0.22 μm syringe filters

The high-performance liquid chromatographic analysis of the anthocyanins extracted from EPE was assessed with a Thermo Finnigan Surveyor HPLC system with a DAD detector, controlled by the Xcalibur software system (Finnigan Surveyor LC, Thermo Scientific, USA). The method of Turturică et al. [39 (link)], with slight modifications, was used. In short, the EPE was filtered through a C18 Sep-Pack cartridge (Cartridge-Waters, USA) to separate the anthocyanins. In order to obtain the chromatographic elution profile, a C18 Synergi 4u Fusion-RP 80A stationary phase column (150 × 4.6 mm, 4 μm) was used, at an optimum column temperature of 25 °C. The mobile phase consisted of two phases: 100% methanol (A) and 10% formic acid (B). The injection volume was 10 μL, at a flow rate of 1 mL/min, whereas the elution took place under the following gradient conditions: 0–20 min, 9%–35% (A); 20–30 min, 35% (A); 30–40 min, 35%–50% (A); and 40–55 min, 50%–9% (A). The samples were filtered through 0.22-μm syringe filters (Bio Basic Canada Inc., ON, Canada) prior to the injection. The detector wavelength was 520 nm. The following HPLC reference substances were used for the identification of anthocyanins: delphininidin-3-glucoside chloride (purity ≥ 95%), delphinidin-3-rutinoside chloride (purity ≥ 95%), and cyanidin-3-rutinoside chloride (purity ≥ 90%) from Sigma Aldrich, Germany.

The high-performance liquid chromatographic (HPLC) analysis of the anthocyanins from the BC samples was conducted on a Thermo Finnigan HPLC system (Thermo Scientific, Waltham, MS, USA). The separation and identification of the main anthocyanin compounds was employed on a Synergi 4u Fusion-RP 80A (150 × 4.6 mm, 4 μm) column, at the 520 nm wavelength, at an oven temperature of 27 °C. The samples were filtered through a C18 Sep-Pack cartridge-Waters and through a 0.22-μm syringe filters (Bio Basic Canada Inc., Toronto, ON, Canada). The mobile elution phase consisted of two main solvents that were 10% formic acid (A) and 100% methanol (B) with a vertical gradient. The injection volume was 10 µL at a flow rate of 1.0 mL/min. The identification and quantification of the compounds were made based on each compound’s calibration curve, and the results were expressed as mg/g d.w.