As wine oxidizes, ethanol is converted to acetaldehyde, but its accumulation is not predictable, due to poorly characterized reactions with alcohols, SO2, thiols, flavanols, and others. Measurement of these components has been thwarted by equilibria into the other forms during sample preparation. NMR spectra can be taken on intact samples and is thus ideal for this situation. Equilibria of acetaldehyde with glycerol, (+)-catechin, and glutathione were studied separately in model wine solutions at pH 3–4 by 1H NMR and 2D (1H–1H) COSY spectra. Glycerol acetals had equilibrium constants between 1.14 ± 0.056 and 2.53 ± 0.043 M–1, whereas ethylidene-bridged (+)-catechin dimers and glutathione thiohemiacetals had more favorable equilibria: from (3.92 ± 0.13) × 103 to (6.13 ± 0.32) × 103 M–2 and from 10.18 ± 0.22 to 11.17 ± 0.47 M–1, respectively. These data can be used to create accurate measures of acetaldehyde in its various forms and, consequently, offer insight into wine oxidation.Keywords: (+)-catechin; acetaldehyde; equilibrium constants; glutathione; glycerol;

Recent developments that have accelerated 2D NMR methods and improved quantitation have made these methods accessible analytical procedures, and the large signal dispersion allows for the analysis of complex samples. Few natural samples are as complex as wine, so the application to challenges in wine analysis look promising. The analysis of carbonyl compounds in wine, key oxidation products, is complicated by a multitude of kinetically reversible adducts, such as acetals and sulfonates, so that sample preparation steps can generate complex interferences. These challenges could be overcome if the compounds could be quantified in situ. Here, two-dimensional (1H–1H) homonuclear and heteronuclear (13C–1H) single quantum correlations (correlation spectroscopy, COSY, and heteronuclear single quantum coherence, HSQC) nuclear magnetic resonance spectra of undiluted wine samples were observed at natural abundance. These techniques achieve simultaneous direct identification and quantitation of acetaldehyde, pyruvic acid, acetoin, methylglyoxal, and α-ketoglutaric acid in wine with only a small addition of D2O. It was also possible to observe and sometimes quantify the sulfite, hydrate, and acetal forms of the carbonyl compounds. The accuracy of the method was tested in wine samples by spiking with a mixture of all analytes at different concentrations. The method was applied to 15 wine samples of various vintages and grape varieties. The application of this method could provide a powerful tool to better understand the development, evolution, and perception of wine oxidation and insight into the impact of these sulfite bound carbonyls on antimicrobial and antioxidant action by SO2.

•We combined 13C isotopic labelling and LC-HRMS based metabolomics.•Phenylalanine-derived compounds in grapes were filtered using untargeted profiling.•Thermal degradation products of known and unknown berry metabolites were detected.•Warm temperature (45 °C) treatment decreased anthocyanins as expected.•Warm temperature produced 5 metabolites that may be anthocyanin degradation products.Anthocyanin degradation has been proposed as one of the primary causes for reduced colour and quality in red wine grapes grown in a warm climate. To study anthocyanin degradation we infused berries with l-phenyl-13C6-alanine and then tracked the fate of the anthocyanins comparing normal (25 °C) and warm (45 °C) temperature conditions. An untargeted metabolomics approach was aided by filtering the MS data using software algorithms to extract all M and M+6 isotopic peak pairs, allowing the analysis to focus solely on the metabolites of phenylalanine. A paired-comparison t-test was performed over the 8 biological replicates revealing 13 metabolites that were statistically different between 25 °C and 45 °C treatments. Most of these features had lower abundances in 45 °C samples, confirming that 45 °C treatment caused anthocyanin degradation. In addition, resveratrol was significantly reduced following heat treatment. However, 5 metabolites increased following the 45 °C treatment. These unidentified metabolites are therefore suspects for anthocyanin degradation products.

Heterocyclic acetals from acetaldehyde and glycerol have been proposed as markers of age and oxidation in fortified wines. Here the analysis of these acetals (cis- and trans-5-hydroxy-2-methyl-1,3-dioxane and cis- and trans-4-hydroxymethyl-2-methyl-1,3-dioxolane) by liquid–liquid extraction and gas chromatography–mass spectrometry was optimized for rapid monitoring of these compounds in aged and oxidized table wines. The method provides a wide range of linearity and reproducibility below 3.0% RSD, while the detection limits, 2.85–13.5 μg/L, were significantly lower than typical levels measured in wines. Stable isotope dilution improved the accuracy of the method. Oxidation of red wines was shown to correlate with increased levels of the acetals. The accuracy and speed of the new method will allow for quantifying oxidation in large experiments such as wine aging or micro-oxygenation trials, and to assess the potential value of these substances as markers of wine oxidation.

•SO2 bound carbonyls can be released with acid instead of base.•Acid treatment combines derivatization with DNPH, reducing complexity.•Heat treatment accelerates the process.•Multiple carbonyls are quantified as the hydrazones by HPLC.•This approach is a new, rapid and simpler method to quantify wine carbonyls.Carbonyl compounds are produced during fermentation and chemical oxidation during wine making and aging, and they are important to wine flavor and color stability. Since wine also contains these compounds as α-hydroxysulfonates as a result of their reaction with sulfur dioxide, an alkaline pre-treatment requiring oxygen exclusion has been used to release these bound carbonyls for analysis. By modifying the method to hydrolyze the hydroxysulfonates with heating and acid in the presence of 2,4-dinitrophenylhydrazine (DNPH), the carbonyl compounds are simultaneously and quickly released and derivatized, resulting in a simpler and more rapid method. In addition, the method avoids air exclusion complications during hydrolysis by the addition of sulfur dioxide. The method was optimized for temperature, reaction time, and the concentrations of DNPH, sulfur dioxide and acid. The hydrazones were shown to be stable for 10 h, adequate time for chromatographic analysis by HPLC-DAD/MS. This method is demonstrated for 2-ketoglutaric acid, pyruvic acid, acetoin and acetaldehyde, wine carbonyls of very different reactivities, and it offers good specificity, high recovery and low limits of detection. This new rapid, simple method is demonstrated for the measurement of carbonyl compounds in a range of wines of different ages and grape varieties.

•Competitive quinone reactions between antioxidants and varietal aroma were studied.•Ascorbate, sulphite and glutathione react quickly and should prevent 3-SH loss.•Quinone reduction and addition products were characterised.•Confirmation of these relative rates needed in wine.Quinones are central intermediates in wine oxidation that can degrade the quality of wine by reactions with varietal thiols, such as 3-sulfanylhexanol, decreasing desirable aroma. Protection by wine preservatives (sulphur dioxide, glutathione, ascorbic acid and model tannin, phloroglucinol) was assessed by competitive sacrificial reactions with 4-methyl-1,2-benzoquinone, quantifying products and ratios by HPLC–UV–MS. Regioselectivity was assessed by product isolation and identification by NMR spectroscopy. Nucleophilic addition reactions compete with two electron reduction of quinones by sulphur dioxide or ascorbic acid, and both routes serve as effective quenching pathways, but minor secondary products from coupled redox reactions between the products and reactants are also observed. The wine preservatives were all highly reactive and thus all very protective against 3-sulfanylhexanol loss to the quinone, but showed only additive antioxidant effects. Confirmation of these reaction rates and pathways in wine is needed to assess the actual protective action of each tested preservative.

Current evidence shows that monomeric flavonoids are known to be only slightly absorbed in the small intestine, but the metabolism of oligomeric and polymeric proanthocyanidins (PAC) in the colon is poorly understood. The objective of this study was to optimize the analysis of grape seed extract (GSE) in feces and use that method to assess the presence of PAC in the colon after ingestion of GSE. Rats were fed a diet ad libitum containing 0.25% (w/w) GSE for 10 days. Feces were collected daily and colonic contents at sacrifice on day 10, respectively. The recovery of fecal PAC using a solid-phase extraction (SPE) method was >70%. PAC were separated by normal-phase HPLC with fluorescence detection, and subsequent peak confirmation was done by MS-ion trap. The concentration of colonic contents at day 10 was 13.9 mg/kg for monomer, and those for oligomers (dimers–hexamers) were 33.4, 84.6, 87.2, 57.3, and 35.7 mg/kg, respectively. The concentration of monomeric and oligomeric PAC in daily feces was similar among days. In the mass balance analysis, approximately 11% of ingested PAC was recovered in the feces. These findings indicate that ingested PAC were present in the colon as the intact parent compounds and thus may contribute to the health of the gastrointestinal tract.

Phenolic compounds in Vitis vinifera contribute important flavor, functionality, and health qualities to both table and wine grapes. The plant phenolic metabolic pathway has been well characterized, however many important questions remain regarding the influence of environmental conditions on pathway regulation. As a diagnostic for this pathway's regulation, we present a technique to incorporate a stable-isotopic tracer, l-phenyl-13C6-alanine (Phe13), into grape berries in situ and the accompanying high throughput analytical method based on LC–DAD–MS/MS to quantify and track the label into phenylalanine metabolites. Clusters of V. vinifera cv. Cabernet Sauvignon, either near the onset of ripening or 4 weeks later, were exposed to Phe13 in the vineyard. Phe13 was present in berries 9 days afterwards as well as labeled flavonols and anthocyanins, all of which possessed a molecular ion shift of 6 amu. However, nearly all the label was found in anthocyanins, indicating tight regulation of phenolic biosynthesis at this stage of maturity. This method provides a framework for examining the regulation of phenolic metabolism at different stages of maturity or under different environmental conditions. Additionally, this technique could serve as a tool to further probe the metabolism/catabolism of grape phenolics.Graphical abstractHighlights► l-Phenyl-13C6-alanine was incorporated into grape berries, intact on the vine, at two stages of maturity. ► Labeled anthocyanins were synthesized by the berry and quantified by LC–UV/Vis–MS/MS. ► Extremely tight regulation of phenylpropanoid pathways was observed.

Quinones are key reactive electrophilic oxidation intermediates in wine. To address this question, the model 4-methyl-1,2-benzoquinone was prepared to study how it reacts with wine nucleophiles. Those investigated included the varietal volatile thiols 4-methyl-4-sulfanylpentan-2-one (4MSP), 3-sulfanylhexan-1-ol (3SH), and 2-furanmethanethiol (2FMT); hydrogen sulfide (H2S); glutathione (GSH); sulfur dioxide; ascorbic acid (AA); and the amino acids methionine (Met) and phenylalanine (Phe) in the first kinetic study of these reactions. Products were observed in fair to quantitative yields, but yields were negligible for the amino acids. The reaction rates of 4-methyl-1,2-benzoquinone toward the nucleophiles were quantified by UV–vis spectrometry monitoring the loss of the quinone chromophore. The observed reaction rates spanned three orders of magnitude, from the unreactive amino acids (Met and Phe) (KNu = 0.0002 s–1) to the most reactive nucleophile, hydrogen sulfide (KH2S = 0.4188 s–1). Analysis of the kinetic data showed three categories. The first group consisted of the amino acids (Met and Phe) having rates of essentially zero. Next, phloroglucinol has a low rate (KPhl = 0.0064 s–1). The next group of compounds includes the volatile thiols having increasing reactions rates K as steric inhibition declined (K4MSP = 0.0060 s–1, K3SH = 0.0578 s–1, and K2FMT = 0.0837 s–1). These volatile thiols (4MSP, 3SH, 2FMT), important for varietal aromas, showed lower K values than those of the third group, the wine antioxidant compounds (SO2, GSH, AA) and H2S (KNu = 0.3343–0.4188 s–1). The characterization of the reaction products between the nucleophiles and 4-methyl-1,2-benzoquinone was performed by using HPLC with high-resolution MS analysis. This study presents the first evidence that the antioxidant compounds, H2S, and wine flavanols could react preferentially with oxidation-induced quinones under specific conditions, providing insight into a mechanism for their protective effect.

Plant-derived polyphenolic compounds have received much attention for their ability to sequester high -energy free radicals in a great variety of food-related and biological systems, protecting those systems from oxidative change. The ability of these compounds to scavenge free radicals has always been attributed to their phenolic functionality, from which a hydrogen atom can be easily abstracted. In this study, the cinnamates and the ubiquitous hydroxycinnamates were found to equally suppress the formation of oxidation products in wine exposed to the Fenton reaction (catalytic Fe(II) with hydrogen peroxide). Mechanistic investigations led to the unexpected discovery that the α,β-unsaturated side chain of cinnamic acids could efficiently trap 1-hydroxyethyl radicals, representing a newly discovered mode of antioxidant radical scavenging activity for these broadly occurring compounds in a food system. The proposed pathway is supported by prior fundamental studies with radiolytically generated radicals.

Protein oxidation in meat is considered to decrease meat tenderness due to protein disulfide cross-link formation of thiol-containing amino acid residues. An LC-MS method for detection of thiol–quinone adducts (RS–QH2) in myofibrillar proteins was developed to investigate the interaction between phenols, as protective antioxidants, and proteins from meat under oxidative conditions using aqueous solutions of (i) cysteine (Cys), (ii) glutathione (GSH), (iii) bovine serum albumin (BSA), or (iv) a myofibrillar protein isolate (MPI). The aqueous solutions were incubated at room temperature (30 min) with 4-methyl-1,2-benzoquinone (4MBQ) prepared from oxidation of 4-methylcatechol (4MC) by periodate resin or incubated at room temperature (5 h) with 4MC and Fe(II)/H2O2. GSH, BSA, and MPI were hydrolyzed (6 N HCl, 110 °C, 22 h) after incubation, and the cysteine–quinone adduct, Cys–QH2 (m/z 244.2) was identified according to UV and mass spectra after separation on an RP-C18 column. The thiol–quinone adduct was present in all thiol systems after incubation with 4MBQ or 4MC oxidized by Fe(II)/H2O2. Direct reaction with 4MBQ resulted in each case in increased Cys–QH2 formation compared to simultaneous oxidation of thiol source and 4MC with Fe(II)/H2O2. The covalent bonds between quinones and thiol groups may act as a potential antioxidant by inhibiting disulfide protein cross-link formation.

The fate of hydrogen peroxide in a model wine system was studied under a competitive scenario in the presence of ferrous ions and sulfur dioxide. The metal-catalyzed reduction of hydrogen peroxide (H2O2), referred to as the Fenton reaction, yields hydroxyl radicals capable of oxidizing ethanol to acetaldehyde and is now thought to be a key step in nonenzymatic wine oxidation. It appears that sulfur dioxide (SO2) exerts its protective function in wine by scavenging hydrogen peroxide in oxidizing wine, thereby diverting peroxide from the Fenton route. In this study, the factors affecting the rate and outcome of hydroxyl radical-mediated ethanol oxidation were examined under wine conditions. The exclusion of oxygen in the model wine led to conditions wherein ferric ions (50 μM) were rapidly reduced, presumably by 1-hydroxyethyl radicals. This resulted in the complete stoichiometric conversion of H2O2 (300 μM) to hydroxyl radicals, giving an equimolar concentration of acetaldehyde (∼300 μM). Surprisingly, the yield of acetaldehyde was markedly depressed in the presence of oxygen. The addition of a model phenol, 4-methylcatechol (4-MeC; 12 mM), did not protect the ethanol from hydroxyl radical-mediated oxidation under the conditions tested but rather appeared to slightly increase the rate of the Fenton reaction, perhaps by forming a complex with the added iron. The competition for H2O2 in the presence of Fe(II) ions and SO2 was also examined, and the effect of added 4-MeC, as well as dissolved oxygen, was investigated. Higher concentrations of 1-hydroxyethyl radicals, which were trapped by N-tert-butyl-α-phenylnitrone (PBN) and detected by electron paramagnetic resonance spectroscopy, were observed when oxygen was excluded and when 4-MeC was included.

Gut microflora metabolize anthocyanins to phenolic acids and aldehydes. These metabolites may explain the relationship between anthocyanin consumption and reduced incidence of colon cancer. Here, all six major metabolites, along with a Cabernet Sauvignon anthocyanin extract, were incubated with Caco-2 cells at concentrations of 0−1000 μM over 72 h to determine effects on cell proliferation and for 24 h to assess cytotoxicity effects and at 140 μM for 24 h to measure induction of apoptosis. These measurements were based on colorimetric methods. Gallic acid and 3-O-methylgallic acid inhibited cell proliferation and lacked cytotoxicity at low concentrations. The aldehyde metabolite and anthocyanin extract also inhibited cell proliferation at low concentrations and had low cytotoxicity at a wide range of concentrations. Of the four substances that effectively reduced cell proliferation, the aldehyde was the best inducer of apoptosis. In addition, these same four treatments degraded quickly in growth media, suggesting the involvement of subsequent oxidation products in the reduction of cell viability. These results indicate that the anthocyanin microfloral metabolites gallic acid, 3-O-methylgallic acid, and 2,4,6-trihydroxybenzaldehyde reduce cell proliferation in Caco-2 cells more effectively than anthocyanins and may offer protection against colon cancer after their formation in the gut.

Free radicals are thought to be key intermediates in the oxidation of wine, but their nature has not been established. Electron paramagnetic resonance spectroscopy was used to detect and identify several free radical species in wine under oxidative conditions with the aid of spin traps. The 1-hydroxylethyl radical was the sole radical species observed when α-(4-pyridyl-1-oxide)-N-tert-butylnitrone was used as a spin trap in a heated (55 °C), low-sulfite (15 mg L−1) red wine. This radical appears to arise from ethanol oxidation via the hydroxyl radical, and this latter species was confirmed by using a high concentration (1.5 M) of the 5,5-dimethylpyrroline-N-oxide spin trap, thus providing the first direct evidence of the Fenton reaction in wine. Hydroxyl radical formation in wine was corroborated by converting hydroxyl radicals to methyl radicals by its reaction with dimethyl sulfoxide. The novel spin trap 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide was also used in this study to identify sulfite radicals in wine for the first time. This spin trap has also been shown to trap hydroperoxyl radicals, the generation of which is predicted in wine; however, no evidence of this species was observed.

The quantity and characterization of extracted flavan-3-ol monomers and procyanidins was determined in seeds from Vitis vinifera cv. Cabernet Sauvignon berries, over the course of ripening and at different levels of vine water status. The per berry extractive yield of all polyphenols decreased with maturity, and followed second-order kinetics. The flavan-3-ol monomers decreased most rapidly, followed by the procyanidin extension units and finally, the terminal units. The relative proportion of procyanidin extension units did not vary with maturity. During fruit ripening, the mean degree of polymerization of extracted procyanidins is unchanged when analyzed intact by HPLC, but decreases by thiolytic degradation. The proportion of extracted procyanidins resistant to acid catalyzed thiolysis increased with maturity. Changes in vine water status affected polyphenol amounts, indicating that cultural practices can be used to influence composition. Oxidation of the seed polyphenols during fruit ripening, could explain these observations.