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. 0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.06.023 208 D. Calvo et al. / Analytica Chimica Acta 524 (2004) 207–213 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.