Structural analysis of heparin by methylation and g.l.c.-m.s.: preliminary results

Heparin is a complex mixture of polysaccharides differing in biological activity and structure, and attempts to relate this activity to structure have suffered, owing to a lack of sufficiently sensitive and specific analytical methods. Application of methylation analysis to determination of the structure of heparin is described. Carboxyl-reduced heparin was converted into its pyridinium salt, this was dissolved in Me2SO, and free OH and NH groups were methylated with dimethylsulfinyl anion. Sulfate groups were removed by solvolysis, and after dialysis, the polymer was acetylated and depolymerized by acetolysis. The resulting monosaccharides were converted into alditol acetates, which were separated by capillary, gas-liquid chromatography, and identified by both electron impact and chemical ionization mass spectrometry. Seventeen different monosaccharides were identified in the hydrolyzate. All of the expected internal hexosaminyl and glycosyluronic residues were identified. Although several sugars were identified as nonreducing termini, only a hexosamine 6-sulfate was identified as a reducing-terminus sugar. The results indicate that methylation analysis of heparins and other complex, sulfated glycosaminoglycans is feasible.