Rapid and sensitive method for measurment of hyaluronic acid and isomeric chondroitin sulfates using high-performance liquid chromatography

Abstract

A high-performance liquid chromatographic method for the separation and analysis of the unsaturated tetrasaccharide and hexasaccharide from Streptomyces hyaluronidase (S.HAase) enzyme digestion products of hyaluronic acid (HA) and standard unsaturated disaccharides 2-acetamido-2-deoxy-3-O-(β- D-gluco-4-enepyranosyluronic acid)- d-galactose (ΔDi-0S), 2-acetamido-2-deoxy-3-O-(β- d-gluco-4-enepyranosyluronic acid)-4-O-sulfo- d-galactose (ΔDi-4S) and 2-acetamido-2-deoxy-3-O-(β- d-gluco-4-enepyranosyluronic acid)-6-O-sulfo- d-galactose (ΔDi-6S) is described. An amino phase chemically bonded to silica with a particle diameter of 6 μm was used as the column. The composition and the pH of the mobile phase were systematically varied to determine the optimal chromatographic conditions for separation and analysis of the compounds. For HA, a complete separation was accomplished in less than 12 min with a practical detection limit of 100 ng. Separation of the disaccharides also required less than 15 min with detection limits of 10 ng for ΔDi-0Sand 25 ng each for ΔDi-4S and ΔDi-6S. This chromatographic method represents a significant improvement over existing methods. It allows the simultaneous separation and analysis of HA and chondroitin sulfate isomers (after digestion of the latter with chondroitinase) at a higher speed, and with more sensitivity and efficiency.