*2.4. Phenolic Compounds*

The obtained extracts were re-suspended in the same solvent in a concentration of 5 mg/mL, filtered (0.2 μm), and finally injected in the HPLC equipment (Dionex Ultimate 3000 UPLC, Thermo Scientific, San Jose, CA, USA), using a diode-array detector (280, 330, 370, and 520 nm wavelengths) and linked to an electrospray ionization mass spectrometry (Linear Ion Trap LTQ XL, Thermo Scientific, San Jose, CA, USA) working in negative (non-anthocyanin compounds) and positive (anthocyanin compounds) mode, according to methodology described by Bessada et al. [10] and Gonçalves et al. [11]. Data were collected and analyzed using the Xcalibur® program (Thermo Finnigan, San Jose, CA,

USA). Separation was achieved using a Water Spherisorb S3 ODS-2 reverse phase C18 column (3 μm, 4.6 mm <sup>×</sup> 150 mm, Waters, Milford, MA, USA) for non-anthocyanin compounds and with an AQUA® reverse phase C18 column (5 μm, 150 mm × 4.6 mm, Phenomenex, Torrance, CA, USA) for anthocyanin compounds, working at 35 ◦C, using previously described gradients [10,11].

The compounds present in the samples were determined according to their UV-Vis and mass spectra and retention times in comparison with authentic standards, and also using information present in the literature. For the quantification, a 7 level calibration curve was obtained of different standard compounds. The contents in the individual phenolic compounds were expressed in mg per g of extract and in mg per g of colourant.
