Abstract
A high-performance liquid chromatographic (HPLC) method with a carbon column was studied for the analysis of unsaturated disaccharides produced from glycosaminoglycans (GAGs). Most of the unsaturated disaccharides were separated on a carbon column (Carbonex, Tonen, Tokyo) under isocratic conditions using sodium carbonate buffer (pH 10.8) containing 3% or 3.5% acetonitrile. The disaccharides eluted were monitored by fluorescence detection using 2-cyanoacetamide as a postcolumn derivatizing reagent with excitation at 335nm and emission at 395nm. The unsaturated disaccharides produced from heparan sulfate family generally eluted in the order of tri-sulfated, di-sulfated, mono-sulfated and non-sulfated disaccharides, while the representative unsaturated disaccharides from chondroitin sulfate family were separated in a unique elution order: ΔDi-diSB, ΔDi-4S, ΔDi-diSE, ΔDi-0S, ΔDi-6S, ΔDi-HA and ΔDi-diSD). The elution order of unsaturated disaccharides found with the proposed HPLC method using a carbon column differs greatly from that obtained by the conventional systems using chemical-bonded type silica columns.
