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In: Középiskolai Kémiai Lapok (KöKéL) vol. 49. issue 1. ISSN 0139-3715 (print), 2498-5198 (online)

Wine quality largely depends on the phenolic compounds, which contribute to the wine colour, bitterness and astringency. Furthermore, phenolic compounds demonstrate the antioxidant potential of wines, which is mainly due to the flavonoids. In this study, phenolic composition, colour and antioxidant activity of Vitis Vinifera red wines Vranec, Merlot and Cabernet Sauvignon from three vintages, 2006, 2007 and 2008, produced in the Republic of Macedonia, have been evaluated. Separation of the individual antioxidant compounds, including monomeric anthocyanins, pyranoanthocyanins, flavonols, hydroxycinnamic acid derivatives and stilbenes has been performed using HPLC-DAD-ESI-MS/MS technique [1]. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay has been applied for the determination of the antioxidant activity of the wines and the wine colour was recorded using the CIELab uniform colour space. The major constituents belonging to the five different families found in the wines from all varieties were malvidin-3-glucoside, quercetin-3-glucuronide, trans-caftaric acid, resveratrol-3-glucoside and carboxy-pyrano-malvidin-3-glucoside (vitisin A). Comparing the wines from 2008 vintage, Vranec contained the highest amount of anthocyanins, pyranoanthocyanins, flavonols, hydroxycinnamic acid derivatives and stilbenes, followed by Merlot and Cabernet Sauvignon. A correlation of the antioxidant activity to each of the five groups of phenolic compounds was observed, confirming that the wine antioxidant potential is due to a complex mixture of phenolics.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as a new valuable technique with a great impact on wine analysis, has been used for qualitative screening of anthocyanins and derived pigments in wine samples from Vranec, Merlot and Cabernet Sauvignon varieties. MALDI-TOF-MS analysis of the wine samples was performed after solid-phase extraction of the pigments using Zip-Tip C18 stationary phase. Solution of acetonitrile/0.1% trifluoroacetic acid (1:1) was used for preconditioning and elution of pigments, while washing was made with 0.1 % trifuoroacetic acid. 2,4,5-trihydroxyacetophenone (dissolved in acetonitrile/H2O = 1/1) was used as a matrix for wine MALDI screening. MALDI-MS results confirmed the presence of glucoside, acetylglucoside and p-coumaroylglucoside derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin in the wine samples. Furthermore, pyranoanthocyanins formed by reactions of anthocyanins with pyrivic acid and acetaldehyde, as well as flavan-3-ol-anthocyanin adducts and ethyl-bridged pigments have been detected in the samples. Identification of the pigment peaks was performed on the basis of the targeted fragmentation of the ions of interest (their M+ signals) under positive ion mode.

Grape polyphenols are characterized by a large range of structures diversely distributed in every part of the berry. The knowledge of the phenolic composition of grapes and its evolution during ripening is thus of crucial importance in relation to food quality. Among the phenolic compounds, the anthocyanins are responsible for the red color of the grapes, which are located in the skins, while flavanols and catechins intervene in their taste (astringency) and bitterness. In this study, the contents of total phenolics, total flavan-3-ols and total anthocyanins were determined in the pulp, skins and seeds of two red, Vranec and Merlot, and two white grape varieties, Smederevka and Chardonnay applying standardized spectrophotometric methods. Extraction of phenolic compounds was performed with acetone/water (80/20, v/v). The obtained results showed that the concentration of total phenols and flavan-3-ols was highest in the skins and seeds of Vranec grapes, while the pulp contained low amounts of these compounds. Chardonnay seeds contained higher amounts of phenolics and flavan-3-ols compared to Smederevka grapes. HPLC-MS analysis was performed in order to identify the phenolic components present in the grape, applying mobile phase consisting of 1 % (v/v) acetic acid in water (solvent A) and 1 % (v/v) acetic acid in methanol (solvent B). The presence of 15 anthocyanins (monoglucosides, acetylgucosides and coumaroylglucosides), 7 flavonols, 5 phenolic acids and derivatives and 3 flavan-3-ols was confirmed in the skins of red grapes.

The content of total phenolics, total anthocyanins, total flavonoids and total flavan-3-ols in pulp, skins and seeds of Vranec, Merlot, Smederevka and Chardonnay grapes has been determined at different stages of berry development: veraison, technological ripeness and late harvest. Anthocyanins accumulate in the berry at the beginning of the veraison and increase during the ripening as it was observed for Vranec grapes, but also, they can decline during berry development, as observed for Merlot grapes. The content of flavan-3-ols was highest in the veraison (noticed for Vranec, Merlot and Chardonnay seeds) because their synthesis begins when the berry starts to grow up and goes on till the beginning of the veraison, followed by decreasing in the next stage of ripeness. Individual identification of the components from different phenolic groups (anthocyanins, phenolic acids and derivatives, flavonols and flavan-3-ols) present in skin, seed and pulp extracts was performed by HPLC-MS using ESI-IT-MS spectrometric data, UV spectra and retention times of the available standards, applying gradient elution with 1 % (v/v) acetic acid in the mobile phase. Malvidin-3-glucoside is the dominant component from the group of anthocyanins and among the phenolic acids and derivatives, caftaric acid dominates in grapes.

Phenolic compounds are one of the major quality parameters in the grape and thus in the resulting wine. They contribute to sensory characteristics of wines, particularly color and astringency, and possess a wide range of biochemical and pharmaceutical effects, including antioxidant, antimutagenic, antimicrobial and anticarcinogenic effects. These constituents can be divided into two groups: non-flavonoids (hydroxybenzoic and hydroxycinnamic acids and stilbens) and flavonoids (anthocyanins, favonols and flavan-3-ols). MALDI-TOF and HPLC-DAD-ESI/MS methods were developed for simultaneous identification of phenolic acids, flavonols, anthocyanins, flavan-3-ols and stilbenes in Macedonian wines and grapes. A rapid MALDI-TOF method was developed for wine and grape fingerprinting without preparation of the samples for identification of anthocyanins introducing a new matrix, fullerene C70. Different MALDI matrices were tested: -cyano-4-hydroxycinnamic acid (CHCA); 2,5-dihydroxy benzoic acid (2,5-DHB); sinapic acid (SA); fullerene (C70) and measurements of some samples without matrix were done. It was found that fullerene can be used for identification of anthocyanins in wines and grapes and the sandwich method with this matrix was applied for all sample measurements. A simple HPLC-MS method was applied for simultaneous identification of phenolic compounds present in wine and grape samples. Analyses were performed on a C18 column with gradient elution using a binary mobile phase consisting of 1 % acetic acid in water and 1 % acetic acid in methanol. Identification of the components was based on retention times, UV-Vis and mass spectra by comparison with a few commercial standards. The optimized HPLC method was used for phenolics analysis in grapes and wines from Vranec, Merlot, Chardonnay and Smederevka varieties (harvest 2007). The obtained results confirm the great utility of ESI-MS coupled with HPLC for simultaneous analysis of non-flavonoid and flavonoid compounds in wine and grape samples, since coelution is not a problem in so far as they have different molecular masses.

A rapid MALDI-TOF-MS method introducing a new matrix, fullerene [C70], has been developed for fingerprinting and for analyzing the anthocyanin content of wine and grape samples. Anthocyanins are red pigments, localized in the skin of the grapes, in the first external layers of the hypodermal tissue and exclusively in the vacuoles. They are based on five anthocyanins (cyanidin, peonidin, petunidin, delphinidin and malvidin). Those compounds are present in aglycone forms in the grapes skin and glycoside forms in the wines. Malvidin-3-glycoside is the main component responsible for the red color of the wine. The wine and grape samples have been analyzed without preparation. Different MALDI matrices have been tested: -cyano-4-hydroxycinnamic acid, 2,5-dihydroxy-benzoic acid, sinapic acid, fullerene [C70] and measurements of some samples without matrix have been done. It was found that fullerene C70 gave satisfactory results for identification of the anthocyanins. Sandwich method has been applied for measurements of grapes and wines from Vranec, Merlot, Chardonnay and Smederevka varieties (harvest 2007). The wine samples have been obtained by different technological wine-making techniques in order to check the effect of two doses of SO2, two yeasts (Saccharomyces cerevisiae, the most commonly used) from different manufactures (Macedonian yeast-Vinalko and French yeast-Levuline CHP) and the maceration time of 3, 6 and 10 days (only for red wines).