Chromatographic analysis and sequencing approach of heparin oligosaccharides using cetyltrimethylammonium dynamically coated stationary phases.

C(18) and C(8) bonded stationary phases dynamically coated with cetyltrimethylammonium (CTA) and strong anion exchange (SAX) were developed to obtain separations of oligosaccharide mixtures resulting from chemical or enzymatic depolymerization of heparin. With this method, the retention of sulfated oligosaccharides is directly adjustable depending on the amount of CTA adsorbed into the column. Oligosaccharides containing up to 20 sulfates were separated with a resolving power superior to that of conventional SAX analysis. The stability of the column coating enables hundreds of injections. Using ammonium methane sulfonate aqueous solutions as ultraviolet transparent mobile phases, it was possible to set up double detection, including selective detection of acetylated oligosaccharides. Analytical gel permeation chromatography was directly coupled to CTA-SAX, obtaining a two-dimensional profile of analyzed oligosaccharidic mixtures. A sequencing method of heparin oligosaccharides using partial depolymerization by heparinases according to their size and sulfation pattern and digest analysis by CTA-SAX was developed. A direct application of this method to the analysis of oligosaccharide mixtures obtained by complete digestion of heparins by heparinases I, II, and III was done. It allowed a reliable quantification of heparin building blocks. We also focused our attention on di- and tetrasaccharidic species containing the 3-O-sulfated glucosamines taken as markers of the active sites for antithrombin III. The method was also applied to more complex mixtures resulting from porcine heparin partially depolymerized with heparinase I. The specificity of the reaction was studied up to decasaccharidic fractions.