Toxoplasmosis is a significant zoonotic parasitic disease. Currently, there is no effective vaccine available to prevent human infection, and treatment primarily relies on chemotherapy. However, the lack of specific therapeutic agents and the limitations of existing drugs highlight the urgent need for novel, safe, and effective anti-Toxoplasma gondii (T. gondii) medications. In this study, we evaluated the toxicity of ICA (N-(pyridin-2-yl)-4-(pyridine-2-yl)thiazol-2-amine) to host cells and assessed its inhibitory and anti-proliferative effects on T. gondii tachyzoites. We further investigated the impact of ICA on the ultrastructure of T. gondii using transmission electron microscopy (TEM). Additionally, we measured alterations in mitochondrial membrane potential, superoxide levels, and ATP levels in T. gondii to assess the effect of ICA on mitochondrial function. Our findings demonstrated that ICA exhibits a safe and effective inhibitory effect on T. gondii, with a selectivity index (SI) of 258.25. Notably, ICA demonstrated a more potent anti-proliferative effect than pyrimethamine (PYR). Ultrastructural observations revealed that ICA induces mitochondrial swelling and membrane rupture in T. gondii. Further investigations confirmed that ICA leads to mitochondrial dysfunction in T. gondii. In conclusion, our results suggest that ICA possesses the potential to serve as a lead compound for the development of novel anti-T. gondii therapies.

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 OXY and LEV antiparasitics, our methodology advances veterinary residue analytics by enabling efficient multi-class surveillance and evidence-based withdrawal period optimization.