Isolation and identification of three new isomer impurities in milbemycin oxime drug substance

In an effort to identification of the unknown impurities in milbemycin oxime (MO) bulk drug, three impurities 1, 2 and 3 were isolated by two-dimensional (2 D) preparation method (Agilent Zorbax-C3 preparative column and Sepax Amethyst C18-H preparative column). Based on the extensive NMR analysis and ESIMS data, the structures of the three impurities were established as 14-desmethyl-14-ethyl-MO A₄ (1), 24-desmethyl-24-ethyl-MO A₄ (2) and 12-desmethyl-12-ethyl-MO A₄ (3), respectively. They are the new isomer impurities of MO D and most likely originate from the oxidation and oximation of natural milbemycin homologs present in the original fermentation broth.