*2.4. Analysis of Volatile Compounds by GC-IT-MS*

Volatile compounds were isolated using headspace solid-phase microextraction (HS-SPME), according to the modified method proposed by Brki´c Bubola, Koprivnjak, Sladonja, Škevin, and Belobraji´c [41]. SPME fiber used was divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS), 1 cm length, 50/30 µm film thickness (Supelco, Bellefonte, PA, USA). Four grams of EVOO sample (or a standard solution) were placed in a 10 mL glass vial containing a micro-stirring bar, and sealed. The headspace in the vial was equilibrated at 40 ◦C for 15 min, and the extraction was carried out at 40 ◦C for 40 min with stirring at 800 rpm. Thermal desorption of analytes was achieved in the GC injection port in splitless mode at 245 ◦C for 3 min. Identification and quantification of volatile compounds was performed using a Varian 3900 GC coupled to a Varian Saturn 2100 T ion trap mass spectrometer (IT-MS) (Varian Inc., Harbor City, CA, USA). A capillary column Rtx-WAX (60 m × 0.25 mm i.d. × 0.25 µm film thickness; Restek, Bellefonte, PA, USA) was used. Initial oven temperature was 40 ◦C, increased to 210 ◦C at 2 ◦C/min, increased to 245 ◦C at 20 ◦C/min, and kept for 20 min. Injector, transfer line and ion trap temperatures were 245, 180, and 120 ◦C, respectively. Mass spectra were acquired in EI mode (70 eV) at 1 s/scan, full scan with a range of 30–450 *m*/*z*. The carrier gas was helium (1.2 mL/min).

Identification was performed by comparing retention times and mass spectra with those of pure standards, and with mass spectra from NIST05 library. Identification by comparison with mass spectra was considered satisfactory if spectra reverse match numbers (RM) higher than 800 were obtained. If in a particular sample the mass spectra were not clear (RM < 800), identification was considered satisfactory if the ratios of a quantifier and three most abundant characteristic ions reasonably matched those in the reference spectra of a given compound. Linear retention indices (relative to C7–C24 n-alkanes) were calculated and compared to those from literature. When standards were available, standard calibration curves based on quantifier ions were used for quantification. Linearity was satisfactory with coefficient of determination higher than 0.99 for all the standards. For other compounds semi-quantitative analysis was carried out, and their concentrations (µg or mg/kg) were expressed as equivalents of the compounds with similar chemical structure for which standards were available, assuming a response factor equal to one.
