Fluorescence high performance liquid chromatography of uronic acids using dansylhydrazine as a prelabelling reagent.

Abstract

The fluorescence high performance liquid chromatography of uronic acids is developed. Various uronic acids, such as glucuronic acid, galacturonic acid, and mannuronic acid, were mixed with 100 μl of 0.75 % trichloroacetic acid-ethanol solution and 100 μl of 1.0 % dansylhydrazine-ethanol solution. The mixture was incubated for 45 min at 40°C and then cooled to room temperature. An aliquot of the resulting solution was injected into a Finepak SIL-NH2 column of the chromatograph. Good separation of uronic acids was obtained within about fifteen minutes by elution system using acetonitrile/acetate buffer (pH 5.6) (90/20 : v/v). The fluorescence of the eluate was monitored at 530 nm, when the excitation was made at 350 nm. Linearity of the fluorescence intensity with the amounts of uronic acids was obtained between 0.1 and 20 nmol. Detection limit was about 50 pmol of uronic acids. The proposed method is applicable to detection of various acids in glycosaminoglycans and other biological substances.