Establishment And Optimization Of Method For Sialic Acid Detection From Animal-originated Foods And The High-throughput Method For Sialylated Conjunction Extraction

Sialic acids are a family of nine-carbon acidic monosaccharides,which normally occupied the terminal position of the glycoconjugation such as glycoproteins and glycolipids.N-acetylneuraminic acid(Neu5Ac)and N-glycolylneuraminic acid(Neu5Gc)are the two major forms of sialic acids.For human,Neu5Ac and its derivatives are endogenous sialic acids and which participate in the development of nervous and poses positive effects on the cognitive ability and the performance ability.Whereas Neu5Gc is exogenous sialic acid and is immunogenic to the body Therefore it is of great importance to investigate the distribution and content of Neu5Ac and Neu5Gc in diet.Additionally,due to the fact that only the sialic acids in glycoconjugates play roles in human,it is also valuable to establish the high-throughput method for glycoconjugates extraction from foods to facilitate the study on the structure in which the sialic acid is presented.This thesis was focused on the method establishment for quantitative study of sialic acid in diet and high-throughput extraction of sialylated conjunction.1.Establishment of UHPLC-based method for sialic acid detectionUHPLC testing condition:Sialic acid was derivatetized with flourescent reagent o-phenylenediamine and separated on UHPLC equipped with C18 reverse column(250 ×4.60 mmol/L,5 ?m)and fluorescence detector.Gradient elution was carried out using water/acetonitrile/methanol for 22 min with flow rate of 1.0 mL/min.Sample was detected at the excitation wavelength 373 nm,emission wavelength 448 nm.The retention time of Neu5Ac and Neu5Gc on UHPLC were 8.7 min and 9.8 min,respectively.Treatment of sample prior to UHPLC detection:Based on the hydrolysis efficiency of sialic acid released from pork sample treated with HCl and HAc,the optimal hydrolysis condition was determined as follows:2 M HAc and heating at 80? for 3 hours2.Qualitative detection of sialic acid in the dietRed meat(pork,beef,lamb and etc.),white meat(poultry,fish and etc.),poultry egg(hen egg,duck egg and etc.)and lower invertebrate animal meat(shrimp,shellfish,squid and etc.)were hydrolyzed and analyzed using the methods developed above.The result was showen that the red meat contained both Neu5Ac and Neu5Gc,white meat and poultry egg only contained Neu5A without Neu5Gc,and lower invertebrate was lack of both.3.Quantitative analysis of sialic acid in the dietNeu5Prop was investigated as internal standard to quantify sialic acid in diet from the viewpoint of hydrolysis efficiency and retention time.Red meat(contains both Neu5A&Neu5Gc)and poultry egg(only conatins Neu5Ac)were selected for the quantification of sialic acid and the result showed that quantitative method of sialic acid with Neu5Prop as internal standard was reliable.4.High-throughput extraction method for sialylated conjunctionSialic acid was normally attached to the terminal position of the glycoconjugation to function in biological process.Analysis of the structure of sialylated conjunction is one of the important contents.Therefore,this chapter was aimed to develop a device using 3D printing technology for glycoconjugate extraction on high-throughput level,and to use this device for glycoconjugate extraction.The results showed that the platform can be used for separation and purification of sialylated conjunction in a high-throughput way.