2.8. Volatile Compounds

The EVOO samples (2 g), spiked with 100 mg of internal standard solution of 4-methyl-2-pentanol dissolved in refined sunflower oil (50 µg/mL), were placed in a 20 mL glass vial, tightly capped with a polytetrafluoroethylene (PTFE) septum, and held for 5 min at 40 °C to allow for the equilibration of the volatiles in the headspace. After the equilibration time, the septum covering each vial was pierced with a solid-phase microextraction (SPME) needle, and the fiber was exposed to the headspace for 40 min. When the process was completed, the fiber was inserted into the injector port of the GC. The temperature and time of the pre-concentration step, performed in a HT280T (HTA s.r.I, Brescia, Italy), were automatically controlled by the software HT-COMSOFT (HTA s.r.I). The SPME fiber (2 cm length and 50/30 μm film thickness) was from Supelco (Bellefonte, PA, USA) and consisted of a stable flex stationary phase divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS). The fiber was previously conditioned following the instructions of the supplier.

The volatiles absorbed by the fiber were thermally desorbed in the hot injection port of a GC for 5 min at 260 °C with the purge valve off (splitless mode) and were then injected onto a TR-WAX capillary column (60 m × 0.25 mm i.d., 0.25 μm coating; Teknokroma, Barcelona, Spain) of a Shimadzu GC-2010 Plus gas chromatograph with a flame ionization detector (FID) (Shimadzu, Kyoto, Japan). The carrier gas was hydrogen with a flow rate of 1.5 mL/min. Detector temperature was 280 °C, and the oven temperature was held at 40 °C for 10 min and then programmed to increase by 3 °C/min to a final temperature of 200 °C, where it was held for 10 min. The data were recorded and processed with GC solution Ver. 2 Workstation Software (Shimadzu, Kyoto, Japan). Each sample was analyzed in triplicate. The identification of the volatile compounds was performed by comparison of the retention times with Sigma standards and quantified by interpolation on the calibration curves. The calibration curves were constructed using sunflower oil spiked with volatile compounds standard (Sigma) in a range from 10 to 1000 µg/mL. The linear regression coefficients (R) were in a range from 0.975 to 0.994.