# From Vine to Wine: Non-Colored Flavonoids as Fingerprints
## 2 Materials and Methods
### Analytical Methods  
High-performance liquid chromatography (HPLC) was employed to separate flavonoid compounds, following a slightly modified method from [PERSON] et al. [16]. The analysis was conducted using a Waters 2690 Separation Module coupled with a Waters 996 Photodiode Array Detector (DAD). Prior to injection, the red wine samples were filtered through a 0.45 \(\upmu\)m membrane, and 15 \(\upmu\)L aliquots were injected into a head of column Nova-Pak C18 reversed-phase column (3.9 \(\times\) 150 mm, 4 \(\upmu\)m particle size; Waters, Milford, MA, USA) maintained at 30 \({}^{\circ}\)C. The mobile phase consisted of Milli-Q water (solvent A) and acetonitrile (solvent B, Sigma-Aldrich, St. Louis, MO, USA), both acidified with 0.2% trifluoroacetic acid (spectrophotometric grade, \(\geqslant\)99%, Sigma-Aldrich). A linear gradient was applied, with a flow rate of 1.5 mL/min. Flavonoid compounds were identified using their retention times and UV-visible spectra (200-700 nm), with direct comparison to commercial standards (Sigma-Aldrich) when available through spiking experiments. Those commercial standards available were individually injected into the HPLC system, and their retention times and spectral characteristics were recorded and used as benchmarks. These peaks from the wine samples were then compared against the benchmarks to confirm identity. Identification of other compounds was based on spectral data and relative retention times reported in the literature for the same methodology and similar experimental conditions, taking into account UV-visible characteristic absorption maxima, elution time, and spectral profiles previously described in similar red wine studies and specific flavonoid databases [2; 17; 24; 2; 18; 2; 2; 21; 22]. The use of HPLC coupled with DAD provides a suitable balance between analytical performance, cost-effectiveness, and ease of implementation in routine laboratories, facilitating its widespread use for qualitative and quantitative analysis in enological studies. Specific chromatographic details from non-visible flavonoids such as retention time, characteristic absorption wavelengths, and detection are presented in Table S1. Calibration curves were constructed over the concentration ranges observed in the wine samples for the flavonoid compounds with available standards. Typically, flavanol \begin{table}
\begin{tabular}{l l l l l l} \hline \hline \multicolumn{6}{c}{**Samples Distribution by Different Characteristics**} \\ \hline Geographical Origin (D.O.) & n & Vine Cultivar & n & Wine aging (years) & n \\ Abona (A.) & 46 & Listan Negro (LN) & 93 & Young (\(\leq\)2) & 125 \\ Tacoronte-Acentejo (TA.) & 45 & Baboso (B) & 30 & Medium (3–5) & 73 \\ Valle de la Orotava (O.) & 27 & Vijariego (V) & 17 & Old (\(\geq\)6) & 7 \\ Ycoden Daute Isora (Y.) & 18 & Negramoll (N) & 13 & & \\ El Hierro (H.) & 18 & Listán Prieto (LP) & 14 & & \\ La Palma (LP.) & 15 & Syrah (S) & 12 & & \\ Gran Canaria (GC.) & 13 & Tintilla (T) & 9 & Total number of samples: & 205 \\ Valle de Gümar (G.) & 10 & Castellana (C) & 7 & & \\ Lanzarote (LZ.) & 7 & Rubi Cabernet (R) & 5 & & \\ La Gomera (GO.) & 6 & Merlot (M) & 5 & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Wine samples distribution with cultivar and DO abbreviations. compounds and rutin detected at 280 nm were quantified using gallic acid as standard and the results were expressed as milligrams of gallic acid per liter (mg GAE/L); flavonoids were detected at 365 nm and they were quantified using quercetin as the standard and the results were expressed accordingly. Limits of detection (LOD) and limits of quantification (LOQ) were calculated as the lowest concentration that yielded a signal to noise ratio of three and ten, respectively. Commercial standards used to spike the samples included catechin, epicatechin, quercetin, and rutin (all \(\geq\)98% purity, Sigma-Aldrich). Calibration curves for these compounds were generated by plotting peak area (absorbance) as a function of concentration (mg/L).