Optimisation and validation of an UPLC-MS/MS method for analysis of veterinary drug residues (coccidiostats & nitroimidazoles) in Bovine liver

The Swedish Food Agency is responsible for the official control of pharmacologically active residues in food of animal origin in Sweden. In this study, an existing analytical method was optimised and developed by addition of the three new analytes, and validated for screening and quantification of 25 coccidiostats and nitroimidazoles in Bovine liver. Sample preparation involved protein precipitation followed by centrifugation, with analysis of the supernatant performed using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Two MS/MS methods were developed to establish a robust analytical strategy: a full multi-residue method employing polarity switching for screening and quantification, and a negative mode-only method used for confirmatory quantification of analytes ionized in negative mode. This approach enabled successful screening and confirmation/ quantification of almost all analytes including new analytes in Bovine liver. Method was validated by analysing 21 different Bovine liver samples in 3 different series (n = 7 x3) in accordance with the performance criteria set by EU Commission Implementing Regulation (EU) 2021/808. All compounds fulfilled the identification criteria for screening while 23/25 compounds fulfilled the criteria for confirmation/ quantification. Linearity of the calibration curves fulfilled regulatory requirements, with correlation coefficients exceeding R² > 0.95 for the full multi-residue method and R² > 0.99 for the negative-only MS/MS method. Specificity, selectivity, precision, trueness, reproducibility and the detection capability for screening (CCβ) were evaluated. Decision limit for confirmation and quantification (CCα) was calculated for each analyte. For two analytes, pronounced matrix effects prevented compliance with performance criteria for confirmation and quantification. However, as these effects resulted primarily in signal enhancement, the analytes remain suitable for qualitative screening within the full multi-residue method. The validated methods are therefore considered suitable for use within official control of Bovine liver at the Swedish Food Agency.