Analysis of Low Molecular Weight Carbohydrates in Food and Beverages: A Review

This review presents a study of new advances in chromatographic methods and capillary electrophoresis for the analysis and quantification of carbohydrates in food and drink that have been made in essentially the last seven years (1995–2002). All the main groups of sugars have been considered: monosaccharides, disaccharides, trisaccharides, oligosaccharides, related compounds such as alditols, alditol glycosides, polyols, amino sugars, deoxy sugars, uronic acids and aldonic acids. The chromatographic methods referred to are High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) and related techniques (AMD-HPTLC: High Performance Thin-Layer Chromatography with Automated Multiple Development). Capillary Electrophoresis (CE) was also discussed.     

高效液相色谱-蒸发光散射法测定食品中的单糖、双糖、低聚果糖和糖醇

建立了食品中常用的木糖、果糖、葡萄糖、蔗糖、麦芽糖、乳糖、蔗果三糖、蔗果四糖、蔗果五糖、赤藓糖醇、木糖醇、甘露糖醇、麦芽糖醇等13种单糖、双糖、低聚果糖和糖醇的高效液相色谱同时分离检测的方法。该法采用NH₂色谱柱,以乙腈-水为流动相梯度洗脱,蒸发光散射检测器检测;13种糖在0.1~5 g/L内均具有良好的线性关系,检出限均在0.1 g/L以下,精密度(RSD)为2.69%~7.21%,回收率为96.1%~105.2%,结果较为理想。将该法用于实际样品检测,结果显示食品标签明示和实际成分相差较大。     

High-performance liquid chromatographic separation of carbohydrates on graphitized carbon columns

Graphitized carbon columns (GCC) for high-performance liquid chromatography are relatively new and have a unique ability to resolve isomeric and closely related compounds. The retention mechanism of carbohydrates on GCC is mainly based on adsorption and the flat surface of GCC packing brings about unique selectivity, but also includes hydrophobic interactions. The chromatographic behaviour of monosaccharides, disaccharides, cyclodextrins (CDs), branched CDs, oligosaccharide alditols, chito-oligosaccharides, N-linked oligosaccharides and glycopeptides has been studied, and it has become apparent that the elution patterns are based on the size and planarity of the molecule (position and configuration of linkage).     

HPAE-PAD – a sensitive method for the determination of carbohydrates

High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was used for the determination of eleven monosaccharides. Three analytical columns with different selectivities were tested, and the resulting separations were compared calculating precision and detection limits. The monosaccharides could be separated on CarboPAC PA10 in one analysis run with the lowest detection limits and a high precision. For the determination of polysaccharides and humic bound carbohydrates in natural organic matter, an hydrolysis step had to be carried out. With the exception of fructose, the recoveries varied between 56% and 83%. The described methods were applied for the determination of bound carbohydrates in a bog lake water and a soil extract without preconcentrating the samples.     

Identification of the composition of oligosaccharides and polysaccharides by pyrlysis/capillary gas chromatography

Pyrolysis/capillary gas chromatography is used for the characterization of the monomer composition of various oligosaccharides and polysaccharides including glucose-containing disaccharides,glucans, a galactomannan and an arabinogalactan. The chromatograms showed many common pyrolysis products, but also unique anhydrosugar products (e.g., 1,6-anhydroglucopyranose, 1,4-anhydroarabinopyranose, 2,6-anhydrofructofuranose) derived from each type of saccharide unit present in the samples. Reasonable values of the monomer composition of the polysaccharide can also be obtained from th pyrograms. The method is rapid and direct, requiring no sample preparation.     

Oligosaccharide sequencing by means of mass spectrometry—I: N-phenylosotriazole acetates

Fragmentation of the acetates of the N-phenylosotriazoles of some mono-, di, tri-, tetra- and pentasaccharides under electron-impact (EI) has been studied. The mass spectra of these derivatives can be used for determination of the sequence of monosaccharide units in oligosaccharides (oligosaccharide sequencing), of molecular weight, of the location of the position of the interglycosidic bond in disaccharides and of the position of the methyl group in partially methylated monosaccharides.     

Rapid quantitative and qualitative confirmatory method for the determination of monofluoroacetic acid in foods by liquid chromatography-mass spectrometry

Many environmental samples contain complex mixtures of organic compounds with different sources, polarities and reactivities. This study reports a method for the analysis of both polar/water-soluble and apolar organic compounds in several kinds of environmental samples. The analytical method consists of extraction with a mixture of dichloromethane:methanol (2:1, v/v), silylation using BSTFA (N,O-bis-(trimethylsilyl)trifluoroacetamide) and analysis by gas chromatography-mass spectrometry (GC-MS), a common device in chemical and environmental laboratories. Fifty individual sugar standards, including monosaccharides, sugar alcohols, anhydrosugars, disaccharides and trisaccharides, were analyzed for the determination of their fragmentation patterns and retention times. Recoveries (at three concentrations) and limits of detection (LOD) were determined for a standard mixture containing glucose (monosaccharide), sorbitol (sugar alcohol), levoglucosan (anhydrosugar) and sucrose (disaccharide), and they varied from 68 to 119% and 130 to 360 ng mL(-1), respectively. The method was used for the analysis of aerosol particle, soil and sediment samples, and demonstrated its feasibility in detecting not only several important environmental sugars (e.g., glucose, fructose, inositol, mannitol, sorbitol, levoglucosan, sucrose, mycose), but also a large range of organic compound classes from other polar components (e.g., dicarboxylic acids) to apolar compounds such as n-alkanes. Therefore, the analytical method presented here demonstrated its usefulness for a better understanding of sources and transport of various organic compounds in different environmental compartments.     

Capillary electrophoresis of complex natural polysaccharides

Complex natural polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules that exhibit a wide range of biological functions and participate and regulate multiple cellular events and (patho)physiological processes. They are generally present either as free chains (hyaluronic acid and bacterial acidic polysaccharides) or as side chains of proteoglycans (PGs; chondroitin/dermatan sulfate, heparin/heparan sulfate, and keratan sulfate) and are most often found in cell membranes and in the extracellular matrix. The recent emergence of modern analytical tools for their study has produced a virtual explosion in the field of glycomics. CE, due to its high resolving power and sensitivity, has been useful in the analysis of intact GAGs and GAG-derived oligosaccharides and disaccharides affording concentration and structural characterization data essential for understanding the biological functions of GAGs. In this review, novel off-line and on-line CE-MS and MS/MS methods for screening of GAG-derived oligosaccharides and disaccharides will be discussed.     

糖类的毛细管电泳及芯片毛细管电泳

 糖类化合物在生物体内发挥多方面的作用。糖研究的复杂性在于其结构的复杂多变。高效毛细管电泳作为一种快速、高效的分离分析手段已广泛应用于糖的研究。芯片毛细管电泳是近几年来发展起来的新的分析技术 ,并已经在生命科学的研究中得到较广泛的应用。就各种糖类化合物的毛细管电泳的分析策略、检测条件及糖类化合物的芯片毛细管电泳进行了阐述 ,共 4 8篇。     

Quantification of Saccharides in Honey Samples Through Surface-Assisted Laser Desorption/Ionization Mass Spectrometry Using HgTe Nanostructures

Quantification of monosaccharides and disaccharides in five honey samples through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using HgTe nanostructures as the matrix and sucralose as an internal standard has been demonstrated. Under optimal conditions (1× HgTe nanostructure, 0.2 mM ammonium citrate at pH 9.0), the SALDI-MS approach allows detection of fructose and maltose at the concentrations down to 15 and 10 μM, respectively. Without conducting tedious sample pretreatment and separation, the SALDI-MS approach allows determination of the contents of monosaccharides and disaccharides in honey samples within 30 min, with reproducibility (relative standard deviation <15%). Unlike only sodium adducts of standard saccharides detected, sodium adducts and potassium adducts with differential amounts have been found among various samples, showing different amounts of sodium and potassium ions in the honey samples. The SALDI-MS data reveal that the contents of monosaccharides and disaccharides in various honey samples are dependent on their nectar sources. In addition to the abundant amounts of monosaccharides and disaccharides, oligosaccharides in m/z range of 650???2700 are only detected in pomelo honey. Having advantages of simplicity, rapidity, and reproducibility, this SALDI-MS holds great potential for the analysis of honey samples.

Thin-layer electrophoretic separation of monosaccharides, oligosaccharides and related compounds on reverse phase silica gel

The electrophoretic mobilities of monosaccharides, oligosaccharides, sugar alcohols and sugar acids were determined in 0.3 M borate buffer, pH 10, using thin-layer electrophoresis on silanized silica gel, pretreated with octanol-1. A rapid separation of a number of sugars, occurring in foods, could be achieved. Using a 0.05-0.1 M neutral solution of barium acetate as electrolyte, thin-layer electrophoresis allowed excellent and rapid separation as well as identification of all common uronic acids which are constituents of many acidic polysaccharides.     

加压毛细管电色谱法高效分离分析葛根多糖中的单糖

葛根多糖具有抗氧化、抗肿瘤等众多生物活性,对葛根多糖进行单糖组成分析对其活性研究具有重要意义。该研究利用响应面分析法考察了超声辅助提取法中液料比、超声温度、超声时间和超声功率对葛根多糖提取率的交互影响,并拟合数据得到多元二次回归方程。同时建立了柱前衍生加压毛细管电色谱检测糖类的方法,对分离8种中性单糖的色谱条件进行了探索与优化,并将此方法应用于两种葛根实际样品的单糖组成测定。响应面分析结果表明,4个试验因素中,超声温度对两种葛根多糖提取率的影响程度最大,其次为液料比,超声时间和超声功率影响程度较小。结合软件预测分析得到的最佳条件及设备实际情况,确定葛根多糖的最佳提取工艺条件为：超声温度90℃,粉葛多糖液料比20 mL/g,柴葛多糖液料比40 mL/g,超声时间30 min,超声功率180 W。优化后的色谱分离条件为：采用Halo-2.7 μm核壳型C18填料毛细管色谱柱,以乙腈-50 mmol/L pH 4.1的醋酸铵水溶液（18：82,v/v）为流动相,在250 nm波长下检测,施加电压-20 kV。在此条件下可以实现24 min内对葡萄糖等8种中性单糖衍生物的快速分离,相比传统液相色谱方法大大提升了分离检测速度和分离柱效。方法学考察表明此方法具有较好的线性关系和良好的重复性。对实际样品分离鉴定表明,粉葛多糖主要由葡萄糖、甘露糖、鼠李糖和岩藻糖组成,4种单糖物质的量之比为1.00：0.16：0.14：0.07;柴葛多糖主要由葡萄糖和甘露糖组成,2种单糖物质的量之比为1.00：0.70。该研究为单糖化合物快速高效分离检测提供了新方法,并为葛根多糖单糖组成分析提供了参考。     

Analysis of microbial extracellular polysaccharides in biofilms by HPLC. Part I: development of the analytical method using two complementary stationary phases

The investigation of microbial extracellular polymeric substances (EPS) is helpful for the implementation of analytical methods which are suitable for biofilm analysis in order to understand the architecture and function of biofilms. A procedure for the qualitative and quantitative determination of various monosaccharides, oligosaccharides and uronic acids as important components of the carbohydrate fraction of microbial EPS by high-performance liquid chromatography (HPLC) and refractive index (RI)/UV detection is presented. Porous graphitic carbon and lead-form cation-exchanger have been examined as stationary phases. Therefore, two complementary HPLC methods are presented. To simulate the conditions of hydrolysis, the influences of various salts, acids and alkalis as matrix components have been investigated. Furthermore, the dependencies on the pH value and temperature of the mobile phase have been thoroughly studied. The results showed that the lead-form cation-exchanger is suitable for the separation of the neutral monosaccharides. However, for direct analysis after acidic hydrolysis with H₂SO₄, HCl or trifluoroacetic acid, an additional purification step, e.g., precipitation or lyophilization, is necessary when the cation-exchanger is used. With the exception of hydrolysis with HCl, the porous graphitic carbon stationary phase can be used without any further purification step and is appropriate for the separation of uronic acids and their γ-lactones. Additionally, the separation of a single monosaccharide and its derivatives is possible. Analytical parameters including the sensitivities, repeatabilities, limits of detection and limits of quantification of both HPLC methods using the RI detector are presented. The optimized method has been applied for the characterization of alginates and is also suitable for other extracellular polysaccharides in biofilms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Expedient Synthesis of Core Disaccharide Building Blocks from Natural Polysaccharides for Heparan Sulfate Oligosaccharide Assembly

The complex sulfation motifs of heparan sulfate glycosaminoglycans (HS GAGs) play critical roles in many important biological processes. However, an understanding of their specific functions has been hampered by an inability to synthesize large numbers of diverse, yet defined, HS structures. Herein, we describe a new approach to access the four core disaccharides required for HS/heparin oligosaccharide assembly from natural polysaccharides. The use of disaccharides rather than monosaccharides as minimal precursors greatly accelerates the synthesis of HS GAGs, providing key disaccharide and tetrasaccharide intermediates in about half the number of steps compared to traditional strategies. Rapid access to such versatile intermediates will enable the generation of comprehensive libraries of sulfated oligosaccharides for unlocking the “sulfation code” and understanding the roles of specific GAG structures in physiology and disease.     

亲水作用色谱在糖类化合物分析中的应用

糖类化合物的结构和组成分析对于探究糖的结构与功能的关系具有重要作用。亲水作用色谱(HILIC)对糖类等极性化合物具有良好的分离效果。该文介绍了适合糖类分析的常用HILIC柱固定相及其分离机制,论述了强洗脱溶剂比例、流动相p H值、缓冲盐浓度和色谱柱温度对HILIC分离效果的影响,并举例说明了HILIC法在单糖组成、糖胺聚糖二糖组成、寡糖聚合度、糖苷、糖脂、糖醇以及N-/O-糖链分析中的应用。     

2,3-Anhydro sugars in glycoside bond synthesis. Highly stereoselective syntheses of oligosaccharides containing alpha- and beta-arabinofuranosyl linkages

The ever-increasing discovery of biologically important events mediated by carbohydrates has generated great interest in the synthesis of oligosaccharides and the development of new methods for glycosidic bond formation. In this paper, we report that 2,3-anhydrofuranose thioglycosides (1, 5) and glycosyl sulfoxides (2, 6), in which the hydroxyl groups C-2 and C-3 are "protected" as an epoxide, glycosylate alcohols with an exceptionally high degree of stereocontrol. The predominant or exclusive product of reactions with this fundamentally new class of glycosylating agent is that in which the newly formed glycosidic bond is cis to the epoxide moiety. We further demonstrate that subsequent nucleophilic opening of the epoxide moiety proceeds under basic conditions to give products in high yield and with good to excellent regioselectivity. The major ring-opened products possess the arabino stereochemistry, and thus this methodology constitutes a new approach for the synthesis of arabinofuranosides. In the epoxide opening reactions of glycosides with the 2,3-anhydro-beta-D-lyxo stereochemistry (e.g., 73), the addition of (-)-sparteine (78) to the reaction mixture dramatically enhanced the regioselectivity in favor of the arabino product. This represents the first example of the use of 78 to influence the regioselectivity of an epoxide ring opening reaction with a non-carbon nucleophile. We have demonstrated the utility of this methodology through the efficient synthesis of an arabinofuranosyl hexasaccharide, 7, which is a key structural motif in two mycobacterial cell wall polysaccharides.     

CarbBuilder: Software for building molecular models of complex oligo‐ and polysaccharide structures

CarbBuilder is a portable software tool for producing three‐dimensional molecular models of carbohydrates from the simple text specification of a primary structure. CarbBuilder can generate a wide variety of carbohydrate structures, ranging from monosaccharides to large, branched polysaccharides. Version 2.0 of the software, described in this article, supports monosaccharides of both mammalian and bacterial origin and a range of substituents for derivatization of individual sugar residues. This improved version has a sophisticated building algorithm to explore the range of possible conformations for a specified carbohydrate molecule. Illustrative examples of models of complex polysaccharides produced by CarbBuilder demonstrate the capabilities of the software. CarbBuilder is freely available under the Artistic License 2.0 from https://people.cs.uct.ac.za/~mkuttel/Downloads.html . © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.     

Development of a carbohydrate silylation method in ionic liquids for their gas chromatographic analysis

This paper reports on the feasibility of silylation of low molecular weight carbohydrates dissolved in different ionic liquids (ILs) for their further analysis by gas chromatography (GC). Derivatization reagents (nature and amounts), temperature and time of reaction and stirring conditions were evaluated for different carbohydrates (i.e., glucose, mannose, fructose and lactose) dissolved in 1-ethyl-3-methylimidazolium dicyanamide [EMIM][DCA]. Evaluation of conformational isomerism of glucose dissolved in [EMIM][DCA] revealed the effect of the time of dissolution in the equilibration of α- and β-furanoses (up to 3% and 6%, respectively, after 70 h of incubation) and that 21 h sufficed to obtain results similar to those provided by the reference method involving pyridine. Once optimized, the proposed derivatization procedure provided satisfactory yields (i.e., close to 100%) using 100 μL of trimethylsilylimidazole (TMSI) at mild conditions (25 °C) for a relatively short time (1 h) for most of the investigated carbohydrates. Under these experimental conditions, linear responses (i.e., R² better than 0.974) were obtained in the tested range of 0.25–1 mg of the derivatized target compounds. Other reagents, such as N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS), were successfully used under ultrasonic conditions for aldose monosaccharides and disaccharides derivatization, while BSTFA was useful for ketose monosaccharides. The possibility of using the proposed method for the derivatization of selected carbohydrates dissolved in different ILs and the efficiency of the method applied to the analysis of carbohydrates present in real samples (fruit juices) have also been investigated.     

Carbohydrates as the next frontier in pharmaceutical research

Synthetic carbohydrates and glycoconjugates are used to study their roles in biological important processes such as inflammation, cell-cell recognition, immunological response, metastasis, and fertilization. The development of an automated oligosaccharide synthesizer greatly accelerates the assembly of complex, naturally occurring carbohydrates as well as chemically modified oligosaccharide structures and promises to have major impact on the field of glycobiology. Tools such as microarrays, surface plasmon resonance spectroscopy, and fluorescent carbohydrate conjugates to map interactions of carbohydrates in biological systems are presented. Case studies of the successful application of carbohydrates as active agents are discussed, for example, fully synthetic oligosaccharide vaccines to combat tropical diseases (e.g., malaria), bacterial infections (e.g., tuberculosis), viral infections such as HIV, and cancer. Aminoglycosides serve as examples of drugs acting through carbohydrate-nucleic-acid interactions, while heparin works by carbohydrate-protein interactions. A general, modular strategy for the complete stereoselective synthesis of defined heparin oligosaccharides is presented. A carbohydrate-functionalized fluorescent polymer has been shown to detect miniscule amounts of bacteria faster than commonly used methods.     

超高效合相色谱-蒸发光散射检测法测定蜂蜜中4种单、双糖的含量

采用超高效合相色谱-蒸发光散射检测( UPC2-ELSD)技术,建立了测定蜂蜜中4种单、双糖含量的方法。其中单糖为果糖和葡萄糖,双糖为蔗糖和麦芽糖。以ACQUITY UPC2BEH(100 mm×3.0 mm,1.7μm)为色谱柱,超临界CO2和甲醇(含0.2%氨水, V/V)作为流动相,流速为1.0 mL/min,柱温为50℃,ELS为检测器,漂移管温度为50℃,N2压力为260.7 kPa(30 psi)。研究表明,4种单、双糖化合物在线性范围内线性关系良好,线性相关系数(R)均大于0.9983;目标物的检出限(S/N≥3)为2.0~4.7 mg/L;3个加标水平下的回收率为86.6%~103.8%,相对标准偏差为1.8%~4.3%。此方法简单、快速、分离效果好、分析成本低,是一种测定蜂蜜样品中单、双糖的优良方法,且检测结果与国家标准方法保持一致。