2.4.2. Spectrophotometric Assays
Spectrophotometric analyses were performed using an Analytik Jena Specord 200 Plus spectrophotometer (Jena, Germany) set at the appropriate wavelength for each assay.
Total polyphenol content was measured by using the Folin-Ciocalteu colorimetric assay and the results were reported in mg/kg of gallic acid equivalents [25
]. Monomeric anthocyanin content was analyzed using the pH-differential method, expressing the results in mg/kg of malvidin-3-glucoside equivalents [26
]. Total flavonoid content was evaluated using a colorimetric assay with aluminum chloride using catechin as standard [27
Additionally, total antioxidant capacities were evaluated by the ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation assay, CUPRAC (cupric reducing antioxidant capacity) method, ORAC (oxygen radical absorbance capacity)—fluorescein (FL), and ORAC—pirogallol red (PGR) assays, as described previously [23
]. In all of the assays, Trolox was used as reference compound and results were expressed in terms of mmol Trolox equivalent antioxidant capacity per kg of sample. All of the measurements were done in triplicate.
HPLC separation, identification, and quantification of specific phenolic compounds were carried out using a Shimadzu HPLC Nexera system (Kyoto, Japan). This equipment consists of a quaternary LC-30AD pump, DGU-20A5R degasser unit, CTO-20AC oven, SIL-30AC auto-sampler, CBM-20A controller system, and UV-Vis diode array spectrophotometer (model SPD-M20A), coupled in tandem with a QTrap LC/MS/MS 3200 Applied Biosystems MDS Sciex system (Foster City, CA, USA). The detector offers wide linearity (2.5 AU) and a noise level of 0.6 × 10−5 AU for a wavelength from 190 to 700 nm. Instrument control and data collection were done using CLASS-VP DAD Shimadzu Chromatography Data System and Analyst software (version 1.5.2) for MS/MS analysis.
Anthocyanins were separated by HPLC using a C18 YMC 5 μm, 250 × 4.6 mm column with a C18 Nova-Pak 4 μm, 22 × 3.9 mm precolumn (Waters, Milford, MA, USA) with a flow rate of 0.3 mL/min at 30 °C. The injection volume was 50 μL. The mobile phase consisted of 0.1% v/v trifluoroacetic acid in water (A) and 100% acetonitrile (B). The gradient program was from 10% to 20% of solvent B in 15 min, followed by 6 min of stabilization, from 20% to 27% in 5 min, followed by 10 min stabilization, from 27% to 100% in 1 min, and from 100% to 10% in 1 min, followed by an isocratic step of 10 min at 10% B.
HPLC separation of flavonols, flavan-3-ols and phenolic acid derivatives was carried out using a Kinetex C18 column (core shell, 150 × 4.6 mm, 2.6 μm) with a SecurityGuard AJO-8768 C18 cartridge (Phenomenex, Torrance, CA, USA). The injection volume was 10 μL. A binary mobile phase of 0.1% v/v formic acid in water and acetonitrile was used at a flow rate of 0.5 mL/min. The acetonitrile gradient ranged from 15% to 25% acetonitrile for 14 min, from 25% to 35% for 11 min, from 35% to 100% for 1 min, from 100% to 15% for 1 min, followed by a stabilization period of 10 min. The column temperature was set at 30 °C for flavan-3-ols and phenolic acid derivatives, and at 40 °C for flavonols.
The analyses of stilbenoids were carried out using a C18 Kromasil 5 μm, 250 × 4.6 mm column (Akzo Nobel, Bohus, Sweden) with a C18 Nova-Pak Waters 22 × 3.9 mm, 4 μm precolumn (Waters, Milford, MA, USA) at 30 °C, using a mobile phase gradient consisting of 0.1% v/v formic acid in water (solvent A) and acetonitrile (solvent B). The injection volume was 25 μL. The flow rate was 0.5 mL/min, and the gradient program was from 15% to 20% of solvent B in 5 min, 20% to 44.5% in 45 min, and 44.5% to 100% in 1 min, followed by an isocratic step of 9 min at 100% and stabilization for 5 min at 15% of B.
The identity of phenolic compounds was assigned by ESI-MS/MS setting the following parameters: negative ionization mode; collision energy, 5 V; ionization voltage, −4000 V; capillary temperature, 450 °C; nebulizer gas, 15 psi. For identification of anthocyanins, a positive ionization mode was used. The identity assignation of compounds was done by comparison of their retention time (tR), UV-Vis spectra and mass (MS/MS) spectra with those of their respective commercially available standards. Quantification was performed using a DAD chromatogram extracted at 280 nm for flavan-3-ols, 306 nm for stilbenoids, 320 nm for phenolic acid derivatives, 360 nm for flavonols, and 518 nm for anthocyanins. For quantitative determinations, calibration curves were made with the commercial standards for flavan-3-ols, stilbenoids, phenolic acids, flavonols, and anthocyanins. Standard solutions spanning the concentration range from 1.0 to 80 mg/L were prepared by appropriate dilution of standard solutions in solvent A. The limits of detection and quantification were three and ten times the noise signal from the chromatograms of low standard concentration.