Determination of 24 variously sulfated galactosaminoglycan- and hyaluronan-derived disaccharides by high-performance liquid chromatography

Abstract

In this report we describe a very sensitive high-performance liquid chromatographic method for the determination of 24 nonsulfated and variously sulfated disaccharides present in chondroitin sulfates, dermatan sulfates, and hyaluronic acid. The method is superior to others in that monosulfated disaccharides at either C-2 or C-3 of the uronic acid moieties and mono-, di-, and trisulfated disaccharides containing N-sulfated galactosamine as well as non-, mono-, and oversulfated disaccharides derived from iduronic acid can be determined. Following chondroitinase digestions of tissue extracts or purified hyaluronic acid, chondroitin sulfate, and dermatan sulfate, the non-, di-, and trisulfate delta-disaccharides, are separated by direct injections into HPLC, whereas the monosulfated delta-disaccharides are chromatographed after a simple reduction of the galactosamine carbonyl group with sodium borohydride. The various sulfated delta-disaccharides are separated on an amino column (Econosphere NH2) and recorded at 231 nm. The column is eluted isocratically with 5 mM sodium dihydrogen orthophosphate, pH 2.55, for nonsulfated delta-disaccharides; 50 mM sodium dihydrogen orthophosphate, pH 2.50, for reduced monosulfated; and 50 mM sodium sulfate-10 mM sodium acetate, pH 5.0, for the separation of di- and trisulfated delta-disaccharides. A linear detector response was obtained for injections up to 50 micrograms of delta-disaccharides. As little as 5-8 ng of nonsulfated, 8-11 ng of monosulfated, 12-15 ng of disulfated, and 25-30 ng of trisulfated delta-disaccharides can be reliably detected. Application of this HPLC method to the analysis of various glycosaminoglycans in conjunction with chondroitinase AC, ABC, or B digestions and sulfatase hydrolysis adds to the knowledge of the structural spectrum of the galactosaminoglycans. It was thus possible to identify 24 different disaccharides in chondroitinase-susceptible glycosaminoglycans, including all C-5 epimeric disaccharides and those sulfated at C-2 or C-3 of the uronic acids and at the amino group of the galactosamine.