Simultaneous separation and identification of oligomeric procyanidins and anthocyanin-derived pigments in raw red wine by HPLC-UV-ESI-MSn

Samples of raw red wine (Primitivo di Manduria, Apulia, Southern Italy) were analysed without any pre-treatment (except 1:2 dilution with water) using HPLC with detection based on UV absorbance and Electrospray Ionisation Sequential Mass Spectrometry (ESI-MSn, with n = 1-3) in a series configuration. In particular, absorbance at 520 nm was monitored for UV detection in order to identify pigments responsible for wine colour. On the other hand, two subsequent stages of MS detection based on positive ions were adopted. The first consisted of an explorative MS acquisition, aimed at the individuation of the m/z ratios for positively charged compounds; the second was based on fragmentation of the detected ions within an ion trap analyser, followed by MS/MS and, if required, MS3 acquisitions. The synergy between UV detection and MSn analysis led to the identification of 41 pigments, which can be classified into five groups: grape anthocyanins, pyranoanthocyanins, vinyl-linked anthocyanin-flavanol pigments, ethyl-bridged anthocyanin-flavanol pigments and flavanol-anthocyanin compounds. Many isomeric and oligomeric structures were found within each group. A further class of compounds, not absorbing in the visible spectrum, could be also characterised by ESI-MSn and corresponded to B-type procyanidins, i.e. proanthocyanidins arising from C4-->C8/C4-->C6 couplings between catechin or epicatechin units. In particular, oligomeric structures (from dimers to pentamers), often present with several isomers, were identified and their fragmentation patterns clarified.     

Structures and colour properties of new red wine pigments

Two new red pigments were synthesized by nucleophilic addition of vinylphenols to malvidin 3-glucoside. The structures of the resulting pyranoanthocyanins were confirmed by electrospray-mass spectrometry and NMR spectroscopy (gHMQC, gHMBC and CIGAR experiments). By means of UV-vis spectroscopy the colour properties of the pigments were characterized; it could be demonstrated that the pyranoanthocyanins retained their red colour at pH 3.6 in model wine and were resistant to bisulfite-mediated bleaching. Finally, HPLC-MS analysis confirmed the presence of both anthocyanin-derived pigments in red wine.     

Analysis of sodium polycarboxylates in detergents by size exclusion chromatography

Summary This paper describes the quantitative analysis and preparative isolation of sodium polycarboxylates in detergents by means of gel permeation chromatography. An analytical monitoring method separates the polymers from other low molecular detergent ingredients within 10 minutes. There is no separation of the various molecular weight polycarboxylate macromolecules themselves. They elute from the column as a single narrow peak at the exclusion volume. A second preparative gel filtration method allows isolation of polycarboxylates in amounts necessary for further characterization. Appropriate sample pretreatments and possible interferences are discussed.     

Comparison of high-performance liquid chromatography separation of red wine anthocyanins on a mixed-mode ion-exchange reversed-phase and on a reversed-phase column

Anthocyanins, which confer the characteristic color to red wine, can be used as markers to classify wines according to the grape variety. It is a complex separation that requires very high chromatographic efficiency, especially in the case of aged red wines, due to the formation of pyranoanthocyanins. A coelution between these kinds of compounds can affect the R_ac/coum ratio of aged wines, and might lead to false results when classifying the wine variety. In 2007, the use of a novel mixed-mode ion-exchange reversed-phase column was reported to separate anthocyanins extracted from grapes of Vitis labrusca with different selectivity than C-18 columns. In the present work, the separation of anthocyanins including pyranoanthocyanins in young and aged Cabernet Sauvignon wines and other varieties is evaluated. The most interesting contributions of this research are the different elution order and selectivity obtained for anthocyanins and pyranoanthocyanins (only formed in wine), compared with those observed in C-18 stationary phases. Also interesting is the separation of the polymeric fraction, which elutes as a clearly separated peak at the chromatogram's end. However, a comparison with a high efficiency C-18 column with the same dimensions and particle size demonstrated that the tested mixed-mode column shows broader peaks with a theoretical plate number below 8000, for malvidin-3-glucoside peak, while it can be up to 10 times higher for a high efficiency C-18 column, depending on the column manufacturer. Under the tested conditions, in mixed-mode phase, the analysis time is almost twice that of a C-18 column with the same dimensions and particle size. A mixed-mode phase with increased efficiency should provide an interesting perspective for separation of anthocyanins in wine, due to its improved selectivity, combined with a useful role in a second-dimension separation in preparative anthocyanin chromatography.     

Separation of pyranoanthocyanins from red wine by column chromatography

With the aim of monitoring the formation of anthocyanin-derived pigments and contributing to the study of their chromatic properties, stability and relative contribution to the colour of red wines, a method for fractionation of the colouring material was set up. The method was based on the distinct reactivity of the different pigment families towards bisulfite (hydrogen sulfite). The wine, acidified and bleached with NaHSO₃, was placed in a Toyopearl^® HW-40(s) gel column and submitted to elution with ethanol. Two fractions with different pigment compositions were collected and analysed by liquid chromatographay diode array detection-mass spectrometry. Compounds present in each fraction were identified according to their UV-visible and MSⁿ mass spectra, showing that the first one was mostly constituted of pyranoanthocyanins, whereas the second basically contained anthocyanins and anthocyanin-flavanol condensation products. A large variety of new pigments were detected, some of which had not been previously reported in red wines, as far as we know. Characteristic MS² and MS³ fragmentation patterns were observed within each family of compounds, which could be further applied for characterisation of unknown pigments in other wines.     

Preparative Purification and Isolation of Pyranoanthocyanins from Red Wine and Evaluation of Their Antioxidant Activity

Untreated silica gel was used as the stationary phase for a simple and cost-effective prepurification of 5-carboxypyranoanthocyanins from red wine enriched by the addition of pyruvic acid. The composition of the fractions obtained was analyzed by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC/MS²) and the results enabled the changes in 5-carboxypyranoanthocyanin profile during preparative separation to be described and explained. Repeated purification on silica gel followed by reversed-phase separation enabled the isolation of 5-carboxypyranomalvidin-3-glucoside in high purity and represents a cheap and robust alternative to the commonly used procedures. Reversed-phase preparative chromatography with an isocratic elution [15 % acetonitrile in water (v/v)] provided sufficient resolution, showing that the addition of an acid is not necessary in anthocyanin separation. The antioxidant capacity of 5-carboxypyranomalvidin-3-glucoside was compared with malvidin-3-glucoside and a group of common polyphenols (e.g., gallic acid, quercetin, rutin, and catechin).     

Effect of oxygenation on polyphenol changes occurring in the course of wine-making

The influence of controlled oxygenation on the colour and phenolic composition of red wine was studied by UV-VIS spectrophotometry, liquid chromatography (LC) coupled to diode array detection (DAD) and electrospray ionisation mass spectrometry, and thiolysis. The comparison between the control and oxygenated wines demonstrated changes in colour characteristics along with a significant increase in concentrations of pyranoanthocyanins, ethyl-bridged compounds and derived pigments both with storage time and with oxidation. Principal component analysis was applied to wine analysis data measured throughout the conservation period. The effect of the storage time and oxygenation was clearly reflected in this analysis.Mass-spectrometric analysis of the wines demonstrated the presence of compounds which are markers of reactions involving acetaldehyde. Two types of mechanisms were observed. The first concerns acetaldehyde condensation reactions and the second, the cycloaddition process between anthocyanins and flavanols mediated by acetaldehyde, generating tannin-pyranoanthocyanins.The presence in wines of trimeric structures resulting from these mechanisms, as well as the results obtained after thiolysis of the fraction containing polymeric species obtained by Fractogel chromatography, confirm that proanthocyanidins react with acetaldehyde in the same way as flavanol monomers.     

A new class of anthocyanin‐procyanidin condensation products detected in red wine by electrospray ionization multi‐stage mass spectrometry analysis

In our previous work, we have identified, in a model wine solution containing malvidin 3‐glucoside, epicatechin and acetaldehyde, a new condensation product – hydroxylethyl‐malvidin‐3‐glucoside‐ethyl‐epicatechin. The objective of this work was to verify the presence of such new condensation products in red wine. For this purpose, red wine was fractionated into various fractions by column chromatography on LiChroprep RP 18 and on Toyopearl 40 (F). The phenolic composition of each fraction was verified by HPLC‐DAD and direct‐infusion ESI‐MSⁿ analysis. In addition to the well‐known anthocyanins and their acetyl and coumaroyl derivatives, and several direct and indirect anthocyanin‐(epi)catechin condensation products, a new class of pigmented products, namely hydroxyethyl‐anthocyanin‐ethyl‐flavanol compounds, have been detected in red wine. The new class of pigmented products would be expected to be the major pigments responsible for the color of aged red wine. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of new analytical methods for selenium speciation in selenium-enriched yeast material

A sequential extraction allowing the discrimination of water-soluble and non-soluble selenium fractions has been developed to evaluate the availability of selenium (Se) in an Se-enriched yeast candidate reference material. The fractionation of selenium-containing compounds in the extracts was achieved on preparative grade 200 Superdex 75 and columns. It showed that water-soluble selenium is present in several fractions with a large mass distribution. Low-molecular- (< or = 10,000) and high-molecular-mass selenocompounds (range 10,000-100,000) were considered separately for further experiments. The analytical approach for low-molecular-mass selenocompounds was based onanion-exchange HPLC with on-line inductively coupled plasma (ICP) MS for quantitative analysis. Selenocystine, selenomethionine, selenite and selenate were quantified in the fractions isolated in preparative chromatography. The study revealed the existence of various unidentified Se species in yeast material. The Se-containing proteins in the yeast material have been further separated and selenium quantified by the combination of gel electrophoresis and electrothermal vaporization-ICP-MS. This new approach allows the separation of the proteins with high resolution by sodium dodecylsulfate-polyacrylamide gel electrophoresis and the sensitive determination of selenium in the protein bands.     

Methods of analysis for anthocyanins in plants and biological fluids

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. They are responsible for most of the red, blue, and purple colors of fruits, vegetables, flowers, and other plant tissues or products. The analysis of anthocyanins is complex as a result of their ability to undergo structural transformations and complexation reactions. In addition, they are difficult to measure independently of other flavonoids, as they have similar structural and reactivity characteristics. Anthocyanins are generally extracted with weakly acidified alcohol-based solvents, followed by concentration (under vacuum), and purification of the pigments. Paper and/or thin-layer chromatography and UV-Vis spectroscopy have traditionally been used for the identification of anthocyanins. Capillary zone electrophoresis, a hybrid of chromatography and electrophoresis, is gaining popularity for the analysis of anthocyanins; however, liquid chromatography (LC) has become the standard method for identification and separation in most laboratories and may be used for both preparative and quantitative analysis. LC with mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are possibly the most powerful methods for the structural elucidation of anthocyanins available, to date. At present, the most satisfactory method for mixture analysis is the multistep method of separation, isolation, and quantification by LC with peak identification by MS and high-field NMR.     

The role of planar chromatography in medicinal plant research

This paper summarizes the role of planar chromatography (PC) in medicinal and aromatic plant (MAP) research and development, and demonstrates the importance of the technique, after extraction, in the analysis of MAP (identification and quantitative determination of the separated compound/s), in the purification and isolation process, and in different types of screening procedure. Special attention is paid to analytical, micropreparative and preparative forced-flow techniques, for example overpressured-layer chromatography (OPLC) and rotation planar chromatography (RPC). The special features of analytical, micropreparative, and preparative layer chromatography (PLC), OPLC, and RPC are compared in tables. Purification and isolation procedures using forced-flow techniques are shown in flowcharts. Some applications, relating to different classes of substance, are presented to demonstrate the versatility of various planar chromatographic techniques.     

Continuous purification of a clotting factor IX concentrate and continuous regeneration by preparative annular chromatography

Preparative continuous annular chromatography, a method to separate proteins in a truly continuous manner, was investigated in an industrial environment. Plasma-derived clotting factor IX concentrate was used as model protein. Separation of vitronectin, a common impurity in commercial available factor IX concentrates, from factor IX was studied and compared to conventional packed bed chromatography in batch mode. As sorbent, Toyopearl DEAE 650M was used. Regeneration was performed simultaneously with the purification of factor IX in continuous mode. All required parameters applied for preparative annular chromatography such as feed flow-rate and elution flow-rate were first estimated from experiments on conventional batch columns. Then preparative annular chromatography and conventional packed beds were compared regarding enrichment, purity and productivity. Three different process scenarios, the optimal batch process,the preparative annular chromatography process and the batch process equivalent to the preparative annular chromatography process were investigated. The productivity of the optimal batch process was higher than that of the preparative annular chromatography and batch process equivalent to the preparative annular chromatography process. Therefore the throughput could not be increased by the use of the continuous chromatographic system.     

Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases

An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 degrees C.     

Tentative identification of polar and mid‐polar compounds in extracts from wine lees by liquid chromatography–tandem mass spectrometry in high‐resolution mode

Sustainable agriculture has a pending goal in the revalorization of agrofood residues. Wine lees are an abundant residue in the oenological industry. This residue, so far, has been used to obtain tartaric acid or pigments but not for being qualitatively characterized as a source of polar and mid‐polar compounds such as flavonoids, phenols and essential amino acids. Lees extracts from 11 Spanish wineries have been analyzed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in high resolution mode. The high‐resolution power of LC–MS/MS has led to the tentative identification of the most representative compounds present in wine lees, comprising primary amino acids, anthocyans, flavanols, flavonols, flavones and non‐flavonoid phenolic compounds, among others. Attending to the profile and content of polar and mid‐polar compounds in wine lees, this study underlines the potential of wine lees as an exploitable source to isolate interesting compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Analytical and preparative native polyacrylamide gel electrophoresis: Investigation of the recombinant and natural major grass pollen allergen Phl p 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot are amongst the most popular methods for allergen characterization, such as comparison of recombinant allergens with their natural counterparts. Native PAGE was evaluated as a possible robust and simple method offering high-resolution capacity for characterization of the major grass pollen allergen Phl p 2. Analytical separation of recombinant Phl p 2 provided a superior quality control in terms of homogeneity and, after Western blotting, immunoglobulin E (IgE) reactivity. Separation of natural Phl p 2 identified two major isoforms which were shown to have different N-terminal sequences and IgE-binding properties. After isolation using preparative native PAGE in combination with electrodialysis, both isoforms were investigated by specific proteolysis and reversed-phase high-performance liquid chromatography (RP-HPLC). The results demonstrate differences in the primary structures and that the recombinant counterpart corresponds exactly to one isoform. Analytical and preparative native PAGE thus proved to be powerful tools for the investigation of allergen isoforms and quality control of recombinant counterparts.     

Liquid chromatographic fractionation of small peptides from wine

Peptides are difficult to isolate from wine because they are present in a complex mixture together with non-peptidic compounds. A method for the isolation, separation and purity assessing of small peptides is proposed. Small peptides (Mr<3000) were isolated from wine by hollow fibre ultrafiltration followed by column chromatography using the gel matrix Sephadex LH20. Fractions obtained by gel filtration on Sephadex LH20 were subjected to HPLC on a porous graphitic carbon column in order to isolate small peptides. Peak purity was then analysed by capillary electrophoresis.     

The Management of Dissolved Oxygen by a Polypropylene Hollow Fiber Membrane Contactor Affects Wine Aging

Background: Numerous oenological practices can cause an excess of dissolved oxygen in wine, thus determining sensory and chromatic defects in the short- to long-term. Hence, it is necessary to manage the excess of oxygen before bottling. Methods: In this study, the management of the dissolved oxygen content by a polypropylene hollow fiber membrane contactor apparatus was performed in two wines from different grape varieties (Aglianico and Falanghina). The wines were analyzed after an 11-month aging. Anthocyanins and acetaldehyde content were evaluated by HPLC. In addition, other phenolic compounds and chromatic characteristics were analyzed by spectrophotometric methods. NMR and HR ESIMS analyses were conducted to evaluate the amount of pyranoanthocyanins and polymeric pigments. Results: After 11 months of aging, in both wines a decrease of free and total SO₂ with respect to initial values was detected. In the wines with the highest dissolved oxygen levels, a more remarkable loss was observed. No significant differences in terms of color parameters were detected. In red wine with the highest oxygen content, a massive formation of polymeric pigments and BSA reactive tannins was observed, as opposed to wines with lower oxygen levels. Conclusion: The study demonstrated that the membrane contactor can prove a successful tool to manage dissolved oxygen in wines as to prevent their oxidative spoilage.     

Analysis of polymeric phenolics in red wines using different techniques combined with gel permeation chromatography fractionation

A multiple-step analytical method was developed to improve the analysis of polymeric phenolics in red wines. With a common initial step based on the fractionation of wine phenolics by gel permeation chromatography (GPC), different analytical techniques were used: high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-mass spectrometry (MS), capillary zone electrophoresis (CZE) and spectrophotometry. This method proved to be valid for analyzing different families of phenolic compounds, such as monomeric phenolics and their derivatives, polymeric pigments and proanthocyanidins. The analytical characteristics of fractionation by GPC were studied and the method was fully validated, yielding satisfactory statistical results. GPC fractionation substantially improved the analysis of polymeric pigments by CZE, in terms of response, repeatability and reproducibility. It also represented an improvement in the traditional vanillin assay used for proanthocyanidin (PA) quantification. Astringent proanthocyanidins were also analyzed using a simple combined method that allowed these compounds, for which only general indexes were available, to be quantified.     

Preparative scale purification of shidasterone, 2-deoxy-polypodine b and 9a,20-dihydroxyecdysone from Silene italica ssp. nemoralis

A suitable combination of preparative scale separation methods results in effective clean-up of the ecdysteroids of Silene italica ssp. nemoralis (Waldst. and Kit.) Nyman. The isolation of minor ecdysteroids from the partially purified extract is based on the use of both droplet counter-current chromatography and low-pressure reversed-phase liquid chromatography. The purification is completed by preparative thin-layer chromatography and preparative high-performance liquid chromatography to obtain the minor ecdysteroids, such as 2-deoxy-20-hydroxyecdysone, shidasterone, 2-deoxy-polypodine B, makisterone C, and 9alpha,20-dihydroxyecdysone.