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 © 2012 Australian Society of Viticulture and Oenology Inc. Sánchez-Ilárduya et al. Flavanol-anthocyanin derivatives mass spectrometry 205 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.