Identifying bromazolam, etizolam, and flualprazolam in blood using gas chromatography–mass spectrometry

Designer benzodiazepines are a commonly misused class of novel psychoactive substances (NPS) that present challenges for forensic toxicology laboratories and public health. Due to the difficulty in detection by routine toxicology methods, it is important to enhance the analytical methods for the identification and quantitation of designer benzodiazepines. The aim of this study was to identify and quantify designer benzodiazepines using gas chromatography coupled with mass spectrometry (GC-MS) in postmortem blood specimens. A method originally validated to identify and quantitate alprazolam was modified to quantitate flualprazolam, etizolam, and bromazolam, as well as identify adinazolam and 4'-chloro-deschloroalprazolam. The method was subsequently used to analyze postmortem specimens (n = 35) from Medical Examiner Districts (n = 4) in Florida that screened positive for one or more designer benzodiazepines from February 2022 to December 2023. Among the screened samples, 21 tested positive for one or more designer benzodiazepines in the postmortem blood; 1 was positive for etizolam (90 ng/mL) and flualprazolam (<5.0 ng/mL), while 20 were positive for bromazolam. Bromazolam was the most frequently detected designer benzodiazepine, with a concentration range of 5.9-352 ng/mL (mean: 59.1 ng/mL; median: 28.5 ng/mL) for 18 decedents and <5.0 ng/mL for two decedents. Polydrug use was confirmed in all decedents. The most prevalent drugs found with bromazolam in the blood were fentanyl, methamphetamine and/or amphetamine, and cocaine. The optimized GC-MS method provides a way to identify and quantitate designer benzodiazepines in postmortem blood and to also qualitatively monitor two newer designer benzodiazepines: 4'-chloro-deschloroalprazolam and adinazolam.