Study On Methods For Purification Of Monosialotetrahexosyl Ganglioside And Determination Of Its Related Substances

Clinical researches show that GM1is beneficial to promote recovery of traumaticand vascular brain injury and protect nerve cell. It’s clinically first-line medicine forcuring cerebrovascular accident, myelencephalon and Parkinson. However, theextraction and purification of GM1is much complicated, GM1raw material is typicallyin short supply. In addition, macromolecular substance and other ganglioside impuritiesare hard to eliminate in purification process. Therefore, establish a simple, efficientmethod of extraction and purification to extract higher-quality and explore a highsensitivity, high resolution detection method for quality control has important meaningto society and economy.In this project, the exchange chromatography（Q SepharoseTMFast Flow）wasoptimized for getting GM1and a certain number of GM1were purified using anionexchange chromatography（Q SepharoseTMFast Flow） from three pilot plant tests. Andthen refer to the national standard, the products were carried on identify reaction andcontrastive research in chemistry and physics levels, and make a preliminary securityresearch. The last step, innovate a detected method for GM1related substances whichprovide the feasibility of GM1quality control and further study for related substances.The research results are showed as followed.①Four jobs including screening for absorption, the elution salts, mobile phase andpurification of technological parameters were completed. The experiments show anionexchange chromatography performs well when choose Q SepharoseTMFast Flow aschromatography packing, chloroform-methanol (15:85,v:v) containing4mM potassiumacetate as mobile phase.②From three pilot plant tests,270g GLs could be extracted from100kg pigbrains making use of Svennerholm extraction method.70g pure GM1product could beobtained after purify with using self-building anion exchange chromatography. Wholepurifing process just holds about36h. Compared to the industry production process,this method has many advantages such as high yield, short purification cycle whichimprove productive efficiency for GM1. The foundation for mass production wasestablished.③Various comparative studies between pure GM1and reference substance havebeen taken including chemistry color response, chromatograph and spectral properties. The compare results showed that physicochemical properties of sample are same withreference substances’, and prove they have same composition.④Related substances and contents in batch three tests are detected under nationallegal standard. The results showed that the limit of impurities in three batch GM1products is up to standard and far below the standard limit, and the GM1contents inbatch three tests are higher than98%and better than standard. In general, there washigh correlation among batch three tests, the self-building Q SepharoseTMFast Flowanion exchange chromatography has good stability in this study.⑤The biological safety tests indicate that products haven’t undue toxicity. Proteincontent range is0.11%~0.13%, which is less than national standard (0.5%). Meantime,the heavy metal detection declares that the product meets with requirements of nationalChinese Pharmacopiea. These results show that this technology is safe.⑥A method for GM1reference substance determination is established with usingRP-HPLC. Moreover, it is been verified the feasibility and rationality. The results showthe method has good specificity, nice repeatability, well accuracy and better linearrelationship (correlation coefficient is0.9999), and it can be use for GM1referencesubstance detection.⑦The GM1reference substances in batch three tests are measured with RP-HPLC.It indicated that total impurity content is7.5%~7.9%which is higher than governmentstandard (5%), and the impurity detected number is about11. According to higherimpurity content and detected number, this method is probable for GM1quality controland further study for related substances.In this research, Q SepharoseTMFast Flow anion exchange chromatography hasimportant economic values and special biological characteristics which increase purifiedyield, shorten purified cycle. The self-building measurement of related substanceprovides much better choice for GM1quality control and further study. It has somescientific value.