Analyses of glycosaminoglycans by gas-liquid chromatography and the nature of hexuronic acids in heparin

Abstract

Glycosaminoglycans in microgram quantities were analyzed by gas-liquid chromatography (GLC). Analyses were made of the total and individual hexuronic acids and hexosamines. Glycosaminoglycans were hydrolyzed with formic acid (90%) at 100° for 20 hr in nitrogen atmosphere. The resulting uronic acids and/or lactones were trimethylsilylated (TMS), and the TMS derivatives were analyzed by GLC on an Apiezon column at 190°. Glucuronic and iduronic acids each showed two peaks on the chromatograms. The areas of the peaks were found proportional to the concentration of the sugar. Uronic acids in several polysaccharides were determined by this method using α-methyl- d-glucoside as an internal standard, and the results were found in good agreement with theoretical values. The method, however, was not found suitable for quantitative analysis of total uronic acids in heparin and heparan sulfates due to incomplete hydrolysis of these polysaccharides even after 40 hr. The nature of uronic acids in heparin was investigated by making use of GLC. Peaks corresponding to the retention times of iduronic acid and/or lactones were observed in the chromatograms of heparin after hydrolysis with formic acid. Effluent fractions from GLC column corresponding to peaks on the chromatograms of hydrolyzed heparin were collected and the identity of these fractions with xylose, iduronic acid, and glucuronic acid was established through their infrared spectra. Although there are limitations in determining the total amount of hexuronic acids in heparin and heparan sulfate by the methods described, these techniques are very useful in resolution of ratios of the different hexuronic acids and detection of trace quantities not heretofore possible.