High Performance Liquid Chromatographie Assay of Disaccharides and Oligosaccharides Produced by the Digestion of Glycosaminoglycans with Chondroitin Sulphate Lyases

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Abstract
In higher performance liquid chromatographic procedures hitherto described. SiO2, NH2 and RP columns were used for the analysis of disaccharides produced by the digestion of glycosaminoglycans with the chondroitin sulfate lyases AC and ABC. The use of a potent anion exchanger offers the following advantages over these columns; superior separation characteristics for nonsulfated disaccharides, and improved column performance, coupled with more stable analytical conditions. Elution with dilute saline solutions permits separation of the 2 nonsulfated disaccharides from chondroitin and hyaluronate. The sequential application of chondroitinase AC and ABC permits the determination of hyaluronate, the chondroitin sulfate isomers and the dermatan sulfate isomers by high performance liquid chromatographic separation of the products of enzymatic hydrolysis. In a previously described method, hyaluronate lyase was used for the determination of hyaluronate. Omission of the hyaluronate lyase step results in superior accuracy in the high performance liquid chromatographic separation of the nonsulfated disaccharides. The enzymatic analysis of human articular cartilage glycosaminoglycans has repeatedly yielded a fraction which is not digestible by chondroitinase AC, but is completely digestible by chondroitinase ABC. More extensive characterization has disclosed that this fraction differs structurally from chondroitin sulfate. Enzymatic characterization indicates that it should presumably be assigned to dermatan sulfate.

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