17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017 PHENOLIC, VOLATILE AND ELEMENTAL COMPOSITION OF MACEDONIAN WINES Violeta Ivanova-Petropulos Faculty of Agriculture, University “ Goce Delčev” - Štip , Republic of Macedonia e-mail: violeta.ivanova@ugd.edu.mk Trajće Stafilov, Marina Stefova, Ernst Lankmayr , Ferenc Kilar
Chemical composition of wine 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
Types of wine By the colour rosé white red Still or sparkling sparkling still 17th CEEPUS Symposium and Summer 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017 School on Bioanalysis, 2-8 July, Ohrid 2017
Winemaking 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
PHENOLIC COMPOSITION OF MACEDONIAN WINES Determine the colour, mouthfeel, astringency and bitterness of wine. Influenece the sensorial characteristics of grape and wine Antioxidant, antimicrobal, anticancerogenic effects, prevention of cardiovascular diseases. HO OH Two groups of polyphenols: Resveratrol R Non-flavonoids: Hydroxybenzoic acids, Hydroxycinnamic acids and derivatives, Stilbenes and stilbene glucosides Flavonoids: Flavanones, Flavonols, Flavones, Flavan-3-ols, Anthocyanidins R1 Anthocyanidins OH Flavan-3-ols OH OH + H O O H O O R2 R OH OH OH OH
Analytical techniques for analysis of phenolics HPLC-DAD-MS MALDI-TOF Wine and grape samples Vitis vinifera varieties, 2007 vintage Red grapes and wines: Vranec and Merlot White grapes and wines: Smederevka and Chardonnay 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
HPLC-DAD-MS C18 column, DAD Binary solvent system: polar solvent (aqueous acetic acid, phosphoric acid, or formic acid) and a less polar organic solvent (methanol or acetonitrile, possibly acidified) HPLC-DAD is limited technique for compounds with similar UV-Vis spectra (flavanols and flavonols) HPLC-MS powerful and more sophisticated technique for characterization of the phenolics Confirmation of the structure of the main phenolic compounds Detection of a novel compounds 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
HPLC analyses Agilent series 1100, equipped with a DAD and ion trap MS t R /min B/ % Column: Phenomenex C18 (60x4,6; 3µm) Gradien elution: A - 1 % acetic acid in water 0 5 B - 1 % acetic acid in metanol 10 20 pH 2,5-3 45 50 Flow rate: 0,2 mL/min; room temperature, 50 80 Injection volume: 5 µL 60 90 Run time: 60 min The ESI parametars : Nebuliyer, 15 psi; Dry gas (N 2 ) flow 5 L/min Dry gas temerature, 325 ºC Scanning range from m/z 50 to m/z 2200 in negative and positive mode 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
UV-Vis Chromatograms Chromatogram at 280 nm Chromatogram at 320 nm Chromatogram at 360 nm Chromatogram at 520 nm 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
MS identification of anthocyanins monoglucosides Intens. 8 x10 5 Intens. vranec12.d: BPC 449 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) x10 6 2 4 1.5 2 [M] + 0 7 x10 vranec12.d: BPC 463 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 4 4 3 2 449 Cyanidin 3-glucoside 1 0 7 x10 vranec12.d: BPC 465 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 2.0 1 463 Peonidin 3-glucoside 1.5 1.0 0.5 0.0 x10 7 vranec12.d: BPC 479 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 465 Delfinidin 3-glucoside 5 3 1.0 4 3 2 1 479 Petunidin 3-glucoside 0 8 x10 vranec12.d: BPC 493 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 2.0 5 1.5 1.0 493 Malvidin 3-glucoside 0.5 0.0 0 10 20 30 40 50 Time [min] 0.5 3 4 1 2 0.0 0 10 20 30 40 50 Time [min]
MS identification of anthocyanins acetylglucosides Intens. Intens. vranec12.d: BPC 491.1 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 6 x10 2 7 2.0 x10 5 1.5 1.0 0.5 0.0 7 x10 vranec12.d: BPC 505.1 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 1.25 4 1.00 0.75 5 0.50 0.25 0.00 6 x10 vranec12.d: BPC 507.1 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 3 1 2 1 0 6 x10 vranec12.d: BPC 521.1 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 8 3 6 4 4 2 0 7 x10 vranec12.d: BPC 535.1 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 6 5 4 2 0 0 10 20 30 40 50 Time [min] 3 [M] + 491 Cyanidin 3-glucoside-acetate 2 505 Peonidin 3-glucoside-acetate 507 Delfinidin 3-glucoside-acetate 4 521 Petunidin 3-glucoside-acetate 1 3 535 Malvidin 3-glucoside-acetate 1 2 0 0 10 20 30 40 50 Time [min]
MS identification of anthocyanins coumaroylglucosides Intens. Intens. vranec12.d: BPC 595.2 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 6 x10 7 6 x10 2 4 4 2 0 7 x10 vranec12.d: BPC 609.2 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 4 2.0 5 1.5 1.0 0.5 0.0 6 x10 vranec12.d: BPC 611.2 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 8 1 6 4 2 0 7 x10 vranec12.d: BPC 625.2 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 1.5 3 1.0 3 0.5 0.0 x10 7 vranec12.d: BPC 639.2 +All MS, -Constant Bkgrnd, Smoothed (6.6,1, GA) 4 4 2 0 0 10 20 30 40 50 Time [min] [M] + 2 5 595 Cyanidin 3-glucoside-coumarate 609 Peonidin 3-glucoside-coumarate 3 611 Delfinidin 3-glucoside-coumarate 1 625 Petunidin 3-glucoside-coumarate 1 2 639 Malvidin 3-glucoside-coumarate 0 0 10 20 30 40 50 Time [min]
17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
HPLC system: Agilent 1100 Series, DAD (G1315B) and a LC/MSD Trap VL (G2445C VL) electrospray ionization mass spectrometry (ESI-MS n ) system. Analysis of anthocyanins and other pigments: Sample preparation : Dilution of wines, 1:4 with HCl 0.1N Solvents: A - water/acetonitrile/formic acid ( 87:3:10 , v/v/v) B - water/acetonitrile/formic acid ( 40:50:10 , v/v/v) Flow rate: 0.63 mL/min t/min B (%) DAD: 520 nm 0 6 The linear gradient 15 30 30 50 for solvent B: 35 60 38 60 17th CEEPUS Symposium and Summer 46 6 School on Bioanalysis, 2-8 July, Ohrid 2017
mAU mv-glc 2000 1750 MERLOT 2008 1500 520 nm 1250 mv-acglc 1000 mv-3-cmglc-4-VP mv-3-acglc-4-VC mv-3-acglc-4-VP 750 mv-3-cmglc-VC mv-3-glc-4-VC mv-3-glc-4-VP mv-cmglc cm-vitisin-A ac-vitisin-A pt-glc vitisin-A 500 pn-glc pn-cmglc dp-acglc pn-acglc pt-acglc dp-glc dp-cmglc cy-cmglc vitisin-B pt-cmglc cy-glc 250 0 10 15 20 25 30 35 min Chromatographic separation of ANTHOCYANINS AND PYRANOANTHOCYANINS 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
Concentration of anthocyanins ANTHOCYANIN PROFILES (molar %) Merlot Merlot Merlot CabSv CabSv CabSv Vranec Vranec Vranec 2006 2007 2008 2006 2007 2008 2006 2007 2008 mv-3-glc 54.51 48.21 48.92 44.91 50.81 51.70 47.58 50.15 48.73 mv-3- 14.09 14.45 20.69 7.08 20.67 20.32 3.13 5.54 7.24 acglc mv-3- 5.72 6.78 6.81 8.41 5.26 6.72 4.57 6.80 7.64 cmglc TA* 47.6 159.7 194.2 351.1 96.1 193.6 16.1 53.6 507.6 * mg/L, as malvidin 3-glucoside; ND, not detected; dp, delphinidin; cy, cyanidin; pt, petunidin; pn, peonidin; mv, malvidin glc, 3-glucoside; acglc, 3-(6"-acetyl)-glucoside; cmglc, 3-(6"-coumaroyl)-glucoside 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
MALDI-TOF-MS analyses Important factors: selection of an appropriate matrix optimal mixing and drying of matrix and sample adjustment of laser strength selection of calibration standards correct interpretation of the spectra Different MALDI matrices were tested: 1. Alpha-Cyano-4-hydroxycinnamic acid (CHCA) 2. Sinapic acid (SA) 3. 2,5-Dihydorxybenzoic acid (2,5-DHB) 4. Fullerene [C70] 5. Measurenments without matrix Sendwich method was applied for all sample measurements 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
MALDI-TOF-MS spectra of wine with different matrices Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] x10 4 x10 4 x10 4 x10 4 1.50 1.50 [M-H]+ 104.1 104.1 [M-H]+ 104 1-Hexanol 301 Peonidin 104 1-Hexanol 303 Delfinidin 1.50 1.50 301 Peonidin 331 Malvidin 1.25 1.25 493 Malvidin 3-glucoside 303 Delfinidin 104.1 104.1 535 Malvidin 3-acetylglucoside 317 Petundin 331.1 331.1 331 Malvidin 1.25 1.25 463 Peonidin 3-glucoside 1.00 1.00 493 Malvidin 3-glucoside 531 Peonidin 3-glucosode pyrivic acid 1.00 1.00 535 Malvidin 3-acetylglucoside 609 Peonidin 3-coumaroylglucoside 639 Malvidin 3-coumaroylglucoside 0.75 0.75 0.75 0.75 0.50 0.50 0.50 0.50 493.1 493.1 0.25 0.25 138.0 138.0 0.25 0.25 358.0 358.0 493.1 493.1 639.1 639.1 118.1 118.1 301.1 301.1 331.1 331.1 176.0 176.0 265.9 265.9 279.1 279.1 138.0 138.0 535.1 535.1 358.0 358.0 535.1 535.1 156.0 156.0 399.1 399.1 176.0 176.0 287.0 287.0 609.1 609.1 663.1 663.1 301.0 301.0 0.00 0.00 0.00 0.00 100 100 150 150 200 200 250 250 300 300 350 350 400 400 450 450 500 500 550 550 100 100 200 200 300 300 400 400 500 500 600 600 m/z m/z m/z m/z CHCA SA (Alpha-Cyano-4-hydroxycinnamic acid) (Sinapic acid) 17th CEEPUS Symposium and Summer School on Bioanalysis, 2-8 July, Ohrid 2017
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