Transition of Determination Method of Potency of Neomycin Sulfate from Microbiological Assay to HPLC-PAD / 中国药学杂志

ABSTRACT

OBJECTIVE: To establish a high performance liquid chromatography combined with pulsed amperometric detection(HPLC-PAD)method for determination of potency of neomycin sulfate. METHODS: An improved HPLC-PAD method from EP method for determination of the content and related substances of neomycin sulfate was established and validated. The study of impurity profile of neomycin sulfate was completed by LC-IT-TOF method with the help of on-line desalination using a suppressor; and the main components in neomycin sulfate were clarified combining the RESULTS of impurity profile and minimum inhibitory concentrations of the main components and impurities. The semi-preparative liquid chromatography-evaporative light scattering detector(ELSD) was self-assembled, highly purified neomycin B and neomycin C were prepared and their structural confirmation was also conducted. The contents of highly purified neomycin B and neomycin C were determined by means of mass balance method. The potencies of highly purified neomycin B and neomycin C were determined by three-dose antibiotic microbial assay and the conversion factors between contents of neomycin B and neomycin C and their potencies were calculated separately and then a formula for the calculation of potency of neomycin sulfate from the content of main components of neomycin B and neomycin C was obtained.At last, a verification experiment for the accuracy of the conversion factor and the formula were designed and a serial of tests were carried out to investigate the interaction and the verification for the actual sample. RESULTS: The improved HPLC-PAD method was superior to the European Pharmacopoeia method in the separation ability and stability, and was suitable for accurate quantification of various components of neomycin sulfate and related substance inspection. The successful removal of trifluoroacetic acid in the mobile phase by the technology of desalination on-line using a suppressor broke a new way for the study of impurity profile of aminoglycoside such as neomycin sulfate. Combining the impurity profile with the RESULTS of MIC it was clarified that the main activity components of neomycin sulfate were neomycin B and neomycin C. Highly purified neomycin B and neomycin C were successfully prepared. A conversion factor for the transition from potency to purity of neomycin sulfate was obtained through experiments and calculations and was verified successfully. CONCLUSION: It is feasible to replace the microbial assay by HPLC-PAD method for determining the potency of neomycin sulfate.