Rapid quantitative determination of sialic acids in glycoproteins by high-performance liquid chromatography with a sensitive fluorescence detection

Analytical Biochemistry
K R Anumula

Abstract

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 bot...Continue Reading

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