# Natural Variation of Volatile Compounds in Virgin Olive Oil Analyzed by HS-SPME/GC-MS-FID
## 2 Materials and Methods
### Analysis of Volatile Compounds  
Analysis of volatile compounds was carried out according to [PERSON] et al. [29]. Olive oil samples (0.5 g) were prepared in 10-mL vials and placed in a vial heater at 40 \({}^{\circ}\)C for a 10 min equilibration time. Volatile compounds from the headspace were adsorbed onto a headspace solid-phase microextraction (SPME) fiber divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 \(\upmu\)m (Supelco Co., Bellefonte, PA, USA). Sampling time was carried out at 40 \({}^{\circ}\)C for 50 min. Desorption of volatile compounds was carried out directly in the GC injector. Volatile compounds were identified on a 7820A/GC-5975/MSD system (Agilent Technologies, Santa Clara, CA, USA), including a DB-Wax capillary column (60 m \(\times\) 0.25 mm i.d., film thickness, 0.25 \(\upmu\)m: J&W Scientific, Folsom, CA, USA) under the following conditions: Injection port operated in splitless mode at 250 \({}^{\circ}\)C; He as the carrier gas at a flow rate of 1 mL/min; column held for 6 min at 40 \({}^{\circ}\)C, then programmed at 2 \({}^{\circ}\)C min\({}^{-1}\) to 168 \({}^{\circ}\)C; the mass detector was operated in the electronic impact mode at 70 eV; source temperature  
\begin{table}
\begin{tabular}{c c c c} \hline
**No.** & **Cultivar** & **No.** & **Cultivar** \\ \hline
1 & Abbadi Abou Gabra & 32 & Lastovka \\
2 & Aggezi Shami & 33 & Lechin de Sevilla \\
3 & Agoourmadakolia & 34 & Lentisca \\
4 & Amygdala Nana & 35 & Levantinka \\
5 & Arbosana & 36 & Maurino \\
6 & [PERSON] & 37 & Merhavia \\
7 & Azapa o Arauco & 38 & Negrinha \\
8 & Blanqueta & 39 & Ouslati \\
9 & Blanqueta-48 & 40 & Palomar \\
10 & Borriolenca & 41 & Patronet \\
11 & Bosana & 42 & Pecoso \\
12 & Canetera & 43 & Pequenña de Casas Ibénez \\
13 & Caninese & 44 & Picholine \\
14 & Chorreao de Montefrio & 45 & Picholine Marroqui \\
15 & Cipresino & 46 & Rapasayo \\
16 & Coratina & 47 & Sabatera \\
17 & Corbella & 48 & Shami \\
18 & Cornicabra & 49 & Torcio de Cabra \\
19 & [PERSON] & 50 & Ulliri i Kuq \\
20 & Elmacik & 51 & Vallesa \\
21 & Figueretes & 52 & Vaneta \\
22 & Galega Vulgar & 53 & Vera \\
23 & Haouzia & 54 & Verde Verdelho \\
24 & Istarska Bjelica & 55 & Verdial de Badajoz \\
25 & Jaropo & 56 & Verdial de Vélez-Malaga \\
26 & [PERSON] & 57 & [PERSON] \\
27 & [PERSON] & 58 & [PERSON] \\
28 & [PERSON] & 59 & [PERSON] \\
29 & [PERSON] & 60 & [PERSON] \\
30 & [PERSON] & 61 & [PERSON] \\
31 & [PERSON] & & \\ \hline \end{tabular}
\end{table}
Table 1: Cultivars selected from the World Olive Germplasm Collection (WOGC, Cordoba, Spain).  
set at 230 \({}^{\circ}\)C; and the mass spectra scanned at 2.86 scans/s in the \(m/z\) 40-550 amu range. Compounds were identified by mass spectrum, comparing to the Wiley/NBS and NIST libraries, retention index in agreement with literature, and co-elution with chemical standard when available. Quantification was performed in triplicate on a HP-6890 GC apparatus (Agilent Technologies, Santa Clara, CA, USA), which was equipped with a similar column and operated under the following operating conditions in order to obtain the same retention times for volatile compounds such as those obtained with the 7820A/GC-5975/MSD system: N\({}_{2}\) as the carrier gas at 17 psi; injector and detector at 250 \({}^{\circ}\)C; oven held for 6 min at 40 \({}^{\circ}\)C and then programmed at 2 \({}^{\circ}\)C min\({}^{-1}\) to 168 \({}^{\circ}\)C. For the quantification of the different compounds, calibration curves obtained by adding known amounts of the different compounds to re-deodorized, high-oleic sunflower oil were used. Limits of quantification and concentration ranges were established according to typical contents in virgin olive oil volatile fractions. Linear regression curves were found for all the compounds, with regression coefficients higher than 0.99. At the beginning, and regularly during the sample analyses, a blank containing no oil and a mixture of volatile standards were run as controls.