Bioanalytical HPLC–DAD method for determining (E)-1-4(trifluoromethyl) benzylidene)-5-(2-4-dichlorozoyl) carbonylhydrazine (LASSBio-1736), a promising antileishmanial compound in canine plasma applied to pharmacokinetic studies

Leishmaniasis remains a significant concern for global public health, with canines serving as primary reservoirs in urban areas, thereby emphasizing the critical importance of their treatment. However, in Brazil, therapeutic options for canine leishmaniasis remain limited, and treatment remains a considerable challenge. This study aims to develop and validate a highly sensitive HPLC-DAD method for the quantification of the leishmanicidal compound LASSBio-1736 (LB) in canine plasma. Protein precipitation was utilized to extract the drug candidate from 100 μL of basified plasma. An internal standard was selected for minimal interference with LB analysis, enabling detection at the same wavelength. Chromatographic separation was performed on a reverse-phase column employing a mobile phase composed of water with 0.4% triethylamine (pH 3), methanol, and acetonitrile in a ratio of 45:15:40 (v/v) under isocratic conditions at a flow rate of 1 mL/min. Plasma samples from both negative and positive canine subjects for Canine Leishmaniasis were analyzed. The method demonstrated a lower limit of quantification of 0.2 μg/mL and exhibited linearity across the range of 0.2-8 μg/mL (R2 > 0.99). The accuracy of the method exceeded 95.11%, with intra- and inter-day relative standard deviations below 19.46% and 10.96%, respectively. No carryover effects were observed in the analyte peaks. Although a matrix effect assessment was conducted in normal, anemic, and hemolyzed plasma samples, it did not impair LB analysis. These findings confirm that the proposed analytical method possesses the requisite linearity, precision, accuracy, and selectivity for quantifying LB in clinical pharmacokinetic studies involving canines diagnosed with leishmaniasis.