Development of a Reversed-Phase HPLC Method by Applying a Scientific Approach and Strategies to Achieve Challenging Separation of Impurity Peaks in Typical Commercial Bulk Batches of Milbemycin Oxime Drug Substance

Abstract Background Typical commercial batches of milbemycin oxime (MO) contain over 25 related substances. Many of these related substance peaks exhibit similar chromatographic properties, often coeluting or being poorly separated under current compendial methods. Therefore, an alternative HPLC method with greater selectivity and resolution for MO and its related substance peaks is highly desirable. Objective This study aimed to develop and validate a new stability-indicating HPLC method for adequately separating all peaks of interest in typical commercial batches of MO. Method: The final HPLC method utilizes a gradient elution on a HALO® C18 column (100 mm × 4.6 mm, i.d. 2.7 µm particle size) maintained at 50°C, with a flow rate of 0.5 mL/min. The composition of mobile phase-A (MPA) for the final method is water–acetonitrile–perchloric acid (70 + 30 + 0.06, v/v/v), and the composition of mobile phase-B (MPB) is isopropanol–methanol–1,4 dioxane–perchloric acid (50 + 45 + 5 + 0.06, v/v/v/v). The injection volume of the new method is 6 µL and the detection wavelength is 240 nm. Results The new HPLC method demonstrated specificity by adequately separating all potential MO-related substances in stress-degraded MO drug substance. It showed good linearity and accuracy in the range of 0.1 to 120% of the target MO analytical concentration. The LOQ and LOD were determined to be 0.1% and 0.03% of the analytical concentration, respectively. The robustness study found no critical parameters affecting the method's specificity or accuracy. Conclusions The new HPLC method offers greater selectivity and resolution compared to compendial methods for the MO drug substance. It is more desirable for batch release and stability studies in both routine and non-routine activities. Highlights An exhaustive equivalency study between the new HPLC method and the compendial methods demonstrated that the new method is superior compared to the European Pharmacopoeia (Ph. Eur.) method for analyzing MO bulk drug substance.