A reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed and validated for assay and determination of related substances peaks of Esafoxolaner, Eprinomectin and Praziquantel including the assay of antioxidant butylated hydroxytoluene (BHT) in a topical finished product. Analytes were separated on a short Zorbax SB-C18 column (50 mm x 4.6 mm I.D., 5 µm particle size) with gradient elution at 40 °C column temperature. Analytes were detected at 245 nm for Praziquantel, Esafoxolaner, Eprinomectin and their degradation products and at 280 nm for Eprinomectin degradation product namely 8a-oxo-B<sub>1a</sub> and BHT. Mobile phase of the method is composed of 5 mM potassium phosphate buffer (pH 6.8) as mobile phase-A and MeOH as mobile phase-B. All analytes of interest were adequately separated within 40 minutes. The assay for Praziquantel, Esafoxolaner, Eprinomectin and BHT was conducted against their corresponding external reference standards. The related substances peaks of each active pharmaceutical ingredient (API) were determined by peak area percent against total peak area of their corresponding API peak in sample solution. Limit of quantitation (LOQ) of the method is 0.2% of target analytical concentration. This method has been demonstrated to be accurate, robust, specific, and stability-indicating.

Simultaneous determination of Esafoxolaner, Eprinomectin, Praziquantel, BHT and their related substances in a topical finished product by a single reversed-phase high performance liquid chromatography (RP-HPLC) method

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ACS Fall 2022

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