Optimization and validation of a QuEChERS-based method with a freezing-out clean-up for pesticide residues in commercial dry food for dogs and cats

Addressing pesticide residues in feed for cats and dogs is essential to assess their potential impact on their health. In this study, we validated a QuEChERS-based multi-residue method for analyzing 211 pesticide residues in cat and dog feed by LC-MS/MS and GC–MS/MS. To overcome the challenges posed by the complex nature of pet feed, we refined the clean-up step, comparing PSA, EMR-Lipid, and freezing approaches. Freeze-out yielded the best results, with 91.9 % of analytes achieving recoveries within 70–120 % and RSDs≤20 %, and two freezing cycles proved sufficient for effective matrix removal while maintaining analyte recoveries. To the best of our knowledge, this is the first validated QuEChERS method to use freezing-out as a standalone clean-up strategy, offering a simplified and cost-effective solution for high-fat matrices. The method was validated in terms of linearity, accuracy, and precision. Matrix-matched calibration curves (R² ≥ 0.99) were used for all analytes. Recoveries were within 70–120 % for all spiking levels in most compounds, with a few analytes showing recoveries between 60–130 % in the extended range allowed by SANTE guidelines. All RSDs were below 20 % by established validation guidelines. The method demonstrated high sensitivity, with most analytes achieving limits of quantification below the generic 10.0 µg/kg MRL established by EU regulations for feed. Notably, over 70 % of analytes achieved LOQs at least ten times lower. All limits of detection were equal to or below 10.0 µg/kg. The method's applicability was demonstrated by analyzing 16 commercial pet feed samples, where 112 residues of 39 pesticides were detected.