A Hydrolysis Method Using Microwaves: Determination of N-Acetyl- and N-Glycolylneuraminic Acids in Biological Systems by Fluorometric High-Performance Liquid Chromatography

✍ Scribed by A. Lagana; A. Marino; G. Fago; B.P. Martinez

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 433 KB
• Volume: 215
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1585

✦ Synopsis

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 \mathrm{M} \mathrm{CH}{3} \mathrm{COOH}) by means of microwave hydrolysis (only (10 \mathrm{~min}) required) and are subsequently converted using 1,2 -diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for (\alpha)-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 (\mathrm{H}{3} \mathrm{PO}_{4}) )-methanol-acetonitrile ( (86: 6: 8), v/v). Fluorescence detection was performed at an excitation wavelength of (373 \mathrm{~nm}) and an emission wavelength of 448 nm. c 1983 Academic Press, Inc.