Prebiotic Potential of a New Sweetener Based on Galactooligosaccharides and Modified Mogrosides.

This study was conducted to investigate the sweetness intensity and the potential fecal microbiome modulation of galactooligosaccharides in combination with enzymatically modified mogrosides (mMV-GOS), both generated through a patented single-pot synthesis. Sweetness intensity was performed in vivo by trained sensory panelists. The impact on the human fecal microbiome was evaluated by in vitro pH-controlled batch fermentation, and bacterial populations and organic acid concentrations were measured by qPCR and GC-FID, respectively. Significant growth (p ≤ 0.05) during the fermentation at 10 h of bacterial populations includes Bifidobacterium (8.49 ± 0.44 CFU/mL), Bacteroides (9.73 ± 0.32 CFU/mL), Enterococcus (8.17 ± 0.42 CFU/mL), and Clostridium coccoides (6.15 ± 0.11 CFU/mL) as compared to the negative control counts for each bacterial group (7.94 ± 0.27, 7.84 ± 1.11, 7.52 ± 0.37, and 5.81 ± 0.08 CFU/mL, respectively) at the same time of fermentation. Likewise, the corresponding significant increase in production of SCFA in mMV-GOS at 10 h of fermentation, mainly seen in acetate (20.32 ± 2.56 mM) and propionate (9.49 ± 1.44 mM) production compared to a negative control at the same time (8.15 ± 1.97 and 1.86 ± 0.24 mM), is in line with a positive control (short-chain fructooligosaccharides; 46.74 ± 12.13 and 6.51 ± 1.91 mM, respectively) revealing a selective fermentation. In conclusion, these substrates could be considered as novel candidate prebiotic sweeteners, foreseeing a feasible and innovative approach targeting the sucrose content reduction in food. This new ingredient could provide health benefits when evaluated in human studies by combining sweetness and prebiotic fiber functionality.

Metal-catalyst-free gas-phase synthesis of long-chain hydrocarbons.

Development of sustainable processes for hydrocarbons synthesis is a fundamental challenge in chemistry since these are of unquestionable importance for the production of many essential synthetic chemicals, materials and carbon-based fuels. Current industrial processes rely on non-abundant metal catalysts, temperatures of hundreds of Celsius and pressures of tens of bars. We propose an alternative gas phase process under mild reaction conditions using only atomic carbon, molecular hydrogen and an inert carrier gas. We demonstrate that the presence of CH2 and H radicals leads to efficient C-C chain growth, producing micron-length fibres of unbranched alkanes with an average length distribution between C23-C33. Ab-initio calculations uncover a thermodynamically favourable methylene coupling process on the surface of carbonaceous nanoparticles, which is kinematically facilitated by a trap-and-release mechanism of the reactants and nanoparticles that is confirmed by a steady incompressible flow simulation. This work could lead to future alternative sustainable synthetic routes to critical alkane-based chemicals or fuels.

High-Yield Synthesis of Transglycosylated Mogrosides Improves the Flavor Profile of Monk Fruit Extract Sweeteners.

Luo Han Guo fruit extract (Siraitia grosvenorii), mainly composed of mogroside V (50%), could be considered a suitable alternative to free sugars; however, its commercial applications are limited by its unpleasant off-notes. In the present work, a central composite design method was employed to optimize the transglycosylation of a mogroside extract using cyclodextrin glucosyltransferases (CGTases) from three different bacteriological sources (Paenibacillus macerans, Geobacillus sp., and Thermoanaerobacter sp.) considering various experimental parameters such as maltodextrin and mogroside concentration, temperature, time of reaction, enzymatic activity, and pH. Product structures were determined by liquid chromatography coupled to a diode-array detector (LC-DAD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sensory analysis of glucosylated mogrosides showed an improvement in flavor attributes relevant to licorice flavor and aftereffect. Consequently, an optimum methodology was developed to produce new modified mogrosides more suitable when formulating food products as free sugar substitutes.

Transglycosylation of Steviol Glycosides and Rebaudioside A: Synthesis Optimization, Structural Analysis and Sensory Profiles.

To improve flavor profiles, three cyclodextrin glucosyltransferases (CGTases) from different bacteriological sources, Paenibacillus macerans, Geobacillus sp. and Thermoanaerobacter sp., were used with an extract of steviol glycosides (SVglys) and rebaudioside A (RebA) as acceptor substrates in two parallel sets of reactions. A central composite experimental design was employed to maximize the concentration of glucosylated species synthesized, considering temperature, pH, time of reaction, enzymatic activity, maltodextrin concentration and SVglys/RebA concentration as experimental factors, together with their interactions. Liquid chromatography coupled to a diode-array detector (LC-DAD), liquid chromatography-mass spectrometry (LC-ESI-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to characterize and identify the chemical structures obtained along the optimization. To assess the impact on the sensory properties, a sensory analysis was carried out with a group of panelists that evaluated up to 16 sensorial attributes. CGTase transglucosylation of the C-13 and/or C-19 led to the addition of up to 11 glucose units to the steviol aglycone, which meant the achievement of enhanced sensory profiles due to a diminution of bitterness and licorice appreciations. The outcome herein obtained supposes the development of new potential alternatives to replace free sugars with low-calorie sweeteners with added health benefits.

Congener-specific determination of hydroxylated polychlorinated biphenyls by polar-embedded reversed-phase liquid chromatography-tandem mass spectrometry.

This paper reports the development of an LC-ESI-MS2 method for the sensitive determination of hydroxylated polychlorinated biphenyls (OH-PCBs) in human serum samples. Congener-specific separation was achieved by using a polar-embedded stationary phase, previously optimized for the working group, which provided better separation of isobaric compounds than the common octadecylsilane phases. MS fragmentation patterns and energies showed differences among OH-PCB congeners, mainly depending on the position of OH-group and the number of chlorine atoms in the molecule, although the most intense transitions were always those corresponding to the neutral loss of an HCl group from the quasi-molecular ion cluster. The method allowed the determination of OH-PCBs with good linearity (dynamic linear range of four orders of magnitude with R2 higher than 0.995) and precision (relative standard deviations of absolute areas lower than 10%), and with better sensitivity than other similar methods previously described in the literature. Matrix effect has been evaluated and reduced to less than 10% by the addition of isotopically labeled standards and a 10-fold dilution of the final sample extract. The low iLODs provided by the developed method (from 1.2 to 5.4 fg µL-1 for all the OH-PCBs studied, except 4'-OHCB108, whose iLOD was 61 fg µL-1) allows dilution without losses of detected peaks. Finally, the applicability of the method has been demonstrated by analyzing human serum samples belonging to an interlaboratory exercise.

Evaluation of different ionic liquid stationary phases for the analysis of carbohydrates by gas chromatography-mass spectrometry.

Five commercial ionic liquid (IL) columns have been evaluated for the first time for the gas chromatography-mass spectrometry (GC-MS) analysis of low molecular weight carbohydrate (LMWC) standards (mono-, di-, and trisaccharides, inositols, and iminosugars). A previous derivatization step was necessary to convert the LMWCs into their volatile and stable derivatives. Compared with conventional GC stationary phases, such as HP-1 and Supelcowax® 10, IL columns have shown a different selectivity in the separation of target compounds. Among the IL columns, only SLB™-IL82 allowed the elution of all the LMWCs studied. Its performance in terms of peak width and asymmetry, evaluated under different oven temperature conditions, was shown to be dependent on the carbohydrate class considered. As an example of application, a SLB™-IL82 column was successfully used to separate the complex mixtures of LMWCs in hyacinth and mulberry extracts. This column is an interesting alternative to the conventional stationary phases used in the GC analysis of LMWCs in real-world samples. Graphical abstract.

Proanthocyanidin Characterization and Bioactivity of Extracts from Different Parts of Uncaria tomentosa L. (Cat's Claw).

Apart from alkaloids, bioactive properties of Uncaria tomentosa L. have been attributed to its phenolic constituents. Although there are some reports concerning low-molecular-weight polyphenols in U. tomentosa, its polymeric phenolic composition has been scarcely studied. In this study, phenolic-rich extracts from leaves, stems, bark and wood (n = 14) of Uncaria tomentosa plants from several regions of Costa Rica were obtained and analysed in respect to their proanthocyanidin profile determined by a quadrupole-time-of-flight analyser (ESI-QTOF MS). Main structural characteristics found for U. tomentosa proanthocyanidins were: (a) monomer composition, including pure procyanidins (only composed of (epi)catechin units) and propelargonidins (only composed of (epi)afzelechin units) as well as mixed proanthocyanidins; and (b) degree of polymerization, from 3 up to 11 units. In addition, U. tomentosa phenolic extracts were found to exhibit reasonable antioxidant capacity (ORAC (Oxygen Radical Absorbance Capacity) values between 1.5 and 18.8 mmol TE/g) and antimicrobial activity against potential respiratory pathogens (minimum IC50 of 133 µg/mL). There were also found to be particularly cytotoxic to gastric adenocarcinoma AGS and colon adenocarcinoma SW620 cell lines. The results state the particularities of U. tomentosa proanthocyanidins and suggest the potential value of these extracts with prospective use as functional ingredients.

Characterization of post-translationally modified peptides by hydrophilic interaction and reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry.

This work explores the use of both hydrophilic interaction liquid chromatography (HILIC) and reverse phase liquid chromatography (RPLC) for the separation and subsequent characterization of bovine caseinomacropeptide (CMP) phosphopeptides and O-glycopeptides using a quadrupole-time-of-flight (QTOF) mass spectrometer with electrospray ionization. Two neutral, ethylene bridged hybrid (BEH) amide and polyhydroxyethyl aspartamide (PHEA), and a zwitterionic, sulfobetaine (ZIC), stationary phases were used for the HILIC mode, whilst an octadecylsilane (C18) stationary phase was employed for the RPLC separation. Overall, developed HILIC-QTOF method using the ZIC or BEH amide stationary phases resulted to be the most efficient methods to separate and characterize post-translationally modified (PTM) peptides without the need of any previous fractionation or derivatization step. The separation of phosphopeptides and differently sialylated O-glycopeptides in the ZIC stationary phase was dominated by an electrostatic repulsion interaction mechanism between the negatively charged phosphate groups or sialic acid moieties and the negatively charged terminal sulfonate group of the stationary phase, whereas the separation of either non-modified peptides or neutral O-glycopeptides both free of basic amino acids was based on a partitioning mechanism. In neutral amide columns, the separation was mainly dominated by hydrophilic partitioning, leading to a higher retention of the post-translationally modified peptides than the unmodified counterparts due to the hydrophilicity provided by the phosphate groups and/or O-glycans. As a consequence, HILIC-ESI-QTOF MS operating in the positive ion mode is a powerful tool for the characterization of underivatized O-glycopeptides and phosphopeptides.

In vitro fermentation of a red wine extract by human gut microbiota: changes in microbial groups and formation of phenolic metabolites.

An in vitro batch culture fermentation experiment was conducted with fecal inocula from three healthy volunteers in the presence and absence of a red wine extract. Changes in main bacterial groups were determined by FISH during a 48 h fermentation period. The catabolism of main flavonoids (i.e., flavan-3-ols and anthocyanins) and the formation of a wide a range of phenolic microbial metabolites were determined by a targeted UPLC-PAD-ESI-TQ MS method. Statistical analysis revealed that catechol/pyrocatechol, as well as 4-hydroxy-5-(phenyl)-valeric, 3- and 4-hydroxyphenylacetic, phenylacetic, phenylpropionic, and benzoic acids, showed the greatest increases in concentration during fermentation, whereas 5-(3'-hydroxyphenyl)-γ-valerolactone, its open form 4-hydroxy-5-(3'-hydroxyphenyl)-valeric acid, and 3,4-dihydroxyphenylacetic acid represented the largest interindividual variations in the catabolism of red wine polyphenols. Despite these changes, microbial catabolism did not produce significant changes in the main bacterial groups detected, although a slight inhibition of the Clostridium histolyticum group was observed.

Behaviour of the isothermal retention indices of n-alkylbenzenes on stationary phases of different polarity.

Isothermal retention indices (I) of benzene, toluene, ethylbenzene, n-propylbenzene and n-butylbenzene were determined at 323-423 K on twelve WCOT capillary columns covering a broad stationary phase polarity spectrum. These I values have been tested carrying out a comparison with the NIST database values. The effect of the stationary phase polarity on I values was studied. But for the poly(3,3,3-trifluoropropylmethyl siloxane) column, a good linear correlation was found. At each temperature, the dependence on I of the alkyl chain length (z) attached to the ring of the n-alkylbenzenes was linear and of similar magnitude for the stationary phases of low to middle polarity, but lower for the more polar ones. Moreover, an important influence of the column temperature on the slope of the I vs. z plots was observed for the only non polysiloxane-type stationary phase studied, i.e., poly(ethylene glycol), due to its higher chain stiffness. Finally, different expressions describing the effect of the temperature on the retention index have been compared. I values of the n-alkylbenzenes in the 323-423 K range increase with increasing column temperature according to the Antoine-type (I=α+ß(γ+T)(-1)) and the extended (I=a+bT(-1)+clnT) models. No significant differences were observed between them, except for the poly[70% bis(3-cyanopropyl) 30% dimethyl silphenylene-siloxane] column, for which the Antoine-type model was slightly better.

Detection of two minor phosphorylation sites for bovine κ-casein macropeptide by reversed-phase liquid chromatography-tandem mass spectrometry.

This work addresses the characterization of phosphopeptides in bovine κ-casein macropeptide by reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry (RPLC-ESI-MS(2)). Two different mass spectrometers, equipped with an ion trap (IT) or a quadrupole time-of-flight (Q-TOF) analyzer, were used to perform an accurate phosphorylation site assignment. A total of 8 phosphopeptides from 26 identified peptides were characterized. MS(2) spectra of phosphopeptides were dominated by the neutral loss of a phosphoric acid molecule (H(3)PO(4)) and sufficient informative fragment ions resulting from peptide backbone cleavages enabling the elucidation of the phosphopeptide sequence. A higher number of sequence informative b and y ions were detected using a Q-TOF instead of an IT analyzer. In addition to the well-established phosphorylation sites at Ser(149) and Ser(127), this study also revealed the presence of two minor phosphorylation sites at Thr(145) and Ser(166). These findings indicate that RPLC-ESI-MS(2) on a Q-TOF analyzer is a useful technique for identifying low-abundance phosphorylation sites in caseins.

Solvation molar enthalpies and heat capacities of n-alkanes and n-alkylbenzenes on stationary phases of wide-ranging polarity.

A comparison of the most usual gas chromatographic methods for the calculation of partial molar enthalpies of solvation (Δ(sol)H(o)) has been carried out. Those methods based on the fitting of lnV(g) or ln(k/T) vs. 1/T and ln(k/T) vs. (1/T and the temperature arrangement, T(a)) are the most adequate ones for obtaining Δ(sol)H(o) values. However, the latter is the only reliable option for Δ(sol)H(o) estimation when commercial WCOT capillary columns are used, since in this case the estimation of some variables involved in the V(g) determination is less accurate or even impossible. Consequently, in this paper, Δ(sol)H(o) obtained from ln(k/T) vs. (1/T+T(a)) fitting at 373.15 and 298.15K for n-alkanes and n-alkylbenzenes on 12 commercial capillary columns coated with stationary phases covering the 203-3608 McReynolds polarity range are reported. Moreover, molar heat capacities of solvation at constant pressure (Δ(sol)C(p)(o)) have also been calculated using this method. A clear influence on Δ(sol)H(o) of the type and content of the substitution group in the stationary phase was observed. In addition, a linear relationship of Δ(sol)C(p)(o) with the van der Waals volume of the n-alkanes and the temperature gradient of density of the stationary phase was found. The effect of the size of the hydrocarbon on both thermodynamic variables was also investigated.

Characterization and optimization by experimental design of a liquid chromatographic method for the separation of hydroxylated polychlorinated biphenyls on a polar-embedded stationary phase.

Traditionally, the determination of hydroxylated polychlorinated biphenyls (OH-PCBs) has been carried out by gas chromatography (GC). However, the gas chromatographic behavior and sensitivity of this type of hydroxylated compounds are not always satisfactory, hence a prior derivatization of the OH-PCBs must be performed. Therefore, the development of liquid chromatographic methods should prove to be a very interesting task aimed at dealing with the instrumental determination of OH-PCBs. Taking into account that octadecylsilane stationary phases are not the most adequate for the separation of isobaric compounds, an amide-type column has been tested. For the development of the method, the Response Surface Methodology was used, based on a Box-Wilson Central Composite experimental design. The initial content of methanol in the mobile phase, the gradient time, and the concentration and the pH value of the buffer were chosen as relevant experimental parameters. A global optimum was obtained by selecting the elution time, the sensitivity and the overall resolution as responses to optimize. The developed method for liquid chromatography presented a very good resolution and sensitivity, and a reasonably short analysis time. In addition, a retention study was conducted in order to survey the different interactions that take place in the separation process, showing that hydrogen bonding is the main interaction between OH-PCBs and the amide-type stationary phase. However, a substantial contribution of dispersion forces was present in methanol contents in the mobile phase below 65%. Besides, the pH value of the mobile phase was found to be the most important parameter to control the hydrogen bond forces and therefore, to regulate the OH-PCBs separation.

Development of a new method using HILIC-tandem mass spectrometry for the characterization of O-sialoglycopeptides from proteolytically digested caseinomacropeptide.

This work addresses the optimization of HILIC (hydrophilic interaction liquid chromatography)-ESI-MS(n) conditions for the comprehensive characterization of O-glycopeptides from proteolytically digested caseinomacropeptide. O-Glycopeptides were satisfactorily analysed on a zwitterionic HILIC column based on their glycan structure and amino acid sequence. The contribution of ionic interactions to the retention of charged glycopeptides was found to be substantial. Thus, O-glycopeptides carrying neutral glycans were more retained than O-sialoglycopeptides because negatively charged sialic acid residues were electrostatically repelled by the stationary phase. In addition, glycopeptides differing only in the position of the linkage of the sialic acid moiety could be separated. The same chromatographic behaviour was observed for model systems constituted by a synthetic tetrapeptide covalently conjugated to neutral and sialylated carbohydrates. Subsequent detection of caseinomacropeptide O-glycopeptides was carried out on an electrospray ion trap tandem mass spectrometer at both positive and negative ionization modes. MS(2) fragmentation at positive ionization mode was valid for determining the glycan structure as the resulting main fragments corresponded to Y(n)-type ions derived from sequential glycosidic bond fragmentation, whilst the fragmentation of the peptide structure was preferably obtained through the formation of b(n)-type ions at the MS(3) stage, allowing the complete structure elucidation of the peptidic chain. Overall, the developed method allowed the identification and characterization of 41 O-glycopeptides covering all the known glycosylation sites without any previous enrichment step. These results point out that HILIC coupled to multistage MS procedures can be a powerful technique for future glycoproteomic applications.

Almond (Prunus dulcis (Mill.) D.A. Webb) polyphenols: from chemical characterization to targeted analysis of phenolic metabolites in humans.

In this paper, a survey of our studies on almond polyphenols including their chemical characterization and further bioavailability in humans is reported. Combination of analytical techniques (LC-DAD/fluorescence, LC/ESI-MS and MALDI-TOF-MS) allowed us, for the first time, the identification of A- and B-type procyanidin, propelargonidin and prodelphinidin polymers in almond skins. Glucuronide, O-methyl glucuronide, sulfate and O-methyl sulfate derivatives of (epi)catechin, as well as the glucuronide conjugates of naringenin and isorhamnetin, and sulfate conjugates of isorhamnetin, together with conjugates of hydroxyphenylvalerolactones were detected in plasma and urine samples after the intake of almond skin polyphenols. In addition, numerous microbial-derived metabolites, including hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, hydroxybenzoic and hydroxyhippuric acids were also identified. Depending of the type of metabolite, maximum urinary excretion was attained at different time in comparison to the control group in the course of the 24-h period of urine excretion, allowing us to establish the onset of microbial metabolism.

Selective linkage detection of O-sialoglycan isomers by negative electrospray ionization ion trap tandem mass spectrometry.

Sialylated O-linked oligosaccharides are involved in many biological processes, such as cell-cell interactions, cell-substance adhesion, and virus-host interactions. These activities depend on their structure, which is frequently determined by tandem mass spectrometry. However, these spectra are frequently analyzer-dependent, which makes it difficult to develop widely applicable analytical methods. In order to deepen the origin of this behavior, two couples of isomers of sialylated O-linked oligosaccharides, NeuAc alpha2-3Gal beta1-3GalNAc-ol/Gal beta1-3(NeuAc alpha2-6)GalNAc-ol and NeuGc alpha2-3Gal beta1-3GalNAc-ol/Gal beta1-3(NeuGc alpha2-6)GalNAc-ol, were analyzed by liquid chromatography/negative electrospray ionization ion trap tandem mass spectrometry (LC/ESI(-)-MS(n)) using both an ion trap and a triple quadrupole mass spectrometer. Results clearly showed that while ions obtained in the triple quadrupole instrument fitted very well with the standard fragmentation routes, in the ion trap several intense ions could not be explained by these rules, specially a fragment at m/z 597. Furthermore, this ion was observed in the mass spectrum of those isomers that sialic acid binds to GalNAc by an alpha2-6 linkage. From the MS(3) spectrum of this ion an unexpected structure was deduced, and it led to propose alternative fragmentation pathways. Molecular mechanics calculations suggested that the found atypical route could be promoted by a hydrogen bond located only in alpha2-6-linked oligosaccharides. It has also been demonstrated that this process follows a slow kinetic, explaining why it cannot be observed using an ion beam-type mass analyzer. In conclusion, ion traps seem to be more appropriate than triple quadrupoles to develop a reliable analytical method to distinguish between isomeric O-linked glycans.

MALDI-TOF MS analysis of plant proanthocyanidins.

Proanthocyanidins or condensed tannins are among the most abundant polyphenols compounds in our diet and may play a key role in the prevention of cardiovascular and neurodegenerative diseases and cancer. These antioxidants are widely distributed in the plant kingdom both in food plants and in non-food plants. The biological activity of plant proanthocyanidins depends on their chemical structure and concentration. However, due to their structural diversity and complexity, the qualitative and quantitative analysis of proanthocyanidins is a difficult task. Mass spectrometry has enabled great advances in the characterization of plant proanthocyanidins. Among these techniques, MALDI-TOF MS has proved to be highly suited for the analysis of highly polydisperse and heterogeneous proanthocyanidins. The objective of the present paper was to assess the potential, limitations and future challenges of the analysis of plant proanthocyanidins by MALDI-TOF MS techniques. Firstly, the fundamental of this technique, including modes of operation, advantages and limitations, as well as quantitative and qualitative operations, have been summarized. Applications of MALDI-TOF analysis to plant proanthocyanidins reported in the last decade (1997-2008) have been extensively covered, including the sample preparation protocols and conditions used for proanthocyanidin analysis, as well as the main findings regarding the determination of the structural features of different plant proanthocyanidin types (procyanidins, propelargonidins, prodelphinidins, profisetinidins and prorobinetinidins). Finally, attempts in the assessment of the molecular weight distribution of proanthocyanidins by MALDI-TOF are described.

Comparative flavan-3-ol profile and antioxidant capacity of roasted peanut, hazelnut, and almond skins.

In the present study, the flavan-3-ol composition and antioxidant capacity of roasted skins obtained from the industrial processing of three commonly consumed tree nuts (i.e., peanuts, hazelnuts, and almonds), as well as fractions containing low and high molecular weight (LMW and HMW) flavan-3-ols, were studied with the aim of assessing their potential as a source of flavonoids. Roasted peanut and hazelnut skins presented similar total phenolic contents, much higher than that of almond skins, but their flavan-3-ol profiles, as determined by LC-ESI-MS and MALDI-TOF MS, differed considerably. Peanut skins were low in monomeric flavan-3-ols (19%) in comparison to hazelnut (90%) and almond (89%) skins. On the other hand, polymeric flavan-3-ols in peanut and almond skins occurred as both A- and B-type proanthocyanidins, but in peanuts the A forms (up to DP12) were predominant, whereas in almonds the B forms (up to DP8) were more abundant. In contrast, hazelnuts were mainly constituted by B-type proanthocyanidins (up to DP9). The antioxidant capacity as determined by various methods (i.e., total antioxidant capacity, ORAC, DPPH test, and reducing power) was higher for whole extracts from roasted hazelnut and peanut skins than for almond skins; however, the antioxidant capacities of the HMW fraction of the three types of nut skins were equivalent despite their different compositions and DPs. Nevertheless, the large variation in flavan-3-ol concentration, structural composition, type of interflavan linkage, and DP found among the three types of nut skins suggests large difference in their expected in vivo biological activities.

Application of liquid chromatography-tandem mass spectrometry for the characterization of galactosylated and tagatosylated beta-lactoglobulin peptides derived from in vitro gastrointestinal digestion.

This article describes a comprehensive characterization of bovine beta-lactoglobulin peptides glycated with an aldohexose (galactose) or a ketohexose (tagatose), derived from in vitro gastrointestinal digestion, by liquid chromatography coupled to positive electrospray ion trap tandem mass spectrometry. In addition to the dissociation pathway previously described for aldohexoses-derived Amadori compounds, i.e. formation of the pyrylium ([M+H](+)-54) and furylium ions ([M+H](+)-84) via the oxonium ion ([M+H](+)-18), another and more direct fragmentation route involving the formation of the imminium ion ([M+H](+)-150) is also reported following extensive glycation rates of beta-lactoglobulin with both carbohydrates. These results indicated that the analysis of digested proteins by LC-ESI-MS(2) on a three-dimensional ion trap monitoring neutral losses is an efficient and direct method to identify peptides glycated not only through the Amadori rearrangement but also via the Heyns rearrangement. Nevertheless, as the predominating MS(2) fragmentation pattern of the glycated peptides is derived from the sugar moiety, the sequence-informative b- and y-ions resulting from peptide backbone cleavage were undetected. To overcome this drawback, and taking advantage of multi-stage fragmentation capabilities of ion traps, the indicative Amadori and Heyns-derived imminium ions were successfully used in MS(3) analyses to identify the peptide backbone, as well as the specific glycation site. In addition, further MS(4) analyses were needed to carry out the characterization of doubly glycated peptides.

Improving the sensitivity of liquid chromatography-tandem mass spectrometry analysis of hexabromocyclododecanes by chlorine adduct generation.

It is well documented and experimentally confirmed that hexabromocyclododecanes (HBCDs) tend to associate with several anions forming different adducts that can affect the sensitivity and the accuracy of the determinations. In the present work, two different approaches for HBCD determination have been optimised and characterised based on their repeatability and intermediate precision, linear calibration ranges, sensitivity, limits of detection and quantification and application to commercial food samples. Both methods involve the use of a triple quadrupole mass spectrometer coupled to a liquid chromatograph and the addition of different ammonium salts to the mobile phase, i.e. ammonium chloride or ammonium acetate, in order to encourage (Cl method) or try to inhibit (Ac method), respectively, the formation of the chlorine adducts of the molecular ion. Precision of the two methods investigated was similar and both approaches presented a comparable behaviour for the analysis of food samples. However, the Cl method showed higher sensitivity and the limits of detection (0.23-0.41 pg on column) and quantification (0.77-1.35 pg on column) were up to 14 times lower than those obtained applying the Ac method. All these facts make the Cl method the best choice for the quantification of HBCDs in food samples with low concentration levels.