Comprehensive study of degradation pathways and formation mechanism of major degradation impurities of gamithromycin active ingredient by UHPLC-HRMS and NMR.

Gamithromycin is a semi-synthetic antibacterial agent used for treating cattle bovine respiratory disease. This study was performed focusing on separating and identifying the degradation impurities of gamithromycin while elucidating its degradation mechanisms. A comprehensive forced degradation analysis was meticulously conducted on the gamithromycin drug substance following VICH/ICH guidelines under a range of stress conditions, including acid, base, oxidation (using HO and KCrO), thermal, and photolytic stress. The analytes were separated by ultra-high performance liquid chromatography using a gradient elution on an Agilent Poroshell HPH-C18 column (50 mm × 2.1 mm, 1.9 µm) at a temperature of 60 °C. The mobile phases employed were 10 mM aqueous NHHCO as mobile phase-A and a mixture of acetonitrile and methanol (7:3, v/v) as mobile phase-B. This study identified six degradation and five additional process impurities in the gamithromycin drug substance using high-resolution mass spectrometry in conjunction with tandem mass spectrometry. For impurity 6, an additional investigation was performed using nuclear magnetic resonance spectroscopy to confirm its structure. Mechanisms for the formation of the degradation impurities were also proposed. These findings enhance the understanding of the stability characteristics of gamithromycin and would provide significant insights for the formulation of drug products that include this drug.