Development and Validation of a Stability-Indicating HPLC Method for Assay of Milbemycin Oxime and Estimation of Its Related Compounds

Abstract

A stability-indicating reversed-phase HPLC method for assay of milbemycin oxime (MO) and estimation of its related compounds has been developed and validated as per VICH and ICH method validation guidelines. The stability-indicating capability of this method has been demonstrated by adequate separation of all process-related impurities including all potential degradation products of milbemycin oxime (MO) that were generated using stress degradation conditions that are prescribed in ICH guideline. The stability-indicating capabilities of this method were further challenged by analyzing aged stability samples of MO active pharmaceutical ingredient (API). Chromatographic separation of MO and its related compounds was achieved using an isocratic elution at a flow rate of 0.5 mL/min on a Supelco Ascentis Express C18 column (100 mm × 3.0 mm, 2.7 µm particle size, 90 Å pore size) maintained at 50 °C. The isocratic mobile phase was composed of 30% v/v of 0.05% phosphoric acid in aqueous solution and 70% v/v of a mixture of methanol and acetonitrile (6:4, v/v). UV detection at 244 nm was employed to monitor the analytes.