A hydrolysis method using microwaves: determination of N-acetyl- and N-glycolylneuraminic acids 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 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 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.
Laganà
A
A
Department of Chemistry, University La Sapienza, Rome, Italy.
Marino
A
A
Fago
G
G
Martinez
B P
BP
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006001:Glycoconjugates / Q000737:chemistry; D006868:Hydrolysis; D008297:Male; D008872:Microwaves; D019158:N-Acetylneuraminic Acid; D009438:Neuraminic Acids / Q000032:analysis* / Q000097:blood; D051381:Rats; D017207:Rats, Sprague-Dawley; D012469:Salivary Glands / Q000737:chemistry; D012794:Sialic Acids / Q000032:analysis* / Q000097:blood; D013050:Spectrometry, Fluorescence
D006001:Glycoconjugates; D009438:Neuraminic Acids; D012794:Sialic Acids; C032592:N-glycolylneuraminic acid; D019158:N-Acetylneuraminic Acid