# Aromatic Characterization of Grasevina Wines from Slavonia and Podunavlje Sub-Regions
## 2 Materials and Methods
### Determination of Volatile Compounds  
Selected volatile organic compounds (VOCs) were determined in the wine samples using an automated robotic system for solid-phase micro-extraction (SPME) in headspace (HS) and injected into a gas chromatograph coupled with a mass spectrometric detector (GC-MS). Esters, C6 alcohols, and volatile phenols were analyzed using two methods adapted from a previously published protocol [8; 9]. Samples were extracted by headspace Solid Phase Micro-Extraction (HS-SPME) using an SPME fiber assembly (50/30 \(\upmu\)m DVB/CAR/PDMS, Stableflex, 24 Ga, Autosampler, Gray (Supelco, St. Louis, MO, USA). To a 20 mL SPME vial, 3 mL of the wine sample was added to 2 g NaCl and 3 mL deionized water, and a 20 \(\upmu\)L solution of internal deuterated standards was added. Terpenes and norisoppenoids were quantified using the same method as in the above-mentioned volatiles with a slightly different temperature program: held at 40 \({}^{\circ}\)C for 5 min, then raised to 65 \({}^{\circ}\)C at a rate of 3 \({}^{\circ}\)C/min, held for 10 min, then raised to 90 \({}^{\circ}\)C at 3 \({}^{\circ}\)C/min, raised to 130 \({}^{\circ}\)C at 10 \({}^{\circ}\)C/min, and finally raised to 240 \({}^{\circ}\)C at 4 \({}^{\circ}\)C/min and held for 10 min. The method validation for the determination of terpenes was performed by checking the recovery, linearity, and repeatability, as shown in Table 1. To study the presence of carryover, two blank samples (6 mL of Milli-Q water) were analyzed, and the calibration sample with the highest calibration point for each compound was analyzed twice, followed by the injection of two blank samples, and this was repeated six times. Carryover was negligible, that is, under 0.1% for all the measured compounds. To quantify the analyzed VOCs, internal standards were spiked accordingly. Each internal standard was separately dissolved in ethanol to obtain a stock solution. Subsequently, two stock mix solutions were prepared separately, one for terpenes and norisoprenoids and one for esters and volatile phenols. The concentration levels in all solutions are presented in Table 2. Internal standard stock solutions and internal standard mix stock solutions were stored at \(-80\)\({}^{\circ}\)C.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Terpenes** & **Quantified** & **Calibration** & **Calibration** & **R\({}^{2}\)** & **Repeatability \({}^{6}\)** & **Recovery \({}^{7}\)** \\  & **Ion (\(\mu\)/z)** & **Curve (k \({}^{5}\))** & **Range (\(\upmu\)/L)** & **(\%)** & **(\%)** \\ \hline
3-caren \({}^{1}\) & 93 & 0.0347 & 0.01–2 & 0.997 & 6.1 & 79 \\ \(\upalpha\)-terpinene \({}^{1}\) & 136 & 0.0198 & 0.01–2 & 0.9999 & 7.0 & 93 \\
1,4-cineole \({}^{1}\) & 111 & 0.0155 & 0.01–2 & 0.9995 & 3.0 & 108 \\ limonene \({}^{1}\) & 136 & 0.0164 & 0.01–2 & 0.9995 & 7.0 & 112 \\ eucalypt \({}^{1}\) & 108 & 0.0085 & 0.01–2 & 0.9997 & 3.0 & 97 \\ \(\updelta\)-terpinene \({}^{1}\) & 136 & 0.0123 & 0.01–2 & 0.9992 & 7.3 & 92 \\ p-cymene \({}^{1}\) & 119 & 0.0072 & 0.01–2 & 0.9997 & 8.2 & 95 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Validation parameters for the HS-SPME-GCMS method for the determination of terpen and norisoprenoids, including a list of quantified compounds with appropriate ions and internal standards used for quantification.