Enzymatic method for the simultaneous determination of hyaluronan and chondroitin sulfates using high-performance liquid chromatography.

A chromatographic method for the simultaneous determination of hyaluronan and chondroitin sulfates was examined. Hyaluronan differs from chondroitin sulfates in the susceptibility to chondroitinase ABC under alkaline conditions. When hyaluronan and chondroitin sulfates were treated with chondroitinase ABC in the buffered solution (pH 9.1), chondroitin sulfates were selectively degraded to the unsaturated disaccharides, whereas hyaluronan was not. Subsequently, hyaluronan in the reaction mixture was digested to the unsaturated tetrasaccharide and hexasaccharide at pH 6.0 by Streptomyces hyaluronidase in the presence of zinc ion (inhibitor for chondroitinase ABC). The separation of the resulting unsaturated disaccharides from chondroitin sulfates and the unsaturated oligosaccharides from hyaluronan was achieved by a reversed-phase ion-pair HPLC. The structural polydispersities of hyaluronan and chondroitin sulfates in the molecular weight, the sulfation position, or the components of uronic acid did not affect their determination. The usefulness of the present method was proved by application to the porcine skin samples.