Validation of a high‐performance liquid chromatography method for detecting flubendazole and 2‐aminoflubendazole in canine plasma

The purpose of this study was to establish a reliable method for the quantification of flubendazole and its metabolite, 2‐aminoflubendazole, in small‐volume canine plasma samples. Following liquid extraction with chloroform, samples were separated by reverse‐phase high‐performance liquid chromatography on an XBridge C18 4.6 × 250 mm column (5 μm). Quantification was performed using ultraviolet detection at 246 nm. A mixture of 5 mM potassium phosphate monobasic and acetonitrile (72:28) was used as the mobile phase. The standard curve ranged from 2.5 to 1000 ng/mL. Intra‐ and interassay variance for flubendazole and 2‐aminoflubendazole was less than 6%, while the recovery ranged from 91 to 101%. The lower limit of quantification was 2.5 ng/mL. This method was successfully validated and applied to the analysis of flubendazole and 2‐aminoflubendazole samples.