**2.2.5 SPME-GC/MS analysis**

Aroma components of products of plant origin are dependent on genetic, agronomic and environmental factors (Benincasa et al., 2003). The complexity of the mass-chromatograms in terms of number of components might represent a drawback when different samples are to be matched. Therefore, in order to consider the minimum set of components that mostly reflect the biogenesis of an oil (Aparicio & Morales, 1998), hexanal (1), 1-hexanol (2), (E)-2 hexenal (3), (E)-2-hexen-1-ol (4) and (Z)-3-hexenyl acetate (5) were chosen as markers of linoleic and linolenic acids specific lipoxygenase oxidation [(path A and B ), Fig. 1].

#### **2.2.5.1 Preparation of samples and standard solutions**

A solution (200 mg/Kg) was prepared by dissolving 0.04 g of each analytes (see paragraph 2.2.5) in 200 g of commercial seeds oil. In the same manner a solution containing the internal standard (ethyl isobutyrate) was prepared.

Volatile and Non-Volatile Compounds of Single Cultivar Virgin Olive Oils Produced

Mass spectra were collected in EI in positive mode.

respectively and m/z 71, 88, 116 for the internal standard.

**2.2.5.3 Quantitative analysis** 

**2.2.5.4 Statistical analysis** 

assumptions for ANOVA.

**3.1 FAMEs analysis** 

**3. Results and discussions** 

in Italy and Tunisia with Regard to Different Extraction Systems and Storage Conditions 7

were taken into account and integrated. Identification of analytes was made by comparison of their mass spectra and retention times with those of authentic reference compounds.

The experimental work was carried out using a Varian 4000 Ion Trap GC/MS system (Varian, Inc. Corporate Headquarters, U.S.A.) equipped with a CP 3800 GC. Volatile components were adsorbed by means of a divilbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber and separation was obtained by means of a capillary column FactorFOUR (Varian VF-5ms). The ion trap temperature was set at 210 °C with an ionization time of 2 ms, reaction time at 50 ms and scan rate at 1000 ms. The transfer line temperature was set at 230 °C. The column was a 30 m Chrompack CP-Sil 8 CB low bleed/MS (0.25 mm i.d., 0.25 m film thickness). The GC oven temperature was initially held at 40 °C for 3 min, then ramped at 1 °C/min to 70 °C and finally ramped at 20 °C/min to 250 °C and held for 8 min. The carrier gas was helium at 1 mL/min. Analyses were performed in splitless mode.

The calibration curves were obtained by covering two concentration range: 0.4-4 mg/Kg with six steps at 0.4, 0.8, 1.5, 3, 4 mg/Kg for each analyte, with 1.5 mg/Kg of internal standard and 5-150 mg/Kg with six steps at 5, 10, 25, 50, 100, 150 mg/Kg for each analyte, with 40 mg/kg of internal standard. Each experimental value corresponds to the average of three replicates.

The quantitative assay was performed by selecting the area of the ionic species as follows: m/z 41, 56, 67, 72, 82 for hexanal; m/z 55, 56, 69 for 1-hexanol; m/z 55, 69, 83, 97 for (E)-2 hexenal; m/z 57, 67, 82 for (E)-2-hexen-1-ol; m/z 67, 82 for (Z)-3-hexenyl acetate,

The data obtained for each compound were subjected to statistical analysis. Statistical treatment was performed by STATGRAPHICS Plus Version 5.1 (Statistical Graphics Corporation , Professional Edition - Copyrigth 1994-2001). The approach chosen to analyse the set of data obtained was Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA). Also, in order to check possible differences between the oils, two-way analysis of variance (ANOVA) was performed considering, as main factors, the nationality of the sample and the type of storage container. Moreover, to evaluate significant differences between averages, Tukey test was performed on the oil quality parameters. Differences were considered statistically significant for P ≥ 0.01 and P ≥ 0.05. The values obtained for free acidity and FAMEs were analyzed after arcsine transformation in order to meet

VOOs under investigation showed the typical profile of fatty acids of the areas of production. In general, the oils were dominated by palmitic acid (C16: 0), stearic acid (C18: 0), oleic acid (C18: 1) and linoleic acid (C18: 2). The observed values do not show a particular pattern that can indicate the mode of extraction and the type of packing. It is well known, in fact, that fatty acids are dependent on genetic factors, soil and climate (Christie, 1998;

Dabbou, et al., 2010; Gharsallaoui, et al., 2011; Manai, et al., 2007).

#### **2.2.5.2 Experimental procedure and instrumentation**

The assay of secoiridoid glycosides, such as oleuropein, in virgin olive oil has been proposed as a marker of quality (De Nino et al., 1999, 2005; Perri et al., 1999). With reference to the works previously mentioned, the chromatogram of volatile compounds was considered a useful target. Only the peaks with a certain threshold value (S/N equal to five) were taken into account and integrated. Identification of analytes was made by comparison of their mass spectra and retention times with those of authentic reference compounds.

The experimental work was carried out using a Varian 4000 Ion Trap GC/MS system (Varian, Inc. Corporate Headquarters, U.S.A.) equipped with a CP 3800 GC. Volatile components were adsorbed by means of a divilbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber and separation was obtained by means of a capillary column FactorFOUR (Varian VF-5ms). The ion trap temperature was set at 210 °C with an ionization time of 2 ms, reaction time at 50 ms and scan rate at 1000 ms. The transfer line temperature was set at 230 °C. The column was a 30 m Chrompack CP-Sil 8 CB low bleed/MS (0.25 mm i.d., 0.25 m film thickness). The GC oven temperature was initially held at 40 °C for 3 min, then ramped at 1 °C/min to 70 °C and finally ramped at 20 °C/min to 250 °C and held for 8 min. The carrier gas was helium at 1 mL/min. Analyses were performed in splitless mode. Mass spectra were collected in EI in positive mode.

#### **2.2.5.3 Quantitative analysis**

6 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

OLIVE LIPIDS

Acyl hydrolase


13-(S)-hydroperoxy linolenic acid 13-(S)-hydroperoxy linoleic acid

COOH

O H

O H

The assay of secoiridoid glycosides, such as oleuropein, in virgin olive oil has been proposed as a marker of quality (De Nino et al., 1999, 2005; Perri et al., 1999). With reference to the works previously mentioned, the chromatogram of volatile compounds was considered a useful target. Only the peaks with a certain threshold value (S/N equal to five)

OOH

**A**

OH H

AAT Isomerase

ADH

ADH

OH

O O

(Z)-3-hexenyl acetate

(E)-2-hexen-ol

O

O H

Isomerase

O H

(E)-2-hexenal

**2.2.5.2 Experimental procedure and instrumentation** 

Fig. 1. Linoleic and linolenic acids specific lipoxygenase oxidation.

COOH COOH

**B**

OOH

COOH

COOH

ADH = alcohol dehydrogenase AAT = alcohol acetyl transferase

Isomerase

COOH

O H

hexanal

1-hexanol

ADH

OH

Lipoxygenase Lipoxygenase

Hydroperoxyde lyase Hydroperoxyde lyase

The calibration curves were obtained by covering two concentration range: 0.4-4 mg/Kg with six steps at 0.4, 0.8, 1.5, 3, 4 mg/Kg for each analyte, with 1.5 mg/Kg of internal standard and 5-150 mg/Kg with six steps at 5, 10, 25, 50, 100, 150 mg/Kg for each analyte, with 40 mg/kg of internal standard. Each experimental value corresponds to the average of three replicates.

The quantitative assay was performed by selecting the area of the ionic species as follows: m/z 41, 56, 67, 72, 82 for hexanal; m/z 55, 56, 69 for 1-hexanol; m/z 55, 69, 83, 97 for (E)-2 hexenal; m/z 57, 67, 82 for (E)-2-hexen-1-ol; m/z 67, 82 for (Z)-3-hexenyl acetate, respectively and m/z 71, 88, 116 for the internal standard.

#### **2.2.5.4 Statistical analysis**

The data obtained for each compound were subjected to statistical analysis. Statistical treatment was performed by STATGRAPHICS Plus Version 5.1 (Statistical Graphics Corporation , Professional Edition - Copyrigth 1994-2001). The approach chosen to analyse the set of data obtained was Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA). Also, in order to check possible differences between the oils, two-way analysis of variance (ANOVA) was performed considering, as main factors, the nationality of the sample and the type of storage container. Moreover, to evaluate significant differences between averages, Tukey test was performed on the oil quality parameters. Differences were considered statistically significant for P ≥ 0.01 and P ≥ 0.05. The values obtained for free acidity and FAMEs were analyzed after arcsine transformation in order to meet assumptions for ANOVA.
