| Oligosaccharides play significant roles in many life processes,such as immunity,canceration,aging and so on.Therefore,it is vitally important for the analysis of oligosaccharides in complex matrices.However,the detection of oligosaccharides can be challenging,due to their intrinsic properties,such as complex structures,lack of chromophores and/or fluorophores and high hydrophilicities.Accordingly,derivatizations are often required to improve their chromatographic separation and instrumental response.In this thesis,a fluorescence derivatization reagent,4-aminomethyl-7-hydroxy-coumarin(AOHC),was used to derivatize oligosaccharides through their reducing ends.After the optimization of various reaction parameters,a highly efficient derivatization method-reductive oxyamination was developed and applied for the sensitive and selective analysis of oligosaccharides.The main results are as follows:Firstly,we developed a reductive oxyamination method for the analysis of N-glycans,using AOHC as the derivatization regent.By using single factor optimization and response surface methodology,the AOHC derivatization conditions were investigated and optimized.Compared with the traditional 2-AB labeling method,our method has the advantages of short reaction time,high sensitivity and ease of operation.Using this method,we successfully detected all the N-glycans in a glycoprotein RNase B.Secondly,we further improved the labeling efficiency using the vacuum centrifuging method instead of the conventional solution reaction.The reaction time was shortened to 30 min and the reaction temperature was lowered to 35℃.Finally,the method was applied for the determination of oligosaccharides in coffee samples.6oligosaccharides were successfully measured with concentrations ranging from11.60-24.16 μg/g.Thirdly,the organic catalysts for the labeling reaction were investigated.Seven catalysts,including p-phenylenediamine,2-amino-5-methoxybenzoic acid,2-amino-5-methylbenzoic acid,3,5-diaminobenzoic acid,aniline,tetrahydropyrrole and triethylamine were investigated for the reaction of AOHC and maltoheptaose.The results show that 2-amino-5-methoxybenzoic acid and p-phenylenediamine have good catalytic effects under mild reaction conditions.The two catalysts were used for the determination of oligosaccharides in infant milk powder samples and 9oligosaccharides were successfully detected. |
