Discovery and quantification of novel diazepam metabolites BP-246 and BP-271 in freshwater fish using HRMS and UPLC-MS/MS

Diazepam, commonly used in freshwater fish transport and aquaculture, poses potential food safety and public health risks due to its residues and metabolites. However, current monitoring efforts primarily focus on the parent drug and its major metabolites, leaving potential toxic transformation products uncharacterized. This study reports two novel metabolites (BP-246, 5-chloro-2-(methylamino)phenyl)(phenyl)methanone, and BP-271, 6-chloro-1-methyl-4-phenylquinazolin-2(1 H)-one) in freshwater fish muscle, a matrix in which these compounds have not been previously reported, using liquid chromatography-Orbitrap high-resolution mass spectrometry (LC-Orbitrap HRMS). Using ProTox3.0 and ADMETlab3.0 for toxicity prediction revealed that BP-246 and BP-271 exhibit high blood-brain barrier permeability, neurotoxicity and ecotoxicity. A sensitive and validated ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous quantification of diazepam, three established metabolites (nordiazepam, oxazepam, temazepam), and the two novel compounds in fish tissue. Sample preparation involved acetonitrile ultrasonication extraction followed by solid-phase extraction (SPE) purification. Matrix-matched calibration curves demonstrated excellent linearity ( R 2 > 0.999) over the range of 0.2–10 µg/kg. Method recovery ranged from 78.5% to 119.9%, with intra-day and inter-day relative standard deviations (RSDs) below 9.9% and 9.1%, respectively. Limits of detection (LOD) and quantification (LOQ) were 0.05–0.06 µg/kg and 0.15–0.20 µg/kg, respectively. The method showed robust selectivity and consistent retention times. When applied to 10 market-sourced, diazepam-positive fish samples, the method successfully revealed differential metabolite profiles, enabling rapid, high-throughput monitoring of diazepam and transformation products in commercial fish products. These findings provide a basis for comprehensive risk assessment and regulatory control of diazepam residues in the food supply chain.