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.
