ISOLATION AND QUANTITATION OF HYALURONAN TETRASACCHARIDE AND HEXASACCHARIDE BY ANION-EXCHANGE HPLC

A high performance liquid chromatographic method for the isolation and quantitation of the tetra- and hexasaccharide derived from enzymatic depolymerization of hyaluronan by bovine testicular hyaluronidase (EC 3.2.135) is described in this paper. This method is well suited for the HPLC analysis of depolymerization reactions of hyaluronan, either alone or in competition with chondroitin sulphate, another glycosaminoglycan which is also a substrate for the hyaluronidase. Using a polymeric-based DEAE column with 50 mM phosphate buffer (pH 3.5) as mobile phase, complete separation of hyaluronan tetra- and hexasaccharide was achieved both in analytical and preparative scale. Under these conditions oligomers higher than hyaluronan-hexasaccharide and the chondroitin sulphate depolymerization products were strongly retained and were not eluted. Their complete elution was obtained in a single desorption step with 1.0 M potassium chloride in the eluent. Changes of the ionic strength as well as of the pH value of the eluent were undertaken to study their influence on the chromatographic behaviour of the oligosaccharides.