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

High Resolution Image Download MS PowerPoint Slide 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 P = 5.69) and levamisole hydrochloride (LEV, log P = 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, R 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, C max = 5377 ng/g, half-life, t 1/2 = 32–48 h) and fecal-dominated excretion (94.58%), while LEV achieved systemic distribution with blood–brain barrier penetration (brain C 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.