2.3. GC-FID, GC–MS Analysis of Volatile Compounds

Agilent 6890 chromatograph interfaced with a flame ionization detector (FID) and Agilent 5973-Network-mass selective detector (MSD) (Wilmington, Delaware, DE, USA) constituted the gas chromatography (GC) system. DB-Wax column (30 m length × 0.25 mm i.d. × 0.5 µm thickness, J&W Scientific, Folsom, CA, USA) were used to separate volatile compounds. An amount of 3 µL of extract was injected in pulsed splitless (40 psi; 0.5 min) mode. Injector and FID detectors were set at 270 °C and 280 °C, respectively. The flow rate of carrier gas (helium) was 1.5 mL·min⁻¹. The conditions of the oven program of the DB-Wax column was 50 °C to 250 °C at 4 °C/min, 10 min hold. As for the mass-selective detector, the identical oven program was used. The MS (electronic impact ionization) conditions were as follows: ionization energy of 70 eV, mass range m/z of 30–300 a.m.u., scan rate of 2.0 scan·s⁻¹, interface temperature of 250 °C, and source temperature of 180 °C. The volatile compounds were analyzed in full scan mode and assigned by comparison of their retention index and their mass spectra on the DB-Wax column with those of a commercial spectra database (Wiley 6, NBS 75k) and the instrument’s internal library made through the aforementioned laboratory researches. After identification, the internal standard method with 4-nonanol was used to determine the mean value of volatile compounds and mean values (µg 100 g⁻¹ dry weight; dw) of the triplicate of GC analyses were calculated for each sample. By using n-alkane (C₈–C₃₂) series, retention indices of the compounds were calculated [12,13].