Sensitive high-performance liquid chromatographic method with fluorometric detection for the determination of heparin and heparan sulfate in biological samples: application to human urinary heparan sulfate.

A sensitive high-performance liquid chromatographic method for the determination of unsaturated disaccharides produced from heparin and heparan sulfate is described. Heparan sulfate was depolymerized using a combination of heparin lyase I (EC 4.2.2.7), heparin lyase II and heparin lyase III (EC 4.2.2.8). Seven unsaturated disaccharides were separated under isocratic conditions within 25 min using acetonitrile-H2O-0.2 M sodium phosphate buffer (pH 7.0)-3.0 M ammonium chloride (32:10:1:1) and were monitored by fluorescence detection using 2-cyanoacetamide as a post-column derivatizing reagent. As little as 2 pmol of a disaccharide could be detected with excitation at 346 nm and emission at 410 nm. This method was applied to the analysis of normal human urine. It was revealed that the concentration of normal human urinary heparan sulfate is 1.53+/-0.36 mg/mg creatinine (n=4).