## 3 Materials and Methods
### LC\(\times\)LC-PDA/ESI-MS Analysis  
LC\(\times\)LC analyses were performed on a Shimadzu LC\(\times\)LC instrument (Kyoto, Japan), consisting of a CBM-20A controller, one LC-Mikros binary pump, one LC-40 BX3 dual-plunger parallel-flow pumps, one LC-30 AD as make-up pump, a CTO-40C column oven, a SIL-40 CX3 autosampler, and an SPD-M40 photo diode array (PDA) detector (1.0 \(\upmu\)L detector flow cell volume). In order to connect the two dimensions, two high speed/high pressure two-position, six-ports switching valves with a micro-electric actuator (model FCV-32 AH, 1.034 bar; Shimadzu, Kyoto, Japan), equipped with two C18 guard columns,were employed. A third LC pump (LC-30 AD) was connected through a t-piece between the outlet of the \({}^{1}\)D and the inlet of switching valve. The LC\(\times\)LC instrument was hyphenated to an LCMS-8050 mass spectrometer, through an ESI source (Shimadzu, Kyoto, Japan).  
Separations were carried out on a \({}^{1}\)D SEQuant ZIC-HILIC column (150 \(\times\) 1.0 mm _I.D._, 3.5 \(\upmu\)m _dp_) (Merck Life Science, Merck KGaA, Darmstadt, Germany) and a \({}^{2}\)D Ascents Express C18 column (50 \(\times\) 4.6 mm _I.D._, 2.7 \(\upmu\)_dp_). (Merck Life Science, Merck KGaA, Darmstadt, Germany).  
Two identical C18 guard columns (5 \(\times\) 4.6 mm _I.D._, 5 \(\upmu\)_dp_) (Merck Life Science, Merck KGaA, Darmstadt, Germany); were used to collect and transfer the fractions from the \({}^{1}\)D into the \({}^{2}\)D.  
\({}^{1}\)D mobile phases: (A) 0.1% formic acid in ACN, (B) 0.1% formic acid in water (pH 3). Gradient: 0 min, 30% B; 40 min, 60% B; 50 min, 100% B; 60 min, 100% B; 61 min, 30% B. Flow rate: 10 \(\upmu\)L min\({}^{-1}\). Column oven: 30 \({}^{\circ}\)C. Injection volume: 20 \(\upmu\)L.  
\({}^{2}\)D mobile phases: employed were (A) 0.1 % formic acid in water (pH 3), (B) 0.1% formic acid in ACN. Segmented-in-fraction conditions: (\({}^{1}\)D 0-12 min), 0.01 min, 10% B; 0.89 min, 40% B; 0.90 min, 10% B; (\({}^{1}\)D 13-17 min) 0.01 min, 0% B; 0.89 min, 40% B; 0.90 min, 0% B; (\({}^{1}\)D 18-51 min) 0.01 min, 0% B; 0.89 min, 25% B; 0.90 min, 0% B; Flow rate: 3 mL min\({}^{-1}\). Modulation time: 1.00 min. Column oven: 30 \({}^{\circ}\)C. PDA conditions were in the range from 200 to 550 nm. Sampling rate was set to 40 Hz, whereas the time constant was acquired at 0.08 s.  
ESI-MS conditions: mass spectral range: _m/z_ 100-2000; event time: 1 s; nebulizing gas (N\({}_{2}\)) flow: 3 L min\({}^{-1}\); drying gas (N\({}_{2}\)) flow: 10 L min\({}^{-1}\); heating gas flow (air): 10 L min\({}^{-1}\); heat block temperature: 400 \({}^{\circ}\)C; desolvation line (DL) temperature: 250 \({}^{\circ}\)C; interface temperature: 300 \({}^{\circ}\)C; interface voltage 3.50 kV; detector voltage: 1.80 kV.  
The LC\(\times\)LC-LCMS-8050 system and the switching valves were controlled by the Shimadzu Labsolution software (ver. 5.93). The LC\(\times\)LC data were visualized and elaborated into two and three dimensions using Chromsquare ver. 2.3 software (Shimadzu, Kyoto, Japan).  
Samples were diluted 1:4 with 0.1% formic acid in MeOH:ACN solution (70:30 _v/v_) prior to LC\(\times\)LC-PDA/ESI-MS analysis.  
For the quantitative analysis of polyphenic compounds, gallic acid, protocatechuric acid, isoquercetin, myricetin, and cyanidin were employed. Standard calibration curves were prepared in a concentration range 10-500 mg L\({}^{-1}\) with seven different concentration levels, run in triplicate.