Dual SPE–HPLC–MS/MS Platform for Cross-Class Antiparasitic Surveillance: Simultaneous Quantification of Oxyclozanide and Levamisole Hydrochloride in Ovine Tissues with Applications to Withdrawal Period Optimization

This study presents a novel high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of oxyclozanide (OXY) and levamisole hydrochloride (LEV) residues in ovine tissues, addressing the critical gap in cross-class antiparasitic drug monitoring. Leveraging dual solid-phase extraction strategies-MAX anion-exchange for lipophilic OXY and MCX cation-exchange for hydrophilic LEV-we achieved efficient purification of these pharmacokinetically divergent compounds from complex matrices (muscle, liver, kidney, and perirenal adipose). The method demonstrated superior sensitivity with limits of detection (1.5 μg/kg) and quantification (2.5 μg/kg) below international maximum residue limits (MRLs), validated through Codex Alimentarius guidelines (CAC/GL 71-2009). Linear responses (2.5-1000 μg/kg, R² > 0.9900) and robust precision (intra-day RSD: 1.44-12.51%; inter-day RSD: 0.29-17.70%) were maintained across spiked concentrations (LOQ, 0.5×, 1×, and 2 × MRLs), with recoveries of 80.94-115.36% confirming matrix-agnostic accuracy. Stability assessments under diverse storage conditions further validated method reliability. Applied to pharmacokinetic profiling in medicated sheep, this protocol established a 28-day withdrawal period for edible tissues, reconciling regulatory compliance with food safety requirements. As the first reported simultaneous quantification platform for <i>OXY</i> and <i>LEV</i> antiparasitics, our methodology advances veterinary residue analytics by enabling efficient multi-class surveillance and evidence-based withdrawal period optimization.