Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography.

A simple, rapid, and highly sensitive fluorometric high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and its mono-O-acetyl derivatives in human and rat sera is described. The neuraminic acids, released by hydrolysis of serum in 2 M acetic acid, are converted with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives without the occurrence of O-acetyl migration and de-O-acetylation. The derivatives are separated isocratically within 25 min by reversed-phase chromatography using a TSK gel ODS-120T column. The limits of detection are 57-192 fmol in a 10-microliters injection volume at a signal-to-noise ratio of 3. This sensitivity permits precise determination of the neuraminic acids in 5 microliters of human and rat sera.