부산대학교 도서관

N-acetylneuraminic acid (Neu5Ac), one of the most common species in sialic acid family, has been proved to possess the crucial bio-functions in tumor biology such as facilitating immune escape, enhancing tumor proliferation and metastasis, promoting tumor angiogenesis etc ... The velvet antler, used as an important traditional medicinal material for hundred years, mainly consists of minerals, proteins, polysaccharides, fatty acid and phospholipids, which can be related to the form of glycoconjugates such as glycoproteins, glycolipids and glycoglycans. In the diverse set of compounds from antlers, the growing attention has been paid on the Neu5Ac. In this study, we have carried out the determination of Neu5Ac in water extract of antler samples based on its derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) using the HPLC fluorescence detection. The DMB reagent as a fluorophore has been used to make derivatization with Neu5Ac to yield a fluorescent adduct with excitation and emission wavelength of 373 nm and 448 nm, respectively. The high performance liquid chromatography parameters include gradient elution: solvent A (0.1 % formic acid) and solvent B (methanol containing 0.1 % formic acid) with program 0-10 min: 0-22 % solvent B, 20-30 min: 22-40 % solvent B; Hector C18 (5µm, 250 x 4.6 mm) column; flow rate 0.5 mL/min. The analytical method has been fully validated with parameters such as linearity range, precision, accuracy, limit of detection and limit of quantification. The Neu5Ac-DMB derivative adduct also has been characterized by HPLC-DAD-ESI-MS/MS.