A highly sensitive high performance liquid chromatographic method with fluorometric postcolumn labeling using 2-cyanoacetamide was developed for determination of unsaturated disaccharides produced enzymatically from chondroitin sulfates (ChS). The calibration curves of the unsaturated disaccharides were linear over the range from 2 pmol to 5 nmol. This method was applied to the analysis of normal rabbit plasma for ChS to verify the sensitivity and practicality.

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Chondroitin sulfate isomers (ChS isomers) were digested with chondroitinase ABC, and the unsaturated disaccharides produced were converted into fluorescent dansylhydrazine derivatives and analyzed by HPLC. The proposed method was applied to the determination of relative amounts of ChS isomers in normal human urine and analytical results showed that chondroitin-6-sulfate and chondroitin-4-sulfate were excreted in large amounts. Moreover, this method was used for the identification of urinary ChS isomers which were extracted from a cellulose acetate strip after carrying out electrophoresis.

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A high performance liquid chromatographic (HPLC) system is described for determination of the unsaturated disaccharide (delta Di-HA) derived from hyaluronic acid (HA) in human urine by digestion with hyaluronidase SD. The effects of eluents on the separation of delta Di-HA and delta Di-0S, which is derived from the reaction of chondroitin with the enzyme, have been studied. The established chromatographic conditions were as follows--column: a stainless steel tube (4 mm i.d. x 250 mm) packed with TSKgel NH2-60; eluent: a mixture of acetonitrile and 0.1 M Tris-HCl buffer containing 0.1 M boric acid and 10 mM sodium sulphate, pH 7.0 (64:36, v/v). The strong fluorescence of unsaturated disaccharide after the reaction with 2-cyanoacetamide in alkaline medium was used for post-column detection. The calibration curve for delta Di-HA was linear in the range 5 pmol-5nmol with a practical detection limit of 2 pmol. The assay coefficients of variation (n = 5) at 200 pmol for delta Di-HA and delta Di-0S were 1.7 and 1.5%, respectively. This HPLC system has been applied to the determination of HA in human urine.