Complementary Untargeted and Targeted Metabolomics for Differentiation of Extra Virgin Olive Oils of Different Origin of Purchase Based on Volatile and Phenolic Composition and Sensory Quality

In order to differentiate the extra virgin olive oils (EVOO) of different origin of purchase, such as monovarietal Italian EVOO with protected denomination of origin (PDO) and commercial-blended EVOO purchased in supermarkets, a number of samples was subjected to the analysis of volatile aroma compounds by both targeted gas chromatography/mass spectrometry (GC-MS) and untargeted profiling by comprehensive two-dimensional gas chromatography/mass spectrometry (GC×GC-TOF-MS), analysis of phenols by targeted high-performance liquid chromatography/mass spectrometry (HPLC-DAD-ESI/MS), and quantitative descriptive sensory analysis. Monovarietal PDO EVOOs were characterized by notably higher amounts of positive LOX-derived C6 and C5 volatile compounds, which corresponded to the higher intensities of all the assessed positive fruity and green odor sensory attributes. Commercial-blended EVOOs had larger quantities of generally undesirable esters, alcohols, acids, and aldehydes, which coincided with the occurrence of sensory defects in many samples. Many minor volatile compounds that were identified by GC×GC-TOF-MS were found to differentiate each of the two investigated groups. The differences between the groups with respect to phenols and taste characteristics were evident, but less pronounced. The results that were obtained in this study have undoubtedly confirmed the existence of the large heterogeneity of oils that are sold declared as EVOO. It was shown that GC-MS, GC×GC-TOF-MS, and HPLC-DAD-ESI/MS analyses have complementary outputs, and that their use in combination has advantages in supporting the results of sensory analysis and objectively differentiating these groups of EVOO.


Introduction
During the previous few decades, the olive oil scientific community and industry have become increasingly linked by the common goal of improving olive oil production and quality [1]. One of the most important permanent aims of this sector is to strengthen and improve the diversification Apart from being the first that aimed to differentiate EVOO on the basis of the origin of purchase, one of the most important novelties of this study is the utilization of a combined untargeted and targeted metabolomics approach utilizing powerful instrumentation and techniques, such as high throughput two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC×GC-TOF-MS) complemented by conventional monodimensional GC-MS for the analysis of volatile compounds, and high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) for the analysis of phenols. As a result, this study reported one of the most detailed and comprehensive qualitative and quantitative analytical characterizations of the volatile profile in EVOO up to date, with many compounds being identified (or tentatively identified) in EVOO for the first time. Additionally, it provided novel evidence regarding the diversity in sensory quality and volatile composition of olive oils sold declared as pertaining to the category of the highest quality grade (EVOO) and confirmed the need to re-evaluate the categorization criteria that were set by the current legislation.

Sensory Attributes
The results of sensory analysis are reported in a spider-web diagram in Figure 1. Monovarietal PDO EVOOs were characterized by higher intensities of the majority of positive odor descriptors, such as apple, green grass/leaf, aromatic herbs, etc. (except tomato and chicory/rucola), as well as general hedonic attributes, such as harmony, complexity, and persistency. Mild intensities of various sensory defects were detected in 19 out of 25 commercial-blended EVOO samples, which cast doubt on the correctness of their categorization and declaration. The average values of each defect intensity should be interpreted with caution, since not all of the samples had the same defect. For this reason, the average of the main perceived defect (the defect with the highest intensity perceived in each sample) was also calculated and is presented in Figure 1. No defects were detected in the monovarietal PDO samples. Interestingly, no significant differences were found for the taste attributes.

GC-MS and Sensory Attributes
The characteristic and unique flavor of EVOO, in particular its green and fruity attributes depend on many volatile compounds [10,11]. The identification and quantification of the compounds, causing both positive odors and off-flavors, is considered to be crucial for EVOO quality control. The list of selected identified and confirmed compounds, sorted by decreasing F-value, is shown in Table  1. Two groups of samples were successfully differentiated by one-way ANOVA. The concentrations

GC-MS and Sensory Attributes
The characteristic and unique flavor of EVOO, in particular its green and fruity attributes depend on many volatile compounds [10,11]. The identification and quantification of the compounds, causing both positive odors and off-flavors, is considered to be crucial for EVOO quality control. The list of selected identified and confirmed compounds, sorted by decreasing F-value, is shown in Table 1. Two groups of samples were successfully differentiated by one-way ANOVA. The concentrations of the majority of C6 and C5 aldehydes, which are regularly listed among the key ones that are responsible for positive green and fruity odors [18,19], were clearly higher in monovarietal PDO EVOOs than in the commercial-blended ones. The most abundant volatile compound in all of the investigated samples, (E)-2-hexanal, was also found in higher amounts in monovarietal PDO EVOOs, although it was not among the ones with the highest discriminative power judging from the F-values. However, (Z)-2-hexenal and (Z)-3-hexenal, carriers of major positive fruity and green notes, together with the isomers of 3-ethyl-1,5-octadiene and 4,8-dimethyl-1,3,7-nonatriene with unknown sensory relevance [20], turned out to be decisive for the differentiation of monovarietal PDO from commercial blended EVOOs. Among other possible causes of the lower amounts of these compounds in commercial blended EVOOs were possibly the changes induced by EVOO aging during storage, as it was shown in earlier studies [21]. In contrast to monovarietal PDO EVOOs that were analyzed relatively fresh, the age of commercial blended EVOO was not declared by the producers/sellers and it was practically unknown, and it was possible that these samples were produced or partially composed from olive oils that were obtained in harvests prior to 2016. Nevertheless, it must be kept in mind that all of the investigated EVOOs were carefully selected and sampled at the same time and they were therefore valid and authentic representatives of both groups of EVOOs offered on the market at that given moment [21]. Identification: the volatile aroma compounds were identified on the basis of the comparison of their mass spectra and retention time with those of pure standards or with mass spectra from a mass spectral database (Std, MS), as well as by retention indexes (RI) matches on a similar phase column (NIST Chemistry WebBook SRD 69, VCF Volatile Compounds in Food 16.1) or by comparing only the mass spectra (MS). An asterisk (*) in a row represents significant differences between mean values at p < 0.05 obtained by one-way ANOVA and least significant difference (LSD) test.
The commercial-blended EVOOs from supermarkets were mostly characterized by the higher concentration of saturated esters, aldehydes, and alcohols (Table 1). Such compounds do not originate from the LOX pathway and they are mostly the result of other, mostly undesirable processes [21][22][23]. Ethyl and methyl acetate, which are responsible for winey-vinegar defect and, together with 2-methylbutanol and 2-phenylethanol, clearly indicated that olives underwent fermentation [12], were the major differentiators of the commercial-blended from the monovarietal PDO EVOOs. Non-LOX C4 and C5 branched compounds are known to derive from the conversion of certain amino acids, while linear acids, esters, and ketones originate from fatty acid metabolism [24]. All of the mentioned processes are commonly linked to more or less degraded raw olive fruit material, due to physical damage, inadequate sanitary conditions, or unsuitable storage of fruit before processing, and they are often found in VOOs with sensory defects [24][25][26][27]. The possibility that particular non-LOX volatile compounds were formed and/or increased in concentration as a result of various oxidative processes during an (unknown) storage period of a number of commercial blended EVOOs, as shown previously by other authors [21], should not be neglected.
PCA analysis clearly divided the samples in two groups ( Figure 2). The majority of the investigated aldehydes and ketones that derive from the LOX pathway, including the major ones, such as (E)-2-, (Z)-2-, and (Z)-3-hexenal, as well as 1-penten-3-one and unsaturated hydrocarbons, were characteristic for monovarietal PDO EVOO samples, and they could have contributed to generating positive green and fruity notes [19] since a positive correlation between their concentrations and the intensities of such sensory attributes was evident (Figure 2b). Sensory defects that were observed in the commercial-blended EVOOs most probably, at least partly, originated from the elevated concentrations of fermentation and oxidation derived volatiles, such as linear alcohols and esters ( Figure 2). PCA analysis clearly divided the samples in two groups ( Figure 2). The majority of the investigated aldehydes and ketones that derive from the LOX pathway, including the major ones, such as (E)-2-, (Z)-2-, and (Z)-3-hexenal, as well as 1-penten-3-one and unsaturated hydrocarbons, were characteristic for monovarietal PDO EVOO samples, and they could have contributed to generating positive green and fruity notes [19] since a positive correlation between their concentrations and the intensities of such sensory attributes was evident (Figure 2b). Sensory defects that were observed in the commercial-blended EVOOs most probably, at least partly, originated from the elevated concentrations of fermentation and oxidation derived volatiles, such as linear alcohols and esters ( Figure 2).

GC×GC-TOF-MS and Sensory Attributes
The first preliminary classification of monovarietal PDO and commercial-blended EVOO samples that were based on untargeted profiling information was obtained by applying PCA on raw

GC×GC-TOF-MS and Sensory Attributes
The first preliminary classification of monovarietal PDO and commercial-blended EVOO samples that were based on untargeted profiling information was obtained by applying PCA on raw data. However, not only selected bidimensional GC peaks with the highest potential for varietal differentiation, but also other peaks were tentatively identified on the basis of mass spectrum and linear retention index matching to improve its effectiveness and specificity and to obtain as much qualitative information as possible. Table 2 reports the list of volatile aroma compounds tentatively annotated in the investigated EVOOs after GC×GC-TOF-MS analysis, in order of decreasing F-ratio. and commercial-blended extra virgin olive oils by headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography-mass spectrometry (HS-SPME-GC×GC-TOF-MS) sorted by descending Fisher F-ratio, compound class, retention index (monodimensional column), and semi-quantitative values in µg/kg relative to internal standard.   Identification: the volatile aroma compounds were identified on the basis of the comparison of their mass spectra with those from a mass spectral database, as well as by retention indexes matches on a similar phase column (NIST Chemistry WebBook SRD 69, VCF Volatile Compounds in Food 16.1), except compounds designated by # -tentatively identified by mass spectra database matches. LRIlit-linear retention index from the literature, LRIexp-linear retention index. An asterisk (*) in a row represents significant differences between mean values at p < 0.05 obtained by one-way ANOVA and least significant difference (LSD) test.

Compounds
GC×GC-TOF-MS analysis extracted many minor compounds as statistically relevant for this study, which were not previously identified by GC-MS, either in this or in earlier studies. Such a result proved that the two techniques have complementary outputs and that their use in combination has serious advantages. Interestingly, several compounds that were most characteristic (the highest F-values) for monovarietal PDO EVOOs were the minor ones, such as curcumene, octanal, 2-hexanol, ocimene, protoamenonine, valencene, etc. (Table 2). On the other hand, GC×GC-TOF-MS in a major part confirmed the GC-MS results, among other findings that higher amounts of standardly reported major LOX-derived C6 and C5 aldehydes, ketones, and alcohols, such as (Z)-2-hexenal, (E)-2-hexenal, 1-penten-3-one, and (Z)-2-penten-1-ol, are characteristic for this group of EVOO. The commercial-blended EVOO group was distinguished by a much larger number of volatile markers, including many of those often co-occurring with negative, defective sensory notes, such as 2-phenyl ethanol, isoamyl alcohol, isoamyl acetate, ethyl-2-methyl benzene, ethyl hexanoate, 3-methyl-2-buten-1-ol, 3-hydroxy-2-butanone, etc. [19,21,22,28], accompanied by a large array of minor and unbeknown compounds ( Table 2). The most characteristic compounds, such as acetic acid, 3-methyl-3-buten-1-ol, 3-hydroxy-2-butanone, 1-octen-3-ol, and others, did not coincide with those that were extracted as the most important by GC-MS (e.g., acetates), showing once again the synergistic potential of the two techniques used. Again, the group of commercial-blended EVOO was seriously deficient in the LOX volatiles that were known to be carriers of positive green and olive fruity flavor notes. Such results were confirmed by PCA: monovarietal PDO EVOOs were sharply separated from the commercial-blended group, owing to the higher amounts of LOX volatiles and more intense positive odor sensory notes ( Figure 3). Although produced from different olive cultivars grown in different geographical areas in Italy, monovarietal PDO EVOOs exhibited a relatively high level of homogeneity, with the exception of the three samples that belonged to the same cultivar/area located in the fourth quadrant of Cartesian plane with high absolute values of the coordinates on both PC1 and PC2 axes, which were even more discriminated from the commercial-blended EVOOs (Figure 3a). It is probable that, in this particular case, the cultivar and/or the geographical area had a greater impact. The commercial-blended EVOOs were mostly grouped by the non-LOX compounds and the occurrence of sensory defects, which were probably in a causal relationship (Figure 3). Indeed, the defects that were observed during the sensory analysis of the commercial EVOOs put these oils in a lower quality category that usually have a lower price than EVOO. The results that were obtained in this study for the particular major volatile compounds were mostly in agreement with the findings from Fiorini et al. (2018) [1], who also utilized their amounts, among other compounds, to differentiate high from low-priced EVOO.

Phenols and Sensory Attributes
In all of the investigated EVOO samples, 13 phenols were identified and quantified (Table 3) while using a wavelength of 310 nm for vanillin and p-coumaric acid, while the wavelength was 280 nm for all other compounds. Moreover, the molecular ions of each compound were used to confirm the identification of the analytes. Generally, the most abundant was p-HPEA-EDA, followed by 3,4-DHPEA-EDA I, 3,4-DHPEA-EA, p-HPEA-EA, and OH-tyrosol. Interestingly, a statistically significant difference was only found for a few phenols: lignans, such as pinoresinol and acetoxypinoresinol, were characteristic for the monovarietal PDO EVOOs, while the commercial-blended group was distinguished by higher amount of p-HPEA-EDA. The reduced data matrix that was obtained through HPLC-DAD-ESI/MS quantitative analysis (the phenols with 0.05 < p and 0.05 < p<0.10) was subjected to PCA ( Figure 4). The two classes of EVOO samples were clearly separated in the score plot (Figure 4a). The commercial-blended ones appeared to be relatively homogeneous in terms of phenol composition, since they were grouped rather close in the center of the plot. Monovarietal PDO

Phenols and Sensory Attributes
In all of the investigated EVOO samples, 13 phenols were identified and quantified (Table 3) while using a wavelength of 310 nm for vanillin and p-coumaric acid, while the wavelength was 280 nm for all other compounds. Moreover, the molecular ions of each compound were used to confirm the identification of the analytes. Generally, the most abundant was p-HPEA-EDA, followed by 3,4-DHPEA-EDA I, 3,4-DHPEA-EA, p-HPEA-EA, and OH-tyrosol. Interestingly, a statistically significant difference was only found for a few phenols: lignans, such as pinoresinol and acetoxypinoresinol, were characteristic for the monovarietal PDO EVOOs, while the commercial-blended group was distinguished by higher amount of p-HPEA-EDA. The reduced data matrix that was obtained through HPLC-DAD-ESI/MS quantitative analysis (the phenols with 0.05 < p and 0.05 < p<0.10) was subjected to PCA (Figure 4). The two classes of EVOO samples were clearly separated in the score plot (Figure 4a). The commercial-blended ones appeared to be relatively homogeneous in terms of phenol composition, since they were grouped rather close in the center of the plot. Monovarietal PDO EVOO samples were scattered in all four quadrants of the Carthesian plane, which indicated a higher degree of diversity. This was even more obvious when all of the phenols were included as variables, resulting with an additional separation of three-sample clusters of particular monovarietal EVOO in PCA representation ( Figure S1). Positive sensory descriptors that were partly related to the EVOO taste, such as harmony and persistency, were characteristic for monovarietal PDO EVOOs (Figure 4), although there was no evidence that the phenols were responsible for that. In fact, for the intensities of attributes that are known to directly originate from phenols, such as bitterness, pungency, and astringency, no notable differences were observed between the two groups of EVOO samples. This result is not completely in accordance with the findings from Fiorini et al. (2018) [1], who found high-priced EVOO to be more abundant in the majority of phenols, including secoiridoids, and characterized by higher intensities of bitterness and pungency in relation to EVOO samples of low price. Table 3. List of phenols found in monovarietal Protected Designation of Origin (PDO) and commercial-blended extra virgin olive oils by high-performance liquid chromatography with diode-array and mass spectrometric detection (HPLC-DAD-ESI/MS) sorted by descending Fisher F-ratio, phenol class, and concentration (mg/kg). An asterisk (*) in a row represents significant differences between mean values at p < 0.05 obtained by ANOVA and least significant difference (LSD) test.

EVOO Samples
After preliminary selection from a larger group of high quality monovarietal EVOOs with PDO, samples that were produced from olives of Italian cultivars harvested in 2016 were collected from different geographical areas in Italy (price range from 20 to 30 €/L), including Reggio Calabria (cultivar: Ottobratica; n = 3), Perugia (cultivar: Moraiolo; n = 3), Ragusa (cultivar: Tonda Iblea; n = 3), Grosseto (cultivar: Frantoio; n = 3), Imperia (cultivar: Taggiasca; n = 1), Brescia (cultivar: Moraiolo; n = 1), Verona (cultivar: Leccino; n = 1), and Riva del Garda (cultivar: Casaliva; n = 3). Furthermore, 25 commercial-blended EVOOs (price range from 3 to 12 €/L) were purchased from Italian grocery stores (supermarkets), which were selected according to Nielsen (New York, NY, USA 2016) data as among the most consumed during 2016 in Italy. All of the samples were stored in dark bottles at a controlled temperature of 15 °C before analysis, and gaseous N2 was added in the headspace to prevent oxidation each time that the bottles were opened.

EVOO Samples
After preliminary selection from a larger group of high quality monovarietal EVOOs with PDO, samples that were produced from olives of Italian cultivars harvested in 2016 were collected from different geographical areas in Italy (price range from 20 to 30 €/L), including Reggio Calabria (cultivar: Ottobratica; n = 3), Perugia (cultivar: Moraiolo; n = 3), Ragusa (cultivar: Tonda Iblea; n = 3), Grosseto (cultivar: Frantoio; n = 3), Imperia (cultivar: Taggiasca; n = 1), Brescia (cultivar: Moraiolo; n = 1), Verona (cultivar: Leccino; n = 1), and Riva del Garda (cultivar: Casaliva; n = 3). Furthermore, 25 commercial-blended EVOOs (price range from 3 to 12 €/L) were purchased from Italian grocery stores (supermarkets), which were selected according to Nielsen (New York, NY, USA 2016) data as among the most consumed during 2016 in Italy. All of the samples were stored in dark bottles at a controlled temperature of 15 • C before analysis, and gaseous N 2 was added in the headspace to prevent oxidation each time that the bottles were opened.

Standards and Solvents
The solvents used for the analysis of phenols in EVOOs were HPLC-MS grade methanol, hexane, isopropanol, and formic acid, which were purchased from Honeywell Riedel-de Haën (Seelze, Germany) and all aqueous solutions, including the HPLC mobile phase, were prepared with water purified while using a Milli-Q system (Millipore, Vimodrone, Milan, Italy). All of the analytical standards used for identification and calibration are listed in Table S1.

GC-MS Analysis of Volatile Aroma Compounds
Three grams of EVOO were put into a 20 mL glass headspace vial, and then spiked with 30 µL of internal standard solution (menthol at 0.057 mg/g; w/w in seeds oil). The headspace in the vial was equilibrated at 40 • C for 5 min. and the volatile aroma compounds were extracted at 40 • C for 30 min. The headspace was sampled using 2 cm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 µm fibre, purchased from Supelco (Bellefonte, PA, USA). The volatile aroma compounds were desorbed in the GC inlet at 250 • C for 4 min. in splitless mode, and the fibre was reconditioned for 7 min. at 270 • C, prior to each analysis. Measurements were made while using a Thermo Trace GC Ultra gas chromatograph coupled to a Thermo Quantum XLS mass spectrometer Thermo Scientific (Milan, Italy), which was equipped with a PAL combi-xt (CTC, Zwingen, Switzerland) autosampler with a SPME option. A VF-wax capillary column (30 m × 0.25 mm × 0.25 µm, Agilent Technologies) was used. The GC oven temperature gradient was starting from 40 • C for 4 min., 6 • C/min. up to 250 • C, and held for 5 min. Carrier gas was helium at the constant flow rate of 1.2 mL/min. The transfer line and the MS ion source were both set at 250 • C. Electron ionization was applied at 70 eV with an emission current of 50 mA. Mass spectra were recorded in centroid full scan mode at a scan time of 0.200 s from 30 to 350 m/z. Thermo Excalibur software (2.2 SP1. 48, Thermo Scientific) was used for all acquisition control and data processing. Figure S1 reports representative chromatograms. Volatile aroma compounds were identified by comparing the retention times and mass spectra with those of standards, and with mass spectra from NIST 2.0, Wiley 8, and FFNSC 2 (Chromaleont, Messina, Italy). Linear retention indexes (relative to C7-C24 n-alkanes) were calculated and then compared to those from the literature. Semi-quantitative analysis was carried out and the concentrations of EVOO volatile aroma compounds were expressed as equivalents of the internal standard menthol in mg/kg of oil.

GC×GC-TOF-MS Analysis of Volatile Aroma Compounds
For GC×GC-TOF-MS analysis, a Gerstel MultiPurpose Sampler autosampler (Gerstel GmbH & Co. KG Mülheim an der Ruhr, Germany) with an agitator and SPME fiber was used to extract the volatiles from the EVOO sample vial headspace.  Figure S2.
For GC×GC-TOF-MS data, LECO ChromaTOF Version 4.22 software was used for all acquisition control and data processing. Automated peak detection and spectral deconvolution with a baseline offset of 0.8 and signal-to-noise of 100 were used during data treatment. With these settings, it was possible to detect 1479 putative compounds. The identification of VOO volatile aroma compounds was performed by comparing the retention times and mass spectra with those of the pure standards, and with mass spectra from NIST 2.0, Wiley 8, and FFNSC 2 (Chromaleont, Messina, Italy) mass spectral libraries, with a minimum library similarity match factor of 750. Additional identification of volatiles was achieved by comparing the experimental linear temperature retention indices with those that were reported in the literature for 1D-GC. In total, 179 volatile aroma compounds were identified.
To account for possible sample-to-sample variation, all of the intensities were normalized to the signal of menthol (internal standard) and corrected for the mass added. The analyses were performed in triplicates, and the average values were used in further data elaboration.

HPLC-DAD-MS Analysis Of Phenols
Samples extraction was made according to [29]. Five grams of oil containing a fixed aliquot of IS (syringic acid = 291 mg/L in MeOH) were dissolved in 5 mL of hexane and then extracted with 5 mL of a methanol: water solution (60:40, v/v) five times. Afterwards, 10 mL of hexane were added to the methanolic extracted solution, vortexed, and centrifuged for 5 min. at 5000 rpm. The methanol solution was collected and evaporated to dryness under vacuum. The extract was reconstituted with 2.5 mL of HPLC-grade methanol and then filtered through a 0.22 µm mPTFE filter before HPLC-DAD-MS analysis.

Sensory Analysis
Quantitative descriptive analysis of monovarietal and commercial EVOO samples was performed by the VOO sensory analysis panel comprised of eight assessors (four female, four male) that were trained for VOO sensory analysis according to the method that was proposed by IOC described in the European Commission Regulation [30]. The panel is accredited according to the EN ISO/IEC 17025:2007 standard from 2012 and continuously recognized by the IOC from 2014 to December 2019.
The panel used a modified profile sheet that was expanded with particular positive odor and taste attributes [30]. Single odor and taste attributes were quantified while using a 10-cm unstructured intensity ordinal rating scale from 0 (no perception) to 10 (the highest intensity). Differently from the standard method, for evaluating different general hedonic quality attributes (complexity, harmony, and persistency of VOO samples), a 10-point overall structured rating scale from 0 (the lowest quality) to 10 (the highest quality) was applied. For overall quality evaluation, the VOOs were graded with points from 1 (the lowest quality) to 9 (the highest quality).

Statistical Data Elaboration
GC-MS, GC×GC-TOF-MS, HPLC-DAD-ESI/MS, and sensory analyses data (concentrations of volatile aroma compounds and phenols) were subjected to one-way analysis of variance (ANOVA), and the average values were compared by Least Significant Difference (LSD) test at the level of p < 0.05. These data, together with the results of sensory analysis (medians of intensities and grades), were further processed by principal component analysis (PCA) in order to better visualize the differences between the two groups of EVOO and explain them on the basis of the concentrations of volatiles and phenols. Three datasets (GC-MS, GC×GC-TOF-MS, and HPLC-DAD-ESI/MS) were separately treated. Prior to PCA analysis, the GC-MS and GC×GC-TOF-MS original datasets were reduced to only include the volatiles with the highest discriminative potency (F-values in one-way ANOVA), combined with those regularly reported as among the most important for EVOO aroma. For the HPLC-DAD-ESI/MS dataset, first PCA was performed including all the identified phenols ( Figure S4), and then it was applied on a reduced dataset including only those for which statistically significant differences were observed (p < 0.05), and those being close to that (0.05 < p < 0.10). Statistical data elaboration was performed by Statistica v. 13.2 software (Stat-Soft Inc., Tulsa, OK, USA).

Conclusions
The combined use of GC-MS and GC×GC-TOF-MS analysis for volatile aroma compounds proved to be a powerful analytical option, providing broad coverage of the volatilome, which is useful for the differentiation of the two classes of EVOO with respect to the origin of purchase: monovarietal PDO EVOO from family farms vs. commercial-blended EVOO from supermarkets, respectively. To our knowledge, this study provided one of the most detailed and comprehensive qualitative and quantitative analytical characterizations of the volatile profile in EVOO up to date, with many compounds being identified (or tentatively identified) in EVOO for the first time. Among them, many potential markers were extracted, despite the known (for PDO) and presumed (for commercial-blended) geographical and pedoclimatic heterogeneity and large variations in olive growing and oil producing parameters among EVOOs. Monovarietal PDO EVOOs were characterized by notably higher concentrations of desirable LOX-derived C6 and C5 volatiles, including the major ones that are known to be crucial contributors to the characteristic and appreciated EVOO green and fruity flavor. Such findings basically confirmed the results of the sensory analysis, which described the monovarietal PDO EVOOs by higher intensities and grades for positive sensory descriptors and attributes. On the other hand, the commercial-blended EVOOs had larger quantities of many volatiles that are known to originate from undesirable chemical and microbiological processes in olive and in olive oil, such as saturated esters, alcohols, acids, and aldehydes, which corresponded to the occurrence of sensory defects in many of the samples from this group. It is worth highlighting that a very large array of minor and unbeknown compounds, not reported or neglected in previous studies, was found to differentiate each of the two investigated EVOO groups, which point to the possibility that they also contributed to the perceived sensory notes. Targeted HPLC-DAD-ESI/MS profiling of phenols succeeded in differentiating monovarietal PDO from commercial-blended EVOOs to a much smaller extent, which was mostly due to the diversity of the concentration in monovarietal oils. Additionally, the differences that were observed during sensory analysis related to the corresponding taste attributes were not large. Nevertheless, the results of this study undoubtedly confirmed the large heterogeneity of oils, which are sold declared as EVOO in Italy, both in terms of chemical composition and sensory attributes, and in a way pointed to the possible reasons behind the existing large span of prices within this quality category.
The approach that was proposed in this study is universal in nature and it could be applied for the characterization and differentiation of various other types of EVOO. The detailed profiles of volatile aroma compounds and phenols that can be obtained by the reported combination of powerful yet complementary techniques may serve experts, producers, and suppliers to better define typical sensory characteristics of EVOO in question, and in this way strengthen their identity and position on the market. From the technological point of view, understanding the compositional origins of the sensory typicity of particular EVOO might allow for more efficient quality management and control in production and more precise information to the consumers.
Supplementary Materials: The following are available online, Figure S1: Chromatograms of volatile aroma compounds found in monovarietal PDO and commercial-blended extra virgin olive oil by headspace solid-phase microextraction combined with gas chromatography/mass spectrometry (HS-SPME-GC-MS). Figure S2: Contour plots (2D-chromatograms) of volatile aroma compounds found in a) monovarietal PDO and commercial-blended extra virgin olive oils by headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography-mass spectrometry (HS-SPME-GC×GC-TOF-MS). Figure S3: Chromatograms of phenols found in a) monovarietal PDO and b) commercial-blended extra virgin olive oils by high-performance liquid chromatography with UV at wavelength of 280 nm and 310 nm respectively. Figure S4