Total Anthocyanin Extraction and HPLC Analysis

The frozen skin samples collected at harvest were ground using a ball mill MM300 (Retch) in the presence of liquid nitrogen. One gram of frozen ground samples was extracted into 5 mL of methanol: HCl (100:1). This extraction was used to determine total anthocyanin content spectrophotometry at 520 nm as described by Boss et al. (1996). Total anthocyanin extract was also used to determine anthocyanin composition using high-performance liquid chromatography (HPLC) as described by Downey and Rochfort (2008). HPLC separation of anthocyanins was utilized by using 10% formic acid in water (Solvent A) with a 10% formic acid in methanol (Solvent B) gradient at a flow rate of 1.0 mL/min as described by Downey and Rochfort (2008). The column temperature was maintained at 40°C for the duration of the analysis. The column selected was a Zorbax SB-C18 (150 mm × 4.6 mm, 5μ; Agilent, Palo Alto, CA) protected by an Agilent C-18 guard column. Samples were run on an Agilent 1200 HPLC system equipped with a diode array detector. The gradient conditions were: 0 min, 18% B; 14 min, 29% B; 16 min, 32% B; 18 min, 41% B; 18.1 min, 30% B; 29 min, 41% B; 32min, 50% B; 34.5 min, 100% B; 35–38 min, 18% B. Anthocyanins were monitored by diode array detection at 520 nm. A tentative identification was made by comparison of the spectra and elution times with previously published results and with three purchased standards (malvidin-3-O-glucoside, cyanidin-3-O-glucoside, and petunidin-3-O glucoside) from Alkemist Labs, Costa Mesa, CA. Quantification was performed using calibration curves of the available standards. We determined the berry skin dry weight in the different treatments, and we did not observe any significant difference among treatments thus, HPLC data is expressed on a fresh weight basis. For those compounds in which no standard was available, estimates of concentration were made by utilizing the standard curve for malvidin-3-O-glucoside.