Designer Benzodiazepines Extracted Using Clean Screen® DAU Columns

Designer benzodiazepines are among the most prevalent Novel Psychoactive Substances (NPS) on the modern drug market. These designer drugs are synthesized to mimic the structure and effects of traditional benzodiazepines. Research and literature on the pharmacological effects of designer benzodiazepines are lacking, but they are generally more potent than their traditional counterparts. The numerous and frequent structural changes made to create designer benzodiazepines create a challenge for forensic toxicology labs. Validation of up-to-date analytical and detection methods is needed to reliably identify these novel substances.

 A group out of the University of Florida developed and validated an extraction and detection method using gas chromatography-mass spectrometry (GC-MS) to identify and quantitate several designer benzodiazepines. Postmortem blood and urine samples were extracted using UCT Clean Screen® DAU columns (200 mg, 10 mL). After conditioning with methanol, water, and phosphate buffer, buffered samples were passed through the column and then washed with water, acetic acid, hexane, and methanol. Analytes were eluted with a mixture of methylene chloride, isopropanol, and ammonium hydroxide (78:20:2 v/v/v) before being evaporated and reconstituted in methanol for analysis. The validated GC-MS method was adapted from a method validated to detect alprazolam, and was updated to quantitate flualprazolam, etizolam, and bromazolam, and qualitatively identify adinazolam and 4’-chloro-deschloroalprazolam. Quantitative analytes were validated using a five-point calibration curve ranging from 5.0 to 100 ng/mL. Separation of the isomers alprazolam and 4’-chloro-deschloroalprazolam was successfully achieved.

 The validated method was then used to analyze postmortem cases that had previously screened positive for one or more designer benzodiazepine from February 2022 to December 2023. Of the 35 cases tested, 21 were confirmed to contain at least one designer benzodiazepine. Bromazolam was the most frequently detected and was found in 20 of the 21 positive cases with concentrations from 5.9 to 352 ng/mL (mean: 59.1 ng/mL). Etizolam and flualprazolam both had one positive case. All positive cases also confirmed positive for additional drugs other than designer benzodiazepines. Fentanyl was the most frequent and was found in 16 of the 21 positive cases. Several cases had other NPSs detected including p-fluorofentanyl and n,n-dimethylpentylone.

This method utilizes UCT’s Clean Screen® DAU columns (200 mg, 10 mL) to successfully identify common designer benzodiazepines and learn their prevalence in postmortem cases in Florida.

Citation: Ballotari M, Truver MT, Baer DC, Brogan SC, Hoyer JL, Crosby MM, et al. Identifying bromazolam, etizolam, and flualprazolam in blood using gas chromatography–mass spectrometry. J Forensic Sci. 2025; 00: 1–9. https://doi.org/10.1111/1556-4029.70003