Simultaneous Determination of 61 Veterinary Drug Residues in Animal Derived Food by Clean-Up LPAS Combined with LC-MS/MS

A rapid analytical method was established for the simultaneous determination of 61 veterinary drug residues, covering 13 different drug categories, in five animal-derived food matrices (pork, pork liver, chicken, egg, and beef). The method integrated Low-Pressure Automated Solid-phase extraction (LPAS) as a clean-up procedure with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detection. The samples were extracted using 8 mL of acetonitrile:water (90:10, containing 0.2% formic acid), followed by purification with LPAS, and were directly analyzed using LC-MS/MS without an evaporation step. The experimental conditions, including LC-MS/MS parameters, extraction solvents and volumes, as well as the clean-up procedure for all 61 veterinary drugs, were systematically optimized. Method validation parameters, such as accuracy, precision, limits of detection (LOD), limits of quantitation (LOQ), linearity, and matrix effects, were thoroughly evaluated to ensure the reliability and robustness of the developed method. The results demonstrated good linearity for all analytes in the concentration range of 0.5 μg/L - 50 μg/L, with correlation coefficients (R2) ≥ 0.995. Recoveries at three spiked levels (5 μg/kg, 10 μg/kg and 50 μg/kg) in the 5 tested matrices ranged between 70.9% and 119.0%, with relative standard deviations (RSD) of 0.1% - 10.9%. The method showed low detection and quantitation limits, with LODs ranging from 0.03 μg/kg to 1.5 μg/kg and LOQs from 0.1 μg/kg to 5.0 μg/kg. When applied to analyze 731 real samples, 13 drug categories were detected in a total of 64 instances, among which 12 categories of restricted drugs were identified 57 times, and two types of prohibited drugs were found seven times; no other veterinary drugs were detected. These findings suggest that thesimplified LPAS clean-up procedure is superior to the conventional QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method, making it highly suitable for batch analysis and monitoring of multiple veterinary drug residues in animal-derived foods.