HPAEC PAD Research Articles

Synthesis of new glycosaminoglycans-like families by regioselective oxidation followed by sulphation of glucoglucuronan from Rhizobium sp. T1

Glycosaminoglycan-like polysaccharides were prepared from Rhizobium sp. T1 polysaccharide using the TEMPO (nitroxyl radical 2,2,6,6-tetramethylpiperidine-1-oxyl radical) mediated oxidation. The structure of this new polyglucuronic acid sodium salt was analyzed by 13C NMR spectra and HPAEC–PAD chromatography. Therefore, new polysaccharide containing only glucuronic acid monomers in both β-(1,3) and β-(1,4) linkage was obtained by the complete TEMPO-mediated oxidation of C6 primary hydroxyl groups of glucose of glucoglucuronan from Rhizobium sp. T1. Sulphation of this β-(1,3),β-(1,4)-polyglucuronic acid sodium salt was carried out using SO3/DMF reagent. These results suggested a new synthetic route using both TEMPO-mediated oxidation and sulphation of polysaccharides from Rhizobium sp. in developing glycosaminoglycans mimic to enhance the profitability of its low-cost production and processing industries. This novel carbohydrate derivative might find use as cheaper surrogates of glycosaminoglycans in the cosmetics and pharmaceutical fields.

Isolation and identification of Di-D-fructose dianhydrides resulting from heat-induced degradation of inulin

Dry heating of inulin up to 200 °C for 30 min resulted in a wide range of degradation products, from which five quantitatively dominating compounds were isolated using flash chromatography and semi-preparative HPLC–RI. It was possible to identify four different DFDAs (di-d-fructose dianhydrides) by means of one- and two-dimensional NMR, namely DFA III (α-D-Fruf-1,2′:2,3′-β-D-Fruf), DFA I (α-D-Fruf-1,2′;2,1′-β-D-Fruf), DFA VII (α-D-Fruf-1,2′;2,1′-α-D-Fruf), and β-D-Fruf-1,2′;2,1′-β-D-Fruf. For the fifth compound, the structure of the difructan α-D-Fruf-1,2′-β-D-Fruf was proposed. Using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), it was possible to quantify those isolated DFDAs in inulin caramels to a total amount of up to 25%, indicating that dry heating of inulin may be a suitable tool to enrich caramels with DFDAs.

Structural investigation of an exopolysaccharide substituted with a lactyl ether group produced by Raoultella terrigena Ez-555-6 isolated in the Chernobyl exclusion zone

Raoultella terrigena strain Ez-555-6, isolated from a root nodule of Medicago sativa harvested in the Chernobyl exclusion zone, produces a non-referenced high-molecular-mass exopolysaccharide (EPS). The structure of this EPS was determined using a combination approach including monosaccharide composition (GLC–FID, HPAEC–PAD), determination of glycosylation sites (GLC–EIMS) and 1D/2D NMR ( 1H, 13C) and ESIMS (HR, MS/MS) studies of oligosaccharides obtained from mild acid hydrolysis. The EPS was found to be a charged pentasaccharide with a repeating unit composed of d-galactose, d-glucose, d-mannose and d-glucuronic acid (1:2:1:1). Lactic acid and O-acetyl substituents were localized on galactose and glucose residues, respectively, as presented in the following structure: [Display omitted]

Fine structure characterization of amylopectins from grain amaranth starch

The aim of this study was to determine the fine structure of amylopectin from grain amaranth. Amaranthus amylopectin was hydrolyzed with α-amylase, and single clusters and a group of clusters (domain) were isolated by methanol precipitation. The domain and the clusters were treated with phosphorylase a and then β-amylase to remove all external chains, whereby the internal structure was obtained. The ϕ,β-limit dextrins were analyzed on Sepharose CL 6B. The average DP (degree of polymerization) and peak-DP values of fractions of clusters were 57 and 82, respectively; the values of the domain were 137 and 309, respectively. The unit chain length profiles were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC–PAD). The results showed that the domain fraction contained ∼2.2 clusters, and single clusters were composed of ∼13 chains. The ϕ,β-limit dextrins of the clusters were further hydrolyzed with α-amylase to characterize their building block composition. The average DP of the branched blocks was ∼11 and they contained on average ∼2.5 chains. Their average chain length, internal chain length, and degree of branching were approximately 4.3, 2.8, and 14, respectively. A cluster consisted of ∼6 branched blocks, and the internal chain length between the blocks was ∼6.8.

Simultaneous determination of two Amadori compounds in Korean red ginseng ( Panax ginseng) extracts and rat plasma by high-performance anion-exchange chromatography with pulsed amperometric detection

A new simple, rapid and sensitive high-performance anion-exchange chromatography method with pulsed amperometric detection (HPAEC–PAD) was developed and validated for the simultaneous determination of two Amadori compounds, arginyl-fructose and arginyl-fructosyl-glucose in Korean red ginseng ( Panax ginseng) extracts, rat plasma. Separation of the two target analytes was efficiently undertaken on CarboPac PA1 anion-exchange column with isocratic elution (400 mM sodium hydroxide and deionized water (90:10, v/v)) at flow rate 0.7 mL/min within 15 min of single chromatographic run. Under optimized conditions, the detection limits (signal-to-noise ratio equal to 3) were 20 and 25 ng/mL for arginyl-fructose and arginyl-fructosyl-glucose, respectively. Calibration curves of peak area for the two analytes were linear over three orders of magnitude with a correlation coefficients greater than 0.999. The accuracy of the method was tested by recovery measurement of the spiked samples which yielded good results of 94.15–102.62%. This method was successfully applied to the quantification of arginyl-fructose and arginyl-fructosyl-glucose in herbal extracts and in the plasma samples from rat.

Rapid determination of collagen in meat-based foods by microwave hydrolysis of proteins and HPAEC–PAD analysis of 4-hydroxyproline

A rapid microwave procedure for protein hydrolysis coupled with High Performance Anion Exchange Chromatography and Pulsed Amperometric Detection (HPAEC–PAD) was developed to quantify the amino acid 4-hydroxyproline in meat and meat-based products. This innovative approach was successfully applied to determine collagen content (4-hydroxyproline×8) as the index quality of meat material employed in the preparation of typical meat sausages (“Mortadella di Bologna PGI” and “Salamini italiani alla cacciatora PDO”) and fresh filled pastas. Microwave hydrolysis showed a precision and accuracy similar to traditional hydrolysis (RSD% from 0.0 to 6.4; relative error 1.4–10.0%) with a reduction in the hydrolysis time from 24h to 20min. HPAEC–PAD allowed detection of 4-hydroxyproline without pre or post-column derivatization and the use of non-toxic eluents.

New enzyme-based method for analysis of water-soluble wheat arabinoxylans

Arabinoxylans (AX) are the predominant cell-wall polysaccharides in wheat flour. Water-extractable AX are essential for dough and bread properties and performance. However, there is no specific and accurate way of determining the content and structure of AX. An enzyme-assisted method employing an efficient enzyme mixture for the total hydrolysis of AX was developed in the present work. Enzymatic hydrolysis (EH) is a gentle method during which no unwanted sugar destruction occurs. Following EH, liberated monosaccharides were analysed by gas chromatography (GC) and liquid chromatography using HPAEC–PAD. The results were compared with acid methanolysis (AM) and acid hydrolysis (AH). EH performed better on commercially isolated AX samples than the reference method AM. Its action in the water extract from wheat flour was also more efficient than that of AM and comparable to the efficiency of AH. HPAEC–PAD revealed a significant amount of fructose in the water extract following EH, originating from fructans in wheat flour not detected in the GC analysis. The wheat flour examined contained 0.29% water-extractable AX. The arabinose/xylose ratio was 0.32. The enzyme-based method developed is applicable for comparison of different wheat flours and can be used in evaluating the effect of processing on the content and structure of water-extractable AX.

Study of molar response of dextrans in electrochemical detection

In this work, a methodological approach is reported, aimed at assessing the electrochemical response of some model gluco-oligosaccharides (dextrans). Such strategy is based on the complementary use of both anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) and capillary zone electrophoresis coupled with UV detection (CZE–UV). Unlike HPAEC–PAD, CZE–UV required derivatization with a chromophoric dye (i.e., 8-aminonaphtalene-1,3,6-trisulphonic acid, ANTS) to enhance UV response and separation selectivity. From the comparison between chromophore response and PAD signal, the reliability of HPAEC–PAD for quantitative evaluation of dextran mixtures containing mainly oligomers with polymerization degree (DP) up to 18 could be proved, due to the fairly constant molar response. For higher DPs (up to 41), a maximum in the trend of the molar responses was observed followed by a steep decrease for DPs higher than about 30–35; indeed, an underestimation of weight-average molecular weight of dextran mixtures containing such oligomers was noticed.

Degradation of polysaccharides and non-digestible oligosaccharides by Bacillus subtilis and Bacillus pumilus isolated from Soumbala, a fermented African locust bean (Parkia biglobosa) food Condiment

In order to select starter cultures for controlled fermentation of African locust beans, Bacillus subtilis and B. pumilus from Soumbala were investigated for their ability to degrade arabinogalactan, stachyose, raffinose and sucrose—the main polysaccharides and oligosaccharides in African locust bean. The methods used were liquefaction of gels of galactomannan for screening, pH measurement and High Performance Anion Exhange Chromatography – Pulsed Amperometric Detection (HPAEC–PAD) for detailed degradation study of the carbohydrates. For B. subtilis isolates, liquefaction of galactomannan gels was observed and HPAEC–PAD showed that arabinogalactan and stachyose were partially degraded while raffinose was fully degraded after 48 h of fermentation. Melibiose, fructose and traces of galactose were detected as residual sugars. For B. pumilus isolates much weaker degradation of the carbohydrates was observed. Two isolates of B. subtilis showed strong ability to degrade the oligosaccharides and they are seen as promising starter cultures for fermentation of African locust bean.

Mode of action and subsite studies of the guluronan block-forming mannuronan C-5 epimerases AlgE1 and AlgE6

AlgE1, AlgE5 and AlgE6 are members of a family of mannuronan C-5 epimerases encoded by the bacterium Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyse the post-polymerization conversion of beta-D-mannuronic acid (M) residues into alpha-L-guluronic acid residues (G). All enzymes show preference for introducing G-residues neighbouring a pre-existing G. They also have the capacity to convert single M residues flanked by G, thus 'condensing' G-blocks to form almost homopolymeric guluronan. Analysis of the length and distribution of G-blocks based on specific enzyme degradation combined with size-exclusion chromatography, electrospray ionization MS, HPAEC-PAD (high-performance anion-exchange chromatography and pulsed amperometric detection), MALDI (matrix-assisted laser-desorption ionization)-MS and NMR revealed large differences in block length and distribution generated by AlgE1 and AlgE6, probably reflecting their different degree of processivity. When acting on polyMG as substrates, AlgE1 initially forms only long homopolymeric G-blocks >50, while AlgE6 gives shorter blocks with a broader block size distribution. Analyses of the AlgE1 and AlgE6 subsite specificities by the same methodology showed that a mannuronan octamer and heptamer respectively were the minimum substrate chain lengths needed to accommodate enzyme activities. The fourth M residue from the non-reducing end is epimerized first by both enzymes. When acting on MG-oligomers, AlgE1 needed a decamer while AlgE6 an octamer to accommodate activity. By performing FIA (flow injection analysis)-MS on the lyase digests of epimerized and standard MG-oligomers, the M residue in position 5 from the non-reducing end was preferentially attacked by both enzymes, creating an MGMGGG-sequence (underlined and boldface indicate the epimerized residue).

Degree of blockiness of amide groups as indicator for difference in physical behavior of amidated pectins

Thickening and gelling properties of commercial amidated pectins depend on the degree of amidation and methyl-esterification, but also the distribution of these groups is of great importance. Methods have been developed during the last few years to determine the distribution of methyl esters over the pectic backbone. We applied the strategies developed for the analysis of high methyl-esterified pectins for studying the distribution of amide groups in amidated pectins. Low methyl-esterified amidated (LMA) pectins were digested before and after removal of methyl esters by an endo-polygalacturonase to determine the degree of blockiness of the substituents. The nature of the substituents (amide groups compared to methyl esters) did not modify the behavior of the enzyme. Oligomers released were separated by using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) at pH 5. Fractions collected after on-line desalting were identified by using MALDI-TOF mass spectrometry. Oligomers were found to elute from the column as a function of their total charge. For the same overall charge and size, oligomers with methyl esters eluted before oligomers with amide groups. Both amide groups and methyl esters of the LMA pectins studied were found to be semirandomly distributed over the pectic backbone, but this may vary according to the amidation process used.

Application of high-performance anion-exchange chromatography with pulsed amperometric detection and statistical analysis to study oligosaccharide distributions – a complementary method to investigate the structure and some properties of alginates

Alginates comprised of essentially alternating units of mannuronic (M) acid-guluronic (G) acid (MG-alginate), and G-blocks isolated from a seaweed where subjected to partial acid hydrolysis at pH 3.5 The chain-length distribution of oligosaccharides in the hydrolysate were investigated by statistical analysis after their separation with high-performance anion-exchange chromatography and pulsed amperometric detection (HPAEC–PAD). Simulated depolymerisation of the MG-alginate provided an estimate of the ratio between two acid hydrolysis rate constants ( p = 8.3 ± 1) and the average distribution of the MM linkages in the original sample of polysaccharide chains. In conclusion, we found HPAEC–PAD together with statistical analysis was a useful method to investigate the fine structure and some properties of binary polysaccharides.

Analysis of sugar phosphates in plants by ion chromatography on a titanium dioxide column with pulsed amperometric detection

This paper describes the development of a practical method for the analysis of sugar phosphates from the model higher plant Arabidopsis thaliana by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD). The extraction method of sugar phosphates from higher plants was first optimized for HPAEC–PAD analysis. In order to improve the resolution in HPAEC–PAD, a column packed with titanium dioxide resin was used. The titanium dioxide column was used as a trap-column for sugar phosphates and nucleotides, for the removal of sample matrices. Sample pretreatment was achieved in-line and automatically using a six-port valve placed after the injection valve.

Effect of food-processing on the degradation of fructooligosaccharides in fruit

The effect of process on fructooligosaccharide degradation in stewed apple-banana and three apple dessert was studied. 1-Kestose was analysed using an accurate analytical method for carbohydrate determination (high-performance anion exchange chromatography with pulsed amperometric detection, HPAEC–PAD). It appears that the content of 1-kestose in raw material was much higher than in the finished product. Furthermore, a banana puree incubated for 30 min over a temperature range (80–110 °C), supposed to favour degradation, appeared stable. This indicate that its the formulation rather than cooking or pasteurization which is responsible for the fructooligosaccharide loss.

Determination of mono- and disaccharides in milk and milk products by high-performance anion-exchange chromatography with pulsed amperometric detection

A simple and sensitive liquid chromatographic method for the separation and quantification of mono- and disaccharides in raw- and processed-milk is described. Samples of cows’, buffalos’, sheeps’ and goats’ milk were analyzed upon clarification and appropriate dilution for the quantification of lactose, galactose, glucose and N-acetylglucosamine (GlcNAc). The separation was accomplished by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), using a gold working electrode and dilute alkaline eluents modified by a millimolar concentration of barium acetate. The eluent composition employed was designed to provide optimum separation with respect to the selected sample, without interference from the matrix components. The analytical method was successfully employed for the determination of mono- and disaccharides naturally occurring in dairy milk, mozzarella cheese and whey samples, with high sensitivity and accuracy.

Physico-chemical and immunological examination of the thermal stability of tetanus toxoid conjugate vaccines

The thermal stability of meningococcal C (MenC)– and Haemophilus influenzae b (Hib)–tetanus toxoid (TT) conjugate vaccines was investigated using spectroscopic and chromatographic techniques and immunogenicity assays in animal models. In this stability study, both the bulk concentrate and final fills were incubated at −20, 4, 23, 37 or 55 °C for 5 weeks or subjected to cycles of freeze-thawing. The structural stability, hydrodynamic size and molecular integrity of the treated vaccines were monitored by circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopic techniques, size exclusion chromatography (FPLC–SEC), and high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC–PAD). Only storage at 55 °C for 5 weeks caused some slight unfolding and modification in the tertiary structure of the carrier protein in the MenC–TT conjugate. Substantial loss of saccharide content from the MenC conjugates was observed at 37 and 55 °C. Unexpectedly, the experimental immunogenicity of MenC–TT vaccine adsorbed to Alhydrogel was significantly reduced only by repeated freeze-thawing, but not significantly decreased by thermal denaturation. Neither the molecular integrity nor the immunogenicity of the lyophilised Hib–TT vaccines was significantly affected by freeze-thawing or by storage at high temperature. In conclusion, the MenC– and Hib–TT conjugate vaccines were relatively stable when stored at higher temperatures, though when MenC–TT vaccine was adsorbed to Alhydrogel, it was more vulnerable to repeated freeze-thawing. When compared with CRM 197 conjugate vaccines studied previously using similar techniques [1–3], the tetanus toxoid conjugates were found to have higher relative thermal stability in that they retained immunogenicity following storage at elevated temperatures.

Development of a high-performance anion-exchange chromatography with pulsed-amperometric detection based quantification assay for pneumococcal polysaccharides and conjugates

A method, using high-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC–PAD), has been developed to determine the concentrations of Streptococcus pneumoniae capsular polysaccharides and polysaccharide conjugates used in formulating a conjugate vaccine for the prevention of pneumococcal infections. In an effort to determine optimum hydrolysis conditions for the analysis, pneumococcal polysaccharides were subjected to three different hydrolysis methods: trifluoroacetic acid (TFA) hydrolysis, methanolysis followed by TFA hydrolysis, or hydrofluoric acid (HF) hydrolysis followed by TFA hydrolysis. For quantification purposes, best results were obtained by methanolysis followed by TFA hydrolysis for uronic acid-containing polysaccharides, and by TFA hydrolysis for all the others. For the quantification of all the polysaccharides (from native to conjugated forms), a monosaccharide reference mixture (Rha, Gal and GlcA) hydrolyzed along with the samples can be used as standards for routine analysis. This is much more convenient than to hydrolyze a well-characterized reference polysaccharide (necessary standard only for type 1 capsular polysaccharide). This method is rapid, very sensitive (less than 10 μg of polysaccharide is required), and may replace advantageously the currently used colorimetric assays used to determine polysaccharides content. Moreover, it can be readily adapted for use with other bacterial polysaccharide preparations as well.

Neutral monosaccharides in surface sediments of the northwestern Mediterranean Sea

This paper is focused on carbohydrates, an abundant chemical class in marine organic matter, mostly found as polysaccharides. An experiment showed that the use of H 2SO 4 1 M (95 °C, 4 h) yielded a good compromise between recovery and degradation of all neutral monosaccharides from two kinds of organic material: surface sediments and trapped particles. Twenty samples of surface sediments (0–0.5 cm) were collected in various physiographic and bathymetric locations on the Northwestern Mediterranean margin. Neutral monosaccharides were determined by an ionic chromatography system with electrochemical detection (HPAEC–PAD). Differences between total neutral monosaccharides (NM TOT) and total carbohydrates, determined by a spectrophotometric method on the same samples, brought further assumptions on the nature of the carbohydrate pool. Total neutral monosaccharides contents varied from 0.46 to 1.04 mg g −1 and their proportions donated to the organic carbon pool (NM TOT–C/OC) barely reached 5%. The spatial distribution of NM TOT and organic carbon (OC) clearly showed that organic inputs, originating from the shallower epicontinental area, spread along canyon axis while undergoing dilution processes. In spite of the high physiographic and bathymetric diversity, surface sediments from most stations were characterized markedly by the homogeneity in the neutral monosaccharide compositions. Two stations, E21 (Ebro prodelta) and EC36 (Balearic slope) showed, however, distinct signatures with high relative abundances of glucose and galactose, respectively. Glucose was found to trace the continental inputs, whereas galactose characterized the marine inputs. The relative “freshness” of the organic material at station EC36, assumed by the high proportion of hexose neutral monosaccharides, confirms the dominance of phytoplankton remains at this station. This work showed that the use of monosaccharides as tracers of the biological source and fate of organic compounds is deeply linked to the quality of the organic material. The monosaccharide signature is only preserved in samples of surface sediments where inputs of fresh organic material are higher than the refractory one.

Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

Capillary isoelectric focusing (CIEF) was used to profile the cellulase composition in complex fermentation samples of secreted proteins from Trichoderma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Cel7A), a major component in these extracts, was purified from different strains and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), and capillary liquid chromatography–electrospray mass spectrometry (cLC–ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. Selective identification of tryptic N-linked glycopeptides was achieved using LC–ESMS on a quadrupole/time-of-flight instrument with a mixed scan function. The suspected glycopeptides were further analyzed by on-line tandem mass spectrometry to determine the nature of N-linked glycans and their attachment sites. This strategy enabled the identification of a high mannose glycan attached to Asn270 (predominantly Man 8GlcNAc 2) and single GlcNAc occupancy at Asn45 and Asn384 with some site heterogeneity depending on strains and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues as indicated by MALDI-TOF and HPAEC–PAD experiments. Additional heterogeneity was noted in the CBH I linker peptide of RUT-C30 strain with the presence of a phosphorylated di-saccharide.

Characterization of the F(ab′) 2 fragment of a murine monoclonal antibody using capillary isoelectric focusing and electrospray ionization mass spectrometry

We characterized a F(ab′) 2 fragment obtained by pepsin cleavage from a murine monoclonal IgG3 by means of electrospray ionization (ESI) mass spectrometry (MS), capillary isoelectric focusing (cIEF), high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC–PAD) and LC–MS peptide mapping. Separation of the fragment by cIEF under nonreducing conditions resulted in a number of distinct peaks. Using reducing conditions the heavy chain and the light chain were separated into two peaks each. Analysis by ESI-MS revealed a high mass heterogeneity of the molecule. Digestion with neuraminidase simplified both the cIEF pattern and the mass spectrum. The cIEF of the reduced molecule showed that the sialic acids were located only on the heavy chain of the F(ab′) 2-fragment. By incubation with O-glycosidase a further reduction of the complexity of the mass spectrum was achieved showing 8 different isoforms. By LC–MS peptide mapping these isoforms could be attributed to the heterogeneity of the pepsin cleavage site in the hinge region of the antibody. The sugars of the O-linked carbohydrate chain were identified by HPAEC–PAD as galactosyl- N-acetyl-galactosamine (GalNAcGal) with terminal N-glycolylneuraminic acid. The glycosylation site was identified by peptide mapping and amino acid sequence analysis as Ser 222.