Midecamycin Substances And Their Analytical Methods

Objective : To design the control method of related substance in mydecamycin(MDM) and meleumycin, establish the HPLC chromatographic condition.To separate the impurities from medecamycin, and analyse the separated impurities and confirm reliability and robustness of the method by precision test and linearity test. After establishing the method, analyse the samples from manufacturer to validate the applicability of the method.Method: Based on the analysis of reported references, a HPLC analysis method was designed and the HPLC conditiongs were optimized, including chromatographic column and mobile phase, column temperature, detection wavelength and solvent. The attributes of the main component compounds were determined with HPLC-MS according to the resulting quasi-molecular m/z and compound fragment m/z. Methodology validation was performed to confirm the feasibility and reliability of the established HPLC method. Simultaneously, national meleumycins were analyzed to determine if the attribute of major related substances was the same as imported meleumycin and confirm if the method is suitable for both national meleumycin and imported meleumycin.Result: Optimal conditions were obtained as follows:Packing material: ODSMobile phase: 0.01mol·L^-1(NH₄)₂Ac/CH₃CN 50/50, pH7.3adjusted by glacial acetic acidColumn temperature: 40℃Detector wavelegnth: 232/280nmFlow rate: 0.8-1.2ml·min^-1The attributes of component compounds were defined by HPLC-MS and shown that the components of national meleumycin were the same as those of imported meleumycin. The LOD and LOQ of mydecamycin A₁ were 50ng/ml and 150ng·ml^-1 respectively. The LOD and LOQ of mydecamycin A₃ is 5.6×10^-4mg·ml^-1 and 7.0×10^-3mg·ml^-1 respectively. Precision test of the method (peak areas of the impurities and variation coefficient of peak height ): RSD was less than 1.4%; intermediate precision test(peak areas of the impurities and variation coefficient of peak height): RSD was less than 3.0%. Resolution for specificity tests demonstrated that the resolution of major component and by-product B was more than 6. Peak homogenicity test (Blanck test) showed that no peaks were observed at the main ingradient or by-product peak positions by injecting the acetonitrile solution without sample into the chromatograp. Standard curve of mydecamycin A₁ was determined as: standard curve equation based on peak area: C=4×10^-6A - 0.0035; standard curve equation based on peak height: C=1×10^-4A-0.0086. The stability of solution is reliable. Recovery test showed that the recoveries of mydecamycin A₁ and mydecamycin A₃ were fine. The content limits of impurities in test sample for imported meleumycin were: less than 6.0% for meleumycin B; less than 4.0% for meleumycin A₂; less than 3.0% for meleumycin A3. The established method was feasible, and the data obtained was reliable. By comparing mydecamycin and national meleumycin, the content of major components of national meleumycin was significantly less than that of mydecamycin and the content of other components of national meleumycin was significantly more than that of mydecamycin. Howerer, major components of them were identical.Conclusion: (1) The established method can not only be used to analyse the content of major component mydecamycin A₁ in mydecamycin(meleumycin), but can also be used to control the content of impurities in the products. (2) The major component and major impurities in both between mydecamycin and meleumycin are identical. (3) The established HPLC method is suitable to determine major component and related substances of mydecamycin and national meleumycine. The method is of great significance to the quality control of mydecamycin(meleumycin).