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

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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 C₁₈ 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 1ml/min. The detection limits of both derivatives were 0.5ng in an injection volume of 5μl. This method was applied to the determination of NANA in human blood and urine.