Abstract: An analytical method was developed for determining total content of sialic acids including N-acetylneuraminic acid (Neu5Ac) and N-glycolyl neuraminic acid (Neu5Gc) in human milk. Sialic acid in human milk was released by acid hydrolysis, and derivatized with 4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride at 50 ℃ for 150 min in a dark environment. The separation of sialic acid was achieved on LiChrosorb RP-18 column (250 mm × 4 mm, 5μm) using methanol-acetonitrile-water (7:8:85) as the mobile phase at a flow rate of 0.9 mL/min. Injection volume and column temperature were set at 10μL and 30 ℃, respectively. The fluorescence excitation and emission wavelengths were 373 nm and 448 nm, respectively. The results showed that the linear range of sialic acid was 50－400μmol/L. The average recovery rate was 94.0%. The precision, stability and repeatability RSDs were 0.4%, 1.0% and 0.8%, respectively. The limits of detection were 0.02μmol/L for Neu5Ac and 0.03μmol/L for Neu5Gc. Therefore, this developed method is characteristics of simple operation, and high reproducibility and sensitivity. It can be widely applied to determine total content of sialic acid in infant formula, cow milk and human milk.

Key words: human milk, sialic acid, fluorescence high performance liquid chromatography