3.3.2. Identification and Quantification of Polyphenols

Polyphenols were extracted from the fruit, juice, and pomace powders in triplicate (*n* = 3) according to Lachowicz et al. [17]. For polyphenolic compounds: wheat bread (1 g) were extracted with 10 mL of mixture containing UPLC-grade methanol (30 mL/100 mL), and acetic acid (1 mL/100 mL of reagent). The extraction was performed twice by incubation for 20 min under sonication (Sonic 6D, Polsonic, Warsaw, Poland) and with occasional shaking. Next, the slurry was centrifuged at 19,000× *g* for 10 min, and the supernatant was filtered through a hydrophilic PTFE 0.20 μm membrane (Millex Samplicity Filter, Merck, Darmstadt, Germany) and used for analysis.

Qualitative (LC-QTOF-MS) and quantitative (UPLC-PDA-FL) analysis of polyphenols (anthocyanins, flavan-3-ols, flavonols, and phenolic acids) was performed as previously described by Lachowicz et al. (2017). Separations of individual polyphenols were carried out using a UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm, Waters Corporation, Milford, MA, USA) at 30 ◦C. The samples (10 μL) were injected, and the elution was completed in 15 min with a sequence of linear gradients and isocratic flow rates of 0.45 mL/min. The mobile phase consisted of solvent A (2% formic acid; *v*/*v*) and solvent B (100% acetonitrile). The program began with isocratic elution with 99% solvent A (0–1 min), and then a linear gradient was used until 12 min, lowering solvent A to 0%; from 12.5 to 13.5 min, the gradient returned to the initial composition (99% A), and then it was held constant to re-equilibrate the column. Characterization of the single components was carried out via the retention time and the accurate molecular masses. Each compound was optimized to its estimated molecular mass [M − H]−/[M + H]+ in the negative and positive mode before and after fragmentation. The data obtained from UPLC-MS were subsequently entered into the MassLynx 4.0 ChromaLynx Application software (Waters Corporation, Milford, MA, USA). On the basis of these data, the software is able to scan di fferent samples for the characterized substances. The runs were monitored at the wavelength of 360 nm for flavonol glycosides. The PDA spectra were measured over the wavelength range of 200–800 nm in steps of 2 nm. The retention times and spectra were compared to those of the pure standard. The calibration curves were run at 360 nm for the standard keampferol-3- *O*-galactoside, quercetin-3- *O*-galactoside, at 320 nm for the standard of chlorogenic, ca ffeic, di-ca ffeic quinic, and *p*-coumaric acids, at 520 nm for the standard cyanidin-3- *O*-galactoside and cyanidin-3- *O*-glucoside and at 280 nm for the standard (−) epicatechin, (+)-catechin, procyanidins B2 and A2, at concentrations ranging from 0.05–5 mg/mL (R<sup>2</sup> = 0.9999). The measurements were performed in triplicates. The results were expressed as mg per 100 g dry matter (d.m.).

### 3.3.3. Analysis of Proanthocyanidins by Phloroglucinolysis

Direct phloroglucinolysis of samples was performed as described by Lachowicz et al. [17]. Samples were weighed in an amount of 5 mg, then 0.8 mL of the methanolic solution of phloroglucinol (75 g/L) and ascorbic acid (15 g/L) were added. After addition of 0.4 mL of methanolic HCl (0.3 mol/L), the vials were incubated for 30 min at 50 ◦C with continuous vortexing in a thermo shaker (TS-100, BioSan, Riga, Latvia). The reaction was stopped by placing the vials in an ice bath with the addition of 0.6 mL of the sodium acetate 0.2 mol/L. Next the vials were centrifuged immediately at 20,000× *g* for 10 min at 4 ◦C. Samples were stored at 4 ◦C before reverse phase HPLC (RP-HPLC) analysis. All incubations were done in triplicate. Phloroglucinolysis products were separated on a Cadenza CD C18 (75 mm × 4.6 mm, 3 μm) column (Imtakt, Kyoto, Japan). The liquid chromatograph was a Waters (Milford, MA, USA) system equipped with diode array and scanning fluorescence detectors (Waters 474) and autosampler (Waters 717 plus). Solvent A (25 mL acetic acid and 975 mL water) and solvent B (acetonitrile) were used in the following gradients: initial, 5 mL/100 mL B; 0–15 min, to 10 mL/100 mL B linear; 15–25 min to 60 mL/100 mL B linear; followed by washing and reconditioning of the column. A flow rate of 1 mL/min and an oven temperature of 15 ◦C with the injection of the filtrate (20 μL) on the HPLC system. The fluorescence detection was recorded at excitation wavelength of 278 nm and emission wavelength of 360 nm. The calibration curves, which were based on peak area, were established using (+)-catechin, (−)-epicatechin, (+)-catechins, and (−)-epicatechin-phloroglucinol adduct standards. The average degree of polymerization was measured by calculating the molar ratio of all the flavan-3-ol units (phloroglucinol adducts + terminal units) to (−)-epicatechin and (+)-catechin, which correspond to terminal units. Quantification of the (+)-catechin, (−)-epicatechin, (+)-catechin, and (−)-epicatechin-phloroglucinol adducts was achieved by using the calibration curves of the corresponding standards (Extrasynthese). All data were obtained in triplicate. The results were expressed as mg per 100 g d.m.
