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 itbondapak-NK, , anion-exchange column. Four steps of isocratic elution with 30% methanol solution containing 10mM ammonium acetate (pH 5.5), 30% methanol solution containing 20mM ammonium acetate (pH 5.5), 10% methanol solution containing 20mM ammonium acetate and 20mM ammonium phosphate (pH 5.5), and 10% methanol solution containing 20mM ammonium acetate and 30mM 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 tetrasaccharides (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 10mM ammonium acetate (pH 5.5)-10% methanol solution containing 20mM ammonium acetate and 0.1M 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
