Study On Impurity Profile For Cephalosporin Generics

Generic varieties of cephalosporin are demanded to be researched on impurity profile for each variety, accroding to guidelines for the study of impurities in ICH and guidelines for standard setting on the related substances of antibiotics in EMA. But there are many varieties of imitations interiorly, and there are many difficulties in the study of the impurity profile of cephalosporin. The work is complicated and the efficiency is poor using the troditional preparation and separation methods. In this paper, the advantages of study on impurity profile of generic drug of cephalosporin and impurity research information of listed species were combined. According to the co-degradation reaction mechanism, column switching and LC/MS/MSn methods were used to analyse the minor impurities contained in the principal component and to research on isomer impurities and polymer impurities in known structure information impurities and unknown structure information impurities. A decision tree for the study of impurities was established and relevant research rules were proposed. The main content is divided into three parts below:1. Column switching-LC/MS/MSn and column switching-HRMS method was used to preform systematically research on cefamandole nafate, Ceftizoxime sodium mixed impurity reference standard, Ceftezole sodium mixed impurity reference standard and Ceftriaxone sodiummixed impurity reference standard. According to the experimental method and the results of the study, a decision tree on the study of known structural information impurities of generic drug of cephalosporin was established:Using impurity profile analysis to determine the number of impurities, over degradation tests to determine the impurity to be process impurity or degradation impurity, using LC/MS technology to determine the molecular weight of target impurities and to infer the possible structure of impurities according to the analysis of fragmentation pattern of raw materials. Finally, the element composition was confirmed by high resolution mass spectrometry, and the structure of the impurity was confirmed.2. Isomer impurities were enriched by preparation liquid chromatography. The isomer impurities in cefamandole nafate, Ceftizoxime sodium mixed impurity reference standard, Ceftezole sodium mixed impurity reference standard and Ceftriaxone sodiummixed impurity reference standard were systematicly studied. According to the experimental method and the results of the study, a decision tree on the study of unknown structure information impurity-isomer impurity of generic drug of cephalosporin was established:Liquid chromatography was used to determined the impurities as isomers. Whether the maximum absorption wavelength is consistent was determined combined with UV spectral data. If not, with blue shift happened, it is presumed to be A-3 isomer impurity. Characteristic fragment ions in LC/MS/MSn confirmed the impurity to be A-3 isomer impurity. If consistent, structural characteristics and degradation tests need to be combined to determine the types of isomer impurities:Photo degradation test produces cis-trans isomer and the structure could be confirmed by the characteristic fragment ions in MS/MS of partial species. RS isomers were produced by alkaline degradation test and side chain isomers were produced by other degradation tests. The impurity structure analysed can not be confirmed by spectroscopic and mass spectrometry techniques. The target impurities can be purified by preparation liquid chromatography and confirmed structure by NMR technique.3. Method for examination of related substances for cephalosporins in Chinese Pharmacopoeia was used and the the ratio of organic phase was adjusted. The polymer impurities in ceftizoxime sodium, cephalexin, cefotaxime sodium, midazolam hydrochloride cefotaxime and cefoxitin sodium was researched by column switching-LC/MS/MSn. The polymerization methods of various types of dimer impurities were speculated and polymeric impurity types were summarized. Method for the test of polymer by eighteen alkyl silica gel column was established. The established method can be used to examine the related substances of dimer impurities after methodology validation.According to the research content, respectively, the decision trees for the study of various types of impurities were established and the research rules were put forward:Rulel. If the retention time, the UV spectrum and the MS information(molecular weight and fragments of MS/MS) of the impurity peak are the same as that of reference standard, the impurity is considered consistent with the structure of the reference standard.Rule2. For process impurities(precursors, initiators, intermediates), if the material could be obtained, rule 1 could be referred to confirm the structure; if couldn’t, the structure should be confirmed according to the fragmentation pattern of MS and HRMS data.Rule3. For degradation impurities, if their degradation reaction mechnisms are clear, such as ring-opening reaction mechanism of β-lactam,3-side chain hydrolysis, accelerated test can be designed to verify whether the degradation products are in compliance with the known degradation mechanism according to the reaction mechanism. Rule 2 should also be used to confirm the structure.Rule4. For isomer impurities, the structures of △3 isomer and trans-isomerism of cephalosporin with the 7-cefotaxime structure can be confirmed by spectrum and mass spectrometry technology. Others need to be confirm the sturcture by combining with NMR and other analytical results. For R and S isomers, at least H1 NMR data are needed to confirm the heterogeneous sites.Rule5. The test method for polymer impurities by eighteen alkyl silica gel column was established. Column switching-LC/MS/MSn can be used to determine the types of impurities(dimer and trime). Column switching-HRMS was used to confirm the structures. But the aggregation site can not be determined.The reserech of this paper is a part of the study of impuriy profile of P-lactam antibiotic. The research results can help researchers to analyse the impurity profile of generic drug of cephalosporins fastly, accurately and effectively. It is beneficial to improve the quality standard domestically, rapid increase the level of generic drug production of domestic pharmaceutical companies and ensure the safety and effectiveness of the use of cephalosporin.