## 2 Materials and Methods
### HPLC Analyses  
Samples used for HPLC analysis were taken from two different bottles of each wine. The wine samples and standard solutions were filtered through a syringe filter with a pore size of 0.45 \(\upmu\)m. All experiments were performed using an Agilent 1100 series liquid chromatograph equipped with a DAD detector and an Agilent Zorbax SB -C18 (4.6 \(\times\) 250 mm, 5 \(\upmu\)m) column preceded by an Agilent Zorbax SB -C18 (10 \(\times\) 4.6 mm, 5 \(\upmu\)m) guard column.  
The HPLC analysis of anthocyanins was performed according to [PERSON] et al. (2000) [28]. Acetonitrile-buffer (5:95) was the mobile phase A, while acetonitrile-buffer (50:50) was the mobile phase B, where 10 mM KH\({}_{2}\)PO\({}_{4}\) + H\({}_{3}\)PO\({}_{4}\) to pH 1.6 was used as a buffer. The oven temperature was 50 \({}^{\circ}\)C. An amount of 20 \(\upmu\)L of 0.45 \(\upmu\)m membrane-filtered samples were injected with a 1 mL/min flow rate. Gradient elution was applied according to the following program: 0-30 min (10-45% B), 30-31 min (45-100% B), 31-34 min (100% B), 34-35 min (100-10%), and 35-45 min (10% B). The wavelength for detection was 518 nm.  
Orthophosphoric acid pH 3.00 was the mobile phase A, while methanol was the mobile phase B for the HPLC analysis of flavonoids, phenolic acids, flavan-3-ols, and stilbenes.  
Figure 1: Map showing the locations of vineyards in the Croatian wine regions where the grape varieties of the studied wine samples are grown. The abbreviations corresponding to the wine samples are listed in Tables 1 and 2.  
The elements were used in the following gradient: 0-7 min (10-30% B), 7-25 min (30% B), 25-50 min (30-70% B), 50-60 min (70-86% B), 60-60.01 min (86-10% B), and 60.01-65 min (10% B). The flow rate was set at 1.0 mL/min. The separation was performed at 35 \({}^{\circ}\)C. The aliquot of the injected sample was 25 \(\mu\)L. The wavelengths for detection were 280, 325, or 360 nm, depending on the absorption maximum of the phenolic component analyzed. Peak assignment and identification were performed by comparing the retention times and UV/VIS spectra of the peaks in the sample chromatograms with those of the standards. Quantification of individual phenolic peaks was performed using the external standard method. Response factors (RF) were calculated daily by using freshly prepared reference solutions. Results were expressed in milligrams of the phenolic compound per liter of wine sample. The validation of this method was performed according to the recommendations of the guideline ICH Q2 [29]. The linearity of the method was evaluated using calibration curves with six concentrations (0.1, 0.5, 1, 2, 4, and 50 mg/L), while for gallic acid and procyanidin B1, an additional concentration of 100 mg/L was used. These concentration ranges were set according to the expected concentration of phenolic compounds in the wine samples. The recoveries were determined, and a linear regression analysis was performed, calculating the determinant coefficient, y-axis intercept, and slope to determine the linearity of the method. The analytical curves showed excellent linear behavior over the entire concentration range studied. The mean recoveries were calculated by comparing the calculated amounts with the amount present in the standard solution. The high mean recoveries for all studied compounds indicate the high accuracy of the method. The limit of detection (LOD) and limit of quantification (LOQ) were determined by analyzing the calibration curves. The following equation was used to calculate the LOD and LOQ values: C = (K \(\times\) SD)/S, where SD is the standard deviation of the y-axis intercept values and S is the mean slope value from the three calibration curves generated. The K values were 3 for LOD and 10 for LOQ [30]. The Supplementary Materials (Table S2) show the corresponding validation parameters.