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 a nd chondroitin sulfates was examined. Hyaluronan differs from chondroitin s ulfates in the susceptibility to chondroitinase ABC under alkaline conditio ns. When hyaluronan and chondroitin sulfates were treated with chondroitina se ABC in the buffered solution (pH 9.1), chondroitin sulfates were selecti vely degraded to the unsaturated disaccharides, whereas hyaluronan was not. Subsequently, hyaluronan in the reaction mixture was digested to the unsat urated tetrasaccharide and hexasaccharide at pH 6.0 by Streptomyces hyaluro nidase in the presence of zinc ion (inhibitor for chondroitinase ABC). The separation of the resulting unsaturated disaccharides from chondroitin sulf ates and the unsaturated oligosaccharides from hyaluronan was achieved by a reversed-phase ion-pair HPLC. The structural polydispersities of hyalurona n and chondroitin sulfates in the molecular weight, the sulfation position, or the components of uronic acid did not affect their determination. The u sefulness of the present method was proved by application to the porcine sk in samples. (C) 1998 Academic Press.