Position Of Glycosidic Linkages Research Articles

Development of a cyclic ion mobility spectrometry-mass spectrometry-based collision cross-section database of permethylated human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are an important class of biomolecules responsible for the healthy development of the brain-gut axis of infants. Unfortunately, their accurate characterization is largely precluded due to a variety of reasons - there are over 200 possible HMO structures whereas only 10s of these are available as authentic analytical standards. Furthermore, their isomeric heterogeneity stemming from their many possible glycosidic linkage positions and corresponding α/β anomericities further complicates their analyses. While liquid chromatography coupled to tandem mass spectrometry remains the gold standard for HMO analyses, it often times cannot resolve all possible isomeric species and thus warrants the development of other orthogonal approaches. High-resolution ion mobility spectrometry coupled to mass spectrometry has emerged as a rapid alternative to condensed-phase separations but largely has remained limited to qualitative information related to the resolution of isomers. In this work, we have assessed the use of permethylation to improve both the resolution and sensitivity of HMO analyses with cyclic ion mobility separations coupled with mass spectrometry. In addition to this, we have developed the first-ever high-resolution collision cross-section database for permethylated HMOs using our previously established calibration protocol. We envision that this internal reference database generated from high-resolution cyclic ion mobility spectrometry-mass spectrometry will greatly aid in the accurate characterization of HMOs and provide a valuable, orthogonal, approach to existing liquid chromatography-tandem mass spectrometry-based methods.

A Multidimensional Mass Spectrometry-Based Workflow for De Novo Structural Elucidation of Oligosaccharides from Polysaccharides.

Carbohydrates play essential roles in a variety of biological processes that are dictated by their structures. However, characterization of carbohydrate structures remains extremely difficult and generally unsolved. In this work, a de novo mass spectrometry-based workflow was developed to isolate and structurally elucidate oligosaccharides to provide sequence, monosaccharide compositions, and glycosidic linkage positions. The approach employs liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based methods in a 3-dimensional concept: one high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) analysis for oligosaccharide sequencing and two ultra high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QqQ MS) analyses on fractionated oligosaccharides to determine their monosaccharides and linkages compositions. The workflow was validated by applying the procedure to maltooligosaccharide standards. The approach was then used to determine the structures of oligosaccharides derived from polysaccharide standards and whole food products. The integrated LC–MS workflow will reveal the in-depth structures of oligosaccharides.

Toward Sequencing the Human Milk Glycome: High-Resolution Cyclic Ion Mobility Separations of Core Human Milk Oligosaccharide Building Blocks.

Human milk oligosaccharides (HMOs) are an unconjugated class of glycans that have been implicated for their role in promoting the healthy development of the brain-gut axes of infants. Production of HMOs is ever-changing and specifically tailored for each infant in response to various biological factors (e.g., cognitive development, diseases, or allergies). While every HMO consists of up to only five monosaccharides, their structures can be composed of many possible glycosidic linkage positions and corresponding α/β anomericities, linear or branched chains, and potential fucosylation/sialylation modifications, thus leading to a tremendous degree of isomeric heterogeneity. With limited availability of authentic standards for every putative HMO structure (estimated to be >200 total), new analytical methods are needed for their accurate characterization. Complete sequencing of the human milk glycome would enable a better understanding of their infant-specific biological roles and potentially lead to their widespread incorporation into infant formula. Herein, we explore the use of our high-resolution cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based platform for the separation of core disaccharide and trisaccharide isomer building blocks as a first step toward the sequencing of larger HMOs. By utilizing the flexible capabilities of the cIMS array, separation pathlengths were extended up to 40 m, thus enabling the resolution of all seven sets of sialylated, fucosylated galactosyllactose and lactosamine HMO building block isomers. Additionally, we assessed the utility of pre-/post-cIMS tandem mass spectrometry (MS/MS) and tandem cIMS (cIMS/cIMS) for the characterization of HMOs based on their diagnostic fragmentation patterns and mobility fingerprints. We anticipate that our presented cIMS-MS-based methodology will enable the better characterization of larger, unknown HMOs when incorporated into an overall workflow that also includes online liquid chromatography and enzymatic hydrolyses.

Comprehensive structural analysis of a set of various branched glucans by standard methylation analysis, 1H NMR spectroscopy, ESI-mass spectrometry, and capillary electrophoresis

Various (semi)-synthetic non-digestible glucans (NDG) (resistant (malto)dextrin, isomaltooligosaccharide, soluble corn fiber, polydextrose) were analyzed by methylation analysis, 1H NMR spectroscopy, ESI-MSn, TLC, and capillary electrophoresis (CE/UV). Partly complementary and partly corroborative results were obtained by these methods yielding a comprehensive overview about the various degrees of complexity and special structural features. Methylation analysis enabled the most detailed quantitative evaluation of glycosidic linkage positions and branching pattern, and detection of furanosyl residues. 1H NMR spectroscopy provided additional information on anomeric configuration. By ESI-IT-MS up to DP 13, for doubly charged up to DP 22 was detected. Glucitol residues in polydextrose as well as anhydrosugar formation in thermally treated glucans could also be recognized from the mass spectra. Furthermore, MS2 of the disaccharide portion gave insights into the linkage positions present in the glucan mixture. Electrophoresis of ANTS-labeled oligosaccharides showed the DP-resolved differences of complexity and was the most powerful method to identify the type of glucan by its CE-fingerprint. The aim was to present complementary analytical data obtained by application of identical conditions and methods to a wide range of NDG with increasing complexity to allow direct structure comparison.

Elucidation of the O-antigen structure of Escherichia coli O63.

The structure of the O-antigen polysaccharide (PS) from the Shiga-toxin producing Escherichia coli O63 has been elucidated using a combination of bioinformatics, component analyses and NMR spectroscopy. The O-antigen is comprised of tetrasaccharide repeating units with the following structure: →2)-β-d-Quip3N(d-allo-ThrAc)-(1→2)-β-d-Ribf-(1→4)-β-d-Galp-(1→3)-α-d-GlcpNAc-(1→ in which the N-acetylated d-allo-threonine is amide-linked to position 3 of the 3-amino-3-deoxy-d-Quip sugar residue. The presence of a predicted flippase and polymerase encoded in the O63 gene cluster is consistent with the Wzx/Wzy biosynthetic pathway and consequently the biological repeating unit has likely an N-acetyl-d-glucosamine residue at its reducing end. A bioinformatics approach based on predictive glycosyltransferase function present in ECODAB (E. coli O-antigen database) suggested the structural element β-d-Galp-(1→3)-d-GlcpNAc in the O-antigen. Notably, multiple gene sequence alignment of fdtA and qdtA from E. coli to that in E. coli O63 resulted in discrimination between the two, confirmation of the latter in E. coli O63, and consequently, together with qdtB, biosynthesis of dTDP-d-Quip3N. The E. coli O63 O-antigen polysaccharide differs in two aspects from that of E. coli O114 where the latter carries instead an l-serine residue, and the glycosidic linkage positions to and from the Quip3N residue are both changed. The structural characterization of the O63 antigen repeat supports the predicted functional assignment of the O-antigen cluster genes.

Mid-infrared spectroscopic analysis of saccharides in aqueous solutions with sodium chloride.

The infrared spectral characteristics of three different types of disaccharides (trehalose, maltose, and sucrose) and four different types of monosaccharides (glucose, mannose, galactose, and fructose) in aqueous solutions with sodium chloride (NaCl) were determined. The infrared spectra were obtained using the FT-IR/ATR method and the absorption intensities respected the interaction between the saccharide and water with NaCl were determined. This study also focused on not only the glycosidic linkage position and the constituent monosaccharides, but also the concentration of the saccharides and NaCl and found that they have a significant influence on the infrared spectroscopic characterization of the disaccharides in an aqueous solution with NaCl. The absorption intensities representing the interaction between a saccharide and water with NaCl were spectroscopically determined. Additionally, the applications of MIR spectroscopy to obtain information about saccharide-NaCl interactions in foods and biosystems were suggested.

Sodium-cationized carbohydrate gas-phase fragmentation chemistry: influence of glycosidic linkage position.

We investigate the gas-phase structures and fragmentation chemistry of two isomeric sodium-cationized carbohydrates using combined tandem mass spectrometry, hydrogen/deuterium exchange experiments, and computational methods. Our model systems are the glucose-based disaccharide analytes cellobiose (β-d-glucopyranosyl-(1 → 4)-d-glucose) and gentiobiose (β-d-glucopyranosyl-(1 → 6)-d-glucose). These analytes show substantially different tandem mass spectra. We characterize the rate-determining barriers to both the glycosidic and structurally-informative cross-ring bond cleavages. Sodiated cellobiose produces abundant Y1 and B1 peaks. Our deuterium labelling and computational chemistry approach provides evidence for 1,6-anhydroglucose B1 ion structures rather than the 1,2-anhydroglucose and oxacarbenium ion structures proposed elsewhere. Unlike those earlier proposals, this finding is consistent with the experimentally observed Bn/Ym branching ratios. In contrast to cellobiose, sodiated gentiobiose primarily fragments by cross-ring cleavage to form various A2 ion types. Fragmentation is facilitated by ring-opening at the reducing end which enables losses of CnH2nOn oligomers. Deuterium labelling and theory enables rationalization of these processes. Theory and experiment also support the importance of consecutive fragmentation processes at higher collision energies.

Formation of type 4 resistant starch and maltodextrins from amylose and amylopectin upon dry heating: A model study

Starch is one of the main components of human diet. During food processing, starch is submitted to high temperatures in the presence or absence of water. Thus, the main goal of this work was to identify structural modifications caused by dry heating in starch polysaccharides (amylose and amylopectin) and structurally related oligosaccharides, maltotetraose (M4) and glucosyl-maltotriose (GM3), simulating processing conditions. The structural modifications were evaluated by methylation analysis, electrospray mass spectrometry (ESI-MS), tandem mass spectrometry (ESI-MS/MS) and anionic chromatography after in vitro enzymatic digestion. Dry heating promoted dehydration, depolymerization, as well as changes in Glc glycosidic linkage positions and anomeric configuration. In oligosaccharides, polymerization was also observed. All these changes resulted in a lower in vitro digestibility, suggesting that dry heating of starch polysaccharides and related oligosaccharides may be associated with the formation of type 4 resistant starch and maltodextrins, non-digestible carbohydrates that are responsible for beneficial effects in human intestinal tract.

Nickel-Catalyzed Proton-Deuterium Exchange (HDX) Procedures for Glycosidic Linkage Analysis of Complex Carbohydrates.

The structural analysis of complex carbohydrates typically requires the assignment of three parameters: monosaccharide composition, the position of glycosidic linkages between monosaccharides, and the position and nature of noncarbohydrate substituents. The glycosidic linkage positions are often determined by permethylation analysis, but this can be complicated by high viscosity or poor solubility, resulting in under-methylation. This is a drawback because an under-methylated position may be misinterpreted as the erroneous site of a linkage or substituent. Here, we describe an alternative approach to linkage analysis that makes use of a nonreversible deuterium exchange of C-H protons on the carbohydrate backbone. The exchange reaction is conducted in deuterated water catalyzed by Raney nickel, and results in the selective exchange of C-H protons adjacent to free hydroxyl groups. Hence, the position of the residual C-H protons is indicative of the position of glycosidic linkages or other substituents and can be readily assigned by heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR) or, following suitable derivatization, by gas chromatography-mass spectroscopy (GC/MS) analysis. Moreover, because the only changes to the parent sugar are proton/deuterium exchanges, the composition and linkage analysis can be determined in a single step.

Effect of Dextransucrase Cellobiose Acceptor Products on the Growth of Human Gut Bacteria

The selective fermentation by human gut bacteria of gluco-oligosaccharides obtained from the reaction between the glucosyl group of sucrose and cellobiose, catalyzed by dextransucrases (DSR) from Leuconostoc mesenteroides , has been evaluated. Oligosaccharides were fractionated according to their molecular weight, and their effect on the growth of different bacterial groups was studied. To determine the structure (position and configuration of glycosidic linkages)-function relationship, their properties were compared to those of DSR maltose acceptor products (DSRMal) and of recognized prebiotic carbohydrates (fructo-oligosaccharides, FOS). Cellobiose acceptor products (DSRCel) showed bifidogenic properties similar to those of FOS. However, no significant differences related to molecular weight or isomeric configurations were found for DSRCel and DSRMal products.

Product distribution from fast pyrolysis of glucose-based carbohydrates

Carbohydrates are the major constituents of biomass. With the growing interest in utilizing bio-oil obtained from fast pyrolysis of biomass for fuels and chemicals, understanding the carbohydrate pyrolysis behavior has gained particular importance. The chemical composition of the bio-oil is an important consideration for its upstream and/or downstream processing. Though the classification of pyrolysis products into overall tar, char and gaseous fraction has evolved as a standard; detailed knowledge of the chemical constituents that determine the quality of bio-oil has received little attention. Furthermore, the speciation arising from primary and secondary reactions has been rarely distinguished. In this study the product distribution arising from the primary reactions during 500 °C fast pyrolysis of several mono-, di- and polysaccharides is studied with the help of micro-pyrolyzer. The study suggests that levoglucosan and the low molecular weight compounds are formed through competitive pyrolysis reactions rather than sequential pyrolysis reactions. It is also shown that the orientation or the position of glycosidic linkages does not significantly influence the product distribution except with 1,6-linked polysaccharide, which showed considerably less formation of levoglucosan than other polysaccharides.

Complete assignments of the 1H and 13C chemical shifts and JH,H coupling constants in NMR spectra of d-glucopyranose and all d-glucopyranosyl- d-glucopyranosides

Complete assignment of the 1H and 13C NMR spectra of all possible d-glucopyranosyl- d-glucopyranosides was performed and the 1H chemical shifts and proton–proton coupling constants were refined by computational spectral analyses (using PERCH NMR software) until full agreement between the calculated and experimental spectra was achieved. To support the experimental results, the 1H and 13C chemical shifts and the spin–spin coupling constants between the non-hydroxyl protons of α- and β- d-glucopyranose ( 1a and 1b) were calculated with density functional theory (DFT) methods at the B3LYP/pcJ-2//B3LYP/6-31G(d,p) level of theory. The effects of different glycosidic linkage types and positions on the glucose ring conformations and on the α/β-ratio of the reducing end hydroxyl groups were investigated. Conformational analyses were also performed for anomerically pure forms of methyl d-glucopyranosides ( 13a and 13b) and fully protected derivatives such as 1,2,3,4,6-penta- O-acetyl- d-glucopyranoses ( 14a and 14b).

Permethylation Linkage Analysis Techniques for Residual Carbohydrates

Permethylation analysis is the classic approach to establishing the position of glycosidic linkages between sugar residues. Typically, the carbohydrate is derivatized to form acid-stable methyl ethers, hydrolyzed, peracetylated, and analyzed by gas chromatography-mass spectrometry. The position of glycosidic linkages in the starting carbohydrate are apparent from the mass spectra as determined by the location of acetyl residues. The completeness of permethylation is dependent upon the choice of base catalyst and is readily confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry mass spectrometry. For the permethylation of beta-cyclodextrin, Hakomori dimsyl base is shown to be superior to the NaOH-dimethyl sulfoxide system, and the use of the latter resulted in selective under-methylation of the 3-hydroxy groups. These techniques are highly applicable to residual carbohydrates from biofuel processes.

Vibrational circular dichroism (VCD) studies on disaccharides in the CH region: toward discrimination of the glycosidic linkage position

The structural features of carbohydrates are a combination of 1) sequence and types of mono-sugars, 2) stereochemistry of their glycosidic linkages, and 3) their glycosidic linkage sites. We performed the first systematic VCD study on glycoside linking site discrimination. VCD spectra, in the CH stretching region from 2000 to 4000 cm(-1), of eleven glucobioses (trehalose (alpha1-alpha1), neotrehalose (alpha1-beta1), isotrehalose (beta1-beta1), kojibiose (alpha1-2), nigerose (alpha1-3), maltose (alpha1-4), isomaltose (alpha1-6), sophorose (beta1-2), laminaribiose (beta1-3), cellobiose (beta1-4), gentiobiose (beta1-6)) suggested a possible new discrimination method for glyco analysis, while VCD spectra in the mid-IR region distinguished the stereochemistry (alpha or beta) of the glycosidic linkage. Both reducing and nonreducing glucobioses showed different VCD spectral features compared to their constituent D-glucose and the anomer-fixed model compounds. Interresidue interaction such as hydrogen bonding was suggested to cause these spectral differences. Interplay between residues is a common phenomenon and thus VCD analysis could be applicable to other di-, oligo- or poly-saccharides. Several isotropic labeled compounds were also measured to support spectral assignment and interpretation.

Effect of monosaccharide composition, glycosidic linkage position and anomericity on the electrophoretic mobility of labeled oligosaccharides

Fluorophore-assisted carbohydrate electrophoresis (FACE) is useful for separation and characterization of oligosaccharides from various sources and for comparing several samples at once. While characterizing fungal surface glycans by FACE we observed that samples and standards of the same mass did not comigrate as expected. Subsequent experiments showed that the samples did not contain contaminating sugars. Therefore, our observation suggested that glycan electrophoretic mobility is affected by factors in addition to molecular mass. This work assesses the contribution of monosaccharide composition, linkage position, and linkage anomericity to glycan mobility. Commercially available (and synthesized when available) bioses of known composition were derivatized with a charged fluorophore, and electrophoretic mobilities compared in a slab gel format. The results indicate that all three parameters mentioned above affect observed migration. Further, no migration patterns emerged to suggest a set of rules for assigning band identity based on mobility alone. These results emphasize the importance of including known, matched, standards to facilitate interpretation of FACE data.

Influences of Monosaccharides and its Glycosidic Linkage on Infrared Spectral Characteristics of Disaccharides in Aqueous Solutions

The infrared spectral characteristics of ten different types of disaccharides (trehalose, kojibiose, nigerose, maltose, isomaltose, trehalulose, sucrose, turanose, maltulose, and palatinose) and five different types of monosaccharides (glucose, mannose, galactose, talose, and fructose) in aqueous solutions (H2O and D2O) were determined. The infrared spectra were collected using the Fourier transform infrared attenuated total reflectance (FT-IR/ATR) method and comparisons between the degrees of absorption band-shift of the saccharide spectra in the H2O solution with those in the D2O solution with respect to the saccharide concentrations were done. The study revealed that the wavenumber shifts in the bands of mono- and disaccharides in the H2O and D2O solutions could be used as an indicator of the level of interaction between the saccharides and water. The study also focused on the glycosidic linkage position and the constituent monosaccharides and found that they have a significant influence on the infrared spectroscopic characterization of disaccharides in an aqueous solution.

Analysis of fluorogenic Smith degradation products of 7-(1,3-disulfonaphtyl)amino-disaccharides for linkage position analysis of carbohydrates.

The linkage position of a glycosidic bond to the reducing-end residue of a pyridylamino (PA-) sugar can be determined sensitively by Smith degradation and HPLC [K. Omichi and S. Hase, (1994) J. Biochem. 115, 429-434]. With the aim of enhancing the sensitivity of this method of linkage position analysis to the fmol-level, use of the 7-(1,3-disulfonaphtyl)amino (DSNA-) group instead of the PA-group as a fluorescent tag was examined. Smith degradation of DSNA-disaccharides with a DSNA-hexose, DSNA-N-acetylglucosamine, or DSNA-N-acetylgalactosamine reducing-end residue was carried out. HPLC and FAB-MS of the fluorogenic Smith degradation products showed that the DSNA-group was stable under the Smith degradation reaction conditions, and that the reaction proceeded in a manner similar to that using PA-disaccharides to give the predicted products. Fluorogenic Smith degradation products specific to the glycosidic linkage position were well separated by reversed-phase HPLC, and were easily assignable by comparing the HPLC elution positions with those of standard compounds. The method was successfully applied to analyzing the structure of an N-linked sugar chain.

An effective strategy for structural elucidation of oligosaccharides through NMR spectroscopy combined with peracetylation using doubly 13C-labeled acetyl groups

The use of NMR spectroscopy for the elucidation of larger carbohydrate structures isolated from natural sources is principally limited by severe overlap of 1H signals, poor sensitivity when experiments involve 13C nuclei, and difficulties in conclusively establishing linkage positions. Peracetylation of oligosaccharides with doubly 13C-labeled acetyl groups provides several major advantages for their structural elucidation when combined with specifically tailored NMR pulse sequences. The 2.5–4.7 Hz J-coupling constants between acetyl carbonyl-13C nuclei and protons of the sugar ring at the sites of acetylation enables these sites to be readily assigned. By inference, glycosidic linkage positions on monosaccharides can be unambiguously determined. This can be used in lieu of permethylation analysis, yet does not require degradation of oligosaccharides. Spectral dispersion in the directly detected (1H) dimension is increased ~2.6–2.7-fold due to the downfield shifting of sugar-ring protons at the positions of acetylation. Peracetylation also introduces three new frequency dimensions for NMR studies, namely the 13CO, 13CMe, and 1HMe frequencies of the acetyl groups. These frequencies can be correlated to sugar protons, either independently or in combination, in alternative 2-, 3-, or 4-D experiments. The use of Hartmann–Hahn coherence transfer combined with zero-quantum dephasing periods permits purely absorptive in-phase multiplets to be extracted and enables accurate scalar couplings between ring protons to be measured, even in multidimensional experiments. Results are illustrated on a nonasaccharide-alditol derived from N-linked glycoproteins and on some smaller structures containing sialic acids and N-acetylhexosamines. Methods for small-scale sample acetylation using the superacylation catalyst, 4-dimethylamino pyridine, are described. A brief historical perspective pertinent to the fundamental contributions of Dr. R.U. Lemieux to the field of carbohydrate NMR is also presented.Key words: NMR, oligosaccharides, peracetylation, doubly 13C-labeled acetyl groups, tailored pulse sequences, heteronuclear Hartmann–Hahn.

Effect of substitution pattern on 1H, 13C NMR chemical shifts and 1JCH coupling constants in heparin derivatives

1H, 13C NMR chemical shifts and 1 J CH coupling constants were measured for derivatives of heparin containing various sulfation patterns. 1H and 13C chemical shifts varied considerably after introducing electronegative sulfate groups. Chemical shifts of protons linked to carbons changed by up to 1 ppm on substitution with O- and N-sulfate or acetyl groups. Differences up to 10 ppm were detected for 13C chemical shifts in substituted glucosamine, but a less clear dependence was found in iduronate. 1 J CH values formed two groups, corresponding to either sulfation or non-sulfation at positions 2 and 3 of glucosamine. O-sulfation caused increases up to 6 Hz in 1 J CH and N-sulfation decreases up to 4 Hz. N-acetylation gave similar 1 J CH values to N-sulfation. At positions 2 and 3 of iduronate the trend was less marked; 1 J CH for O-sulfated positions usually increasing. Introduction of sulfate groups influences chemical shift and 1 J CH values at the position of substitution, but also at more remote positions. 1 J CH at the glycosidic linkage positions varied between free-amino and N-sulfated compounds, by up to 9 Hz. These results and changes in chemical shift values suggest that iduronate residues and the glycosidic linkages are affected, indicating overall conformational change. This may have important implications for biological activities.

Identification of flavonoid diglycosides in yellow lupin (Lupinus luteus l.) with mass spectrometric techniques

Various mass spectrometric techniques were used for the structural elucidation of flavonoid glycosides isolated from green parts of yellow lupin plants (Lupinus luteus). A methodological approach is proposed consisting of (1) extraction and purification of flavonoid glycosides from plant tissues, (2) registration of liquid secondary ion mass spectra of intact compounds and (3) gas chromatographic/mass spectrometric analyses of those compounds subjected to several types of chemical modification. On the basis of these data, it was possible to define the molecular mass of the glycosides, the structure of the aglycones, the configuration of sugar moieties and the position of glycosidic linkages between sugars in diglycosides or between aglycone and sugar moieties. Analysis of the mass spectrometric data permitted structural identification of four flavonoid diglycosides, where the aglycones had flavone, isoflavone and flavonol structures. They were recognized as: apigenin 7-O-(2″-O -rhamnopyranosyl)glucopyranoside (1), genistein 4′,7-O-diglucopyranoside (2), kaempferol 3-O -(6″-rhamnopyranosyl)glucopyranoside (3) and 3′- or 4′-methylquercetin 3-O-(6″-O -rhamnopyranosyl)glucopyranoside (4). Complete structural evaluation of flavonoid glycosides was possible with only submilligram quantities of samples needed to register various mass spectra. It was not possible, however, to define the anomeric configuration of the C-1 carbons in investigated glycosides. Copyright © 1999 John Wiley & Sons, Ltd.