Analysis of fluorescently labeled sugars by reversed-phase ion-pairing high-performance liquid chromatography

Reducing sugars, including monosaccharides, disaccharides, and a trisaccharide, are derivatized by reductive amination with 7-amino-1,3-naphthalene disulfonic acid. Reversed-phase ion-pairing high-performance liquid chromatography is then used to separate these visibly fluorescent, charged conjugates. Isocratic elution with triethylamine-acetic acid from a phenyl column, a C18 column, and C18 and phenyl columns in series gives good separations of a mixture of monosaccharides and a mixture of disaccharides and trisaccharides. Resolution of certain monosaccharides is enhanced by replacing triethylamine with a chiral amine and using gradient elution. Further enhancement of resolution is achieved by adding phenylboronic acid, an agent capable of complexing with the vicinal diol functionality present in many sugars. The trimethylamine-acetic acid eluant permits detection by either ultraviolet absorbance or fluorescence, and the addition of a chiral ion-pairing agent or a phenylboronic acid complexing agent necessitates fluorescence detection. A reversible Schiff base form of the fluorescent sugar conjugate is prepared; it is sufficiently stable to perform fractionations but sufficiently unstable to be converted to a fluorescent label and reducing sugar.