Identification and characterization of major degradation products of Eprinomectin drug substance including degradation pathways using LC-HRMS and NMR.

Eprinomectin (EPM) is a semi-synthetic potent antiparasitic drug widely used in veterinary medicine. In this study, a comprehensive forced degradation study was carried out on EPM drug substance as per ICH guidelines. Generation of adequate quantities of major degradation products of EPM via forced degradation studies was necessary for identification, structure elucidation, and understanding its degradation mechanism and degradation pathways. EPM drug substance was subjected to acid, base, oxidation (HO and KCrO), thermal (solid and solution state), and photolytic (solid and solution state) stress degradation. The degradation products (DPs) formed in the stressed degraded samples were successfully separated using a gradient elution on a HALO C18 column (100 × 4.6 mm, 2.7 µm). Mobile phase A consisted of water and mobile phase B consisted of ethanol/isopropanol (98/2, v/v). A total of six major DPs of EPM drug substance formed under various stress conditions. The chemical structures of DPs were determined using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and characterized through comparison of their fragmentation profile with EPM B using tandem mass spectrometry (MS/MS). Additionally, two solvates (methanol adduct B #1 and methanol adduct B #2) were observed during the acid-stressed degradation study of EPM in presence of methanol. To confirm the chemical structure, these products were isolated with semi-preparative HPLC and characterized by using a combination of LC-MS/MS and nuclear magnetic resonance spectroscopy. The elucidated chemical structure of the degradation products of EPM was also justified through mechanistic explanations. Identification and characterization of the DPs including degradation mechanism(s) of EPM should facilitate the understanding of the stability behavior of EPM drug substances as well as aid in the design of new formulations made with EPM.