ABSTRACT
OBJECTIVE: To establish an HPLC-HRMS/MS method for analyzing the structures and sources of the related substances in etimicin sulfate. : The chromatographic separation was achieved on a broad pH range column with a basic elution system composed of[H2O-ammonia-glacial acid(96:3.6:0.4)]-methanol(70:30). Twenty percent of the eluent was detected under positive electrospray ionization(ESI) by Q-Exactive mass spectrometry. The fragmentation pathways were elucidated according to the HRMS and HRMS/MS fragmentation of etimicin and some known compounds. Then the unknown related substances were identified by analyzing their HRMS and HRMS/MS fragmentation with the help of the rule. RESULTS: Sixty-five related substances were detected by the HPLC-HRMS/MS method in the two samples from different companies,among which 38 were detected in the product of company A, 59 in that of company B, and 32 were detected for both companies. Fifty-four related substances were identified or deduced, and the other 11 were not able to be identified due to limited information. Based on the significant difference of impurity spectra between the two enterprises, the key parameters of the synthesis process were analyzed. CONCLUSION: An HPLC-HRMS/MS method, which showes excellent accuracy, is developed to identify the related substances in etimicin sulfate. The resources and structures of the related substances are analyzed, which will be helpful to the process optimization.