Chemoenzymatic Synthesis of N-Propionyl-Neuraminic Acid Derivative and Its Application in the Determination of Sialic Acid Content in Poultry Eggs

To realize the accurate quantification of N-acetylneuraminic acid(Neu5Ac)in egg yolk and egg white of poultry,and eliminate the matrix effect caused by substances other than analytes in egg samples,5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside-N-propionyl-sialic acid(X-Gal-α-2,6-Neu5Prop)was synthesized by chemical enzymatic method using mannosamine as substrate. The hydrolysis characteristics and stability of the derivative under acidic conditions were studied. The content of Neu5Ac in egg yolk and egg white of quail,goose,guinea fowl,ostrich,duck,pigeon and turkey was determined by using the derivative as internal standard. The glycoconjugates of sialic acid were dissociated into free sialic acid after being treated in acidic condition. The samples were analyzed by high performance liquid chromatography with fluorescence detector(HPLC-FLD).It was found that the hydrolysis rate of X-gal-α-2,6-Neu5aAc and X-gal-α-2,6-Neu5Prop at the same concentration were similar under the condition of 2 mol/L acetic acid solution at 80 ℃,which could be completely converted into free sialic acid after 90 min. The stability of Neu5Ac and Neu5Prop were similar at 10 h. The Neu5Ac content of the egg whites showed a wide variation across species. Quail egg whites had the lowest concentration of Neu5Ac(0.13 mg/g),while the ostrich had the highest(2.20 mg/g),which was seventeen times higher nearly. And Neu5Ac content of chicken egg yolk and albumen was found to be in agreement with previous reports. The method of analysis described herein could eliminate the matrix effect in biological samples and quantify the content of Neu5Ac accurately,with facile sample preparation requirements and was well suited to the routine analysis of foodstuffs.