Migration Order of Wine Anthocyanins in Capillary Zone Electrophoresis

Analytica Chimica Acta 524 (2004) 207–213

Migration order of wine anthocyanins in capillary zone electrophoresis

David Calvo, Rubén Sáenz-López, Puriﬁcación Fernández-Zurbano, Mar´ıa Teresa Tena∗

Department of Chemistry, University of La Rioja, Madre de Dios, 51. Logroño, La Rioja E-26006, Spain Received 17 November 2003; accepted 9 June 2004 Available online 10 July 2004

Abstract

Capillary zone electrophoresis (CZE) is an advantageous alternative to liquid chromatography (LC) in the analysis of wine anthocyanins in terms of separation efficiency, analysis time and reagent consumption. Thirteen wine anthocyanins of a Tannat wine, including acylated and non-acylated anthocyanin monoglucosides, pyranoanthocyanins and flavanol derivates, were separated using a 46 cm × 75 ␮m (i.d.) fused-silica capillary and a 50 mM sodium tetraborate buffer of pH 8.4 with 15% (v/v) methanol as modifier. Some of the pigments were isolated by LC fractionation and concentrated by lyophilisation. The reconstituted fractions were analysed by CZE in order to identify the compounds in the electrophoregram and to determine their migration times. The migration order found for the wine anthocyanins is discussed taking into account the molecular charge and mass, and the possibility of complex formation with tetraborate molecules. © 2004 Elsevier B.V. All rights reserved.

Keywords: Anthocyanins; Wine; Capillary zone electrophoresis

1. Introduction characterisation of anthocyanin aglycon and sugar moieties, as well as more complex structures of polymeric pigments. Wine anthocyanins are the pigments responsible for Very few capillary electrophoresis methods for the sepa- colour in red wines. They are all based on a cationic ration of anthocyanins have been reported [7]. They describe flavylium structure, differing by the degree and position the separation of mixtures of standards [8,9], strawberry, el- of hydroxylation. This structure is shown in Fig. 1(A). derberry [10] or blackcurrant anthocyanins [11]. There are five anthocyanidin precursors of wine antho- Recently, a method based on capillary zone electrophore- cyanins, namely: cyanidin (R1=OH, R2=H), peonidin sis (CZE) has been proposed for separating the five wine (R1=OMe, R2=H), malvidin (R1=OMe, R2=OMe), petu- anthocyanin monoglucosides and quantifying the three that nidin (R1=OMe, R2=OH) and delphinidin (R1=OH, are commercially available as pure compounds [12]. The R2=OH). These anthocyanidins are glucosylated in the C3 results obtained in the analysis of different wines by CZE position, giving them chemical stability. In addition, they were well correlated with those obtained by the standard LC can be acylated with acetic, caffeic and p-coumaric acid method. Moreover, the CZE method showed good sensitiv- at glucose C6 position. Moreover, anthocyanins can be ity and reproducibility, and provided minimal set-up time, present in aged red wines combined with flavanols, such as reduced costs and reagent consumption, as well as higher catechin, and tannins. separation efficiencies in a shorter analysis time. Therefore, The analysis of wine anthocyanins in wine and grapes is it seems to be an advantageous alternative to LC for the usually carried out by liquid chromatography (LC) with pho- analysis of wine anthocyanins. todiode array or mass spectrometry (MS) detection [1–6]. CZE has also been shown to be efficient enough to sepa- Nowadays, LC–MS is the best analytical tool for studying rate red wine polymeric pigments [13]. These compounds, anthocyanidin structures. The MS/MS approach allows the responsible for the colour stability of aged red wine, can be separated from free anthocyanins in less than 20 min, but this analysis time might be reduced by electroosmotic flow ∗ Corresponding author. Tel.: +34 941 299627, fax: +34 941 299621. inversion and a change of polarity. This is therefore our fu- E-mail address: [email protected] (M. Teresa Tena). ture objective.

Fig. 1. Chemical structures of (A) anthocyanin-3-O-glucosides and (B) malvidin-3-O-glucoside pyruvic acid adduct.

In this paper, a total of 13 wine anthocyanins were iden- trospray ionisation source operated in positive ion mode tified in the CZ electrophoregram. The wine anthocyanins and a Hewlett–Packard liquid chromatograph (Palo Alto, were previously separated and identified by LC, and some CA, USA) were used. of them were isolated by LC fractionation, and the fraction A Nucleosil 120 C18 column (20 cm × 0.46 cm, 5 ␮m) obtained analysed by CZE in order to obtain their migration from Teknokroma (San Cugat del Vallés, Spain) was used, times. and the mobile phase was a mixture of a 5% (v/v) formic acid aqueous solution and acetonitrile. The gradient program be- gan with 10% acetonitrile, increasing to 30% in 65 min, then 2. Experimental to 50% in 10 min and finally to 100% in 2 min. The flow-rate and the injection volume were 1 ml min−1 and 20 ␮l, respec- 2.1. Reagents and samples tively. The chromatograms were recorded at 520 nm. ␮ −1 HPLC-grade methanol and ethanol, disodium tetraborate A flow-rate of 35 l min was introduced in the electro- and potassium metabisulfite supplied by Merck (Darmstadt, spray ionisation interface by splitting the mobile phase into Germany); phosphoric and hydrochloric acid by Carlo Erba a 35:965 ratio between the UV detector and MS detector. The chromatographic effluent was mixed with nitrogen at (Rodano, Italy); sodium hydroxide by Prolabo (France); tar- −1 ◦ taric acid by Sigma (St. Louis, MO, USA) and Milli-Q (Mil- 30 l min at 150 C in the electrospray ionisation interface. lipore, Molsheim, France) ultrapure water were used. The cone voltage was variable from 40 to 80 V, depending Synthetic wine consisted of 12% (v/v) ethanol aqueous on the m/z ratio selected. The compounds were chemically solution with pH 3.5 (apparent), containing 6 g l−1 tartaric ionised by proton transfer, the positive ions generated were introduced into the mass spectrometer and the abundance acid. + All the solutions were filtered through a 0.45 ␮m nylon of selected m/z ratios corresponding to M ions of antho- filter and sonicated for 15 min before use. cyanins were recorded. The anthocyanins identified in the wine sample and their retention times are listed in Table 1. The sample was a 2002 vintage Tannat red wine from + Chile. The Cabernet wine and pomace were obtained from Peaks 9 and 11 (m/z M 809) were identified as dimers of Bodegas Ontañón (Logroño, La Rioja, Spain). malvidin-3-O-glucoside and catechin, with the anthocyanin The wine sample was centrifuged at 5000 rpm for 5 min moiety ethyl-linked by its C6 or C8 position to C8 position at room temperature using a 5804 Eppendorff centrifuge of catechin, but this m/z value is also characteristic of a dimer (Hamburg, Germany), transferred to a topaz bottle, kept at between malvidin-3-O-glucoside and epicatechin. Besides, 4 ◦C under nitrogen and filtered through a 0.45 ␮m nylon the elution order coincides with that reported by Revilla filter before use. et al. [3], but for petunidin-3-O-(6-O-acetyl)glucoside that In order to obtain concentrated fractions, 10 ml of wine eluted between the two malvidin-3-O-glucoside catechin was freeze dried at −80 ◦C for 10 h using a 26-G model Tel- dimers. star Cryodos (Terrassa, Spain) prior to LC fractionation. The freeze-dried wine was dissolved in 10 ml of methanol, then 2.3. LC fractionation the methanolic solution containing the anthocyanins was dried under a nitrogen stream and finally the solid residue The 10-fold concentrated freeze-dried wine solution was reconstituted by 1 ml of synthetic wine. injected into a Waters liquid chromatograph (Milford, MA, USA) and the anthocyanins separated under the same con- 2.2. LC–MS analysis ditions described above, but with an injection volume of 100 ␮l. The chromatograph consisted of two 515 HPLC An Engine 5989-B Hewlett–Packard quadrupole mass pumps, an on-line degasser, a 717 Plus autosampler and a spectrometer equipped with a 59987 Hewlett–Packard elec- photodiode array detector. The chromatogram was recorded D. Calvo et al. / Analytica Chimica Acta 524 (2004) 207–213 209

Table 1 Retention times (tR) of the anthocyanins separated and identified by LC–MS and fractions taken tR (min) Compound Fraction Time interval (min) 13.32 Delphinidin-3-O-glucoside – – 17.12 Cyanidin-3-O-glucoside – – 20.30 Petunidin-3-O-glucoside – – 22.95 Petunidin-3-O-glucoside and pyruvic acid derivative F1 22.75–23.20 24.12 Peonidin-3-O-glucoside – – 26.42 Malvidin-3-O-glucoside – – 29.64 Malvidin-3-O-glucoside and pyruvic acid derivative F2 29.35–29.90 30.72 Delphinidin-3-O-(6-O-acetyl)glucoside F3 30.40–31.00 34.92 Malvidin-3-O-glucoside and catechin dimer F4 34.70–35.20 37.07 Petunidin-3-O-(6-O-acetyl)glucoside F5 36.85–37.35 38.65 Malvidin-3-O-glucoside and catechin dimer F6 38.45–38.95 41.95 Peonidin-3-O-(6-O-acetyl)glucoside F7 41.55–42.25 43.77 Malvidin-3-O-(6-O-acetyl)glucoside – – 56.87 Malvidin-3-O-(6-O-p-coumaroyl)glucoside – – at 520 nm and the spectrum from 250 to 650 nm for each (effective length) fused-silica capillary and with a diode peak was also collected. A typical chromatogram is shown array detector. in Fig. 2. The capillary was conditioned before injection by first The column effluent was collected when some of the an- washing with 0.1 M sodium hydroxide for 2 min, then with thocyanins were eluted. The seven fractions taken and their ultrapure water for 2 min and finally with the running buffer time interval are shown in Table 1. The LC fractions were for 5 min. The buffer vials were replenished automatically freeze dried under the same conditions described for the after each run in order to use fresh buffer solution each time wine sample, reconstituted with 600 ␮l of methanol and the and improve the reproducibility of the migration times. methanolic solution of each fraction was analysed by CZE. The running buffer was a 50 mM sodium tetraborate solution at pH 8.4 with 15% (v/v) methanol as modifier. A 2.4. CZE separation voltage of 25 kV was used and the capillary was maintained at 10 ◦C. The sample (30 nl) was injected by hydrodynamic Capillary zone electrophoresis was carried out using an injection of 50 mbar × 6 s. The separation was carried out in Agilent CE instrument (Waldbronn, Germany) furnished positive polarity mode, injection at the anode and detection with a standard cassette containing a 75 ␮m (i.d.) × 46 cm at the cathode, with a positive electroosmotic flow.

Fig. 2. Liquid chromatogram at 520 nm of a 2002 vintage Tannat red wine. The LC conditions are described in the Section 2. Peak identiﬁcation: 1, delphinidin-3-O-glucoside; 2, cyanidin-3-O-glucoside; 3, petunidin-3-O-glucoside; 4, derivative of petunidin-3-O-glucoside and pyruvic acid; 5, peonidin-3-O-glucoside; 6, malvidin-3-O-glucoside; 7, derivative of malvidin-3-O-glucoside and pyruvic acid; 8, delphinidin-3-O-(6-O-acetyl)glucoside; 9, dimer of malvidin-3-O-glucoside and catechin; 10, petunidin-3-O-(6-O-acetyl)glucoside; 11, dimer of malvidin-3-O-glucoside and catechin; 12, peonidin-3-O-(6-O-acetyl)glucoside; 13, malvidin-3-O-(6-O-acetyl)glucoside; 14, malvidin-3-O-(6-O-p-coumaroyl)glucoside. 210 D. Calvo et al. / Analytica Chimica Acta 524 (2004) 207–213

The electrophoregrams were recorded at 599 nm, and the migration time for this anthocyanin was 9.80 min. The spectrum from 200 to 599 nm was also collected for each malvidin-3-(6-O-p-coumaroyl)glucoside was identified peak. All the samples and fraction solutions were injected by LC–MS in a Cabernet pomace extract. The com- in duplicate and the results expressed as mean values. pound was concentrated enough in the extract to obtain −1 Potassium metabisulphite was added (250 mg of SO2 l ) a LC fraction which could be analysed by CZE with- to the sample and LC fractions before CZE analysis in order out prior concentration. The migration time obtained for to avoid differences in the electrophoretic separation caused malvidin-3-(6-O-p-coumaroyl)glucoside was 9.47 min. The by SO2. migration time of the remaining compounds was obtained by CZE analysis of the lyophilised LC fractions. The electrophoregram obtained for each fraction is shown in Fig. 3. 3. Results and discussion As expected, the migration times of the first five anthocyanins increased with decreasing molecular mass: Table 2 lists the migration times of the 13 wine antho- malvidin-3-O-(6-O-p-coumaroyl)glucoside (M = 639) cyanins detected in the CZ electrophoregram of a Tannat red < malvidin-3-O-(6-O-p-acetyl)glucoside (M = 535) wine. < peonidin-3-O-(6-O-p-acetyl)glucoside (M = 505) < The migration times of non-acylated wine anthocyanin malvidin-3-O-glucoside (M = 493) < peonidin-3-O- monoglucosides have been reported previously [12]. Since glucoside (M = 301). As regards the other anthocyanins, the anthocyanins are anions at the pH of the running buffer, the charge increased due to complex or adduct formation they migrate to the cathode (and to the detector) because and thus the migration time also increased. the mobility of the positive electroosmotic flow is higher. The LC fraction F1 corresponded to a derivative Therefore, anthocyanins with a lower molecular mass have of petunidin-3-O-glucoside and pyruvic acid, but it a higher charge/size and show longer migration times. In ad- also contained peonidin-3-O-glucoside, which eluted dition, cyanidin-, delphinidin- and petunidin-3-O-glucoside very close, as shown in the chromatogram in Fig. 2. have ortho-hydroxy groups and form complexes with tetrab- Petunidin-3-O-glucoside pyruvic acid adduct displayed a orate molecules, which increase their charge and then their migration time of 11.60 min. In the electrophoregram, the charge/size ratio. In contrast, the non-orthohydroxylated trace of peonidin-3-O-glucoside appeared at 10.49 min anthocyanins, malvidin- and peonidin-3-O-glucoside, can- (Fig. 3). not form complexes. This accounts for the fact that Mateus et al. [14] characterised this pigment and others malvidin-3-O-glucoside showed the shortest migration related by LSI–MS and NMR spectroscopy. The proposed time, followed by peonidin-3-O-glucoside, which preceded chemical structure is shown in Fig. 1(B). Vivar-Quintana petunidin-3-O-glucoside and delphinidin-3-O-glucoside, et al. [15] also reported pyranoanthocyanins in wine. although they have higher molecular mass, and finally If the migration times of petunidin-3-O-glucoside cyanidin-3-O-glucoside. (12.22 min) and its adduct with pyruvic acid (11.60 min) are The malvidin-3-(6-O-acetyl)glucoside was identified compared, it seems that the cycloaddition of pyruvic acid to by LC–MS in a Cabernet red wine. The compound was the anthocyanin prompts a decrease in the charge/size ratio. isolated by LC fractionation and the fraction analysed This decrease might be caused for one of the following two by CZE. Neither wine nor fraction lyophilisation was reasons: (a) the resulting molecule has increased molecular needed since the concentration was high enough. The mass but the same charge, or (b) the carboxylic group is negatively charged, but this hinders the formation of tetrab- Table 2 orate complexes responsible for the increased charge/size Migration times (tm) of wine anthocyanins separated by CZE ratio of petunidin-3-O-glucoside. Peak Compound tm (min) In contrast, the derivative of malvidin-3-O-glucoside number and pyruvic acid displayed a migration time of 11.50 min, 1 Malvidin-3-O-(6-O-p-coumaroyl)glucoside 9.47 longer than that of malvidin-3-O-glucoside (10.34 min). 2 Malvidin-3-O-(6-O-acetyl)glucoside 9.80 The migration time was obtained by CZE of fraction F2, 3 Peonidin-3-O-(6-O-acetyl)glucoside 9.95 4 Malvidin-3-O-glucoside 10.34 which contains only the malvidin-3-O-glucoside pyruvic 5 Peonidin-3-O-glucoside 10.63 acid adduct (Fig. 3). The cycloaddition of pyruvic acid to 6 Malvidin-3-O-glucoside and catechin dimer 10.90 malvidin-3-O-glucoside introduces a carboxylic group in 7 Malvidin-3-O-glucoside and catechin dimer 11.25 the anthocyanin molecule, which increases the compound 8 Petunidin-3-O-(6-O-acetyl)glucoside 11.35 charge and its charge/size ratio. Therefore, the adduct mi- 9 Malvidin-3-O-glucoside and pyruvic 11.50 acid derivative grated slower to the cathode than malvidin-3-O-glucoside. 10 Petunidin-3-O-glucoside and pyruvic 11.60 No signals were detected in the electrophoregram of frac- acid derivative tion F3, probably because the concentration was too low. 11 Petunidin-3-O-glucoside 12.22 Fraction F4, corresponding to a malvidin-3-O-glucoside 12 Delphinidin-3-O-glucoside 12.42 catechin dimer, showed two signals at 10.34 and 10.90 min, 13 Cyanidin-3-O-glucoside 12.64 as can be seen in Fig. 3. The former was produced by D. Calvo et al. / Analytica Chimica Acta 524 (2004) 207–213 211

Fig. 3. Electrophoregrams obtained for the LC fractions. The CZE conditions are described in the Section 2. malvidin-3-O-glucoside from dimer degradation and the lat- These pigments result from condensation mediated by ter corresponded to the dimer. Therefore, the dimer displays acetaldehyde, where malvidin-3-O-glucoside is linked to a higher charge/size ratio than malvidin-3-O-glucoside, catechin through an ethyl bridge [3]. In model solution, which means that the molecular charge increase is more im- it has been shown that two isomers are formed from cat- portant than the molecular mass increase. In fact, catechin echin and malvidin-3-O-glucoside [16]. The anthocyanin has ortho-hydroxy groups that can form complexes with would be ethyl-linked by its C6 or the C8 position to tetraborate. The other dimer of malvidin-3-O-glucoside the C8 position of the catechin moiety. The reaction be- and catechin, isolated in fraction F6, showed a migra- tween malvidin-3-O-glucoside and catechin starts with the tion time of 11.31 min (Fig. 3), also higher than that of protonation of acetaldehyde, followed by addition to a nu- malvidin-3-O-glucoside. cleophilic position of the catechin; the dehydration of the 212 D. Calvo et al. / Analytica Chimica Acta 524 (2004) 207–213

Fig. 4. Electrophoregram at 599 nm of a 2002 vintage Tannat red wine. For CZE conditions, see Section 2. Peak identification: 1, malvidin-3-O-(6- coumaroyl)glucoside; 2, malvidin-3-O-(6-acetyl)glucoside; 3, peonidin-3-O-(6-acetyl)glucoside; 4, malvidin-3-O-glucoside; 5, peonidin-3-O-glucoside; 6, malvidin-3-O-glucoside catechin dimer; 7, malvidin-3-O-glucoside catechin dimer; 8, petunidin-3-O-(6-acetyl)glucoside; 9, malvidin-3-O-glucoside and pyruvic acid derivative; 10, petunidin-3-O-glucoside and pyruvic acid derivative; 11, petunidin-3-O-glucoside; 12, delphinidin-3-O-glucoside; 13, cyanidin-3-O-glucoside. resulting protonated adduct yields a new carbocation, which The migration time of malvidin-3-O-glucoside and undergoes a nucleophilic attack by the anthocyanin. peonidin-3-O-glucoside is explained by their charge/size The dimers showed different migration times, but were ratios. Petunidin-, delphinidin- and cyanidin-3-O-glucoside nevertheless very close (10.9 and 11.3 min) because they with ortho-hydroxy groups can form complexes with tetrab- have the same charge and mass. The different molecular orate molecules, which increase their charge and their shape might account for the differences in electrophoretic charge/size ratio. Therefore, they show longer migration mobility. In fact, their chromatographic behaviour is more times. different than their electrophoretic behaviour. Acetic and coumaric acid acylated anthocyanin monoglu- Petunidin-3-O-(6-O-acetyl)glucoside was isolated in frac- cosides have lower migration times than non-acylated ones tion F5 and displayed a migration time of 11.35 min, as because acylation causes a charge/size ratio decrease as the shown in Fig. 3. Its migration time was shorter than that of molecular mass increases, while the molecular charge re- petunidin-3-O-glucoside (12.22 min) because the molecular mains the same. mass increased and the molecular charge remained the same. The migration times of the two malvidin-3-O-glucoside Hence, the charge/size ratio decreased. The same explana- catechin dimers and the pyruvic acid derivative corre- tion is valid for peonidin-3-O-(6-O-acetyl)glucoside, which sponded to an increase in the compound charge because showed a migration time of 9.95 min (see electrophoregram catechin has ortho-hydroxy groups that can form complexes of fraction F7 in Fig. 3), whereas the migration time of with tetraborate, and because the cycloaddition of pyru- peonidin-3-O-glucoside was 10.63 min. vic acid to malvidin-3-O-glucoside introduces a carboxylic As can be seen from Fig. 4, which shows the electrophore- acid group into the anthocyanin molecule. In contrast, gram of the wine sample, the 13 anthocyanins identified were the petunidin-3-O-glucoside and pyruvic acid cycloaddi- completely resolved in less than 4 min and with an analysis tion adduct has a shorter migration time than petunidin time of less than 13 min. Our current research is aimed at re- monoglucoside because the negatively charged carboxylic ducing the analysis time by inversion of the electroosmotic group might hinder the formation of tetraborate complexes, flow. thus diminishing the molecule charge/size ratio.

4. Conclusions Acknowledgements

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