This study developed a novel analytical method integrating liquid-liquid extraction with solid-phase extraction for simultaneous detection of oxyclozanide (OXY) and levamisole hydrochloride (LMS) residues in milk using ultra-high-performance liquid chromatography tandem MS (UPLC-MS/MS). Method validation demonstrated exceptional sensitivity with detection limits of 1.00 ng/mL and quantitation limits of 3.00 ng/mL for both analytes. Low matrix effects (CV <8.00%) and satisfactory recoveries (OXY: 84.00%-108.77%, relative SD [RSD]: 2.20%-8.00%; LMS: 89.50%-119.60%, RSD: 2.10%-8.36%) were achieved across multiple validation parameters, while sample stability remained consistent under varied processing conditions (CV <10.00%). Analysis of 120 commercial milk samples revealed no detectable residues, confirming both the reliability of this monitoring framework and the compliance of Chinese dairy products with international safety standards. As the first reported method enabling concurrent detection of this antiparasitic drug combination, the established protocol provides crucial technical support for veterinary drug residue surveillance, ensuring safer application of compound oxyclozanide suspension in livestock husbandry.

This study aimed to establish a more sensitive and reliable quantitative method for determining rafoxanide (RFX) in ovine plasma. The method was applied in an 84-day long-term comparative pharmacokinetic trial to evaluate the performance of a rafoxanide nanosuspension (RFX-NS) versus its conventional suspension (RFX-S) in sheep. Characterization of the two formulations revealed the following results: RFX-NS had an average particle size of 484.93 ± 43.11 nm, a uniform size distribution (PDI 0.06 ± 0.07), and a Zeta potential of −43.59 ± 0.67 mV, which were significantly superior to those of RFX-S (particle size 2379.67 ± 121.71 nm, PDI 0.93 ± 0.10, Zeta potential −38.10 ± 0.55 mV), demonstrating excellent physical stability. In vitro dissolution tests indicated a higher dissolution rate for RFX-NS (97.6% at 60 min). By integrating formulation characterization, in vitro dissolution assessment, and pharmacokinetic studies, this research provided a comprehensive analysis of the differences between the two formulations. Pharmacokinetic results showed that, compared to RFX-S, RFX-NS had a significantly reduced apparent volume of distribution (Vz/F: 856.02 ± 274.00 vs. 1404.17 ± 285.1 mL/kg, p < 0.01), a shorter elimination half-life (t_1/2: 4.05 ± 1.18 vs. 5.32 ± 0.94 days, p < 0.05), and a relative bioavailability of 128.98%. These findings suggest that RFX-NS is an improved novel formulation superior to the conventional suspension, as it enhances drug efficacy and efficiency by improving drug dissolution and absorption.