In vitro and in vivo human metabolism profiling of designer benzodiazepines using high-resolution mass spectrometry

Designer benzodiazepines continue to emerge on the recreational drug market, and are particularly implicated in poly-drug intoxications, posing challenges for clinical and forensic toxicology. It is therefore important to characterize biomarkers that are useful for tentative confirmation of designer benzodiazepine (mis)use. In vitro human metabolism studies were thus conducted for at least three hours and analysis was performed using ultrahigh performance liquid chromatography tandem high-resolution mass spectrometry to identify their metabolites and proscribed consumption markers. Where bio-sample of reported benzodiazepine intoxication was available, they were analyzed with the same method. The main phase I metabolism pathway involved hydroxylation, particularly at the 4 carbon position of the diazepine ring in fluclotizolam, flubrotizolam, and desalkylgidazepam, the latter yielding 3 hydroxy desalkylgidazepam as its main metabolite. Alternative hydroxylation sites and subsequent phase II glucuronidation were observed for fluetizolam and bretazenil, while gidazepam metabolism was characterized by transformations of its hydrazine chain. These findings underscore the structural dependence of benzodiazepine metabolism and highlight the complexity of identifying consumption markers for compounds undergoing extensive hepatic clearance. Human hepatocyte models successfully simulated both phase I and II metabolism, enabling detection of novel metabolites and pathways within three hours of incubation. Such data are critical for establishing reliable biomarkers of use, though comprehensive pharmacokinetic parameters, including renal and hepatic clearance and protein binding, remain limited. Importantly, active metabolites such as 3 hydroxy desalkylgidazepam, from our in silico molecular modelling, may contribute to prolonged pharmacodynamic effects, warranting further in vitro and in vivo investigations. High resolution mass spectrometry combined with hepatocyte models provides a robust approach for metabolite profiling and biomarker identification. These methodologies are essential for confirming designer benzodiazepine intoxication, and thus supporting harm reduction strategies, and informing public health interventions in response to evolving new psychoactive substances.