Dual SPE-UPLC-MS/MS method for multi-residue determination of anthelmintics in bovine tissues

This study establishes a sensitive Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) method for multi-residue determination of Compound Oxyclozanide Suspension components in bovine tissues using 20 Simmental cattle (10 males, 10 females). A novel dual extraction-purification protocol addressed veterinary drug monitoring challenges: Oxyclozanide (OXY) and Niclosamide (NIC) were isolated via acetonitrile-acetone (6:4) liquid-liquid extraction with CNWBOND MAX SPE cleanup, while Levamisole Hydrochloride (LEV) and Mebendazole (MED) required ethyl acetate extraction followed by Oasis MCX SPE purification. Validated across liver, kidney, muscle, and fat matrices, the method achieved recoveries of 84.13–106.45 % with intra−/inter-day precision (RSDs <12.55 %) at quantification ranges of 2.5–1250 μg/kg (OXY) and 2.5–220 μg/kg (LEV). Residue kinetics revealed peak tissue concentrations of 11,560 μg/kg (OXY) and 6452 μg/kg (LEV) in liver, followed by 8983 μg/kg (OXY) and 6201 μg/kg (LEV) in kidney, with significantly lower levels in muscle (720.10 μg/kg OXY; 1685 μg/kg LEV) and fat (277.40 μg/kg OXY; 377.10 μg/kg LEV). Critical depletion analysis confirmed final residues in all tissues fell below EU and China maximum residue limits (MRLs) at calculated withdrawal periods. Tissue-specific intervals were determined as 23.72 days (liver), 22.00 days (kidney), 20.80 days (muscle), and 24.82 days (fat) for OXY, and 18.72, 26.25, 27.86, and 21.35 days for LEV, respectively. To ensure international regulatory compliance, a harmonized 28-day withdrawal period is proposed based on the most conservative tissue-specific data. This work establishes pivotal pharmacokinetic benchmarks and validates a sensitive multi-residue analytical framework, significantly advancing the science-based application of Compound Oxyclozanide Suspension in veterinary therapeutics. • Problem addressed. Environmental risks from veterinary antiparasitic residues drove UPLC-MS/MS method development for bovine liver/fat/kidney/muscle analysis. • Originality of the approach. Developed the first method for simultaneous detection of levamisole (imidazole) and oxyclozanide (salicylanilide) in bovine tissues (liver, kidney, fat, and muscle), overcoming their divergent physicochemical properties. • Contribute to the work to create new knowledge in the field. MaxPeak column technology eliminates oxyclozanide carryover via metal adsorption reduction, enabling robust UPLC-MS/MS quantitation. • Relevance of the work to advance research and impact on society. Enables monitoring levamisole/oxyclozanide residues in cattle, supporting food safety, human health, and sustainable farming.