Analysis of carbohydrates in plants by high-performance anion-exchange chromatography coupled with electrospray mass spectrometry

A mass spectrometer was coupled to high-performance anion-exchange chromatography (HPAEC) with the help of electrochemical neutralization of the eluent and post-column addition of lithium chloride for carbohydrate analysis. Parallel selective channels (single ion monitoring) were used to decrease the detection limits and separate unresolved peaks. The mass specific detection allowed the simultaneous analysis of a wide range of sugar alcohols, mono-, di- and oligosaccharides. Carbohydrates extracted from leaves of poplar submitted to drought stress were analyzed using pulsed amperometric detection (PAD), then mass spectrometry. It allowed the confirmation of peak attribution and the identification of salicin as a major compound in the extracts. Different responses to water deficit and re-hydration were obtained for several carbohydrates, suggesting different roles in osmoprotection processes.     

Hydrophilic interaction chromatography/electrospray mass spectrometry analysis of carbohydrate-related metabolites from Arabidopsis thaliana leaf tissue

This work describes the development and application of an on-line liquid chromatography/mass spectrometry (LC/MS) method using hydrophilic interaction chromatography (HILIC) coupled to negative ion mode electrospray ionisation ion trap mass spectrometry (ESI-MS) for the analysis of highly polar carbohydrate-related metabolites commonly found in plants, ranging from reducing and non-reducing sugars and sugar alcohols to sugar phosphates. Using this method, separation and detection of a mixture of eight authentic standard compounds containing glucose (Glc), sucrose (Suc), raffinose, verbascose, mannitol, maltitol, glucose-6-phosphate (Glc6P) and trehalose-6-phosphate (Tre6P) were achieved in less than 15 min. The method is rapid, robust, selective, and sensitive, with limits of detection (LODs) ranging from 0.2 microM obtained for neutral sugars, to 1.0 microM obtained for sugar alcohols, and 2.0 microM obtained for negatively charged sugar phosphates. We have studied the negative ion collision-induced dissociation (CID) fragmentation behaviour of the non-reducing raffinose family oligosaccharides (RFOs) raffinose, stachyose, and verbascose. Mainly Bi and Ci glycosidic and Ai cross-ring structurally informative cleavages are observed. We have applied this HILIC/ESI-MS method for the analysis of Arabidopsis thaliana wild-type Columbia-0 (Col-0) and its starchless phosphoglucomutase mutant (pgm1) leaf extracts. The method was used to quantify Glc, Suc, raffinose, and Glc6P in A. thaliana extracts. Data obtained using this HILIC/ESI-MS method were compared with those obtained using a comparable porous graphitic carbon-based LC/ESI-MS method.     

Corona Discharge Ion Mobility Spectrometry of Ten Alcohols

Ion mobility spectra for ten alcohols have been studied in an ion mobility spectrometry apparatus equipped with a corona discharge ionization source. Using protonated water cluster ions as the reactant ions and clean air as the drift gas, the alcohols exhibit different product ion characteristic peaks in their ion mobility spectra. The detection limit for these alcohols is at low concentration pmol/L level according to the concentration calibration by exponential dilution method. Based on the measured ion mobilities, several chemical physics parameters of the ion-molecular interaction at atmosphere were obtained, including the ionic collision cross sections, diffusion coefficients, collision rate constants, and the ionic radii under the hard-sphere model approximation.     

Determination of sugar alcohols in confectioneries by high-performance liquid chromatography after nitrobenzoylation

A method was developed for the determination of sugar alcohols, meso-erythritol, xylitol, D-glucitol, D-mannitol, maltitol and parachinit by high-performance liquid chromatography (HPLC). The sugar alcohols were converted into strong ultraviolet (UV)-absorbing derivatives with p-nitrobenzoyl chloride. HPLC was performed on a phenyl column, using acetonitrile-water (67:33) as mobile phase and UV detection (260 nm). The calibration curves for all sugar alcohols tested were linear in the 10-250 microg/ml range. The average recoveries of the sugar alcohols from four sugarless confectioneries spiked at 5 and 10% levels of six sugar alcohol standards ranged from 73.2 to 109.0% with relative standard deviations ranging from 0.7 to 9.0%. The detection limit of the developed method was 0.1% for the above sugar alcohols contained in the samples.     

Determination of "net carbohydrates" using high-performance anion exchange chromatography

For labeling purposes, the carbohydrate content of foods has traditionally been determined by difference. This value includes sugars, starches, fiber, dextrins, sugar alcohols, polydextrose, and various other organic compounds. In some cases, the current method may lack sufficient specificity, precision, and accuracy. These are subsequently quantitated by high performance anion exchange chromatography with pulsed amperometric detection and expressed as total nonfiber saccharides or percent "net carbohydrates." In this research, a new method was developed to address this need. The method consists of enzyme digestions to convert starches, dextrins, sugars, and polysaccharides to their respective monosaccharide components. These are subsequently quantified by high-performance anion exchange chromatography with pulsed amperometric detector and expressed as total nonfiber saccharides or percent "net carbohydrates." Hydrolyzed end products of various novel fibers and similar carbohydrates have been evaluated to ensure that they do not register as false positives in the new test method. The data generated using the "net carbohydrate" method were, in many cases, significantly different than the values produced using the traditional methodology. The recoveries obtained in a fortified drink matrix ranged from 94.9 to 105%. The coefficient of variation was 3.3%.     

Characterization of low-molecular-weight metal species in plant extracts by using HPLC with pulsed amperometric detection and cyclic voltammetry

An analytical separation scheme is presented for the isolation of low-molecular-weight metal species (< 10 kDa) in plants. After ultrafiltration of the aqueous plant extracts isotachophoresis or gel chromatography is used for pre-separation and HPLC at a cyclodextrin-phase for further separation of metal containing fractions. Trace metals are detected off-line by using either adsorptive stripping voltammetry for platinum or AAS for zinc and magnesium. It is shown that platinum and zinc detection closely correlate with pulsed amperometric detection (PAD), while magnesium behaves differently. PAD is carried out after post-column addition of sodium hydroxide, thus enabling a sensitive and selective detection of carbohydrates. It is demonstrated that cyclic voltammetry is a useful tool for further characterization of the PAD-detectable metal species. By comparison of the cyclic voltammograms of carbohydrate standards, including carbohydrate-metal species, with the respective voltammograms obtained from HPLC fractions, carbohydrate species isolated from the samples can be considered as being either partly oxidized sugars (sugar alcohols or sugar acids) or glycosidically bound sugars.     

Characterization of low-molecular-weight metal species in plant extracts by using HPLC with pulsed amperometric detection and cyclic voltammetry

An analytical separation scheme is presented for the isolation of low-molecular-weight metal species (< 10 kDa) in plants. After ultrafiltration of the aqueous plant extracts isotachophoresis or gel chromatography is used for pre-separation and HPLC at a cyclodextrin-phase for further separation of metal containing fractions. Trace metals are detected off-line by using either adsorptive stripping voltammetry for platinum or AAS for zinc and magnesium. It is shown that platinum and zinc detection closely correlate with pulsed amperometric detection (PAD), while magnesium behaves differently. PAD is carried out after post-column addition of sodium hydroxide, thus enabling a sensitive and selective detection of carbohydrates. It is demonstrated that cyclic voltammetry is a useful tool for further characterization of the PAD-detectable metal species. By comparison of the cyclic voltammograms of carbohydrate standards, including carbohydrate-metal species, with the respective voltammograms obtained from HPLC fractions, carbohydrate species isolated from the samples can be considered as being either partly oxidized sugars (sugar alcohols or sugar acids) or glycosidically bound sugars. Received: 26 January 1998 / Revised: 19 June 1998 / Accepted: 24 June 1998     

Phase Transfer Promoted Ruthenium Oxide Oxidations of Carbohydrates II

ABSTRACT

The use of a phase transfer catalyst, benzyltriethylammonium chloride (BTEAC), is described in conjunction with the ruthenium dioxide/periodate : water/chloroform system for the oxidation of carbohydrate alcohols to the corresponding ketone, aldehyde, or carboxylic acid. The method was found to be applicable to carbohydrates appropriately protected as acetals, ethers, or containing a benzoyloxy group not positioned to readily undergo β-elimination. While the method was very suitable for the oxidation of carbohydrate secondary alcohols to ketones, it was found to be less suitable for the oxidation of a carbohydrate primary alcohol to the corresponding aldehyde or carboxylic acid. Evidence presented suggests that under the mildly basic conditions of the reaction, ruthenium tetraoxide is converted to ruthenate and perruthenate ions in the aqueous solution and then the perruthenate ion is carried by the phase transfer catalyst into the organic layer where oxidation of the substrate occurs. A number of examples illustrating the scope of the method are presented.     

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。     

Sample preparation and current applications of liquid chromatography for the determination of non-structural carbohydrates in plants

This article summarizes the current methods of determination of non-structural carbohydrates (NSCs) in plant samples based on liquid chromatography (LC). NSCs comprise several types of carbohydrates: sugar alcohols (e.g., sorbitol), monosaccharides (e.g., glucose and fructose), disaccharides (e.g., sucrose), oligosaccharides (e.g., raffinose) and polysaccharides [e.g., starch and polyfructans (e.g., inulin)]. NSCs are important in plant metabolism and have to be strictly distinguished from all sorts of structural carbohydrates (e.g., polysaccharide cellulose) that make up the backbone of the plants. Consequently, preservation of structural carbohydrates is a crucial step during sample preparation for NSC determination and is therefore addressed.Sugar alcohols, monosaccharides, disaccharides and those oligosaccharides that are easily soluble in polar solvents can be analyzed directly by high-performance LC. They are also referred to as free carbohydrates (FCs).However, polysaccharides are generally submitted to hydrolyzation into monomers prior to their quantitative analysis. This can be done either chemically, using acids, or enzymatically - both methods are discussed. For identification and quantification of the NSCs after LC separation, the following detectors are used: pulsed amperometry, refractive index, evaporate light scattering and finally, mass spectrometry.     

Enhancement of leaf gas exchange and primary metabolites under carbon dioxide enrichment up-regulates the production of secondary metabolites in Labisia pumila seedlings

A split plot 3 by 3 experiment was designed to investigate and distinguish the relationships among production of primary metabolites (soluble sugar and starch), secondary metabolites (total phenolics, TP; total flavonoids, TF) and leaf gas exchange of three varieties of the Malaysian medicinal herb Labisia pumila Blume, namely the varieties alata, pumila and lanceolata, under three levels of CO? enrichment (400, 800 and 1,200 μmol mol?1) for 15 weeks. The treatment effects were solely contributed by CO? enrichment levels; no varietal differences were observed. As CO? levels increased from 400 to 1,200 μmol mol?1, the production of carbohydrates also increased steadily, especially for starch more than soluble sugar (sucrose). TF and TP content, simultaneously, reached their peaks under 1,200 μmol exposure, followed by 800 and 400 μmol mol?1. Net photosynthesis (A) and quantum efficiency of photosystem II (f(v)/f(m)) were also enhanced as CO? increased from 400 to 1,200 μmol mol?1. Leaf gas exchange characteristics displayed a significant positive relationship with the production of secondary metabolites and carbohydrate contents. The increase in production of TP and TFs were manifested by high C/N ratio and low protein content in L. pumila seedlings, and accompanied by reduction in cholorophyll content that exhibited very significant negative relationships with total soluble sugar, starch and total non structural carbohydrate.     

Determination of sugar compounds in atmospheric aerosols by liquid chromatography combined with positive electrospray ionization mass spectrometry

We here report a method for the determination of sugar compounds of known presence in atmospheric aerosols using liquid chromatography (LC) combined with positive electrospray ionization mass spectrometry (MS). The target analytes include C(3)-C(6) monosaccharide alcohols (glycerol, erythritol, xylitol, mannitol), C(5)-C(6) monosaccharides (xylose, glucose, and levoglucosan), a disaccharide (sucrose), and a trisaccharide (melezitose). A mobile phase consisting of 20% 10 mM aqueous ammonium acetate, 8% methanol, and 72% water was found to provide abundant [M+NH(4)](+) adduct ions when coupled with electrospray ionization. Use of a polymer-based amino analytical column resolved the target compounds from the bulk solvent and provided limited separation among the target compounds. The target analytes were quantified using their [M+NH(4)](+) ions. Sample pretreatment was greatly simplified in comparison with the more commonly used gas chromatographic methods. It involved extraction of aerosol filters in methanol, evaporation of the solvent, and reconstitution with 5 mM ammonium acetate in water prior to the LC-MS analysis. The analyte recoveries were measured at the levels of 100, 500 and 1000 microg/L to be in the range of 78-102%, 94-112%, and 92-110%, respectively. The detection limits were lower than 10 pmol/injection for the tested target compounds except for xylose. Xylose had a detection limit of 95 pmol/injection. The method was applied to analyze 30 atmospheric aerosol samples to demonstrate its feasibility. The LC-MS method made possible the detection of trisaccharides as aerosol constituents for the first time.     

Qualitative and quantitative analysis of enantiomers by mass spectrometry: Application of a simple chiral chloride probe via rapid in-situ reaction

A tandem mass spectrometry method for high-sensitivity qualitative and quantitative discrimination of chiral amino compounds is conducted. The method is based on a chemical derivation process that uses a simple reagent, L-1-(phenylsulfonyl)pyrrolidine-carbonyl chloride, as the probe. The method is applicable in both organic solutions and biological conditions. Twenty-one pairs of enantiomer containing amino acids, amino alcohols, and amines are used to produce diastereomers using the probe via in situ reaction for 20 s at room temperature. The resulting diastereomers are successfully recognized based on the relative peak intensities of their fragments in positive mode, with the chiral recognition ability values ranging from 0.35 to 3.83. The L/D ratio of Pro spiked at different concentrations (enantiomeric excess) in both acetonitrile and dog plasma is determined by establishing calibration curves. This method achieves a lower limit of quantification of 50 pmol in analyzing amino acids using an extract ion chromatograph. The relative standard deviation for both qualitative and quantitative results is <5%. Thus, the present method is demonstrated as a new and practical technique of rapidly and sensitively determining enantiomers of amino compounds.     

Determination of post-culture processing with carbohydrates by MALDI-MS and TMS derivatization GC-MS

Biological materials generally require stabilization to retain activity or viability in a dry form. A number of industrial products, such as vaccines, probiotics and biopesticides have been produced as dry preparations. The same methods and materials used for stabilizing commercial microbial products may be applicable to preserving biothreat pathogens in a dry form. This is a likely step that may be encountered when looking at samples from terrorism attempts since only spores, such as those from Bacillus anthracis, are inherently stable when dried. The stabilizers for microbial preparations generally include one or more small carbohydrates. Different formulations have been reported for different industrial products and are often determined empirically. However sugar alcohols (mannitol and sorbitol) and disaccharides (lactose, sucrose and trehalose) are the common constituents of these formulations. We have developed an analytical method for sample preparation and detection of these simple carbohydrates using two complementary analytical tools, MALDI-MS and GC-MS. The native carbohydrates and other constituents of the formulation are detected by MALDI-MS as a screening tool. A longer and more detailed analysis is then used to specifically identify the carbohydrates by derivatization and GC-MS detection. Both techniques were tested against ten different types of stabilization recipes with Yersinia pestis cell mass cultured on different media types used as the biological component. A number of additional components were included in these formulations including proteins and peptides from serum or milk, polymers (e.g. poly vinyl pyrrolidone - PVP) and detergents (e.g. Tween). The combined method was characterized to determine several figures of merit. The accuracy of the method was 98% for MALDI-MS and 100% for GC-MS. The repeatability for detection of carbohydrates by MALDI-MS was determined to be 96%. The repeatability of compound identification by GC-MS was determined by monitoring variation in retention time, which is vital for identification of isomeric carbohydrates. The figures of merit illustrate an effective and accurate method for mono and disaccharide detection independent of formulation. This meets our primary goal for method development as small carbohydrates are among the most common stabilizers employed.     

Quantification of sugars and sugar phosphates in Arabidopsis thaliana tissues using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry

This work reports the development and optimisation of a negative ion mode on-line LC-ESI-MS/MS method for the sensitive targeted analysis of the key glycolytic intermediates, sugars and sugar phosphates from plants, using a porous graphitic carbon (PGC) stationary phase and an MS compatible mobile phase. Using this newly developed method, separation and detection of a solution of standard compounds is achieved in less than 20min. Target metabolite compounds were identified in plant extracts from their characteristic retention times, and product ion spectra. This on-line PGC-ESI-MS/MS method shows good linearity over the concentration range 0-100microM, selectivity, short analysis time, and limits of detection of 0.1microM for disaccharides trehalose (Tre), sucrose (Suc), and maltose, and 1.5microM for hexose phosphates fructose-6-phosphate (Fru6P), glucose-1-phosphate (Glc1P), and glucose-6-phosphate (Glc6P), and phosphoenolpyruvate (PEP). This paper describes details of our method and its application to the simultaneous quantitative analysis of soluble sugars and sugar phosphates from Arabidopsis thaliana tissues. We have demonstrated the utility of our method for the analysis of biological samples by applying it to the simultaneous quantitation of changes in soluble sugars and sugar phosphates in A. thaliana Columbia-0 (Col-0) and its starchless phosphoglucomutase (pgm) mutant over a 12-h light/12-h dark growth cycle.     

Electrochemical detection of sugar-related compounds using boron-doped diamond electrodes

Electrochemical detection of sugar-related compounds was conducted using a boron-doped diamond (BDD) electrode as a detector for flow-injection analysis (FIA). Sugar-related compounds oxidize at high applied potentials, for which the BDD electrode is suitable for electrochemical measurements. Conditions for an FIA system with a BDD detector were optimized, and the following detection limits were achieved for sugar-related compounds: monosaccharides, 25-100 pmol; sugar alcohols, 10 pmol; and oligosaccharides, 10 pmol. The detection limit for monosaccharide D-glucose (Glu) was 105 pmol (S/N = 3). A linear range was acquired from the detection limit to 50 nmol, and the relative standard deviation was 0.65% (20 nmol, n = 6). A high-performance liquid chromatography (HPLC) column was added to the system between the sample injector and the detector and detection limits to the picomole level were achieved, which is the same for the HPLC system and the FIA system. The electrochemical oxidation reaction of Glu was examined using cyclic voltammetry with the BDD detector. The reaction proved to be irreversible, and proceeded according to the following two-step mechanism: (1) application of a high potential (2.00 V vs. Ag/AgCl) to the electrode causes water to electrolyze on the electrode surface with the simultaneous generation of a hydroxyl radical on the surface, and (2) the hydroxyl radical indirectly oxidizes Glu. Thus, Glu can be detected by an increase in the oxidation current caused by reactions with hydroxy radicals.     

Dual ambient plasma source ionization mass spectrometry for the rapid detection of trace sterols in urban water

A direct analytical method based on dual ambient plasma ion source tandem mass spectrometry was used for the simultaneous determination of four sterols in the environment. This technology has very high sensitivity and the method detects the four sterols in methanol–water (1:3) solutions with limits of detection (LOD) and limits of quantification (LOQ) ranging from 1.2 ng/L to 6.9 ng/L and 7.6 ng/L to 10.0 ng/L, respectively. The method was also used to test water quality at three locations within the city and successfully detected all four sterols at very low concentrations. The dual plasma source tandem mass spectrometry technique is extremely simple, rapid, sensitive and highly efficient compared to other traditional methods, providing a useful screening tool for sterols in water.     

Versatile Functionalization of Carbohydrate Hydroxyl Groups through Their O-Cyanomethyl Ethers

O-Cyanomethyl ethers of carbohydrates are shown to be versatile intermediates for the preparation of sugar amines, carboxylic acids, amides, and amidine salts. This methodology for the functionalization of carbohydrates can thus provide a new array of analogs for the study of carbohydrate binding proteins. In addition, the resulting O-aminoethyl and O-carboxymethyl carbohydrates can be coupled to amino acids under standard conditions used in solid-phase peptide synthesis, providing a method for the construction of glycopeptides in which the carbohydrate moiety can be linked through any of its hydroxyl groups to the C- or the N-terminus of a given peptide.     

Boric acid as a mobile phase additive for high performance liquid chromatography separation of ribose, arabinose and ribulose

A new high performance liquid chromatographic (HPLC) method is described for the analysis of ribose, arabinose and ribulose mixtures obtained from (bio)chemical isomerization processes. These processes gain importance since the molecules can be used for the synthesis of antiviral therapeutics. The HPLC method uses boric acid as a mobile phase additive to enhance the separation on an Aminex HPX-87K column. By complexing with boric acid, the carbohydrates become negatively charged, thus elute faster from the column by means of ion exlusion and are separated because the complexation capacity with boric acid differs from one carbohydrate to another. Excellent separation between ribose, ribulose and arabinose was achieved with concentrations between 0.1 and 10 gL(-1) of discrete sugar.     

Electrochemistry and chromatographic detection of monosaccharides,disaccharides, and related compounds at an electrocatalytic chemically modified electrode

Many carbohydrate compounds undergo electro-oxidation at modest positive potentials at chemically modified electrodes (CMEs) made by adding cobalt phthalocyanine (CoPC) electrocatalyst to a conventional carbon paste mixture. The active carbohydrates which were oxidized at the CME at +0.4 to 0.5 V vs. Ag/AgCl under the very basic pH conditions required, included not only monosaccharides but also oligosaccharides, deoxy sugars, and even simple polyalcohols such as glycerol. When used for carbohydrate detection after liquid chromatography, the CoPC CME yielded detection limits in the 10–50 pmol range. Electrode stability and selectivity was such that no sample preparation other than dilution and particulate filtration was required for carbohydrate determinations in sample matrices including food products and physiological fluids.