Study On Quality Control Methods Of Polysaccharides Sufate

Sulfated polysaccharides are complex polysaccharide moleculars containing sulfate;it is highly electronegative.Its structure is composed of a monosaccharide or disaccharide with the same type or different type of sugars bound together by glycosidic linkages;forming a polysaccharide polymer of the same or opposite sex.Sulfated polysaccharides have anticoagulant,antithrombotic,antiviral,anti-inflammatory,antioxidant and other biological activities.Because of its great potential and good physical and chemical properties,it has attracted extensive attention in the field of biomedical science.It has been proved that the biological activity is closely related to the structure,the position and degree of substitution,the molecular weight,the composition of monosaccharides and the number of hydroxyl groups are the key factors affecting the activity,and are also the key parameters for the quality control of sulfated polysaccharides.In this paper,the key parameters of quality control of sulfated polysaccharides from different sources were studied in the following aspects:Chapter Ⅰ: The significance of this research was introduced,and the research progress of polysaccharide sulfate was summarized,including the source,the relationship between structural characteristics and biological activity,and the analysis methods of sulfated polysaccharides.Chapter Ⅱ: A high performance anion exchange chromatography/pulsed amperometric detection(HPAEC-PAD)method was established for the separation and analysis of nine monosaccharides and four uronic acid mixtures.The chromatographic condit-ions were optimized by sigle fator test.The results showed that when the concentration of Na OH was 50mmol/L,the flow rate was 0.6 m L/min,and the column temperature was30℃,the separation of the nine mixed monosaaccharides could basically reach the baseline.The results showed that the linear range of nine monosaccharides and four uronic acids was 0.10～32 μg/m L and 0.20～64μg/m L,respectively.The detection limits were 0.02～1.15 μg/m L and the quantitation limits were 0.07～3.84 μg/m L.The repeatability RSD value was less than 5%.The method has good precision and linear relationship,and can be used for quantitative analysis of monosaccharide composition of sulfated polysaccharides.Chapter Ⅲ: A method for the determination of molecular weight and molecular weight distribution of sulfated polysaccharides by size exclusion chromatography(SEC),relying on multiple angle laser scattering(MALS)/refractive index(RI)detectors was established.The key factors of the experiment were systematically investigated.In this study,four chromatographic columns were selected for analysis and they were used in series to compare the separation status of different sulfated polysaccharides on different chromatographic columns.According to the separation chromatogram of laser and differential,molar mass distribution and cumulative molar mass distribution trend line,the chromatographic column suitable for different sulfated polysaccharides was screened out,and the molecular weight and molecular weight distribution values of each sulfated polysaccharides were obtained.Chapter Ⅳ: An inductively coupled plasma mass spectrometry(ICP-MS)method was developed for the rapid determination of total sultate in sulfated polysaccharides.Using ICP-MS with collision pool,sulfate ion standard solution as reference,sulfur isotope 34 as monitoring element,inert gas helium(He)as collision gas,adding the internal standard solution online,the sulfate content in sulfated polysaccharides was detected.The linear range of sulfate ions determined by this method was 0～9.6 mg/L,the correlation R was 1.0000,the sample repeatability RSD was 1.30%(n=6),and the average spiked recovery rate was in the range of 93.07%～106.76%,the RSD value was 4.27%(n=9).The results show that the method has high specificity,high sensitivity and rapid analysis.The method was used to determine the content of total sulfate in sulfated polysaccharides samples from different sources,which can effectively and accurately determine the content of total sulfate in sulfated polysaccharides.Chapter Ⅴ: Fourier transform infrared spectroscopyc(IR)and nuclear magnetic resonance spectroscopy(NMR)were used to further analyze the fine structure of sulfated polysaccharides.The compositon of monosaccharide and sulfate root sites in sulfated polysaccharides were classified in detail,and their similarities and differences and identification methods were compared.It can play a role in the better study of the molecular basis between the structural characteristics and biological activity of sulfated polysaccharides,and provide more refercence for the quality control methods of sulfated polysaccharides.The determination methods of monosaccharides,sulfate content and molecular weight and the results of fine structure analysis established in this paper can lay a foundation for the further study of deconstructive effect relationship and biological activity,and provide a simple,accurate and reliable analytical method for the quality control and formulation of new quality standards of sulfated polysaccharides.