Analysis by high-performance liquid chromatography of hyaluronic acid and chondroitin sulfates

The use of high-performance liquid chromatography for the quantification of glycosaminoglycan disaccharides has been hampered by the inability to isocratically resolve the chondroitinase digestion products 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose (delta Di-HA) and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS). To overcome this limitation, we have developed a solvent system capable of resolving delta Di-HA, delta Di-OS, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S). Integrator responses were linear from 1 microgram down to 25 ng for delta Di-HA, delta Di-OS, and delta Di-4S and down to 100 ng for delta Di-6S. This method was used to examine changes in the content of urinary hyaluronic acid and chondroitin sulfates isolated from normal individuals and from patients with Lowe Syndrome, Werner Syndrome, and Hutchinson-Gilford Progeria Syndrome. We confirmed that the HPLC method gave results comparable to colorimetric methods.