3.5. Volatile Compounds Analysis by GC–FID (Gas Chromatography–Flame Ionization Detector) and SPME–GC–MS (Solid Phase Microextraction–Gas Chromatography–Mass Spectrometry)

Selected volatile compounds were analysed using GC–FID as described by Satora et al. (2008) [20]. The analysis was carried out on the Hewlett Packard 5890 Series II chromatograph system (Agilent Technologies, Santa Clara, CA, USA). The separation was conducted with an HP-INNOVAX capillary column (crosslinked polyethylene glycol stationary phase; 30 m × 0.53 mm ID with 1.0 μm film thickness). Temperature of detector and injector was 250 °C and the column was heated using the following program: 35 °C for five minutes at an increment of 5 °C/min to 110 °C, then 40 °C/min to 220 °C and maintaining a constant temperature for three minutes. The carrier gas was helium at a 20.0 mL/min flow. Hydrogen flow speed was 33.0 mL/min, and that of air was 400 mL/min. The qualitative and quantitative identification of volatile compounds and internal standard (anethole, ethyl nonanoate, and 4-methylo-2-pentanol) was based on the comparison of retention times and peak surface area read from sample and standard chromatograms. Concentrations of volatile components were recalculated based on 100% (v/v) ethanol and were expressed as mg/L. All tests were carried out three times.

In the SPME–GC–MS method, 2 mL of saturated saline with an internal standard solution (5 mg of 4-methyl-2-pentanol/L and 0.05 mg of ethyl nonanoate/L, Sigma-Aldrich, Saint Louis, MO, USA) and 0.05 mL of spirit were added into a 10 mL vial. The SPME device (Supelco Inc., Bellefonte, PA, USA) coated with polydimethylsiloxane (100 μm) fiber was first conditioned by inserting it into the GC injector port at 250 °C for 1 h. For sampling, the fiber was inserted into the headspace under stirring (300 rpm) for 30 min at 60 °C. Subsequently, the SPME device was introduced in the injector port of the Agilent Technologies 7890B chromatograph system (Agilent Technologies) equipped with LECO Pegasus HT, High Throughput TOFMS (time-of-flight mass spectrometry), and was kept in the inlet for 3 min. The SPME process was automated using the GERSTEL MultiPurpose Sampler (MPS).

Separation was conducted with a Rtx-1ms capillary column (Crossbond 100% dimethyl polysiloxane, 30 m × 0.53 mm × 0.5 μm). The detector temperature was 250 °C, and the column was heated using the following program: 40 °C for three minutes at an increment of 8 °C/min to 230 °C, then maintaining a constant temperature for 9 min. The carrier gas was helium at a 1.0 mL/min constant flow. EIMS electron energy 70 eV; ion source temperature and connection parts: 250 °C. Analyte transfer was performed in splitless mode; the MSD (mass spectrometer detector) was set to scan mode from m/z = 40 to m/z = 400.

Volatiles were identified using mass spectral libraries and linear retention indices, calculated from a series of n-alkanes from C6 to C30. The amount of components was determined semi-quantitatively by measuring the relative peak area of each identified compound, according to the NIST database, in relation to that of the internal standard (ethyl nonanoate for esters, 4-methyl-2-pentanol for other components). This semi-quantification approach was already performed in many previous scientific studies [49,50,51].