Flubromazolam-Derived Designer Benzodiazepines: Toxicokinetics and Analytical Toxicology of Clobromazolam and Bromazolam.

Flubromazolam is widely known as highly potent designer benzodiazepine (DBZD). Recently, the two flubromazolam-derived new psychoactive substances (NPS) clobromazolam and bromazolam appeared on the drugs of abuse market. Since no information concerning their toxicokinetics in humans is available, the aims of the current study were to elucidate their metabolic profile and to identify the isozymes involved in their phase I and phase II metabolism. In vitro incubations with pooled human liver S9 fraction were performed and analyzed by liquid chromatography coupled to orbitrap-based high-resolution tandem mass spectrometry (LC-HRMS-MS). Biosamples after the ingestion of bromazolam allowed the identification of metabolites in human plasma and urine as well as the determination of bromazolam plasma concentrations by LC-HRMS-MS using the standard addition method. In total, eight clobromazolam metabolites were identified in vitro as well as eight bromazolam metabolites in vitro and in vivo. Predominant metabolic steps were hydroxylation, glucuronidation and combinations thereof. Alpha-hydroxy bromazolam glucuronide and bromazolam N-glucuronide are recommended as screening targets in urine. Bromazolam and its alpha-hydroxy metabolite are recommended if conjugate cleavage is part of the sample preparation procedure. The bromazolam plasma concentrations were determined to be 6 and 29 µg/L, respectively. Several cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) isozymes were shown to catalyze their metabolic transformations. CYP3A4 was involved in the formation of all phase I metabolites of both NPS, while UGT1A4 and UGT2B10 catalyzed their N-glucuronidation. Several UGT isoforms catalyzed the glucuronidation of the hydroxy metabolites. In conclusion, the determined bromazolam plasma concentrations in the low micrograms per liter range underlined the need for sensitive analytical methods and the importance of suitable urine screening procedures including DBZD metabolites as targets. Such an analytical strategy should be also applicable for clobromazolam.