Determination of olive oil volatile compounds

The extraction of volatile compounds dominating the headspace of olive oil samples was done using a divinyl benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber of 50/30 μm purchased by Supelco (Bellefonte, PA, USA). Before the analysis of samples the fiber was cleaned daily using the method of the “clean” program. During the “cleaning” of the fiber, oven temperature was held at 80 °C for 0 min, and then increased to 260 °C at a rate of 10 °C/min (2 min hold). The inlet temperature was 270 °C. The auxiliary temperature was 280 °C and that of the MS source 230 °C.

Approximately, 4 g of olive oil was placed in 20 mL screw-cap vials equipped with polytetrafluoroethylene (PTFE)/silicone septa and 100 μL of the internal standard (4-methyl-2-pentanone of initial concentration of 500 μg/L) was added. 4-methyl-2-pentanone was chosen as an internal standard during the optimization of the method given that it did not occur in any of the olive oil volatiles compounds and did not cause any co-elution problems. The vials were vortexed and maintained in a water bath at 45 °C under stirring at 600 rpm during the extraction procedure with the fiber. The HS-SPME extraction procedure included the optimized conditions: 15 min equilibration time, 15 min sampling/exposure time of the fiber, weight of sample 4 g, vial volume 20 mL, and as reported above, constant extraction temperature of the water bath at 45 °C (Supplementary Table 5).

A gas chromatograph (GC) unit (Agilent 7890 A) coupled to a mass spectrometry (MS) detector (Agilent 5975) was used for the analysis of olive oil volatile compounds. A DB-5MS [cross-linked (5%-Phenyl)-methylpolysiloxane)] capillary column (J & W Scientific, Agilent Technologies, Santa Clara, CA, USA), with dimensions of 60 m × 320 μm i.d., × 1 μm film thickness was used, with helium as the carrier gas (purity 99.999%), at a flow rate of 1.5 mL/min. The temperature for the injector and MS-transfer line were maintained constant at 260 °C and 270 °C, respectively. The oven temperature was held at 40 °C for 4 min and was further increased to 160 °C at a rate of 4 °C/min for 2 min, increasing further to 250 °C at a rate of 8 °C/min for 2 min. Electron impact mass spectra were recorded at the mass range of 29–500. The ionization energy of the electron ionization system was 70 eV. A split ratio 2:1 was used.

The identification of olive oil volatile compounds was done based on the Wiley 7 NIST (National Institute of Standards and Technology) mass spectral library (NIST 2005) (J. Wiley & Sons Ltd., West Sussex, England). For the determination of the linear retention index values, the mixture of n-alkanes (C₈–C₂₀) was used (Supplementary Table 6). The calculation of retention time indices was carried out for volatile compounds eluting between n-octane and n-eicosane according to Kováts formula:

where t_n and t_n+1 are the retention times of heading and trailing n-alkanes and t_i is the retention time of the volatile compound of interest [40].

Results were expressed as semi-quantitative data according to the formula:

where E_analyte refers to the peak area of analyte, E_IS refers to the peak area of internal standard (IS), and C_IS: final concentration of IS.

To eliminate any kind of contamination that could cause memory effects, affecting thus, the obtained results, blank runs were carried out before and after the analysis of olive oil samples.