Quantitation of the sulfated disaccharides of heparin by high performance liquid chromatography

Heparin was converted by treatment with nitrous acid primarily into sulfated disaccharides. The mixture of disaccharides was reduced with sodium boro³H]hydride and the disaccharides were purified by preparative paper electrophoresis and paper chromatography. Four disaccharides were obtained. On the basis of their paper electrophoretic mobilities and the products formed at intermediate stages of their acid hydrolysis, the disaccharides were identified as 4-O-(2-O-sulfo-α-l-idopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol, 4-O-(2-O-sulfo-α-l-idopyranosyluronic acid)-2,5-anhydro-d-mannitol, 4-O-(α-l-idopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol, and 4-O-(β-d-glucopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol. The purified disaccharides were used as standards in the development of a high-performance liquid chromatography procedure for their separation and quantitation on a Partisil-10 SAX anion-exchange column. The three monosulfated disaccharides were resolved by isocratic elution with 40 mm KH₂PO₄. The KH₂PO₄ concentration was tehn increased to 400 mm to elute the disulfated disaccharide. Column effluents were collected in $\text{1}\text{2}\text{-}\text{ml}$ fractions, and the recovery of each ³H-labeled product was determined by scintillation counting. When sodium boro-³H]hydride with a specific activity of 315 mCi/mmol was used in the reduction of the heparin deamination products, the disaccharides gave 28,500 cpm/nmol in the effluent peaks. Quantitative recoveries of the ³H-disaccharides were obtained. It was demonstrated that the method developed using the purified disaccharides gave reproducible and quantitative results in direct assays of aliquots of boro³H]hydride-reduced heparin deamination mixtures.