Studies On New Methods For Structure Analysis Of Carbohydrate Using GC-MS

Gas chromatography mass spectrometry (GC-MS) as an analytical tool has been widely used for structural determination of carbohydrate, giving information about monosaccharide composition, the way how the sugar components are linked, and the absolute configuration of monosaccharide, as well as the substitution of hydroxy groups. We have developed two new methods for GC-MS analysis of carbohydrate.1. Mixtures of partially O-methylated alditol acetate standards (PMAAs) of galactofuranose were synthesized rapidly according to a new method. Galactose was treated with methanolic hydrogen chloride to give methyl galactosides, the products were then converted into their trimethylsilyl ethers and analyzed by gas chromatography as well as gas chromatography mass spectrometry. The reaction conditions were optimized with respect to the concentration of hydrogen chloride, reaction time and reaction temperature. The results suggested that for the preparation of methyl galactofuranosides, mild reaction conditions should be used. When galactose was heated under reflux with methanolic hydrogen chloride (0.004 mol/L) for 4 h, 79.9% methyl galactofuranosides could be retrieved. Methylation of methyl glycosides was carried out in the presence of BaO/Ba(OH)₂·8H₂O. Resulting partially methylated glycosides (PMGs) were assayed by thin layer chromatography (CHCl₂:MeOH 10:1) and as times that were optimum for formation of mono-O-methyl derivatives and later for higher degrees of methylation. Over a period of 4 h, the mother liquors from this reaction contained unsubstituted, mono-, di-, tri-and tetra-O-methyl derivatives, which were converted to partially O-methylated alditol acetates, via successive hydrolysis, reduction and acetylation, and then subjected to GC-MS. The resulting PMAAs could be identified by their retention time and EIMS profiles and almost all of the PMAA structures were obtained in the rapid synthesis, except for 2,5-Me₂Gal. These PMAAs can be used as GC-MS standards for simultaneous identification of galactofuranoic units in complex carbohydrates.Thus, A simple, efficient, and very useful procedure for the simultaneous synthesis of partially O-methylated alditol acetate standards of galactofunose can be developed. It is of particular importance for the structural analysis of complex galactofuranose-containing carbohydrates.2. GC-MS was used to determine the degree of substitution (DS) and substituent distribution of carboxymethyl starch (CMS) etherificated in isopropyl alcohol. CMS was converted into hydroxyethyl starch (HES) by carboxyl-group reduction. The product was then converted into partially O-methylated alditol acetates of glucose and hydroxyethyl glucose by per-methylation, hydrolysis, reduction, and acetylation, and which can be identified by GC-MS using their characteristic GC retention times and EIMS fingerprints. The DS was calculated on the base of their peak areas in GC: DS_totle= 0.41, DS_C2= 0.30, DS_C6= 0.11. The etherificating reaction preference of C-2, C-6, and C-3 among anhydroglucose is C-2 > C-6 > C-3.