5.9. Chromatographic conditions for anthocyanin analysis

Column Zorbax Eclipse Plus C18 150 × 2.1 mm, 3.5 µm (SFF‐CXX, P/N 959763‐902) and Precolumn Zorbax Eclipse Plus‐C18 12.5 × 4.6 mm, 5 µm (SFF‐C002, P/N 820950‐936) were assembled over P/N 820888‐901. HPLC conditions: injection volume 5 µl; mobile phase A Water HPLC‐grade (0.2% trifluoroacetic acid); mobile phase B methanol (0.2% trifluoroacetic acid); column temperature 50°C; Detector DAD (diode array detector) (Peak width > 0.1 mm (2 s); storage of all 190–700 nm step 2 nm; slit 4 nm; margin for negative absorbance 100 mAu. ITMS conditions: ionization source ESI positive; ion trap analyzer (capillary 3,500 V, target mass 493 m/z, comp stability 100%, trap drive level 100%, scan 100–900 m/z, ICC smart target 500,000, max accu time 200 ms, average 5). The anthocyanidin monoglucosides of the skin extracts and wines were chromatographed by HPLC using a Beckman Ultra sphere (C18) ODS (250 × 4.6 mm i.d.) column, and detection was carried out at 520 nm. The solvents were A, H₂O/HCOOH (9:1), and B, CH₃CN/H₂O/ HCOOH (3:6:1). The gradient was 20%–85% B for 70 min, 85%–100% B for 5 min, and then isocratic for 10 min at a flow rate of 1 ml/min. The content in free anthocyanins was determined using a calibration curve (based on peak area), which was established using malvidin 3‐glucoside. Standard solutions were subjected to the same procedure [concentration (mg/L) = 803.7 × (do − d) + 15.13].

The contents of free anthocyanins were determined using calibration curves (based on peak area), which were established using malvidin 3‐glucoside. Standard solutions were subjected to the same procedure (y = 0.7968x + 7.5756, R ² = .9774). The anthocyanidin‐3‐monoglucosides and respective acetylated and coumaroylated glycosides were identified based on their UV‐Vis spectra and retention times. The anthocyanidins were identified by HPLC by comparison with internal standards. The calibration curves were obtained by injecting standards with different concentrations of malvidin 3‐glucoside (Sigma). The range of linear calibration curves was from 0.1 to 1.0 mg/L for the lower concentration compounds (R ² > .996), 0.1 to 5.0 mg/L for intermediate concentration compounds (R ² > .987), and 10.0 to 200.0 mg/L for the higher concentration compounds (R ² > .987). Unknown concentrations were determined from the regression equations, and the results were expressed in mg of malvidin 3‐glucoside per berry. Repeatability of this method from extraction to HPLC analysis for four samples of the same batch of grape skins had a coefficient of variation <7%.