A comprehensive study to identify major degradation products of avermectin active ingredient using high resolution LCMS and NMR

Avermectin (AVM) is the parent molecule of the family of macrocyclic lactone compounds that are generated by the bacterium <i>Streptomyces avermitilis</i> which exhibits a broad spectrum of activity and potency as a parasiticide. In this study, a comprehensive forced degradation study was carried out on AVM following ICH guidelines to identify and characterize its degradation products (DPs). AVM was subjected to acidic, alkaline, oxidative, thermal, and photolytic stress conditions. Forced degraded samples were analyzed using a gradient elution on an ACE UltraCore 2.5 SuperC18 column (150 × 4.6 mm, 2.5 µm particle size) to achieve sufficient separation of the active ingredient, impurities, and degradation products. AVM was susceptible to degradation under all conditions tested, with 7 major degradation products and one critical process impurity identified. Several minor degradation products and process impurities were also identified. The DPs were identified using liquid chromatography-high resolution mass spectrometry in comparison with the tandem mass spectrometry fragmentation profile of AVM B_1a. Additionally, the identity of monosaccharide B_1a, 8a-OH B_1a, and 26-epimer B_1a were elucidated through 1D and 2D nuclear magnetic resonance spectroscopy. Understanding the stability and degradation profile of Avermectin will allow for improved drug product formulations of not only Avermectin active ingredient but also be applicable to all compounds of the <i>avermitilis</i> family.