The vines of the varieties Cabernet Franc, Merlot, Sangiovese and Syrah were planted in 2003, and the clones used were 986, 181, VCR23 and VCR1, respectively. The rootstock find more used was Paulsen 1103 (Vitis berlandieri Planch × Vitis rupestris Scheele); the vertical shoot positioning

trellis system training was used; the row and vine spacing was 3.0 × 1.2 m and the vineyard yield was between 6 and 7 t/ha. The wines were all produced under the same conditions in the commercial winery of São Joaquim – SC through a traditional skin-contact technique. The berries were separated from the stalks, crushed and maintained in a stainless steel vat. The maceration period was 15 days, with one or two daily pumping events at 22–28 °C. The must was separated from the solid parts and transferred to other stainless steel vats. Prior to initiating the alcoholic fermentation, a commercial sulphating agent (12 g 100 kg−1 of must, corresponding to 60 mg L−1 of free SO2) (Noxitan, Pascal Biotech, Paris), Saccharomyces cerevisae strain (20 g 100 kg−1) (Fermol Rouge, Pascal Biotech, Paris) and commercial enzymes with pectolytic Fasudil chemical structure activity (2–4 g h L−1) (Pectinex SPL/Ultra, Pascal Biotech, Paris)

were added to the musts. Malic acid consumption by lactic acid bacteria occurred spontaneously within 60–75 days. Once alcohol fermentation had finished the wines were stored in French oak wood for approximately 1 year. Before bottling, Noxitan (35 mg L−1 of free SO2, on average) was added. The wine samples from 2007 and 2006 vintages were analysed after 1 and 2 years of aging in bottle, respectively. The wines were stored at 10 °C prior to analysis. The wine was purified and concentrated using the method described by Pastor del Rio and Kennedy (2006) with the following modifications.

Ten millilitres of wine, dealcoholised under reduced pressure at 30 °C, were applied on the C18-SPE cartridge (1 g, Waters, Milford, MA) previously activated with 4 mL of methanol followed by 10 mL of water. The applied sample was washed with 50 mL of water, eluted Methisazone with 40 mL of methanol, evaporated, and then dissolved in 2 mL of methanol. The sample preparation and analysis were carried out in triplicate for each wine. The PA subunit composition, percentage of galloylation (%G), percentage of prodelphinidins (%P), and mean degree of polymerisation (mDP), were determined after acid-catalysis in the presence of excess phloroglucinol (phloroglucinolysis) (Kennedy & Jones, 2001). A solution of 0.2 N HCl in methanol, containing 100 g L−1 phloroglucinol and 20 g L−1 ascorbic acid, was prepared. One hundred microlitres of concentrated and purified wine sample (item 2.3) was reacted with 100 μL of the phloroglucinol reagent at 50 °C for 20 min and then combined with 1000 μL of 40 mm aqueous sodium acetate to stop the reaction. The final solutions were filtered through 0.22 μm, 13 mm PTFE syringe tip filters (Millipore, Bedford, MA) into LC vials and immediately injected into a HPLC-DAD–MS system.