An Integrated QuEChERS LC–MS/MS Method for Screening, Confirmation, and Quantification of Nitroimidazole Residues in Food

BACKGROUND: Nitroimidazoles are prohibited veterinary drugs, for which sensitive and reliable analytical methods are required for regulatory residue control in food, particularly across diverse and complex matrices. OBJECTIVE: This study aimed to develop an integrated QuEChERS-LC-MS/MS method for screening, confirmatory identification, and quantitative determination of nitroimidazole residues within a single harmonized analytical workflow. METHODS: Samples were prepared using a unified QuEChERS procedure followed by dispersive solid-phase clean-up and LC-MS/MS analysis with isotope-labelled internal standards. Screening was conducted using a predefined screening target concentration (STC) as a validation reference level. Samples meeting relative retention time requirements and exhibiting responses exceeding a conservative screening cut-off were classified as suspected positives and subjected to confirmatory evaluation based on ion ratio criteria, followed by quantitative determination within the same analytical run. The method was validated for muscle, offal, fat, milk, egg, and aquatic products. Decision limits (CCα) and detection capabilities (CCβ) were established in accordance with regulatory guidelines. RESULTS: Excellent linearity was obtained (r2 ≥ 0.99) over the range of 0-2.5 µg/L. Mean recoveries ranged from 84 to 116%, with RSDs 0.1-8.1%. Limits of quantification were 0.2-0.4 µg/kg across all matrices, and matrix effects were adequately controlled (91-110%). CONCLUSION: The validated method provides a robust and harmonized analytical workflow suitable for screening, confirmation, and quantification of nitroimidazole residues across a wide range of food matrices, supporting regulatory residue control. HIGHLIGHTS: A single harmonized QuEChERS-LC-MS/MS workflow was developed for the screening, confirmatory identification, and quantitative determination of four nitroimidazoles and their metabolites in animal and aquatic foods. The method applies an STC-based decision strategy with confirmatory identification criteria and was validated across multiple matrices, achieving low LOQs. Performance was evaluated in accordance with Commission Implementing Regulation (EU) 2021/808 and verified through international proficiency testing.