Determination of monosaccharides in glycoproteins by reverse-phase high-performance liquid chromatography

This report examines monosaccharide composition and content using the reverse-phase high-performance liquid chromatographic separation of p-aminobenzoic ethyl ester derivatives of neutral and amino sugars released from glycoproteins. After acid hydrolysis of glycoproteins with 2 M trifluoroacetic acid, sugars are derivatized with p-aminobenzoic ethyl ester (ABEE), which strongly absorbs uv light at 254 nm in the presence of sodium cyanoborohydride. Reverse-phase (RP)-HPLC of the ABEE-sugar derivatives is performed on Pico.Tag column in an isocratic mode. RP-HPLC conditions are optimized by using ternary mixture as a mobile phase and 45 degrees C as a column temperature. This procedure is very useful for the simultaneous analysis of neutral and amino sugars in a single chromatographic step using RP-HPLC without reacetylation of deacetylated amino sugars, which were produced by acid hydrolysis, and additional chromatography to remove side products. Monosaccharide composition and contents determined on glycoproteins by this method were very comparable to those previously obtained by other techniques.