Chromatographic separation of 1-phenyl-3-methyl-5-pyrazolone-derivatized neutral, acidic and basic aldoses

Abstract Analysis, by HPLC, of reducing monosaccharides as their 1-phenyl-3-methyl-5-pyrazolone derivatives is attractive owing to its sensitivity of detection and the generation of single derivatives of each aldose molecule [Honda et al., Anal. Biochem. 180 (1989) 351]. The present studies establish conditions for reversed-phase chromatographic analyses of hydrolysates containing neutral, basic and acidic reducing monosaccharides. In particular, glucuronic and galacturonic acids, glucosamine and galactosamine are separated completely both from one another and from the aldoses of glycoproteins commonly found. Analyses of blank hydrolysates provide the baseline for background amounts of these carbohydrates and analyses of a variety of glycoproteins illustrate the effectiveness of these separations. Elution times were established for derivatives of all tetroses, pentoses and hexoses, as well as a variety of deoxy and phosphorylated aldoses.