A Method To Distinguish Glycosaminoglycans By High Performance Liquid Chromatography And Its Applications

In this thesis, a method was founded to distinguish different sorts ofglycosaminoglycans (GAGs) by reversed phase-high performance liquidchromatography (RP-HPLC). According to different monosaccharide composition ofdifferent sorts of glycosaminoglycans and their derivatives, pre-column derivationstrategy was used to effectively separate ten monosaccharide standards (glucuronicacid, GlcA; Iduronic acid, IdoA; galacturonic acid, GalA; glucosamine, GlcN;mannosamine, ManN; galactosamine, GalN; mannose, Man; glucose, Glc; galactose,Gal; xylose, Xyl) simultaneously on a C18column. Different acidolysis-resistdisaccharides derived from incomplete acid hydrolyzates of different sorts ofglycosaminoglycans were used to distinguish heparin/heparan sulfate (Hp/HS),chondroitin sulfate/dermatan sulfate (CS/DS) from hyaluronic acid (HA). Theestablished method was successfully applied to the determination of the molar ratio ofGlcN to ManN in enoxaparin sodium and the structural analysis of chondroitinsulfate/dermatan sulfate hybrid chain (hybrid chondroitin sulfate, HyCS) purifiedfrom marine fish gill tissues. This method provides a new approach for mass balanceof low molecular weight heparin and identification of marine complex GAGs.The first aim of the thesis is to set up a method to distinguish different sorts ofglycosaminoglycans by RP-HPLC. Firstly, on the basis of previous work in ourlaboratory, the chromatography condition of pre-column derivation HPLC wasoptimized to separate10monosaccharide standards (GlcA, IdoA, GalA, GlcN, ManN,GalN, Man, Glc, Gal and Xyl) simultaneously. The optimum condition was as follows:under the column temperature of30°C and the flow rate of1.0mL/min, the elutionwas conducted at15%(v/v) acetonitrile in0.1mol/L phosphate buffer (pH6.0) for25min, and17%(v/v) acetonitrile in0.1mol/L phosphate buffer (pH6.0) for another 25min. Then the optimized condition was applied to the analysis of incomplete acidhydrolyzate of glycosaminoglycans. Results showed that three sorts ofacidolysis-resist components in the hydrolyzate could differentiate Hp/HS, CS/DSfrom HA simultaneously. The structures of the acidolysis-resist components wererespectively identified as GlcNα1→4IdoA/GlcA, GlcAβ/IdoAα1→3GalN andGlcAβ1→3GlcN by mass spectrometry and high performance anion exchangechromatography. Meanwhile, the acidolysis-resist disaccharides could also be appliedto the quantitative analysis of Hp, CS and HA, with detection limit down to nanogramlevel.The second aim of the thesis is to determine the molar ratio of GlcN to ManN inenoxaparin sodium. Firstly, the structure of eight enoxaparins was characterized bysize exclusion chromatography-multiangle laser light scattering (SEC-MALLS),strong anion exchange (SAX)-HPLC and nuclear magnetic resonance (NMR), and theresults showed that they confirmed to the USP35standard. Then the optimalhydrolysis time for obtaining GlcN and ManN was determined, and the reasonablemolar ratio of GlcN to ManN for eight qualified enoxaparins was16.4-19.1.The third aim of the thesis is to identify the HyCS purified from marine fish gilltissues using by the RP-HPLC method, and disaccharide composition analysis,polyacrylamide gel electrophoresis and NMR methods were used to validate thestructure of the identified HyCS. Results indicated that the identification of structuresof HyCSs from fish gill tissues was correct and reliable. These HyCSs were mainlycomposed of covalent hybrid of CSA repetitive unit (→4GlcAβ1→3GlcNAc4Sβ1→),CSC repetitive unit (→4GlcAβ1→3GlcNAc6Sβ1→) and DS repetitive unit(→4IdoAα1→3GlcNAc4Sβ1→), and the DS unit accounted for3.9-24.6%.