Fluorometric Determination of N-Acetyl and N-Glycolyl Neuraminic Acids by High Performance Liquid Chromatography as Their 4′-Hydrazino-2-stilbazole Derivatives

A sensitive and specific method for the determination of N -acetylneuraminic acid and N -glycolylneuraminic acid was developed by using high performance liquid chromatography with fluorometric detection. Both neuraminic acids were derivatized with 4′-hydrazino-2-stilbazole, a fluorogenic reagent for α-keto acid. The derivatives were separated by reversed phase chromatography on a Radial PAK Cartridge C_18 Column (Waters) with methanol-aqueous 1.5% triethylamine solution adjusted the pH to 2.3 with phosphoric acid (35:65 v/v) as a mobile phase at a flow rate of 1 ml/min. The detection limits of both derivatives were 0.5 ng in an injection volume of 5 μl. This method was applied to the determination of NANA in human blood and urine.