A green sorptive extraction method (HiSorb-TD-GC-MS) for determining the extra virgin olive oil (EVOO) aroma profile

Thermal desorption

The HiSorb probe was then placed into a clean, empty TD tube (Markes International Ltd., UK) and desorbed using the TD unit. The parameters of TD-GC-MS are listed in Table 3. The analysis of VOCs was performed in an Agilent GC-7890B coupled to quadrupole mass spectrometer MSD-5977B (Agilent Technologies, USA). The separation of compounds was carried out on an SPB™-624 (60 m × 250 μm, 1.4 μm film thickness) capillary column with cyanopropylmethyl phenylmethyl polysiloxane stationary phase (Supelco). Helium (99.999 %, Linde) was used as carrier gas at a flow rate of 1.7 mL/min. The oven temperature was held at 35 °C for 5 min, then increased to 180 °C at 4 °C/min and remained for 20 min. The overall GC run time was 61.25 min. The temperatures of MS transfer line, quadrupole, ion source were set at 250, 150, 230 °C, respectively. The electron impact was 70 eV and the scan range was set at 35–350 atomic mass units (amu) in full scan mode. Each sample was analysed in triplicate.

Qualitative and quantitative analysis

The identification of compounds was based both on validated and putative levels (mass spectra matching against the library spectra database of NIST20 with a minimum matching factor over 80 %). The VOCs were semi-quantified by normalised the peak area of each compound in respect to the area of internal standard (4-methy-2-pentanone). The concentration of compounds was calculated according to Equation (1) and expressed in micrograms per kg (mg/kg) equivalent of 2-methyl-4-pentanone:

whereas, C x : corresponds to the compound concentration in mg/kg, A x : corresponds to the area under the curve (AUC) per compound, A I S : corresponds to the area under the curve (AUC) of 2-methyl-4-pentanone (mg/kg) serving as IS, and C I S : corresponds to the concentration of IS 2-methyl-4-pentanone (mg/kg) added initially to the sample.

Evaluation of the HS-HiSorb-TD-GC-MS method

Extraction temperature

The extraction temperature affects the extraction efficiency of the analytes. Due to its nature, olive oil is sensitive to temperature, so high extraction temperatures should be avoided to prevent the oxidation of the sample and the release of unwanted oxidation VOCs. Therefore, various extraction temperatures (25, 40, and 60 °C) were examined during the sampling process. This experiment was performed using a 2.5 g sample without stirring, and the applied sampling time was 120 min. As shown in Fig. 1(a), by increasing the extraction temperature, the total area of all VOCs increased. The increase in extraction temperature to 60 °C resulted in a higher number of compounds with 7–12 carbon atoms in their molecular structure (C7–C12), in contrast to oil samples heated at lower temperatures. The VOCs with C7–C12 are less volatile than the C5–C6 ones; this is mainly the reason why their areas were higher at higher extraction temperatures. Also, these compounds are related to olive oil oxidation processes (Fig. 1(b)) and therefore must be kept to a minimum during sample heating [22]. This observation agrees with previous studies in which the extraction temperature was set lower, at 55 °C. Therefore, 40 °C was selected as the optimum extraction temperature, as this is the closest temperature to the human body [23, 24].

Fig. 1:

Effect of extraction temperature (a) on the total area of VOCs, (b) on the total area of VOCs by category (C5- and C6-compounds derived from the LOX biochemical pathway, whereas C7- to C12-compounds are related to oxidation processes).

Extraction time

The exposure time of the sorbent probe in the headspace of the sample also affects the number of analytes extracted. Several adsorption times (30, 60, 90, and 120 min) of the HiSorb-PDMS probe were evaluated. The total area, as well as the number of compounds detected, showed an upward trend with the increasing of adsorption time, as shown in Fig. 2(a) and (b), respectively. Hence, the time of 120 min was selected as the optimum sampling time.

Fig. 2:

Effect of adsorption time (a) on the total area of all VOCs, (b) on the number of detected VOCs.

Stirring rate

The stirring rate during the extraction process creates a positive effect on the mass transfer from the sample to the headspace of the vial and to the adsorbent material. In addition, it assists homogenize the sample and the headspace phase, reducing the equilibration time. Therefore, the diffusion of VOCs and especially of semi-VOCs from the matrix to the adsorbent material is enhanced. For the optimisation of stirring, four different speeds were studied: 0 (no stirring), 200, 400, and 800 rpm. The total area of VOCs showed a maximum value at the stirring rate of 800 rpm, so it was selected as the optimum stirring speed (Fig. 3).

Fig. 3:

Effect of stirring rate on the total area of VOCs.

Sample amount

The effect of sample amount on extraction efficiency was also examined. In Fig. 4, the total area of extracted VOCs for two different amounts of sample (2.5, and 5 g) is plotted. As illustrated, the amount of extracted compounds (total area) did not change significantly as the amount of sample increased; this observation was also noticed by other authors [25]. Considering this and the limited availability of the sample, the smallest volume (2.5 g) was selected for the experiments.

Fig. 4:

Effect of sample amount on total area.

In summary, the optimum HS-HiSorb extraction conditions that were selected for the subsequent VOCs analysis of the olive oil samples were 40 °C extraction temperature, 120 min of adsorption time, 800 rpm of stirring rate, and 2.5 g of olive oil sample.

Aldehydes

Aldehydes are usually the most abundant chemical class in the aroma of olive oil. Most aldehydes, especially those with 5 and 6 carbons in their molecular structure (C5- and C6-), are produced by the LOX biosynthetic pathway, which is activated during the crushing of olive fruit tissue by the release of several fruit enzymes. Some aldehydes also arise from other endogenous biochemical pathways, such as the metabolism of amino acids, carbohydrates, vitamins, and steroids. Others derive from exogenous sources, such as abiotic and biotic stress.

(E)-2-hexenal was the most abundant aldehyde found on average of the examined samples, with a concentration ranging from 0.91 ± 0.05 mg/kg to 4.97 ± 0.64 mg/kg. Its highest concentration was observed in the O12 sample. (E)-2-hexenal contributes positively to the organoleptic characteristics of olive oil, providing the sensation of green, fruity, and bitter almond. In particular, considering each sample separately, it was observed that (E)-2-hexenal was the most abundant aldehyde for most samples (10 out of 21), whereas other samples showed nonanal (6) and hexanal (2) as the most dominant aldehydes. Only in one sample (O11), 2-undecenal was the most abundant aldehyde.

The concentration range of nonanal and hexanal was 0.73 ± 0.16 mg/kg to 4.80 ± 3.06 mg/kg and 0.48 ± 0.14 mg/kg to 3.70 ± 1.29, respectively. Nevertheless, (E)-2-hexenal was still found in high concentrations in all samples. These results are in agreement with previous studies, which showed that (E)-2-hexenal is the major aldehyde in the olive oils of European cultivars [26], [27], [28], [29], [30], [31], [32], [33]. Furthermore, according to a study by Fernandez et al. where Argentinian cultivars were examined, hexanal was revealed as the most abundant aldehyde for the Arbequina cultivar, while for the Empeltre and Farga cultivars it was (E)-2-hexenal [7]. Similarly, P. Reboredo-Rodríguez et al. found (E)-2-hexenal as the major aldehyde, followed by hexanal [17]. Regarding nonanal, its high concentration is associated with a bitter taste [34]. In Fig. 7, the concentration of aldehydes is plotted for each sample.

Fig. 7:

Concentration of aldehydes (mg/kg) in the examined olive oil samples.

Hydrocarbons

Hydrocarbons were detected in a significant proportion in the aromatic profile of the examined olive oils, representing on average 41.08 %. Methyl-cyclodecane was the major hydrocarbon, with a concentration ranging from 1.45 ± 0.41 mg/kg to 13.55 ± 2.76 mg/kg. In addition, several aliphatic and aromatic hydrocarbons were detected. The major aliphatic hydrocarbons detected in the majority of samples were heptane, pentane dimers, and octane. Pentene dimers are produced during olive oil production via side reactions of the LOX pathway. Aromatic hydrocarbons can be highly modified oils due to their lipophilic nature and their wide distribution in the environment. Four aromatic hydrocarbons were detected in the examined samples: toluene, p-xylene, ethylbenzene, and mesitylene. Their origin is attributed to various endogenous and exogenous factors such as the biological processes in the fruit, the extraction system, contamination from fuel vapors, etc. [35]. Figure 8 shows diagrammatically the concentrations of hydrocarbons (mg/kg) in the examined samples.

Fig. 8:

Concentration of hydrocarbons (mg/kg) in the analysed samples.

Acids

The carboxylic acids were the third most prominent chemical class and contributed on average 7.16 % to the aromatic profile of the samples. A total of six carboxylic acids were detected, including acetic acid, butanoic acid, hexanoic acid, octanoic acid, benzoic acid, and nonanoic acid [32]. Figure 9 shows the concentrations of the acids (mg/kg) determined in the examined samples. Carboxylic acids with 2–3 carbon atoms derive from microbial processes such as the acetic acid that is produced during anaerobic fermentation by certain bacteria, which convert glucose directly into acetic acid. High acid concentrations are associated with olive oil oxidation and yield sensory defects such as rancid taste. The main carboxylic acid found in the examined samples was nonanoic acid.

Fig. 9:

Concentration of acids (mg/kg) in the examined samples.

Terpenes

The terpenes contributed 6.61% of the total aroma profile and were considered the fourth most abundant chemical class in the anaysed olive oil samples. Their total content ranged from 0.49 ± 0.10 mg/kg to 3.47 ± 0.73 mg/kg. The following terpenes were identified and semi-quantified in most of the examined samples: D-limonene, beta-ocimene and (E)-4,8-dimethylnona-1,3,7-triene (Fig. 10). These compounds were also detected in virgin olive oil samples from Crete [36]. According to previous studies, terpenes depend on the variety and are important indicators of olive oil quality and geographic origin [37, 38].

Fig. 10:

Concentration of terpenes (mg/kg) in the examined samples.

Table 4 shows the semi-quantified VOCs concentrations expressed in mg/kg, based on the IS of 4-methyl-2-pentanone. The total concentration of VOCs (TVOCs) in the samples ranged from 8.73 to 39.81 mg/kg.

Repeatability and calibration curves

Table 5 presents the intra-day results, indicating satisfactory repeatability for the examined compounds as SD ranged from 2.22–15.86 %. The repeatability of the HiSorb-TD-GC-MS method was evaluated for selected compounds, at three different concentrations (1, 10, and 100 ppb_v).

The results of the calibration curves of compounds (E)-2-hexenal, 1-penten-3-one, hexanal, and nonanal are summarized in Table 6. The linearity range of the examined VOCs was estimated, and the results were satisfactory, with a coefficient of determination (R²) greater than 0.97, except for nonanal (R² = 0.94) which is emitted in relatively small amounts.

The analytical results in Tables 5 and 6 show the importance of sample pretreatment in the analytical process. HiSorb-PDMS is more suitable for non-polar VOCs (it was the first commercially available high sorptive product from Markes International Ltd. in 2016), while other multiphase sorptive products are available nowadays to extend the sensory applications. This observation is in agreement with previous SPME studies that found a low sensitivity of PDMS to the polar molecules in olive oil [39]. In this context, recent studies have highlighted the application of three-phase coatings (DVB/CAR/PDMS) for the identification of VOCs in EVOO [9, 36, 40].