A hydrolysis method using microwaves: determination of N-acetyl-and N-glicolylneuraminicacids in biological systems by fluorometric high-performance liquid chromatography

A simple, rapid, and extremely sensitive method for determining N-acetyl- and N-glycolylneuraminic acids in serum and in submandibular, sublingual, and parotid glands using high-performance liquid chromatography with a fluorometric detector is described. The neuraminic acids contained in the samples are released in the presence of 2 M CH3COOH by means of microwave hydrolysis (only 10 min required) and are subsequently converted using 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for α-ketoacids, into highly fluorescent derivatives. In order to optimize the release of sialic acids and to minimize the effect of destruction, the following analytical variables were investigated: temperature, time, and concentration of the acid. Within 12 min after derivatization, the compounds were separated on a reversed-phase column by means of isocratic elution using a mobile phase of water (pH 3 with H3PO4)-methanol-acetonitrile (86:6:8, v/v). Fluorescence detection was performed at an excitation wavelength of 373 nm and an emission wavelength of 448 nm.