Separation and Quantification by Ion-Association Capillary Zone Electrophoresis of Unsaturated Disaccharide Units of Chondroitin Sulfates and Oligosaccharides Derived from Hyaluronan

Abstract

An analytical method has been developed for assay of unsaturated disaccharides of chondroitin sulfates and of oligosaccharides (tetra- and hexasaccharides) of hyaluronan, using ion-association capillary zone electrophoresis. Samples were applied at the anode (the usual polarity), using a borate buffer modified by an ion-pairing reagent, tetrabutylammonium (TBA) phosphate, and the effect of the concentration of the ion-pairing reagent on various electrophoretic parameters (electroosmotic flow, electrophoretic mobility of products, capacity factors) was observed. Increasing concentrations of the reagent led to a decrease of ζ potential, probably due to specific adsorption of the quaternary ammonium ion onto the capillary wall. The authors propose a mechanism of separation, in which anionic borate complexes are formed and interact with TBA ion inside the capillary tube. The capillary electrophoretic system described is potentially a powerful method for specific measurement of the concentrations in joint tissues of chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronan, whose relative abundances may vary in various diseases or after local treatments with, for example, antiinflammatory drugs, chondroprotective agents, or orthopedic implants.