Sample Clean-Up and Analysis of N-Acetyl and N-Glycolylneuraminic Acids in Blood Serum and Tissue Specimen by HPLC

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Abstract
A procedure for sample clean-up, isolation of glycoconjugates, and analysis of the two major sialic acids, Neu5Ac and Neu5Gc, as per-O-benzoylated derivatives in biologic samples is described. In blood sera, the bulk of low molecular weight molecules was efficiently removed by precipitation of sialic acids containing glycoconjugates with saturated ammonium sulphate, pH 10, at 0°C, centrifugation, and gel chromatography on a Sephadex G-25. The tissue specimen was homogenized with water at 0°C and following centrifugation the water-soluble sialic acids containing glycoconjugates were recovered in the supernatant. Sialic acids were liberated from their isolated macromolecules by acidic hydrolysis with 25 mM trifluoroacetic acid at 80°C for 2 h. Constituents with positive charged groups and neutral monosaccharides were removed by ion-exchange chromatographies on Dowex 50X8 and Dowex 1X8, whereas sialic acids were eluted with 2M formic acid. Following derivatization with benzoic anhydride in the presence of p-dimethylaminopyridine, the per-O-benzoylated derivatives were completely separated and analyzed on a Supelcosil LC-18 column by isocratic elution with acetonitrile-water (67:33, v/v) and detection at 231 nm. Quantitation was performed using peak areas obtained from external standards treated under the same hydrolytic conditions as samples. Application of the method in C57BI mouse lung showed that N-glycolylneuraminic acid is the major sialic acid and that the amounts of sialic acids in rat serum are in excellent agreement with those previously reported.

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