Non-targeted screening of alprazolam and flualprazolam metabolites in in vitro metabolism of different species by high-resolution mass spectrometry.

Flualprazolam (FALP), a new psychoactive benzodiazepine structurally related to alprazolam (ALP), is increasingly misused, yet its interspecies metabolism remains unclear. In this study, the in vitro phase I and II metabolism of ALP and FALP was systematically investigated across multiple species under optimized liver microsome incubation conditions (1.5 mg mL-1, 2 h), using a non-targeted screening strategy with high-resolution mass spectrometry (HRMS). ALP and FALP exhibited similar metabolic profiles, with 11 metabolites each, including 7 phase I and 4 phase II metabolites, of which 6 were newly discovered. Species-specific differences were observed, with humans and mice primarily producing 4- and α-hydroxylated metabolites, while edible animals such as fish, bovine, sheep, and pig mainly generated 4-hydroxylated metabolites. This interspecies discrepancy suggests that the currently designated residue markers for ALP in animal-derived foods may not accurately reflect its actual metabolic fate. Notably, newly identified benzene-ring hydroxylated metabolites were more abundant in rats and livestock, and toxicity predictions indicated that these metabolites, including M1, M5, and M7, may pose higher ecotoxicological or developmental risks than the parent drugs, with greater predicted toxicity than the α-hydroxylated metabolites currently used as residue markers. These findings provide detailed insights into interspecies metabolic patterns, emphasize the need to consider species-specific metabolites in residue monitoring, and inform the toxicological assessment of benzodiazepines in food and environmental contexts.