Carbohydrate Labeling Reagent In High Performance Liquid Chromatography - Mass Spectrometry

The carbohydrate material is the richest kind of organic compound in nature reserves, and the most important living thing besides the protein and the nucleic acid. But the biology research of carbohydrate material fall behind by far, mainly because of the varieties of sugar type, the complication of structure, also many isomers and the microscopic nonuniformity. For familiar sacchrides and oligosaccharides, the merely diversities lie in the arrangement of -CHOH group, therefore it is a great challenge for conventional separation methods. Moreover, carbohydrates analysis by HPLC coupled with ultraviolet (UV) detectors is made invalid because of the absence of effective chromophores or fluorescence. There are two problems in analysis of carbohydrate compounds: difficulties of separation and lack of sensitive detectors. Derivatization is often employed in biochemistry processing to label the carbohydrate component with color groups. By partially changing structure of sugars the high performance liquid chromatographic separation become possible and ultraviolet or the fluorescence examination can be used following the separation. At present the numerous labeling reagents had been employed in the carbohydrate compound analysis, but there are various shortcomings in the practical application. Therefore development of highly sensitive labeling reagent for saccharide analysis is extremely important. This article has put emphasis on the synthesis of two labeling reagents for the determination of carbohydrates, spectrum research, optimization of the derivation process, and the chromatographic separation. Novel methods for saccharide analysis using new labeling reagents with high performance liquid chromatography-mass spectrum (HPLC-MS) have been established, and also have been applied to analyze the actual sample.Chapter one: The present situations of carbohydrate compound analysis are summarized, including difficulties in analysis work resulting from unique structure features of the carbohydrate, modern separation technologies employed in the carbohydrate analysis, the detecting methods of carbohydrate compound, the commonly used labeling reagent, and mass spectrum analysis for carbohydrate compound.Chapter two: Two kind of label reagents: l-(2-naphthyl)-3-methyl-5-pyrazolone (NMP), l,2-benzocarbazole-9-ethoxy carbonyl hydrazine (BCECH) are designed and synthesized, and spectrum research are studied. By comparison with traditional reagent, possibilities of enhancement on separation and sensitivities are discussed.Chapter three: a novel method for saccharide analysis using NMP as labeling reagent with high performance liquid chromatography-mass spectrum (HPLC-MS) has been established, and the monosaccharide composition of pollen polysaccharide sample is determined with this method. By the contrast research with the traditional reagent, regularities of pyrazolones in HPLC-ESI-MS analysis are summarized: a proposed derivation mechanism according to the mass-spectrometric data; rules of chromatographic order and the corresponding explanation; the decomposition pattern of electrospray mass spectrum and attempts of explanation; the systematic comparison parozolones reagents.Chapter four: Studies of derivatization in the presence of the acidic catalyst for carbohydrate compound analysis are presented. The reductive amination reaction with 1-amino anthracene in HPLC-MS is discussed. Meanwhile identification by mass spectrum and separation are also concerned. With l,2-benzocarbazole-9-ethoxy carbonyl hydrazine as labeling reagent, derivation conditions for the familiar saccharides are optimized, and separation of 7 saccharides is achieved(including 5 monosaccharides, 1 disaccharide and 1 trisaccharide). At the same time, identification with mass spectrum is also investigated. The potential for further separation is discussed and strategies for resolving difficulties are proposed.