Quantitation of hyaluronic acid and chondroitin sulphates in rabbit synovial fluid by high-performance liquid chromatography of oligosaccharides enzymatically derived thereof.

A high-performance liquid chromatographic (HPLC) method for quantifying unsaturated hexasaccharide and tetrasaccharide from Streptomyces hyaluronidase enzyme digestion products of hyaluronic acid was developed using a gel-permeation column packed with a sulphated polystyrene-divinylbenzene gel. For the oligosaccharides, the separation was accomplished in less than 7 min with a detection limit of 65 ng. An unsaturated non-sulphated disaccharide prepared from hyaluronic acid (delta Di-HA) and an unsaturated sulphated disaccharide (delta Di-4S) were analyzed by a HPLC method using a combination of two different gel-permeation columns. The separation of the disaccharides required less than 17 min at a flow rate of 0.7 ml/min with detection limits of as little as 4 ng for delta Di-HA and 5 ng for delta Di-4S. Both chromatographic methods were used for assay of a major component of hyaluronic acid and trace amounts of chondroitin sulphates in rabbit synovial fluid. The resulting contents of hyaluronic acid were compared to the values of polymeric hyaluronic acid directly measured by a HPLC method using two gel-permeation columns packed with a poly(hydroxyalkyl methacrylate) gel and the amounts of hyaluronic acid converted from uronic acid content determined by a colorimetric method.