An improved method for preparation of perbenzoylated ganglioside-derived sialic acids and nanogram detection of N-acetyl- and N-glycolylneuraminic acid by high performance liquid chromatography.

An improved method for the preparation of perbenzoylated ganglioside-derived sialic acids is described. After mild acid hydrolysis, isolation of sialic acids can be achieved by Folch partition (Method A) or by anion exchange chromatography (Method B). Perbenzoylated sialic acids were freed from benzoylation reagents by a second Folch partition. Total recoveries of both methods were found to be greater than or equal to 90%, calculated from metabolically labelled gangliosides. Derivatized N-acetylneuraminic and N-glycolylneuraminic acids were separated and quantified by isocratic high performance liquid chromatography using a RP18 column as the stationary phase and methanol:water (8:2) as the mobile phase. Both sialic acids were completely separated and eluted as single peaks within 15 min, monitored by UV detection. As little as 20 ng of neuraminic acid could be detected, the detector being linear up to 5 micrograms tested.