Assay of afoxolaner and determination of its related substances in commercial bulk batches of afoxolaner by reversed-phase HPLC method based on a short octadecyl column

• First reported RP-HPLC method to analyze afoxolaner and its related substances. • First reported stability-indicating method to analyze afoxolaner and its related substances. • Application of rapid and automated chromatographic method development tools. • The method was successfully validated according to the ICH guidelines. • This proposed method is simple, efficient, sensitive, and QC-friendly for routine analysis of afoxolaner. Afoxolaner is a new insecticidal and acaricidal active pharmaceutical ingredient (API) belonging to the isoxazoline family, widely prescribed for the control of fleas and ticks in dogs. A stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for the assay of afoxolaner and determination of its related compounds in bulk API lots of afoxolaner. The chromatographic separation of afoxolaner and its related compounds was achieved by gradient elution on a Zorbax-SB C18 column (50 mm × 4.6 mm i.d., 5 µm particle size) maintained at 40 °C. Mobile phase-A is composed of water and mobile phase-B is composed of acetonitrile/methanol (50/50, v/v). Analytes were monitored by UV detection at 225 nm with a flow rate of 2.0 mL/min. The stability-indicating capability of the method has been demonstrated by adequate separation of all the process related impurities and degradation products of afoxolaner generated by stress degradation of afoxolaner bulk drug substance under various stress conditions. This method was also successfully validated as per the current ICH guidelines for afoxolaner and Q6S07 (afoxolaner related substance) with respect to specificity, linearity (R 2 > 0.999), detection limit (∼0.21 µg/mL), quantitation limit (∼0.70 µg/mL), accuracy, precision, and robustness. Due to its speed, high degree of selectivity, and accuracy, the proposed method is suitable and highly desirable in quality control laboratories for routine analysis of afoxolaner.