Ultrasensitive Tracking of Dual Antiparasitics in Living Tissues: UPLC-MS/MS Reveals Synergistic Distribution of Oxyclozanide and Levamisole Hydrochloride

This study addresses the critical gap in tissue distribution kinetics of veterinary anthelmintic combination therapies by developing a novel dual-mode dynamic-switching ultraperformance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-MS/MS) method with ultrahigh sensitivity (limit of detection, LOD = 0.5 ng/g). The method enables synchronous quantification of oxyclozanide (OXY, log<i>P</i> = 5.69) and levamisole hydrochloride (LEV, log<i>P</i> = 2.32) in complex biological matrices through optimized ESI+/ESI- ionization and antiadsorption chromatographic separation (BEH C18 column). Validated per VICH GL49 and EMA/ICH guidelines, the assay demonstrated excellent linearity (2-800 ng/g, <i>R</i>² > 0.990), precision (relative standard deviation, RSD ≤ 10.32%), and recovery (91.41-102.49%) across nine tissues. Spatiotemporal profiling in rats revealed distinct pharmacokinetic synergies: OXY exhibited prolonged gastric retention (maximum concentration, <i>C</i>_max = 5377 ng/g, half-life, <i>t</i>_1/2 = 32-48 h) and fecal-dominated excretion (94.58%), while LEV achieved systemic distribution with blood-brain barrier penetration (brain <i>C</i>_max = 501 ng/g) and renal clearance (57.88%). The three-dimensional complementarity (spatial targeting, temporal kinetics, and clearance pathways) provides a mechanistic basis for enhanced anthelmintic efficacy and informs the optimization of withdrawal periods. This work establishes a paradigm for the preclinical evaluation of veterinary drug combinations.