Construction and Comparison of UPLC-QE-Orbitrap-MS and UPLC-MS/MS Methods for the Detection of Diazepam Residues in Complex Aquatic Matrices

A standardized pretreatment protocol was established for simultaneous determination of diazepam and its metabolites—nordazepam, oxazepam, and temazepam—in aquatic products using liquid chromatography–mass spectrometry. Samples were extracted with 0.2% formic acid in acetonitrile (solid–liquid ratio 1:5, m/v), purified via MCX solid-phase extraction, eluted with 5% ammoniated methanol, and concentrated under reduced pressure. The residue was reconstituted in 0.2% formic acid–50% acetonitrile aqueous solution. Chromatographic and mass spectrometric conditions were optimized on two platforms: UPLC-QE-Orbitrap-MS and UPLC-MS/MS, with quantification based on internal standards. Both platforms showed excellent linearity across 0.2–200 ng/mL (R2 > 0.997), with detection and quantification limits as low as 0.1 μg/kg and 0.2 μg/kg, respectively. Following Codex Alimentarius guidelines (CAC/GL-71), 330 matrix samples (intra-batch n = 6, inter-batch n = 5) were validated, showing strong inter-platform agreement (Pearson r > 0.990, p < 0.001). Intra-batch RSDs ranged from 1.86% to 14.64%, and inter-batch RSDs from 1.10% to 11.41%. Recoveries ranged from 73.8% to 117.9% (p > 0.05). The dual-platform detection system developed herein demonstrates high sensitivity, strong matrix interference resistance, and excellent reproducibility, enabling accurate trace quantification of diazepam and its metabolites in heterogeneous aquatic samples.