Validation of qualitative and quantitative methods for 40 benzodiazepines and Z-drugs in hair by UPLC-MS/MS

In recent years, the analysis and identification of benzodiazepines and Z-drugs have presented significant challenges in forensic medicine and pharmaceutical analysis due to their widespread use. Notably, some of these substances are classified as new psychoactive substances (NPS) by the UNODC. Hair was chosen as a biological matrix for monitoring long-term drug intake in this study. We aimed to develop and validate a rapid and sensitive method for simultaneous determination of 40 benzodiazepines and Z-drugs in hair using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). All the analytes were detected within 11 min. The method was validated with linear calibration curves over the range of 5-500 pg mg^-1 (coefficient of determination, r² > 0.99). The limits of detection (LOD) were 1-3 pg mg^-1, and the limits of quantification (LOQ) were 5 pg mg^-1. The method demonstrated acceptable selectivity, accuracy, and precision (< 20%). Matrix effects ranged from -29.8-23.5%, and recoveries ranged from 77.9% to 119.9%. The method was successfully applied to six authentic hair samples, detecting nine substances: Nitrazepam, Temazepam, Lorazepam, 7-Aminoflunitrazepam, Clobazam, Lormetazepam, α-Hydroxyalprazolam, 7-Aminoclonazepam, and Zolpidem. Benzodiazepines concentrations ranged from 5.6 to 436.1 pg mg^-1, while zolpidem concentrations ranged from 11.1 to 113.8 pg mg^-1. This method provided a reliable tool for detecting benzodiazepines and Z-drugs in hair and addresses the requirements of routine forensic practice.