Highly sensitive determination of N-acetyl- and N-glycolylneuraminic acids in human serum and urine and rat serum by reversed-phase liquid chromatography with fluorescence detection

A simple, rapid and highly sensitive high-performance liquid chromatographic method for the determination of N-acetyl- and N-glycolylneuraminic acids in serum and urine is described. The neuraminic acids, released by hydrolysis of serum and urine, are converted in dilute sulphuric acid with 1,2-diamino-4,5-dimethoxybenzene, a fluorogenic reagent for α-keto acids, to highly fluorescent derivatives. The derivatives are separated isocratically within 8 min by reversed-phase chromatography using a Radial-Pak cartridge C18 column and detected fluorimetrically. The limit of detection is 40 fmol (12 pg) for both neuraminic acids in 10-μl injection volume [0.3 nmol (90 ng)/ml) of serum or urine]. This sensitivity permits the precise determination of the neuraminic acids in 5 μl of serum or urine. The method was applied to the determination of the neuraminic acids in sera from normal subjects and cancer patients, normal urine and rat serum.