Purity of glycosaminoglycan-related compounds using capillary electrophoresis.

Abstract

High performance capillary electrophoresis was used to determine impurities in glycosaminoglycans. The counterion of glycosaminoglycans was analyzed with indirect UV-detection using a 40 mM 4-aminopyridine buffer. Calcium, lithium, potassium and sodium could be resolved. A linear correlation between the area under the curve and the concentration of sodium (r2 = 0.98) and calcium (r2 = 0.99) was found. Using enzymatic depolymerization, chondroitin sulfates were cleaved to disaccharides. The resulting disaccharides, with the structure 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid (delta UA) 2 x (1-->3)-D-GalNY6X (X = H, sulfate and Y = acetyl, sulfate) for dermatan sulfate, were detected selectively at 230 nm using capillary electrophoresis. Dermatan sulfate disaccharides were analyzed using a 50 cm long fused silica capillary (75 microns ID). The buffer used was 10 mM sodium tetraborate and 50 mM SDS, pH 8.8. The detection was at 230 nm. Using the main peak delta UA (1-->3)-D-GalNAc4S as standard, between 1 and 80% dermatan sulfate in heparin preparations were analyzed. The disaccharide showed a linear correlation of the peak area versus the concentration with a correlation coefficient r2 = 0.98. The methods are useful in characterizing the identity and concentration of the counterion of glycosaminoglycans after chondroitinase degradation.