Metabolism and detection of designer benzodiazepines: a systematic review

Synthesis and illicit use of designer benzodiazepines are growing concerns, with these new psychoactive substances (NPS) posing serious health consequences and new hurdles for toxicologists. Consumption marker identification and characterization is paramount in confirming their use. The benzodiazepine core structure is a fusion of benzene and a seven-membered heterocycle with two nitrogen atoms forming the diazepine ring. Minor variations on the core structure produce different classes of benzodiazepines with marked differences in physiological effects. The present review provides a comprehensive designer benzodiazepines metabolism overview and suggests suitable human consumption biomarkers for toxicology casework. A systematic literature search of PubMed^®, Scopus^TM, Web of Science^TM, and Cochrane databases was conducted independently by two coauthors adhering to PRISMA guidelines. Data from 30 <i>in vitro</i> and <i>in vivo</i> models of designer benzodiazepines metabolism from January 2007 to February 2023 were included. 1,4-benzodiazepines (<i>n</i> = 10), 2,3-benzodiazepines (<i>n</i> = 1), triazolo-benzodiazepines (<i>n</i> = 9), and thieno-triazolo-benzodiazepines (<i>n</i> = 3) study design, sample pretreatment, analytical techniques, and major metabolites detected in various matrices are addressed. Metabolites following hydroxylation and phase II glucuronide conjugation were the most prevalent analytes. <i>N</i>-Glucuronidation of parent azole-fused benzodiazepines, and nitro-reduced and <i>N</i>-acetylated metabolites of nitro-containing designer benzodiazepines were also common. From these data, we propose a generic metabolic pathway for designer benzodiazepines. The sporadic illicit market presents challenges in toxicological casework and necessitates comprehensive biomarker investigations, especially in cases with legal implications. There are few metabolism data for many designer benzodiazepines, emphasizing the need for research focusing on closing these gaps.