Structural elucidation of major degradation products of milbemycin oxime drug substance using LC-MS and NMR.

Milbemycin oxime (MO) drug substance is a 16-membered macrocyclic lactone that exhibits a broad spectrum of biological activity and high potency towards parasites. In this study, a comprehensive forced degradation study was carried out on MO drug substance to identify and characterize its major degradation products (DPs). MO drug substance was subjected to acid, base, oxidation (HO), heat (solid and solution state), and photolytic (solid and solution state) stress degradation as per the ICH guidelines. Chromatographic separation of the drug substance (MO A and MO A) and its DPs was achieved using a gradient elution on a HALO C18 column (100 × 4.6 mm, 2.7 µm). Mobile phase A consisted of water/acetonitrile (60/40, v/v) and mobile phase B consisted of ethanol/isopropanol (50/50, v/v). A total of twelve major DPs were observed for MO drug substance under various stress conditions. These DPs were further identified and characterized using liquid chromatography-high resolution mass spectrometry and comparison of their fragmentation profile with MO A and MO A using tandem mass spectrometry. Of these, HO induced oxidative degradation product (3,4-dihydroperoxide MO A) was isolated using semi-preparative HPLC and characterized by comparison of its nuclear magnetic resonance spectroscopy data with MO A The proposed structures of the DPs have been rationalized by appropriate degradation pathways for MO A and MO A.