Structural Identification Of Natural Sialic Acid Isomers And The Study And Application Of Its Relative Quantitative Analysis Method

Sialic acid(Sia)widely spreads in vertebrates and human,as well as some microbes and invertebrates.It is well known that O-acetylation is a common modification for Sia.For human and mammals,usually less than two O-acetyl groups can be observed in a single Sia,mostly at position C9.However,fish may contain multiple O-acetylated species.The derivatization based on 1,2-diamino-4,5-methylenedioxybenzene(DMB)coupled with LC-fluorescence detection or LC-MS analysis,has been the routine analytical strategy for O-acetylation profiling of Sias.Unfortunately,DMB offers poor chromatographic resolution for some Oacetylated species.Herein,we study another derivatization reagent,4,5-dimethylbenzene-1,2-diamine(DMBA),for LC-MS analysis of Sia isomers.The DMBA method was first evaluated using Sia reference standard,horse serum and crucian carp blood.The results demonstrate that DMBA provides superior chromatographic separation efficiency and comparable MS/MS spectra with DMB.The proposed method was then applied to the analysis of relative abundance of each Sia species in fish tissues,including liver,intestine,muscle and brain,and muscle,gonad of starfish.It was found that the relative amounts of Sia species were significantly different among the four tissues of crucian carp,and between starfish and crucian carp.Thus,DMBA derivatization-based LC-MS methods have great potentials in investigating the O-acetylation and O-methylation level of Sias in various samples,such as human food and bio-pharmaceutical products.