Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate

In this report we describe a convenient and sensitive HPLC method for separating and determining the non- and variously sulphated Delta-disaccharides derived from heparan sulphate, heparin and Fragmin, using heparin- and heparan sulphate lyases. This method is superior to others since it can separate and determine twelve different non-, mono-, di- and trisulphated Delta-disaccharides containing either N-sulphated, N-acetylated or unsubstituted glucosamine in a single HPLC run. The various types of Delta-disaccharides are separated by an ion-pair reversed-phase chromatographic procedure on a Supelcosil LC-18 column, using a binary acetonitrile gradient system with tetrabutylammonium as the ion-pairing reagent. The eluted peaks were recorded by dual wavelength at 232 and 226 nm and a linear detector response was obtained over the entire interval tested, i.e., to 50 mu g of Delta-disaccharides. As little as 0.8-5 ng of Delta-disaccharides can be reliably detected and accurately determined. Following separate digestion with the heparin- and heparan sulphate lyases (heparin lyases I, II and III), the characteristic heparin Delta-disaccharides in the heparan sulphate chain, as well as the heparan sulphate Delta-disaccharides in the heparin polymer, can be identified. Using combined digestions with these three lyases, the glycosaminoglycan chains are degraded almost completely (>90%) to Delta-disaccharides, which are then determined by direct injections into the HPLC system and thus an almost complete spectrum of disaccharide composition can be obtained. By this method, it is possible to analyse and confirm that the heparan sulphate chain is defined as a glycosaminoglycan dominated by GlcNAc(+/-6S)-GlcA disaccharides and by some copolymeric disaccharides, such as GlcNS-IdoA2S and GlcNS6S-IdoA2S, otherwise most common in heparin. Fragmin, which is a controlled depolymerized heparin fragment of M-r 5000, is made up mainly of trisulphated disaccharides of the GlcNS6S-IdoA2S type (88.8%). Using separate digestions with the specific heparin lyases, one can also distinguish between heparin and heparan sulphate.