2.1. Reagents and Solutions
All reagents were of analytical grade, and deionized water was used for extraction procedure and preparation of all aqueous solutions. The reagents used incuded methanol (MeOH, Chem-Lab, Zedelgem, Belgium), ethanol (EtOH, Chem-Lab), ortho-phosphoric acid (H3PO4 85%, Panreac, Barcelona, Spain), formic acid (HCOOH, Panreac), sodium hydroxide (NaOH, Sigma-Aldrich, St. Louis, MO, USA), potassium chloride (KCl, J.T. Baker, Deventer, The Netherlands), hydrochloric acid (HCl 37%, Carlo Erba Reagents, Italy), acetic acid (CH3COOH, Panreac), sodium acetate (CH3COONa, J.T. Baker), sodium carbonate (Na2CO3, Sigma-Aldrich), Folin & Ciocalteu’s phenol reagent (Sigma-Aldrich), gallic acid ((HO)3C6H2CO2H, Sigma-Aldrich), potassium sodium tartrate tetrahydrate (KOCOCH(OH)CH(OH)COONa × 4H2O, Sigma-Aldrich), 3,5-dinitrosalicylic acid ((O2N)2C6H2-2-(OH)CO2H, Sigma-Aldrich), glucose (C6H12O6, Sigma-Aldrich), phenol (C6H5OH, Sigma-Aldrich), sodium sulfite (Na2SO3, Panreac), kuromanin chloride (cyanidin-3-O-glucoside, C21H21O11Cl, ≥96%, Extrasynthese, Lyon, France), and oenin chloride (malvidin-3-O-glucoside, C23H25O12Cl, ≥97%, Extrasynthese). Acetonitrile (ACN, LC-MS grade, Sigma-Aldrich), methanol (MeOH, LC-MS grade, Sigma-Aldrich), and water (H2O, Thermo Fischer Scientific, Waltham, MA, USA) were used as elution solvents during liquid chromatography.
The nine materials investigated were the Amberite XAD 2 (Sigma-Aldrich), Amberlite IRA 400 Cl−
(Sigma-Aldrich), Lewatit MDS TP 208 (Lanxess AG, Cologne, Germany), Lewatit MDS TP 260 (Lanxess AG), Chromosorb G-HP (Sigma-Aldrich), Zeocros CA 150 (PQ Corporation, Rolle, Switzerland), Activated Carbon (Chemviron Carbon, Brussels, Belgium), Oasis HLB (Waters Corporation, Milford, MA, USA), and Isolute C8 (EC) (Biotage, Sweden), Table 1
2.3. Pretreatment of Solid Phase Materials and Evaluation Tests
The resins were pretreated and activated according to the manufacturer’s recommendation. At first, resins were soaked in methanol overnight. After soaking they were packed into plastic syringe tubes with glass microfiber filters (Whatman, Little Chalfont, UK). The resulting material volume into the SPE columns were between 0.75 and 0.85 cm3. After filling, the SPE columns were rinsed with water, and equilibrated with four bed volumes (BV) of H2O + 5% v/v ortho-phosphoric acid (H3PO4 85%), which was the solvent used during extraction procedure. The pretreatment of ion exchange resins was constituted of four extra steps that were performed between the steps of rinsing with water and before the equilibration of the column. The cation exchange resins were treated with 2 BV of 4% w/v NaOH solution, then rinsed with 4 BV of deionised water. Afterward, these were treated with 2BV of a 4% v/v HCl solution, and then rinsed again with 5 BV of water. The same procedure, but using the NaOH and HCl solutions in reverse order, was applied during the activation of the anion exchange resins.
The collected byproducts’ extract was the substrate used during the evaluation tests on the selected resins. The pretreatment of the materials was followed by a three steps procedure. In the first step, 1.3 BV of sample was passed through each column. The second step was the washing of the column with 1.3 BV of H2O, and the third step was the elution with 1.3 BV of acidified ethanol solution (3% v/v formic acid). After every step, the eluents of the columns were collected and stored for the subsequent analytical procedures.
2.4. Analytical Procedures for Total Anthocyanins, Phenolic Content, and Sugars Determination
Total anthocyanins, phenolic content, and sugars were determined in byproducts extract and at the eluents collected after SPE procedure. The methods used were the pH-differential method, Folin-Ciocalteu (FC) method, and dinitrosalicylic acid method respectively.
pH-differential method: The total anthocyanin content was determined using the pH-differential method [32
] using a UV-vis spectrophotometer (Jenway 6300, Staffordshire, UK). This method is based at the property of anthocyanins to alter their structure in different pH conditions, resulting in a change in their color. In brief, two dilutions of the sample were prepared using buffer solutions, one at pH = 1 and another at pH = 4.5, and the absorbances of these solutions at 510 nm and at 700 nm were measured. Anthocyanin content was calculated as cyanidin-3-O
-glucoside equivalents using an extinction coefficient of 26,900 L·mol−1
and molecular weight of 449.
Folin-Ciocalteu (FC) method: The phenolic content was determined using the Folin-Ciocalteu (FC) method. In brief, a calibration curve was made using gallic acid solutions. In a test tube 0.2 mL of the sample were added, followed by 2.6 mL H2
O, 2 mL Na2
of a (7% w
) solution and 0.2 mL of FC reagent. The mixture left in the dark for 90 min. The absorbance of the mixture at 745 nm was measured [18
Dinitrosalicylic acid (DNS) method: The sugars were determined using the Dinitrosalicylic acid method. In brief, a calibration curve was made using glucose solutions. Α solution containing 1% DNS, 0.2% phenol, 0.05% Na2
, and 1% NaOH was prepared. In a test tube, 1.5 mL of the sample with 1.5 mL of the previous prepared solution were mixed. The mixture was heated for 5 min at 100 °C in a water bath. Afterwards 0.5 mL of a 40% w
potassium sodium tartrate solution was added and the mixture was left at room temperature before the absorbance measurement at 575 nm [33
2.5. LC-PDA-MS Analysis
The obtained extract along with the collected ethanolic elutions of Oasis HLB and Isolute C8 (EC) columns were filtered through a 0.45 μm syringe filter (BGB Analytik, Richmond, VA, USA) and analyzed for their anthocyanin content using an LC-PDA-MS method. A Shimadzu LCMS-2010 EV, using electrospray ionization (ESI) process and a quadrupole mass analyser, equipped with an SPD-M20A PDA (Shimadzu Corporation, Kyoto, Japan) detector, at positive ionization mode was used for the chromatographic separation and identification of the compounds while the chromatographic column used was a Dionex, RP-C18, Acclaim 120, 150 × 4.6 mm × 5 μm (Thermo Fisher Scientific, Waltham, MA, USA). A gradient elution program was employed, using water/formic acid (99/1, v/v) and acetonitrile/formic acid (99/1, v/v) as elution solvents, while the sample injection volume was 20 μL. The flow rate was 0.5 mL·min−1 with a 45 min gradient elution program as follows: 0 min, 10% B; 0–5 min, 10%–22% B; 5–14 min, 22%–28% B; 14–35 min, 28%–42% B; 35–40 min, 42%–100% B; 40–45 min, 100%–10% B.