A METHOD TO QUANTITATE TOTAL SIALIC-ACID, GLUCOSAMINE, AND GALACTOSAMINE IN BLOOD-SERUM AND GLYCOCONJUGATES BY HPLC

A sensitive method for determining the total amount of sialic acid, gl ucosamine, and galactosamine in blood serum and glycoconjugates using reversed phase high performance liquid chromatography is described. Si alic acids present in glycoconjugates are released by acidic hydrolysi s with 2 M TFA at 80 degrees C for 2 h, whereas hexosamines are releas ed following hydrolysis with 6 M HCl at 100 degrees C for 8 h. The sia lic acids released under these conditions are identified as a decarbox ylated and deacylated product bearing a primary amino group (amino-pro duct). Following removal of neutral monosaccharides and amino acids by ion-exchange chromatography on Dowex 50x8, the obtained amino-product and hexosamines are converted to Tosyl-derivatives. The obtained deri vatives of amino-product, glucosamine, and galactosamine, are complete ly separated and determined on a Supelcosil LC-18 column by means of i socratic elution using a mobile phase of water-acetonitrile (85:15, v/ v) and detection at 231 nm. The method shows a linearity range up to 2 5 nmol of sialic acids and hexosamines, has a detection limit ranging from 6 to 12 pmol, and is highly accurate when compared with literatur e values. The present method has the advantage of permitting determina tion of total sialic acid, glucosamine, and galactosamine in glycoconj ugates and blood serum using the same HPLC conditions.