High performance liquid chromatography of pyridylamino derivatives of sulfated oligosaccharides in the deamination products of porcine and whale heparins.

Abstract

Eleven purified sulfated oligosaccharides previously isolated from the deamination products of porcine and whale heparins were coupled with a fluorescent compound, 2-aminopyridine. The resulting pyridylamino derivatives were used as standards in the development of high performance liquid chromatography procedures for their separation and quantitation on a μbondapak-NH 2 , anion-exchange column. Four steps of isocratic elution with 30% methanol solution containing 10 m m ammonium acetate (pH 5.5), 30% methanol solution containing 20 m m ammonium acetate (pH 5.5), 10% methanol solution containing 20 m m ammonium acetate and 20 m m ammonium phosphate (pH 5.5), and 10% methanol solution containing 20 m m ammonium acetate and 30 m m ammonium phosphate (pH 5.5) satisfactorily separated the standard pyridylamino derivatives of monosulfated disaccharides (Di-6S(G), Di-6S, and Di-2S), disulfated disaccharide (Di-2,6S) plus monosulfated tetrasac-charides (Tetra-6′S and Tetra-2S) plus monosulfated trisaccharide (Tri-6′S), disulfated tetrasaccharides (Tetra-6,6′S and Tetra-2, 6S), and disulfated tetrasaccharides (Tetra-6,6′S and Tetra-2,6′S), respectively. The pyridylamino derivatives of the deamination products of porcine and whale heparins were separated into 19 peaks, including 11 peaks corresponding to the standards, by the foregoing procedures. Linear gradient elution with 10% methanol solution containing 10 m m ammonium acetate (pH 5.5)—10% methanol solution containing 20 m m ammonium acetate and 0.1 m ammonium phosphate (pH 5.5) directly separated the pyridylamino derivatives of the deamination products of porcine and whale heparins. The proportions of the pyridylamino-saccharides in the 19 peaks from porcine and whale heparins were then calculated in terms of the absorbance at 309 nm in total. The structures of some of the sulfated oligosaccharides in the newly observed peaks are tentatively proposed, based on the elution positions and the elution order with the location of the sulfate groups on the standards together with the previous findings. The present method may be applicable for separation and quantitation of sulfated oligosaccharides produced by deamination with nitrous acid or by digestion with mucopolysac-charidases of sulfated glycosaminoglycans.