Development of methods for identification and quantification of phenolic compounds in wine and grape using spectrophotometry, liquid chromatography and mass spectrometry

Spectrohotometric assays of total phenolics, total anthocyanins, total catechins, total flavonoids, color intesity and hue were performed on twenty four red Vranec and Merlot wines obtained with 3, 6 and 10 days of maceration, containing 30 and 70 mg/L SO2, and eight white Smederevka and Charodonnay wines, with 50 and 100 mg/L SO2. Macedonian yeast, Vinalco and French yeast Levuline were used for fermentation for the red and white wines. Changes of phenolic contents were observed during four stages of storage of the wines: after maceration (for the red wines) or after fermentation (for the white wines), after 2, 6 and 16 months in order to check the effect of maceration time (for red wines), SO2 and time of wine storage. Wines were stored at low and higher temperature to check also the influence of storage temperature on the studied parameters. Folin-Ciocalteu method was used for total phenolics determination, p-DMACA reagent was used for measurements of total falvan-3-ols in the wines and colorimetric method with aluminium chloride was applied for determination of flavonoids. It was found that maceration and storage time and SO2 influence the content of antocyanins, phenolics, flavan-3-ols and flavonoids in the analysed wines, and the yeast did not have significant influence on phenolics contents. An HPLC-DAD-MS study of anthocyanins, phenolic acids, flavan-3-ols and flavonols in Vranec and Merlot wines obtained under different vinification is reported here. The percentage of formic acid in the mobile phase and pH value of the wine samples were optimized in order to transform the chalcone forms of anthocyanins into their flavylium red cations, to increase sensitivity and perform a simultaneous MS identification of different groups of phenolic compounds in wine. Quantification of compounds was performed by direct injection of the wines into HPLC, using mobile phase consisted of water/formic acid (95:5; solution A) and acetonitrile/water/formic acid (80:15:5; solution B) with flow rate of 0.25 mL/min at 38 °C. The proanthocyanidin composition, mean degree of polymerization and concentration of tannins of the same Vranec and Merlot wines, were determined by reversed-phase HPLC after acid-catalysis in the presence of excess of phloroglucinol. Two procedures for preparation of wine samples, solid-phase extraction and precipitation with methanol, were compared. Precipitation with methanol was chosen for sample preparation before phloroglucinol treatment and HPLC-DAD-FLD quantification. An HPLC-MS analysis was used for identification purposes of monomers and phloroglucinol adducts. Principal component analysis was performed in order to check the possible grouping of the wine samples from both, red and white varieties accoriding to spectrophotometric and HPLC data. Matrix-assisted laser desorption/ionization mass spectrometry is a new valuable screening technique for the presence and identification of anthocyanins in wine and grape samples. The MALDI matrices: α-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (SA), 2,5- hydroxybenzoic acid (2,5-DHB) and C70 fullerene were tested in this study. 2,5-DHB was superior with respect to all the matrices used. Fullerene was used for the first time as a possible matrix for the MALDI TOF MS analysis of anthocyanins