Seven impurities were detected in doramectin bulk drug by HPLC analysis. These impurities were named as Imp-I, Imp-II, Imp-III, Imp-IV, Imp-V, Imp-VI, and Imp-VII. All impurities were isolated from doramectin bulk drug by means of preparative HPLC. Among impurities, Imp-IV and Imp-V were unknown and have not been reported previously. Based on the complete spectral analysis, including 1D (H-1, C-13, H-D, DEPT90 and 135), 2D (COSY, HSQC, HMBC, and NOESY) NMR, IR, and ESI-HRMS, the structure of Imp-IV and Imp-V were elucidated. Imp-IV was found to be a furan ring-opening product, while Imp-V lacked amethyl group at the C-14 in doramectin. Identification, isolation, structural characterization, probable formation mechanism, and insecticidal activity of impurities of doramectin were also discussed. (C) 2020 Elsevier B.V. All rights reserved.