Development and validation of a stability-indicating high performance liquid chromatography method based on a core-shell octadecyl column to determine Avermectin and its related substances in bulk Avermectin batches

Abstract

The Avermectins are a family of macrocyclic lactones isolated from the soil bacterium Streptomyces avermitilis that possess broad spectrum activity as anthelmintics and are employed throughout the agricultural and animal health industries. Avermectin is the parent molecule from which several important parasiticides have been derived, but currently the Ph. Eur. and USP do not describe an HPLC monograph for analysis of Avermectin. Here, we detail the development and validation of a reversed-phase HPLC method for determination of Avermectin and its related substances in bulk batches of Avermectin. The separation of all peaks of interest was accomplished by a gradient elution on an ACE UltraCore (core shell) 2.5 Super C18 column (150 mm × 4.6 mm, 2.5 µm particle size) maintained at 45 °C. The mobile phase for the gradient is 5 mM NH₄OAc in water at pH 9.5 (mobile phase-A) and acetonitrile/methanol/dichloromethane (52/40.5/7.5, v/v/v) (mobile phase-B). Avermectin and its related substances were detected and quantified at 245 nm with a quantitation limit of 0.05 % of the analytical concentration. Method validation following the International Conference on Harmonization guidelines was carried out with respect to specificity, linearity, accuracy, intermediate precision, and robustness and all acceptance criteria were met. The new HPLC method will provide reliable analysis of Avermectin and its related substances, while the method development data and forced degradation data herein will provide critical information applicable to the Avermectin family as a whole.