Quantitative microanalysis of oligosaccharides by high-performance liquid chromatography
ABSTRACT The rapid separation and quantitative determination of per-O-benzoyl oligosaccharides were obtained using high performance, liquid chromatography. Oligosaccharides were completely O-benzoylated without concomitant N-benzoylation of acetamidodeoxyhexoses. Benzoylation prior to analysis allowed a quantitative determination of picomolar amounts because the absorbance at 230 nm of these derivatives is directly proportional to the number of benzoyl groups present. Separation by normal and reversed-phase chromatography was demonstrated, and the best resolution was obtained on an Ultrasphere octyl column. Excellent separations of oligosaccharides containing up to 10 sugar residues present in mannosidosis urine and of malto-oligosaccharides containing up to 15 sugar residues present in Karo syrup were achieved within an analysis time of 30 min. Anomers of maltose, maltotriose, and maltotetraose were separated; for this reason, reduction of complex samples prior to analysis is advisable. The effect of linkage configuration on retention time was tested with reduced, α-linked di- and tri-glucopyranosides. The presence of an acetamidodeoxyhexose residue in an oligosaccharide significantly reduced its retention time, whereas branching had the opposite effect. A linear response was obtained for the injection of 1–600 pmol of raffinose, and the detection limit was 0.5 pmol. Derivatization and analysis of raffinose was shown to yield reproducible results within the range 0.01–1 μmol, and, with special precautions to minimize losses, as little as 100 pmol could be analyzed successfully.
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ABSTRACT: Although fungi have always been with us as commensals and pathogens, fungal infections have been increasing in frequency over the past few decades. There is a growing body of literature describing the involvement of carbohydrate groups in various aspects of fungal disease. Carbohydrates comprising the cell wall or capsule, or as a component of glycoproteins, are the fungal cell surface entities most likely to be exposed to the surrounding environment. Thus, the fungus-host interaction is likely to involve carbohydrates before DNA, RNA, or even protein. The interaction between fungal and host cells is also complex, and early studies using whole cells or crude cell fractions often produced seemingly conflicting results. What was needed, and what has been developing, is the ability to identify specific glycan structures and determine how they interact with immune system components. Carbohydrate analysis is complicated by the complexity of glycan structures and by the challenges of separating and detecting carbohydrates experimentally. Advances in carbohydrate chemistry have enabled us to move from the foundation of composition analysis to more rapid characterization of specific structures. This, in turn, will lead to a greater understanding of how fungi coexist with their hosts as commensals or exist in conflict as pathogens.Clinical Microbiology Reviews 05/2004; 17(2):281-310. · 17.31 Impact Factor
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ABSTRACT: The stem bark extract of the medicinal plant Sarcocephalus latifolius was analysed for its qualitative and quantitative carbohydrate content. Preparative high-performance liquid chromatography (HPLC) of the benzoylated sugar fraction, led to the isolation of d-fructose derivatives 1,2,3,4,6-penta-O-benzoyl-α-d-fructofuranose, 1,2,3,4,6-penta-O-benzoyl-β-d-fructofuranose, 1,2,3,4,5-penta-O-benzoyl-β-d-fructopyranose, and 1,3,4,5-tetra-O-benzoyl-β-d-fructopyranose, in addition to α- and β-d-pyranose forms of glucose, xylose, and arabinose perbenzoates, glycerol and d-erythriol perbenzoates, and an inseparable mixture of methyl 1,3,4,6-tetra-O-benzoyl-α,β-d-fructofuranoside, whose occurrence has never been previously reported from a natural source. The corresponding structures and conformations were fully characterised by extensive one and two-dimensional nuclear magnetic resonance (NMR) experiments. Quantification of the natural free sugars in the extract was achieved by HPLC using refractive-index detection.Carbohydrate Polymers - CARBOHYD POLYM. 01/2001; 45(2):155-160.
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ABSTRACT: This review gives an analysis of methods of high-performance liquid chromatography for the separation of various polysaccharides.Chemistry of Natural Compounds 01/1999; 35(1):1-13. · 0.60 Impact Factor