Rapid Quantitative Determination of Sialic Acids in Glycoproteins by High-performance Liquid Chromatography with a Sensitive Fluorescence Detection

Sialic acids were specifically labeled with o-phenylenediamine-2HCl (OPD) to yield stable fluorescent quinoxaline derivatives. The sialic acids were released from the glycoprotein in a NaHSO4 solution (0.25 M 80 degrees C, 20 min) and derivatized in the same solution with the OPD (10 mg/ml (final conc.) 80 degrees C, 40 min). Various sialic acids derivatized with the OPD were separated on a C-18 reversed-phase Ultrasphere-ODS column using the solvent systems and the detector conditions used for the determination of monosaccharides derivatized with anthranilic acid as reported earlier. The common N-acetyl- and N-glycolylneuraminic acids were separated within 20 min, and the other N- and O-acylated sialic acids were separated in 40 min. The OPD derivatives of mono-, di-, and triacylated sialic acid were separated into their respective groups in the present separation. The fluorescence maxima for the OPD-N-acetylneuraminic acid were 232 nm excitation and 420 nm emission and the limit of quantitation was < 2 pmol. The relative standard deviation was less than 3.0% for the sialic acid determinations using the glycoproteins. A common single HPLC system is used for complete carbohydrate composition analysis of glycoproteins, since both the sialic acid and the monosaccharide methods use the same solvent systems, column, and detector settings. Furthermore, in contrast to high-performance anion-exchange chromatography with pulsed amperometric detection, these methods are easy to set up for an analysis and offer the highest sensitivity for analyzing samples available in microgram amounts.