validation in Forensic Toxicology and applied to authentic cases as proof of concept.

The best calibration model for all analytes was a linear model with 1/x weighting. The limits of quantification were 5 or 25pg/mg, depending on the analyte, and calibration functions were linear up to 200pg/mg. Imprecision was<20% (n=15) and bias was from −13 to 18% (n=15). All the analytes yielded high extraction efficiencies>70% but displayed ion suppression between −63% and −24% (n=10). No carryover was observed. All samples showed good stability after 48h at 10°C in the autosampler, except for nifoxipam, etizolam, flubromazepam and diclazepam, which showed significant loss in signal. The method was applied to 19 authentic cases. Five 3-cm segments from three different cases were positive for flualprazolam (<LOQ – >200pg/mg) and/or etizolam (47.4–88.5pg/mg).

Few publications have focused on the analysis of a limited number of designer benzodiazepines in hair. As a result, limited information is available regarding hair concentrations of these type of substances. The present method is the most comprehensive method to date including 17 analytes. In the analysis of authentic cases, etizolam concentrations were within the range of previous reports; however, no hair concentrations for flualprazolam have been previously described in the literature.

The present validated method has proven to be fast, sensitive, specific, and capable of determining 17 designer benzodiazepines in hair using LC-MS/MS.