Analysis of glycosaminoglycans by high-performance liquid chromatography

Glycosaminoglycan chains were liberated from proteoglycan by successive digestion with protease and endo-beta-xylosidase. The glycosaminoglycan chains were then labeled with a fluorescent reagent, 2-aminopyridine, by reductive amination. The resulting pyridylamino-glycosaminoglycans, including hyaluronic acid, heparan sulfate, chondroitin sulfate/dermatan sulfate and heparin, were separated by ion-exchange high-performance liquid chromatography using a TSK gel SAX analytical column with a limit of sensitivity in the picomol range. With the combined use of a dermatan sulfate-degrading enzyme, chondroitinase B, chondroitin sulfate and dermatan sulfate were identified and quantified, separately. About 50 mg of wet animal tissue was enough for analysis of each glycosaminoglycan with satisfactory results. This method facilitates rapid separation and microanalysis of glycosaminoglycans.