Xcalibur software system

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 chromatographic analysis of the anthocyanins from black rice flour was performed as described earlier by Bolea et al. [24 (link)]. HPLC analysis was performed using a Surveyor HPLC system, controlled by an Xcalibur software system (Finnigan Surveyor LC, Thermo Scientific, Waltham, MA, USA). The anthocyanins detected in black rice flour were analyzed at a wavelength of 520 nm. The column used for this analysis was a C18 BDS Hypersil (150 mm × 4.6 mm, 5 mm). The gradient used for the elution of the anthocyanins was: 0–20 min: 9–35% (A), 20–30 min: 35% (A), 30–40 min: 35–50% (A), 40–45 min: 50–9% (A), with an injection volume of 10 µL, and the flow rate maintained at 1.000 mL/min.