# Pairing Red Wine and Closure: New Achievements from Short-to-Medium Storage Time Assays
## 2 Materials and Methods
### Determination of the Volatile Profile of Wines  
The volatile composition of the wines was analyzed using headspace solid-phase microextraction (HS-SPME) coupled with comprehensive two-dimensional gas chromatography and mass spectrometry with a time-of-flight analyzer (GC \(\times\) GC-ToFMS). In brief, 4 mL of wine, 50 \(\mu\)L of the internal standard 3-octanol (810.81 ug/L), and 1.2 g of sodium chloride were placed in a 12 mL vial and equilibrated at 40.0 \(\pm\) 0.1 \({}^{\circ}\)C for 5 min. The extraction of volatile compounds was performed using a 50/30 \(\upmu\)m fused silica fiber coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS). The fiber was inserted into the headspace for 20 min while continuously stirring the sample at 250 rpm. After extraction, the SPME fiber was manually introduced into the GC \(\times\) GC-ToFMS injector, at 250 \({}^{\circ}\)C. A 0.75 mm I.D. glass liner was used, and splitless injection mode was performed (30 s). The system used was a LECO Pegasus 4D GC \(\times\) GC-ToFMS (LECO, St. Joseph, MI, USA), consisting of an Agilent GC 7890A (Agilent Technologies, Inc., Wilmington, DE, USA) equipped with a dual-stage jet cryogenic modulator (licensed by Zoex) and a secondary oven. A DB-FFAP column (30 m \(\times\) 0.25 mm I.D., 0.25 \(\upmu\)m film thickness, J&W Scientific Inc., Folsom, CA, USA) was used in the first dimension (\({}^{1}\)D), and an Equity-5 column (0.79 m \(\times\) 0.25 mm I.D., 0.25 \(\upmu\)m film thickness, Supelco, Inc., Bellefonte, PA, USA) in the second dimension (\({}^{2}\)D). The carrier gas was helium, flowing at a constant rate of 2.50 mL min\({}^{-1}\). The oven temperature program began at 40 \({}^{\circ}\)C for 3 min, increased to 150 \({}^{\circ}\)C at 6 \({}^{\circ}\)C min\({}^{-1}\), and then increased to 230 \({}^{\circ}\)C for 2 min at 20 \({}^{\circ}\)C min\({}^{-1}\). The secondary oven was set at 5 \({}^{\circ}\)C higher than the primary oven. The MS transfer line and source temperatures were both maintained at 250 \({}^{\circ}\)C. The modulation period was 3 s, with a modulator \begin{table}
\begin{tabular}{c c c c c c} \hline
**Compound** & **Concentration Range (mg/L)** & **Calibration Curve a** & **r2** & **LOD (mg/L)** & **LOQ (mg/L)** \\ \hline Gallic acid & 2.60–83.20 & y = 1025.2x \(-\) 820.43 & 0.9998 & 1.84 & 5.58 \\ Caffeic acid & 0.82–61.20 & y = 2697.2x \(-\) 6.4998 & 1.0000 & 0.32 & 0.98 \\ Ellagic acid & 0.71–35.40 & y = 1197.2x \(-\) 416.12 & 0.9999 & 0.37 & 1.12 \\ Vanillin & 0.88–44.00 & y = 2492.8x \(-\) 916.61 & 0.9999 & 0.82 & 2.48 \\ Catechin & 5.10–66.30 & y = 323.99x \(-\) 416.38 & 0.9994 & 2.47 & 7.48 \\ Narigenin & 1.08–37.80 & y = 1484.1x \(-\) 734.29 & 0.9996 & 1.20 & 3.64 \\ Quercetin & 0.27–32.40 & y = 2080.7x \(+\) 355.79 & 1.0000 & 0.06 & 0.18 \\ Cyanidin chloride & 1.00–80.00 & y = 846.03x \(-\) 375.37 & 1.0000 & 0.51 & 1.56 \\ \hline \end{tabular}  
* y = peak area, x = concentration in mg/L, r2—correlation coefficients, LOD—Limit of Detection, LOQ—Limit of Quantification.  
\end{table} 
Table 2: Calibration data used for the quantification of phenolic compounds in red wines from Douro and Burgenland regions. offset 15 \({}^{\circ}\)C above the primary oven and modulation pulses alternating between 0.80 s (hot) and 1.70 s (cold). The time-of-flight (ToF) analyzer was operated with a spectrum storage rate of 100 spectra per second. The mass spectrometer was set to electron ionization mode (70 eV), scanning an \(m/z\) range of 30-300. Automated data processing software ChromaTOF(r) (V4.72.0, LECO) was used to process total ion chromatograms, at a signal-to-noise threshold of 100. The data obtained were transferred into Guineu software (version 0.9, the software source code is published under GNU General Public License that can be downloaded from the internet [[https://code.google.com/p/guineu/](https://code.google.com/p/guineu/)]([https://code.google.com/p/guineu/](https://code.google.com/p/guineu/)) (accessed on 14 October 2024).