3.5.3. Determination of Volatile Compounds by SBSE-GC-MS

Wine volatile compounds were determined according to the method described by Campayo et al. (2020) [28]. Their extraction was carried out by stir-bar sorptive extraction (SBSE). Later analysis was performed using an automated thermal desorption unit (TDU, Gerstel, Mülheim and der Ruhr, Germany) mounted on an Agilent 7890A gas chromatograph system (GC) coupled to a quadrupole Agilent 5975C electron ionization mass spectrometric detector (MS, Agilent Technologies, Palo Alto, CA, USA). The GC system was equipped with a fused silica capillary column (BP21 stationary phase, 30 m length, 0.25 mm I.D. and 0.25 μm film thickness) (SGE, Ringwood, Australia), and the carrier gas was helium with a constant column pressure of 20.75 psi. The GC oven temperature was programmed to 40 °C (held for 2 min), raised to 80 °C (5 °C/min, held for 2 min), raised to 130 °C (10 °C/min, held for 5 min), raised to 150 °C (5 °C/min, held for 5 min), and then raised to 230 °C (10 °C/min, held for 5 min). The MS was operated with an ionization energy of 70 eV. The temperature of the MS transfer line was maintained at 230 °C. MS data acquisition was carried out in positive scan mode. The MS identification and quantification were performed in the single ion-monitoring mode using the characteristic m/z values of the volatiles with the aid of the NIST library and confirmed with the mass spectra and retention time of their pure standards (Sigma-Aldrich, Steinheim, Germany). 3-Methyl-1-pentanol was used as internal standard. Quantification was based on calibration curves of the respective standards at five different concentrations (R² = 0.95–0.99). 2-Methyl-1-butanol was quantified together with 3-methyl-1-butanol with the calibration curve of the latter. All analyses were conducted in duplicate on each wine replicate (n = 2). The specific contribution of each volatile compound to the overall wine aroma was determined by calculating the odour activity value (OAV) as the ratio between the concentration of the compound and its odour threshold [54].