Mass Spectrometry Fragmentation Pattern of Coloured Flavanol

Sánchez-Ilárduya et al. Flavanol-anthocyanin derivatives mass spectrometry 203

Mass spectrometry fragmentation pattern of coloured ﬂavanol-anthocyanin and anthocyanin-ﬂavanol derivatives

in aged red wines of Rioja_190 203..214

M.B. SÁNCHEZ-ILÁRDUYA, C. SÁNCHEZ-FERNÁNDEZ, M. VILORIA-BERNAL, D.M. LÓPEZ-MÁRQUEZ, L.A. BERRUETA, B. GALLO and F. VICENTE Analytical Chemistry Department, Science and Technology Faculty, Basque Country University, PO Box 644, 48080 Bilbao, Spain Corresponding author: Dr Luis A. Berrueta, fax +34 94 601 3500, email [email protected]

Abstract Background and Aims: During wine ageing, a great variety of reactions take place, resulting in an immense variety of products whose structure sometimes remains unknown. The aim of this work is the study of different fragmentation patterns of flavanol-anthocyanin derivatives formed along the wine ageing; these patterns are useful for elucidating the different structures of these compounds and other new related ones. Methods and Results: Several wines from the Protected Denomination of Origin Rioja have been studied by an analytical method that combines column chromatography and high-performance liquid chromatography with diode array and mass and tandem mass spectrometric detections. Thirty-five coloured flavanol-anthocyanin compounds formed by direct reaction or by acetaldehyde-mediated condensation have been identified. For direct reaction derivatives, two different fragmentation patterns (one of them not previously reported) have been observed depending on the position of flavanol in the coloured derivative. Several compounds have been identified in aged wines for the first time to the authors’ knowledge, like (+)-gallocatechin-cyanidin-3-glucoside and (+)-catechin- cyanidin-3-glucoside Conclusions: The developed analytical procedure has allowed the identification of some compounds for the first time, and two different fragmentation patterns have been observed depending on the position of flavanol in the pigment. Significance of the Study: The establishment of different fragmentation patterns allows the structural elucidation of unknown compounds.

Keywords: anthocyanin-derived pigment, colour, ﬂavanol, MS, wine

Introduction In recent years, different anthocyanin-derived pigments Polyphenols play an important role in the nutritional, organo- have been identified; they can be classified in two groups: pyra- leptic and commercial properties of agrofoods. In wine, they noanthocyanins and pigments originated by reactions between have importance in the final quality because of their influence anthocyanins and flavanols. On one hand, pyranoanthocyanins on characteristics such as colour, astringency or bitterness are obtained by a cycloaddition reaction of some compounds (Monagas et al. 2005) and they can also help in the differentia- present in wine with the flavylium form of anthocyanins, giving tion among grape variety and, sometimes, among growing con- rise to the formation of a new pyranic ring. Pyranoanthocyanins ditions of fruit. can be formed with pyruvic acid, acetaldehyde, acetoacetic Wine phenolics belong to two main groups: non-flavonoid acid, vinylphenols, hydroxycinnamic acids and vinylflavanols compounds (namely, hydroxybenzoic and hydroxycinnamic (Hayasaka and Asenstorfer 2002, Pozo-Bayón et al. 2004). acids and their derivatives, stilbenes and phenolic alcohols) These anthocyanin-derived pigments cause hypsochromic shifts and flavonoid compounds (namely anthocyanins, flavanols, fla- in the visible absorption maxima of the initial anthocyanins, vonols, flavanonols and flavones). providing a brick-red hue to the wine. Anthocyanins are water-soluble pigments that are respon- On the other hand, reactions between anthocyanins and sible for flower and fruit colour. In red grapes, anthocyanins flavanols can proceed directly (Remy et al. 2000, Hayasaka and provide the red colour to the grape skin and also to the pulp in Kennedy 2003, Ribéreau-Gayon et al. 2006), be mediated by some variety. Anthocyanins and flavanols are the major pheno- acetaldehyde (Rivas-Gonzalo et al. 1995, Francia-Aricha et al. lics in red wines. Colour evolution during vinification and 1997, Es-Safi et al. 1999, Lee et al. 2004) or be mediated by ageing has been attributed to the progressive changes of phe- other aldehydes (Es-Safi et al. 2002, Pissarra et al. 2004). These nolic compounds extracted from the grapes. The original grape condensations cause bathochromic shifts in the visible absorp- anthocyanins, which are responsible for the initial red colour of tion maxima of the initial anthocyanins, providing a bluish-red young red wines, are involved in irreversible reactions towards hue to the wine. more stable pigments. These pigments are responsible for the Within the pigments formed by direct reaction, two different different hues of the more aged wines. mechanisms have been described depending on the position of doi: 10.1111/j.1755-0238.2012.00190.x © 2012 Australian Society of Viticulture and Oenology Inc. 204 Flavanol-anthocyanin derivatives mass spectrometry Australian Journal of Grape and Wine Research 18, 203–214, 2012

R OH R HO O OH OH + HO O OH R H OH OH OH OH HO O OH OH F+ OH OH R Flavanol oligomer Figure 1. Formation scheme of OH F-A+ pigments. A, anthocyanin; F, HO O ﬂavanol; glc, glucoside. R OH R OH OH R1 HO O OH OH -H (Epi)catechin OH OH HO O -OH (Epi)gallocatechin R C4 OH 2 OH O-glc R1 R2 Anthocyanidin OH R1 OH Colourless F-AOH -OH -OH Delphinidin C8 OH HO O -OH -H Cyanidin R2 -OCH3 -OH Petunidin C6 O-glc -H2O -OCH3 -H Peonidin OH R -OCH3 -OCH3 Malvidin Hydrated anthocyanin AOH OH HO O OH

OH R1 OH OH

HO O R2 O-glc OH

Red-coloured F-A+ the flavanol: F-A+ and A+-F formation. The F-A+ formation The formation of most of anthocyanin-derived pigments mechanism (Figure 1) begins with the acid cleavage of the occurs in the first months of ageing, as the oxidative conditions in interflavanic bond of a procyanidin (flavanol oligomer), giving a oak barrels favour their formation (Atanasova et al. 2002, Alcalde- carbocation F+ which reacts as an electrophile through the C4 Eon et al. 2006). Both anthocyanin-flavanol derived pigments, with the C6 or C8 of the hydrated form of the anthocyanin, that direct ones and ethyl-linked ones, show less stability during ageing acts as nucleophile. This mechanism leads to a colourless com- than pyranoanthocyanins. The pyranic ring in pyranoanthocya- pound (F-AOH) which easily dehydrates to the coloured flavy- nins provides a protection against the nucleophilic attack from lium form F-A+ (Macz-Pop et al. 2006). In the formation of A+-F water or bisulfite, increasing their stability (He et al. 2006). pigments (Figure 2), the anthocyanin in flavylium form A+ acts The aim of this work was the identification of the coloured as electrophile through the C4. The hydroxyl groups of C5 and flavanol-anthocyanin derivatives that provide bluish hues to C7 of flavanol have mesomeric effect and give nucleophilic aged wines. Wine colour, together with other sensorial charac- characteristics to the C6 and C8 of flavanol. A nucleophilic teristics, is a very important wine quality parameter. A study addition of the flavanol takes place onto the flavylium form of mass spectrometry (MS) fragmentation patterns of these of the anthocyanin, yielding a colourless compound with the compounds, present in aged red wines of the Protected Denomi- anthocyanin in flavene form. This flavene can be oxidized, nation of Origin (PDO) Rioja has been carried out in order resulting in a coloured flavylium A+-F pigment or in a colourless to establish different fragmentation patterns that ensure the compound A-F with a type-A bond (Salas et al. 2003). correct identification of these derivatives and allow the struc- Acetaldehyde-mediated condensation of anthocyanin- tural characterization of unknown compounds. flavanol begins with the protonation of the acetaldehyde, leading to the formation of a carbocation (Figure 3). This carbocation is added to a nuclephilic position (C8) of the phloroglucinol ring of a Materials and methods flavanol, followed by a dehydration which yields a new carboca- Reagents tion. This carbocation suffers a nucleophilic attack by an antho- Methanol and acetonitrile (Romil Chemical Ltd, Heidelberg, cyanin to give rise the final acetaldehyde-mediated anthocyanin- Germany) were of high-performance liquid chromatography flavanol condensation pigment (Pissarra et al. 2004). (HPLC) grade. Water was ultrapurified on a Milli-Q system

Anthocyanin A+ R1 R1 OH OH

HO O HO O R2 R2 R C4 O-glc O-glc OH OH R OH Figure 2. Formation scheme of OH HO O A+-F pigments. A, anthocyanin; F, OH ﬂavanol; glc, glucoside. HO C8 O OH OH R C6 OH OH OH Colourless flavene A-F -H (Epi)catechin Flavanol -OH (Epi)gallocatechin

R1 R2 Anthocyanidin

-OH -OH Delphinidin R1 R 1 -OH -H Cyanidin OH OH -OCH3 -OH Petunidin HO O R HO O 2 R2 -OCH3 -H Peonidin R O-glc O-glc R -OCH3 -OCH3 Malvidin OH OH OH OH O O HO O OH OH

OH OH OH OH

Colourless A-type A-F Red-coloured flavilyum A+-F

(Millipore, Bedford, Massachusetts, USA). Trifluoroacetic commercial cartridges (IST, Hengoed Mid, Glam, UK, 150 mL acid (TFA) provided by Merck (Darmstadt, Germany) was of capacity) were filled with 40 g of C18-modified silica (IST, par- spectroscopic grade. NaH2PO4 and Na2HPO4 (Fluka, Steinheim, ticle size 60 mm). Both top and bottom of solid phase were Germany) were of analysis grade. All solvents used were covered with 20 mm polyethylene frits (IST). The cartridges were previously filtered through 0.45-mm nylon membranes (Lida, activated with 120 mL of methanol, washed with 2 ¥ 120 mL of Kenosha, Wisconsin, USA). Malvidin-3-glucoside (Extrasyn- ultrapurified water and preconditioned with 120 mL of phos- thèse, Lyon, France) was used as standard. phate buffer (1 mol/L, pH = 7.0). Two hundred millilitres of red wine were de-alcoholized in a Büchi (Büchi Labortechnik AG, Fractionation of wine by column chromatography (CC) Flawil, Switzerland) R200 rotary evaporator at 25°C during 30 min. Two 60 mL aliquots of de-alcoholized wine were loaded Wines used in the study were from Vitis vinifera cv. Tempranillo in two preconditioned cartridges. Elution began with 200 mL of grapes of 1995 (wine 1), 2001 (wine 2) and 1998 (wine 3) 0.125 mol/L phosphate buffer (pH = 7.0) to elute phenolic acids, vintages from the Spanish PDO Rioja and were aged (according followed by 200 mL of ethyl acetate for the elution of monomer to PDO Rioja regulations) for no less than 36 months of which and oligomer flavanols and, finally, 250 mL of methanol for more than 12 must be in oak barrels. Ten millilitres of wine were the elution of anthocyanins and anthocyanin-derived pigments. filtered through a 0.45-mm Pall (Ann Arbor, Michigan, USA) The two methanolic extracts from both cartridges were mixed acrodisc Polytetrafluoroethylene (PTFE) filter and were injected and evaporated to dryness in the rotary evaporator and redis- using a four-way Rheodyne valve (Cambridge, UK) in a 250-¥- solved in 15 mL of methanol. The rest of the procedure continued 15 mm Internal diameter (i.d.) glass column packed with as the previous one (CC procedure), loading 10 mL of the redis- Toyopearl HW-40S (Tosoh Bioscience, Stuttgart, Germany). solved extract in the glass column packed with Toyopearl Methanol was used as elution solvent at a flow rate of 0.5 mL/ HW-40S. min using a HPLC pump, and 30 fractions of 6 mL for each wine were collected. These fractions were concentrated to dryness under a stream of nitrogen in a Zimark TurboVap-LV evaporator HPLC-diode array detector (DAD)-electrospray ionization (Hopkinton, Massachusetts, USA) at 25°C, redissolved in 400 mL (ESI)-collision-induced dissociation (CID)-MS/MS identification of the initial HPLC mobile phase (88% of a TFA : H2O 0.5:99.5 The separation and identification of the anthocyanin-derived v/v solution and 12% acetonitrile, in volume) solution and pigments were performed by liquid chromatography in a Waters filtered through a 0.45-mm PTFE membrane before analysis. (Milford, Massachusetts, USA) Alliance 2695 instrument coupled to a DAD model 2996 and a mass spectrometer Micro- mass Quattro micro (Milford). A reversed-phase Phenomenex Fractionation of wine by solid-phase extraction followed (Torrance, USA) Luna C18(2) column (150 ¥ 4.6 mm i.d., by CC (SPE + CC) 3 mm) with a Waters (Milford) Nova-Pack C18 guard column In order to preconcentrate the wine sample, an SPE procedure (10 ¥ 3.9 mm i.d., 4 mm) was used. Eluents were TFA/water was adapted from the literature (Sun et al. 2006). Two empty (0.5:99.5, v/v) (phase A) and acetonitrile (phase B). A gradient

H+ CH3-CHO CH3-CH-OH Acetaldehyde

CH3-CH-OH

R R R H C H OH OH H3C OH OH 3 C CH H+ C8 HO O HO O HO O OH OH OH H2O Figure 3. Formation scheme of OH OH OH OH OH OH A-ethyl-F pigments. A, Ethyl-flavanol cation anthocyanin; F, ﬂavanol; glc, glucoside.

R1 OH R OH OH HO O -H (Epi)catechin R2 HO O-glc HO -OH (Epi)gallocatechin O OH OH R1

Hydrated anthocyanin AOH HO H3CHC OH R OH R1 R2 Anthocyanidin R HO O R H C OH 2 3 CH -OH -OH Delphinidin O-glc HO O OH OH -OH -H Cyanidin

OH -OCH3 -OH Petunidin OH Dehydration -OCH3 -H Peonidin

-OCH3 -OCH3 Malvidin

OH HO

HO O OH R1

HO H3CHC OH R HO O R2 O-glc OH

A-ethyl-F

Table 1. Chromatographic, Ultraviolet-visible and mass spectrometry data and proposed identities of the monoglucosylated anthocyanins detected as well as fractions where each compound was found.

+ n RT (min) Compound [M] Fragment lmax Fractions (m/z) ion (m/z) (nm) Wine 1 Wine 2 Wine 3 Wine 2 Wine 3 CC CC CC SPE + CC SPE + CC

1 10.90 Dp-3-glc 465 303 524 8–10 6–21 6–12 9–10 10–11 2 16.30 Cy-3-glc 449 287 516 7–8 6–14 7–11 9 — 3 19.50 Pt-3-glc 479 317 525 5–10 6–30 6–12 8–10 8–10 4 28.20 Pn-3-glc 463 301 516 8 6–30 6–9 8–9 8–11 5 32.38 Mv-3-glc 493 331 526 5–10 4–30 6–26 7–30 5–30 6 44.50 Dp-3-(6-ac)-glc 507 303 527 — 9–11 — — — 7 57.83 Cy-3-(6-ac)-glc 491 287 522 — 8–9 — — — 8 62.13 Pt-3-(6-ac)-glc 521 317 527 — 6–10 — — — 9 71.53 Pn-3-(6-ac)-glc 505 301 517 — 6–9 — — — 10 74.17 Mv-3-(6-ac)-glc 535 331 529 5–7 6–11 7 8 8 11 66.48 Dp-3-(6-cis-p-coum)-glc 611 303 n.d. — 11–12 — — — 12 74.40 Dp-3-(6-trans-p-coum)-glc 611 303 531 10–11 11–20 — — 12 13 82.32 Cy-3-(6-trans-p-coum)-glc 595 287 523 — 10–14 — 11 11–12 14 76.70 Pt-3-(6-cis-p-coum)-glc 625 317 n.d. — 10–11 — — — 15 84.25 Pt-3-(6-trans-p-coum)-glc 625 317 532 9–10 10–16 11 9–11 11–12 16 92.23 Pn-3-(6-trans-p-coum)-glc 609 301 522 8–9 9–17 10–11 10–11 10 17 86.03 Mv-3-(6-cis-p-coum)-glc 639 331 n.d. 8 8–16 — — — 18 93.35 Mv-3-(6-trans-p-coum)-glc 639 331 533 7–10 6–30 7–14 8–30 8–30 19 80.68 Mv-3-(6-caf)-glc 655 331 534 — 10–15 — — —

—, not detected; ac, acetyl; CC, column chromatography; Cy, cyanidin; Dp, delphinidin; glc, glucoside; Mv, malvidin; m/z, mass to charge ratio; n.d., not determined; Pn, peonidin; Pt, petunidin; RT, retention time; SPE, solid-phase extraction.

Table 2. Chromatographic, Ultraviolet-visible and mass spectrometry data and proposed identities of the ﬂavanol-anthocyanin direct reaction products detected as well as fractions where each derivative was found.

+ n RT (min) Compound Fragment[M] max l Fractions (m/z) ion (m/z) (nm) Wine 1 Wine 2 Wine 3 Wine 2 Wine 3 CC CC CC SPE + CC SPE + CC

20 4.95 (+)-catechin-Dp-3-glc 753 591, 573, 465, 439, 345, 303 n.d. 11 12–16 — — — 21 6.61 (-)-epicatechin-Dp-3-glc 753 591, 573, 439, 303 532 — 11–13 — — — 22 6.17 (+)-catechin-Cy-3-glc 737 575, 557, 423, 329, 287 526 — 11–14 — — — 23 6.90 (+)-catechin-Pt-3-glc 767 605, 587, 479, 453, 359, 317 533 9–11 10–17 10–13 10–11 — 24 10.80 (-)-epicatechin-Pt-3-glc 767 605, 453 n.d. — 11–13 — — — 25 9.42 (+)-catechin-Pn-3-glc 751 589, 571, 437, 343, 301 526 9 10–17 10–12 10–12 12 26 3.03 (Epi)catechin-(C4-C6)-Mv-3-glc 781 619, 493, 467, 373, 331 532 — 17–20 — 12 12 27 10.37 (+)-catechin-Mv-3-glc 781 619, 601, 493, 467, 373, 331 532 8–11 7–25 9–14 8–15 10–13 28 20.63 (-)-epicatechin-Mv-3-glc 781 619, 601, 467, 373, 331 532 9 8–13 10 9–12 10–12 29 13.28 Mv-3-glc-(+)-catechin 781 619, 601, 577, 493, 457, 331 532 — 23–25 — — 16 30 25.93 Mv-3-glc-(-)-epicatechin 781 619, 601, 577, 493, 457, 331 n.d. — 23–25 17–19 — — 31 40.35 (Epi)catechin-Mv-3-(6-ac)-glc 823 619, 601, 467, 373 533 — 10–11 — 9 — 32 50.87 (Epi)catechin-Dp-3-(6-p-coum)-glc 899 591 534 — 17–18 — — — 33 54.13 (Epi)catechin-Cy-3-(6-p-coum)-glc 883 575 n.d. — 14 — — — 34 61.35 (Epi)catechin-Pt-3-(6-p-coum)-glc 913 605, 453, 359 533 — 13–16 — — — 35 64.70 (Epi)catechin-Pn-3-(6-p-coum)-glc 897 589, 571, 437, 343 527 — 12–16 — 12–13 —

36 67.25 (+)-catechin-Mv-3-(6-cis-p-coum)-glc 927 619, 467, 373, 331 n.d. — 12–13 — — 207 spectrometry mass derivatives Flavanol-anthocyanin — 37 69.82 (+)-catechin-Mv-3-(6-trans-p-coum)-glc 927 619, 601, 493, 467, 373, 331 536 9–11 11–24 — 9–16 12 38 71.53 (-)-epicatechin-Mv-3-(6-trans-p-coum)-glc 927 619, 601, 373 n.d. — 12–13 — — — 39 3.22 (Epi)gallocatechin-Dp-3-glc 769 607, 589, 439, 345, 303 532 — 13–17 — — — 40 3.50 (Epi)gallocatechin-Cy-3-glc 753 591, 423, 287 n.d. — 12–14 — — — 41 3.77 (Epi)gallocatechin-Pt-3-glc 783 621, 603, 495, 453, 359, 317 533 — 11–17 — — — 42 4.85 (Epi)gallocatechin-Pn-3-glc 767 605, 587, 437, 343, 301 n.d. — 10–14 — — — 43 4.85 (Epi)gallocatechin-Mv-3-glc 797 635, 617, 509, 467, 373, 331 531 9–10 9–18 10–12 — — 44 46.07 (Epi)gallocatechin-Mv-3-(6-p-coum)-glc 943 635, 617, 467, 373 532 — 12–18 — — — 45 17.90 (Epi)catechin-(epi)catechin-Mv-3-glc or (epi)catechin-Mv-3-glc-(epi)catechin 1069 907, 781, 619, 467, 373 537 12–13 11–23 — 13–16 — 46 77.25 (Epi)catechin-(epi)catechin-Mv-3-(6-p-coum)-glc or (epi)catechin-Mv-3-(6-p-coum)-glc-(epi)catechin 1215 927, 907, 619, 331 542 — 17–18 — — —

47 11.43 (Epi)catechin-(epi)gallocatechin-Pn-3-glc† 1055 893, 767, 605 n.d. — 15–18 — — — 48 11.43 (Epi)catechin-(epi)gallocatechin-Mv-3-glc† 1085 923, 797, 635 536 — 14–21 — — —

†There are more possible structures with the (epi)gallocatechin joined to the anthocyanin. —, not detected; ac, acetyl; CC, column chromatography; Cy, cyanidin; Dp, delphinidin; glc, glucoside; Mv, malvidin; m/z, mass to charge ratio; n.d., not determined; Pn, peonidin; Pt, petunidin; RT, retention time; SPE, solid-phase extraction. 208 Flavanol-anthocyanin derivatives mass spectrometry Australian Journal of Grape and Wine Research 18, 203–214, 2012

OH OCH3 HO O OH OH

OH OCH3 HO O -288 u OCH3 OH OH O HO O glc OCH3 OH m/z 493 O-glc OH OH Figure 4. Mass spectrum and HO O m/z 781 OH main MS fragmentations of a ﬂavanol-anthocyanin derivative OCH 3 ( )-catechin-Mv-3-glc. ESI, OH OH + OH electrospray ionization; glc, HO O OCH3 glucoside; M, molecular ion; OH m/z, mass to charge ratio. OH m/z -162 u 601 OH OH OCH3 OH

-18 u O O OCH3 OH 1/4, -126 u OH m/z 493 OH 1 HO O OH HO O 2 OH OCH3 4 OCH3 OH 3 OH 1/3, -152 u OH OH HO O OCH3 HO O OCH3 OH OH OH OH m/z 619 m/z 467 2/4, -246 u

-288 u

OCH 3 OCH3 OH OH OH HO O OCH HO O 3 OCH3 OH OH OH OH m/z 331 m/z 373 program was employed: 0–45 min, linear gradient from 12 to were set to 0.8 mL/min and 30°C, respectively. Vial samples 15% B; 45–100 min, linear gradient from 15 to 25% B; 100– were kept in the injector at 4°C. Fifty microlitres of each fraction 140 min, linear gradient from 25 to 40% B; 140–145 min, iso- were injected. The chromatograms were monitored at 530 nm cratic 40% B; 145–150 min, linear gradient from 40 to 100% B; (optimum for anthocyanins), and complete spectral data were 150–155 min, isocratic 100% B; 155–160 min, linear gradient accumulated in the range 250–600 nm each second. Mass from 100 to 12% B; 160–175 min, isocratic 12% B for recondi- spectra were obtained on a Micromass Quattro micro triple tioning the column. The ﬂow rate and column temperature quadrupole mass spectrometer equipped with a Z-spray ESI

Figure 5. ESI(+)-CID-MS/MS product ions spectrum of (+)-catechin-Mv-3-glucoside, taking as precursor the corresponding to the aglycone ion. ESI, electrospray ionization; glc, glucoside; M, molecular ion; m/z, mass to charge ratio.

source and coupled to the exit of the DAD by a T split introduc- Flavanol-anthocyanin reactions can be direct or indirect and ing about 250 mL/min of the eluent into the spectrometer. Nitro- can take place with (epi)catechins and (epi)gallocatechins. Both gen was used as desolvation gas at 300°C and at a flow rate of types of flavanols are found in wine, as well as their oligomers 450 L/h, and no cone gas was used. A potential of 3.2 kV was and polymers, also called procyanidins and condensed tannins used on the capillary for positive ion mode. The source block (Escarpa and González 2001). temperature was held at 120°C. MS spectra, within the Mass to charge ratio (m/z) range 50–1500, were recorded in positive Direct flavanol-anthocyanin reactions mode at different cone voltages (15, 30, 45 and 60 V). CID Direct flavanol-anthocyanin reaction can yield flavanol- MS/MS product ion spectra were recorded using argon as col- anthocyanin (F-A) or anthocyanin-flavanol (A-F) adducts lision gas at 1.5 ¥ 10-3 mbar and different collision energies in depending on the spatial disposition of each molecule, but for the range 10–35 eV depending on the type of pigment. the case of coloured derivatives, anthocyanin should be in its flavylium form (A+). Moreover, the interflavanoid bond in F-A Results and discussion and A-F compounds can be formed between the carbon at The monoglucosylated anthocyanidins (acylated and not position C4 of the upper unit and those at positions C6 or C8 of acylated) are the precursors of the anthocyanin-derived pig- the lower unit (Salas et al. 2004, Alcalde-Eon et al. 2006), but ments, which appear during vinification and ageing. In the these isomers cannot be distinguished by MS (Fossen et al. analysed wines, five non-acylated monoglucosides (compounds 2004, Macz-Pop et al. 2006). On the other hand, distinction 1–5) and 14 acylated (compounds 6–19) were identified. Table 1 between flavanol isomers (+)-catechin or (-)-epicatechin (or shows retention times, m/z-values for the molecular ion and between (+)-gallocatechin or (-)-epigallocatechin) is possible in the ion corresponding to the aglycone (the anthocyanin mol- some cases by their elution order: the (+)-catechin isomer elutes ecule after losing the sugar unit), visible absorption maxima and before the corresponding (-)-epicatechin isomer (González- in which fractions of the three wines each anthocyanin has been Paramás et al. 2006). In this work, when this distinction was not detected. Acylated derivatives corresponded to acetyl derivatives possible, the pigment was named as (epi)catechin or (epi)gallo- (compounds 6–10), p-coumaroyl derivatives for compounds catechin derivative. Despite these attempts, further research is 11–18 and a caffeoyl derivative (compound 19). Anthocyanins needed to achieve a complete identification of these com- 11 and 12, 14 and 15, and 17 and 18 are isomers, eluting first the pounds. Their isolation and nuclear magnetic resonance analy- cis isomer because it is more polar because of its spatial configu- sis should be the next steps in their ultimate structural ration (García-Beneytez et al. 2003, Boido et al. 2006). Differ- elucidation. ences in visible absorption maxima can be observed as a result Table 2 shows the chromatographic and spectrometric of a substitution effect in the chromophore group of anthocya- characteristics of the different flavanol-anthocyanin derivatives nins. Absorption maxima observed for both disubstituted detected formed by direct reaction. Pigments 20–44 are F-A+ or anthocyanins cyanidin-3-glucoside and peonidin-3-glucoside A+-F adducts with a monomer of flavanol ((+)-catechin or (-)- were lower (516 nm) than the corresponding for trisubstituted epicatechin for compounds 20–38 and (+)-gallocatechin or (-)- ones: delphinidin-3-glucoside (524 nm), petunidin-3-glucoside epigallocatechin for 39–44). All these pigment mass spectra (525 nm) and malvidin-3-glucoside (526 nm). Acylation of exhibit an aglycone ion because of loss of the sugar unit (-162 sugar unit also results in bathochromic shifts in visible absorption u for glucosides, -204 u for acetylglucosides or -308 u for maxima. These displacements are of 2–3 nm in the case of p-coumaroylglucosides), these ions still have the flavanol part acetylation, 6–7 nm for the p-coumaroylated derivatives and attached. For F-A+ pigments, a retro Diels–Alder (RDA) frag- 8 nm for the caffeoyl derivative of malvidin. mentation is observed for the aglycone ion through the cleavage The pigments were collected in several fractions, simplifying of the 1 and 3 bonds of the flavanol C-ring (loss of 152 u for the HPLC separation and allowing the identification of less- (epi)catechin or of 168 u for (epi)gallocatechin). Other frag- concentrated pigments. Figure S1 illustrates an example of this mentations for F-A+ adducts were the cleavage of the 2 and 4 kind of chromatograms, showing the successful separation of 19 bonds of the flavanol C-ring (loss of 246 u for (epi)catechin or monoglucosides. of 262 u for (epi)gallocatechin) and the cleavage of the 1 and 4

OCH3 OH OCH3 HO O OH OCH3 HO O -288 u O-glc OCH3 OH OH O-glc HO O OH OH Figure 6. Mass spectrum and m/z 493 main MS fragmentations of an OH OH OCH anthocyanin-ﬂavanol derivative, 3 Mv-3-glc-( )-catechin. ESI, m/z OH + 781 electrospray ionization; glc, HO O OCH3 glucoside; M, molecular ion;

OH m/z, mass to charge ratio. OH OH HO O -162 u OH

m/z OH 601 -18 u

OCH3

OCH3 OH OH HO O OCH3 HO O OCH 3 OH 2/4, -42 u OH OH OH OH OH HO O 1 OH HO O OH 2 OH OH m/z 577 4 OH 3

m/z 619 -288 u

OCH3 OH

HO O OCH3

OH OH m/z 331 bonds of the C-ring of flavanol, losing a phloroglucinol ring (loss (compounds 36 and 37) as in the case of their precursor of 126 u for both (epi)catechin and (epi)gallocatechin). anthocyanins. This fragmentation pattern is characteristic for deriva- In addition, new compounds like (+)-catechin-cyanidin-3- tives with the flavanol as upper unit (F-A+) (Salas et al. 2003, glucoside and (+)-gallocatechin-cyanidin-3-glucoside have been González-Paramás et al. 2006, Pati et al. 2006) and it was detected for first time to our knowledge in red wines. observed for pigments 20–28 and 31–44. Both isomers with Figure 4 shows an example of this fragmentation pattern, (+)-catechin and (-)-epicatechin are detected for the anthocya- the mass spectrum of (+)-catechin-malvidin-3-glucoside and the nidins delphinidin, petunidin and malvidin. Compounds 31–38 proposed chemical structures for the fragment ions can be and 44 are acetylated or p-coumaroylated derivatives. The observed. For this compound, ESI(+)-CID-MS/MS experiments p-coumaric acid cis and trans isomers in (+)-catechin-malvidin- selecting as precursor ion the corresponding to the aglycone 3-(6-p-coumaroyl)-glucoside derivatives were distinguished ion (Figure 5) confirms that the fragment ion at m/z 493 is

originated from the loss of 126 u from the aglycone, as it is CC

+ described in literature (Nave et al. 2010). This fact together with the loss of 246 u would conﬁrm a F-A+ structure. +

densation In the MS spectra of compounds identiﬁed as A -F pigments (Figure 6), the fragment ion corresponding to the RDA fragmentation was not observed. The cleavage of the 2 and 4 link-

CC SPE ages of C-ring of ﬂavanol originates a loss of 42 u, as it is

+ observed for 29 and 30 malvidin derivatives (m/z 577). This fragment ion could also be interpreted as a procyanidin, but not only its characteristic fragment ions because of its monomers (m/z 289 or m/z 291) are not present but also the rest of fragmentations are in agreement with an A+-F structure. For these molecules, the loss of ﬂavanol unit is of 288 u. The cleavage

Fractions of phloroglucinol ring (cleavage of the 1 and 4 bonds of the flavanol C-ring) from flavanol unit would generate a fragment ion at 455 u, which is not observed; however, mass spectra of compounds 29 and 30 show a fragment ion at m/z 457. These fragmentation patterns obtained herein showed that when the flavanol-anthocyanin B-type interflavanic bond is broken, a loss of 288 u, corresponding to the complete loss of the flavanol unit, is observed for both A+-F and F-A+ compounds. This fact is opposite to previous descriptions (González-Paramás et al. umn chromatography; glc, glucoside; Mv, malvidin; m/z, mass to charge ratio; 2006, Pati et al. 2006), they refer a loss of 290 u in the case of A+-F CC CC CC SPE +

Wine 1 Wine 2 Wine 3 Wine 2 Wine 3 pigments. We found that the loss of flavanol unit in A -F pigments produces a loss of 288 u and losses of 290 u are seen for non-coloured anthocyanin-flavanol A-type bond associations because of the loss of flavanol unit (data not shown). + max The results show that F-A isomers elute before than the l (nm) A+-F, as seen in the literature (Pati et al. 2006). Compounds 27 and 28, F-A+ type structures pigments, elute at 10.37 and 20.63 min, respectively, and the corresponding A+-F pigments 29 and 30 elute at 13.28 and 25.93 min, which is a consequence of being the A+-F conformation less polar. Although flavanol-anthocyanin reactions can take place ion (m/z) Fragment between C4 of the upper unit and either C6 or C8 of the lower unit, the compounds formed by C4–C8 linkage are more probable (González-Paramás et al. 2006). Taking this into account, the compounds 27 and 28, which are in higher concentrations (data + no shown), and the corresponding derivatives observed for the

(m/z) other anthocyanins are formed by the linkage between C4 of the flavanol ((+)-catechin and (-)-epicatechin, respectively) and C8 of the anthocyanin. On the other hand, linkage in compound 26 (detected in lower concentration) is assigned between the C4 of flavanol and the C6 of anthocyanin. Differences in retention times also support these assignments. Compounds 27 and 28 elute in rather similar retention times (10.37 and 20.63 min, respectively) so the spatial configuration should be similar. On the contrary, the presence of compound 26 in very early retention times (3.03 min) indicates a different structure. )-epicatechin 809 519, 357 541 8 — — — — )-catechin 2† 809 647, 519, 357 542 6–8 — — — — )-catechin 1† 809 647, 519, 357 n.d. 8 — — — — - + + Pigments 45–48 are adducts with two flavanol units: two units of (epi)catechin for compounds 45 and 46 (the compound 46 is a p-coumaroylated derivative of compound 45) and one (epi)cat- -coum)-glc-8-ethyl-(epi)catechin 955 665, 647, 357 544 8 8–10 — 10 11

p echin and one (epi)gallocatechin for compounds 47 and 48.Inthe mass spectrum of compound 45, fragment ions at m/z 467 and 373 (RDA fragmentation and loss of 246 u from aglycone ion at m/z 619) involve that one flavanol is the upper unit in the molecule. Thus, this compound could be (epi)catechin-(epi)catechin-Mv-3- glc or (epi)catechin-Mv-3-glc-(epi)catechin. Fragment ions at m/z 767 and 605 for compound 47 and at m/z 797 and 635 for compound 48 suggest that at least, the (epi)gallocatechin is joined to the anthocyanin, but further information about the pigment Chromatographic, Ultraviolet-visible and mass spectrometry data and proposed identities of the flavanol-anthocyanin acetaldehyde-mediated con RT (min) Compound [M] structure could not be obtained. The molecular ion of compound 47 could correspond to the same derivative with peonidin. Peoni- din derivatives elute just before of the corresponding to malvidin. 53 89.88 Mv-3-(6- 5254 68.57 61.33 Mv-3-glc-8-ethyl-( Mv-3-glc-8-ethyl-(epi)gallocatechin 825 519, 357, 331 n.d. 8 — — — — 51 65.41 Mv-3-glc-8-ethyl-( 4950 52.85 60.17 Pt-3-glc-8-ethyl-(epi)catechin Mv-3-glc-8-ethyl-( 795 505, 343 540 8 — — — — n.d., not determined; Pt, petunidin; RT, retention time; SPE, solid-phase extraction. Table 3. n †Compounds 50 and 51 are positional isomers with different spatial configuration of the asymmetric carbon of the ethyl bridge. —, not detected; CC, col products detected as well as fractions where each derivative was found. The same fragmentation pattern also agrees with a structure with

OH Figure 7. Mass spectrum and HO main MS fragmentations of an HO O OH ethyl-bridge ﬂavanol- OCH3 anthocyanin condensation HO H3CHC OH product, Mv-3-glc-8-ethyl- HO O OCH (+)-catechin. ESI, electrospray 3 ionization; glc, glucoside; M, OH molecular ion; Mv, malvidin; OH m/z, mass to charge ratio. m/z 647

-162 u

OH HO OCH HO 3 O OH OH OCH3 HO H CHC OH HO O 3 OCH -290 u 3 HO O OCH3 O-glc OH O-glc m/z 519 OH

m/z 809 -162 u

OCH3 OH

HO O OCH3

OH OH

m/z 357 two (epi)catechin units linked to the anthocyanidin petunidin, reaction can take place between C6 or C8 carbons but probably this pigment only differs from the peonidin derivative in the occurs between C8 carbons because of a higher stabilization of position of an hydroxyl group, which explains their similar polari- the charge (De Freitas and Mateus 2006). Compound 54 is a ties. Because this compound elutes just before of the malvidin derivative with (epi)gallocatechin and compounds 49–53 are derivative 48, it has been assigned as the peonidin homologue, but (epi)catechin derivatives, being the peak 53, a p-coumaroylated further research is needed to get a complete identification. pigment. In this work, 29 pigments derived from direct flavanol- Compounds 50, 51 and 52 are isomers, but they could anthocyanin reactions were identified in different fractions, be differentiated by their retention times. Pigments 50 and 51 as it can be observed in Figure S2, this figure collects the are derivatives with (+)-catechin, they are positional isomers chromatograms at 530 nm of fractions 11, 12, 14, 17 and 24 of involving different spatial configuration of the asymmetric wine 2. carbon of ethyl bridge (Atanasova et al. 2002, Alcalde-Eon et al. 2004). On the other hand, pigment 52 has a different spatial Flavanol-anthocyanin condensations mediated by acetaldehyde configuration of the flavanol unit, being a derivative with (-)- Peaks 49–54 were pigments formed by anthocyanin-flavanol epicatechin. Compound 49 is a similar derivative with the agly- condensations mediated by acetaldehyde (Table 3). This kind of cone petunidin instead of malvidin.

Figure 8. UV-vis normalized absorption spectra of ( ) Mv-3-glc, ( ) Catechin- Mv-3-glc and ( ) Mv-3-glc-8- ethyl-catechin. Mv, malvidin; glc, glucoside.

The main MS fragmentations of these compounds (Figure 7) S-PE07UN34, S-PE08UN05, S-PE09UN03, IT413-10). Maria B. are the loss of the ﬂavanol unit. In case of (epi)catechin deriva- Sánchez-Ilárduya, Cristina Sánchez-Fernández, Maria Viloria- tives, it involves a loss of 290 u and of 306 u for (epi)gallocat- Bernal and Diana M. López-Márquez thank Universidad del País echin ones. The ulterior loss of sugar (-162 u for glucosides Vasco/Euskal Herriko Unibertsitatea and Agricultural Depart- or -308 u for p-coumaroylglucosides) is also observed for ment of Basque Government for their PhD grants. Authors also these compounds. The loss of glucose or p-coumaroylated thank the wineries Faustino, Cune, Coto de Rioja, Torre de Oña glucose is the main loss in all kinds of coloured pigments except and Unión de Cosecheros de Labastida and the association of for these ones. Figure S3 collects the chromatogram at 530 nm wineries ABRA for their collaboration. Technical and human of fraction 8 of wine 1, showing the six acetaldehyde-mediated support provided by SGIker (UPV/EHU, MICINN, GV/EJ, ESF) is derivatives. gratefully acknowledged.

Spectrophotometric analysis Ultraviolet-visible spectra of these flavanol-anthocyanin com- References pounds are similar to those of the precursor monoglucosylated Alcalde-Eon, C., Escribano-Bailón, M.T., Santos-Buelga, C. and Rivas- anthocyanins, because the same chromophore group is still Gonzalo, J.C. (2004) Separation of pyranoanthocyanins from red wine by present. These flavanol-anthocyanin pigments with the antho- column chromatography. Analytica Chimica Acta 513, 305–318. Alcalde-Eon, C., Escribano-Bailón, M.T., Santos-Buelga, C. and Rivas- cyanin in flavylium form show bathochromic shifts in compari- Gonzalo, J.C. (2006) Changes in the detailed pigment composition of son with the visible absorbance maxima of their corresponding red wine during maturity and ageing. A comprehensive study. Analytica anthocyanin precursors (Figure 8). This effect is more pro- Chimica Acta 563, 238–254. nounced for the ethyl-bridge compounds (Fulcrand et al. 1996, Atanasova, V., Fulcrand, H., Cheynier, V. and Moutonet, M. (2002) Effect of oxygenation on polyphenol changes occurring in the course of wine- Es-Safi et al. 1999). Thus, these compounds contribute to the making. Analytica Chimica Acta 458, 15–27. bluish hue of wine. Boido, E., Alcalde-Eon, C., Carrau, F., Dellacassa, E. and Rivas-Gonzalo, In addition, for these compounds, the effect of the B-ring J.C. (2006) Aging effect on the pigment composition and color of Vitis substitution in the anthocyanin can be also observed: trisubsti- vinifera L. Cv. Tannat wines. Contribution of the main pigment families to tuted anthocyanin derivatives show higher visible absorption wine color. Journal of Agricultural and Food Chemistry 54, 6692–6704. De Freitas, V. and Mateus, N. (2006) Chemical transformations of antho- lambda maxima than disubstituted ones. The number of fla- cyanins yielding a variety of colours (Review). Environmental Chemistry vanol units influences the shift of visible absorbance maxima Letters 4, 175–183. because two flavanol derivatives show higher bathochromic Escarpa, A. and González, M.C. (2001) An overview of analytical chemistry shifts than pigments with one flavanol unit. of phenolic compounds in foods. Critical Reviews in Analytical Chemistry 31, 57–139. Es-Safi, N.E., Cheynier, V. and Moutounet, M. (2002) Role of aldehydic Conclusions derivatives in the condensation of phenolic compounds with emphasis on This article gives comprehensive information about the different the sensorial properties of fruit-derived foods. Journal of Agricultural and coloured flavanol-anthocyanin derivatives detected in aged Food Chemistry 50, 5571–5585. red wines of Rioja and provides new knowledge about different Es-Safi, N.E., Fulcrand, H., Cheynier, V. and Moutounet, M. (1999) Studies on the acetaldehyde-induced condensation of (-)-epicatechin and fragmentation patterns depending on the position of the fla- malvidin-3-O-glucoside in a model solution system. Journal of Agricul- vanol unit in the pigment. This knowledge allows the structural tural and Food Chemistry 47, 2096–2102. elucidation of unknown compounds. Fossen, T., Rayyan, S. and Andersen, Ø.M. (2004) Dimeric anthocyanins New compounds not reported before have been identified, from strawberry (Fragaria ananassa) consisting of pelargonidin-3- glucoside covalently linked to four flavan-3-ols. Phytochemistry 65, they are dimers with (+)-catechin or (+)-gallocatechin and 1421–1428. cyanidin-3-glucoside. Francia-Aricha, E.M., Guerra, M.T., Rivas-Gonzalo, J.C. and Santos- Buelga, C. (1997) New anthocyanin pigments formed after condensation Acknowledgements with flavanols. Journal of Agricultural and Food Chemistry 45, 2262– 2266. This research was supported by the Agricultural, Industrial Fulcrand, H., Cameira dos Santos, P.J., Sarni-Manchado, P. and Cheynier, and Education Departments of Basque Government (pro- V. (1996) Structure of new anthocyanin-derived pigments. Journal of jects numbers PA08/09, PA09/11, PA10/13, S-PE06UN14, Chemical Society 1, 735–739.

García-Beneytez, E., Cabello, F. and Revilla, E. (2003) Analysis of grape Rivas-Gonzalo, J.C., Bravo-Haro, S. and Santos-Buelga, C. (1995) and wine anthocyanins by HPLC-MS. Journal of Agricultural and Food Detection of compounds formed through the reaction of malvidin-3- Chemistry 51, 5622–5629. monoglucoside and catechin in the presence of acetaldehyde. Journal of González-Paramás, A.M., Lopes da Silva, F., Martín-López, P., Macz- Agricultural and Food Chemistry 43, 1444–1449. Pop, G., González-Manzano, S., Alcalde-Eón, C., Pérez-Alonso, J.J., Salas, E., Fulcrand, H., Meudec, E. and Cheynier, V. (2003) Reactions of Escribano-Bailón, M.T., Rivas-Gonzalo, J.C. and Santos-Buelga, C. anthocyanins and tannins in model solutions. Journal of Agricultural and (2006) Flavanol-anthocyanin condensed pigments in plant extracts. Food Food Chemistry 51, 7951–7961. Chemistry 94, 428–436. Salas, E., Atanasova, V., Poncet-Legrand, C., Meudec, E., Mazauric, J.P. Hayasaka, Y. and Asenstorfer, R.E. (2002) Screening for potential pigments and Cheynier, V. (2004) Demonstration of the occurrence of flavanol– derived from anthocyanins in red wine using nanoelectrospray tandem anthocyanin adducts in wine and in model solutions. Analytica Chimica mass spectrometry. Journal of Agricultural and Food Chemistry 50, 756– Acta 513, 325–332. 761. Sun, B., Leandro, M.C., de Freitas, V. and Spranger, M.I. (2006) Hayasaka, Y. and Kennedy, J.A. (2003) Mass spectrometric evidence for the Fractionation of red wine polyphenols by solid-phase extraction and liquid formation of pigmented polymers in red wine. Australian Journal of Grape chromatography. Journal of Chromatography A 1128, 27–38. and Wine Research 9, 210–220. He, J., Santos-Buelga, C., Mateus, N. and de Freitas, V. (2006) Isolation and quantification of oligomeric pyranoanthocyanin-flavanol pigments from red wines by combination of column chromatographic techniques. Manuscript received: 11 December 2011 Journal of Chromatography A 1134, 215–225. Revised manuscript received: 13 March 2012 Lee, D.F., Swinny, E.E. and Jones, G.P. (2004) NMR identification of ethyl-linked anthocyanin-flavanol pigments formed in model wine fer- Accepted: 24 March 2012 ments. Tetrahedron Letters 45, 1671–1674. Macz-Pop, G.A., González-Paramás, A.M., Pérez-Alonso, J.J. and Rivas- Gonzalo, J.C. (2006) New flavanol-anthocyanin condensed pigments and anthocyanin composition in Guatemalan beans (Phaseolus spp). Journal Supporting information of Agricultural and Food Chemistry 54, 536–542. Additional Supporting Information may be found in the online Monagas, M., Bartolomé, B. and Gómez-Cordovés, C. (2005) Updated knowledge about the presence of phenolic compounds in wine. Critical version of this article: Reviews in Food Science and Nutrition 45, 85–118. http://onlinelibrary.wiley.com/doi/10.1111/j.1755- Nave, F., Teixeira, N., Mateus, N. and de Freitas, V. (2010) The fate of flavanol-anthocyanin adducts in wines: study of their putative reaction 0238.2012.00190.x/abstract patterns in the presence of acetaldehyde. Food Chemistry 121, 1129– 1138. Figure S1. Chromatograms recorded at 530 nm corresponding Pati, S., Losito, I., Gambacorta, G., La Notte, E., Palmisano, F. and Zam- to fractions 8 and 11 of wine 2, fractionated by Toyopearl bonin, P.G. (2006) Simultaneous separation and identification of oligo- column chromatography methodology, showing the 19 mono- meric procyanidins and anthocyanin-derived pigments in raw red wine by glucosides. HPLC-UV-ESI-MS. Journal of Mass Spectrometry 41, 861–871. Pissarra, J., Lourenςo, S., González-Paramás, A.M., Mateus, N., Santos- Figure S2. Chromatograms recorded at 530 nm corresponding Buelga, C. and de Freitas, V. (2004) Formation of new anthocyanin-alkyl/ to fractions 11, 12, 14, 17 and 24 of wine 2, fractionated by aryl-flavanol pigments in model solutions. Analytica Chimica Acta 513, 215–221. Toyopearl column chromatography methodology, showing the Pozo-Bayón, M.A., Monagas, M., Polo, M.C. and Gómez-Cordovés, C. 29 flavanol-anthocyanin derivatives. (2004) Occurrence of pyranoanthocyanins in sparkling wines manufac- tured with red grape varieties. Journal of Agricultural and Food Chemistry Figure S3. Chromatogram recorded at 530 nm corresponding 52, 1300–1306. to fraction 8 of wine 1, fractionated by Toyopearl column chro- Remy, S., Fulcrand, H., Labarbe, B., Cheynier, V. and Moutounet, M. matography methodology, showing the six derivatives formed (2000) First confirmation in red wine of products resulting from direct by acetaldehyde-mediated condensation. anthocyanin–tannin reactions. Journal of the Science of Food and Agri- culture 80, 745–751. Please note: Wiley-Blackwell is not responsible for the content Ribéreau-Gayon, P., Glories, Y., Maujean, A. and Dubourdieu, D. (2006) or functionality of any supporting materials supplied by the Aging red wines in vat and barrel: phenomena occurring during aging. In: Handbook of Enology. The Chemistry of Wine, Stabilization and Treat- authors. Any queries (other than missing material) should be ments.(John Wiley & Sons Ltd: Chichester) pp. 387–428. directed to the corresponding author for the article.

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