Determination of N-acetylneuraminic acid and N-glycolylneuraminic acid in glycoproteins by high-performance liquid chromatography without derivatization.

A simple and sensitive method for the determination of N-acetylneuraminic acid and N-glycolylneuraminic acid in glycoproteins by high-performance liquid chromatography has been investigated. The best condition to release sialic acid from glycoproteins involved incubation at 80 degrees C for 1 h in vacuo in 0.01 M hydrochloric acid with removal of the hydrochloric acid by evaporation without decomposing the sialic acids. Of the substances examined, N-acetylglycine was the best internal standard, because it is absent from glycoproteins, has an appropriate retention time on high-performance liquid chromatography (HPLC) and is stable during the procedures. The sample solution containing the internal standard was analysed directly by isocratic HPLC without pre-column or post-column labelling, and elution was monitored by ultraviolet absorbance at 205 nm. This method can determine 0.08-10 nmol of sialic acid accurately within 12 min and was successfully applied to bovine vitronectin.