**1.4. Identification and assay of minor component of olive oil**

The quality of olive oil is often associated with the presence of microcomponents whose healing effects have been proved in some special cases.52, 53 Virgin olive oils contains also phenolic substances responsible for their stability against oxidation.54 Phenolic fraction includes simple phenols, tyrosol, and hydroxytyrosol, derivatives of hydroxybenzoic and hydroxycinnamic acids, aglycons of some glucosides, namely oleuropein, demethyloleuropein, ligstroside, and verbascoside.55, 56

Absolute analytical methods for mass spectrometric detection as well as for quantification of such bio-active molecules, such as oleopentadialdehydes have been developed. As previously mentioned, the presence of dialdehydes in olive oil can be easily ascertained by a simple LC-MS approach (scheme and figure above). However *false positive* can be easily formed during the ionization process due to extreme reactivity of the sampled molecules with polar solvents such as water or methanol.57 Such novel method provides an in situ chemical derivatization of the whole set of molecules into stable alkyloxime derivates, allowing the concomitant use of a stable isotope standard which improves both the precision and the accuracy of the measurements, thus reducing the drawbacks may arising from the calibration procedure, sample preparation, and matrix effects.

The method was applied to a set of different Italian virgin olive oils showing the presence of **5** and **6** in the range of 70÷166 and 23÷132 ppm, respectively. The same approach was exploited in the assay of hydroxytyrosol (1) and tyrosol (2), the strong antioxidant present in large amount in virgin olive oil, by LC–MS/MS under MRM condition and isotope dilution method, using d2-labelled internal standards, **3** and **4**, obtained by simple synthetic procedures (Chart 1). This active principles ranged from 10 to 47 and 5 to 25 ppm in experimental and commercial virgin olive oil, respectively.58

modifications to the Folch procedure have been published.51

**1.4. Identification and assay of minor component of olive oil** 

from the calibration procedure, sample preparation, and matrix effects.

experimental and commercial virgin olive oil, respectively.58

extraction for total fat determination.49

ligstroside, and verbascoside.55, 56

The ideal objective of any extraction method is to extract the largest possible amount of matrix constituents without altering their identity. to measure "total fat" various methods have been approved by the regulatory agencies of most major countries.45 Most of the older methods involve solvent extraction and gravimetric mass measurement of the lipid residue. Several apparatus have been developed since 1939 for automatic extraction (e.g., Soxhlet, Soxhterm, Soxtec, Butt, etc).46 For total fat determination ethyl ether (diethyl ether) is often the solvent of choice as it is relatively non-polar and extracts mostly the non-polar lipids (triacylglycerols, sterols, tocopherols, etc) while poorly extracting the polar lipids (glycolipids and phospholipids).47, 48 Methods have been developed to use supercritical fluid

However, because of the diversity of the matrix content, methods are designed to efficiently extract specific molecule classes. The extraction of total lipids in foods is often considered to be a simple procedure; to extract total lipids (non-polar and polar) more polar solvents must be used as extractants (e.g., hexan or petroleum ether, chloroform, methanol, isopropanol, water). The most popular method for total lipid extraction is the Folch method.50 Lipids are extracted from a sample by using chloroform-methanol (2:1 by volume). Many

The quality of olive oil is often associated with the presence of microcomponents whose healing effects have been proved in some special cases.52, 53 Virgin olive oils contains also phenolic substances responsible for their stability against oxidation.54 Phenolic fraction includes simple phenols, tyrosol, and hydroxytyrosol, derivatives of hydroxybenzoic and hydroxycinnamic acids, aglycons of some glucosides, namely oleuropein, demethyloleuropein,

Absolute analytical methods for mass spectrometric detection as well as for quantification of such bio-active molecules, such as oleopentadialdehydes have been developed. As previously mentioned, the presence of dialdehydes in olive oil can be easily ascertained by a simple LC-MS approach (scheme and figure above). However *false positive* can be easily formed during the ionization process due to extreme reactivity of the sampled molecules with polar solvents such as water or methanol.57 Such novel method provides an in situ chemical derivatization of the whole set of molecules into stable alkyloxime derivates, allowing the concomitant use of a stable isotope standard which improves both the precision and the accuracy of the measurements, thus reducing the drawbacks may arising

The method was applied to a set of different Italian virgin olive oils showing the presence of **5** and **6** in the range of 70÷166 and 23÷132 ppm, respectively. The same approach was exploited in the assay of hydroxytyrosol (1) and tyrosol (2), the strong antioxidant present in large amount in virgin olive oil, by LC–MS/MS under MRM condition and isotope dilution method, using d2-labelled internal standards, **3** and **4**, obtained by simple synthetic procedures (Chart 1). This active principles ranged from 10 to 47 and 5 to 25 ppm in


**Scheme 3.** Derivatization of tyrosol (Tyr) and hydroxytyrosol dialdehydes with unlabelled and labeled methoxyamine.

**Scheme 4.** Labelled and unlabelled hydroxytyrosol and tyrosol.

A renowned antioxidant present in olive oil is the secoiridoid oleuropein (OLP). A number of studies have recognized that a diet rich in olive oil, particularly unrefined oils, provides a healthy prevention of artery wall thickening as a consequence of low-density lipoprotein (LDL) oxidation process. This beneficial effect has been associated to the presence of oleic and linoleic monounsaturated fatty acids and to the action of potent antioxidants such as tocopherols and the "polyphenols".59 OLP, a secondary metabolite of terpenoid origin, is the main iridoid of the "phenolic pool" 60 of *Olea europaea*, whose activity is likely associated to its *o*-dihydroxybenzene (catechol) moiety. The same moiety is shared by hydroxytyrosol which is formed by enzymatic degradation of the intact secoiridoid and exhibits similar

redox activity. Other phenolic compounds, such as tyrosol, caffeic acid, etc., account for the radical scavenger effect of virgin olive oil;61 however, attention has been paid to the actual content of oleuropein in foodstuffs due to its therapeutic action.62 An ESR study performed on the pure molecule has demonstrated that OLP can likely be exhibited in particular physiological conditions a pro-oxidant activity.63

Modern Methodologies to Assess the Olive Oil Quality 257

Cassanese

A comprehensive MS characterization of OLP, showed the advantage of sampling [M+NH4]+ ammonium adduct either for the sensitivity of the analytical method and for the peculiar gas-phase chemistry of the species as shown by low and high energy collision experiments. It was shown that the loss of ammonia neutral and the consequent formation of the [M+H]+

The analytical data suggested, therefore, that the [M+NH4]+ species of oleuropein might correspond to a mixture of different structures and react, in the gas phase, through the elimination of neutral ammonia to give a transient [M+H]+ which possesses enough internal energy to undergo further fragmentation. Oleuropein is extremely reactive in the experimental conditions leading to the preparation of olive oil from drupes, mainly due to the action of glycosidase and esterase enzymes. Therefore its assay in oils derived from different cultivars and different procedures could be considered a marker of process and of good practise. The validated process previous discussed was therefore applied to the monitoring of OLP in oils

**Figure 14.** Ripening-phase-dependent oleuropein content in virgin olive oils of Cassanese and Carolea

Carolea 0,000

pigmentation mean

pigmentation 100 % deep

The importance of oleuropein has prompted the development of an absolute method for its

assay in olive oil, based on APCI-MS/MS and isotope dilution.66

100 % green 100 % superficial

species was controlling the unimolecular dissociations (Figure 13).64

formed from different cultivars at different ripening stage. (Figure 14).65

cultivars. Oleuropein assay in olive oil.

0,050 0,100 0,150 0,200 0,250 0,300 0,350 0,400 0,450

**Scheme 5.** R1= CH3- OLPd0; R1= CD3- OLPd3

**Figure 12.** ESI (-) MSMS spectra of compound HTyr (A) and labelled HTyr (B,insert).

**Figure 13.** ESI tandem mass spectra of OLP commercial standard. Product ion spectra (A) from [M+H]+ and (B) from [M+NH4]+ precursors.

A comprehensive MS characterization of OLP, showed the advantage of sampling [M+NH4]+ ammonium adduct either for the sensitivity of the analytical method and for the peculiar gas-phase chemistry of the species as shown by low and high energy collision experiments. It was shown that the loss of ammonia neutral and the consequent formation of the [M+H]+ species was controlling the unimolecular dissociations (Figure 13).64

The analytical data suggested, therefore, that the [M+NH4]+ species of oleuropein might correspond to a mixture of different structures and react, in the gas phase, through the elimination of neutral ammonia to give a transient [M+H]+ which possesses enough internal energy to undergo further fragmentation. Oleuropein is extremely reactive in the experimental conditions leading to the preparation of olive oil from drupes, mainly due to the action of glycosidase and esterase enzymes. Therefore its assay in oils derived from different cultivars and different procedures could be considered a marker of process and of good practise. The validated process previous discussed was therefore applied to the monitoring of OLP in oils formed from different cultivars at different ripening stage. (Figure 14).65

**Figure 14.** Ripening-phase-dependent oleuropein content in virgin olive oils of Cassanese and Carolea cultivars. Oleuropein assay in olive oil.

The importance of oleuropein has prompted the development of an absolute method for its assay in olive oil, based on APCI-MS/MS and isotope dilution.66

**Scheme 5.** R1= CH3- OLPd0; R1= CD3- OLPd3

256 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

**Figure 12.** ESI (-) MSMS spectra of compound HTyr (A) and labelled HTyr (B,insert).

**Figure 13.** ESI tandem mass spectra of OLP commercial standard. Product ion spectra (A) from [M+H]+

and (B) from [M+NH4]+ precursors.

physiological conditions a pro-oxidant activity.63

redox activity. Other phenolic compounds, such as tyrosol, caffeic acid, etc., account for the radical scavenger effect of virgin olive oil;61 however, attention has been paid to the actual content of oleuropein in foodstuffs due to its therapeutic action.62 An ESR study performed on the pure molecule has demonstrated that OLP can likely be exhibited in particular

The sensitivity and accuracy of the method allowed the discrimination of olive oil samples with very similar content of OLP. In particular filtered and not-filtered products from different southern Italian regions, located at latitude ranging between (38° 57' N) and (42° 28' N), were discriminated by the amount of OLP which was in the range of 0.093÷0.225 and 0.116÷0.344 ppm, respectively.

Modern Methodologies to Assess the Olive Oil Quality 259

**Figure 16.** LTP-MS full scan spectrum of crude virgin olive oil in the positive (+) and negative

The peculiarity of the method is also represented by the possibility of analyzing each component present in the full scan spectrum with conventional MS/MS devices. Further, another ion source related to the ambient mass spectrometry techniques, named paper spray, has been used for olive oil rapid analysis. The latter, allowed for qualitative determination of several analytes as well as for their assay adding a suitable labeled internal standard. Currently, this technique is applyed also to the *is situ* derivatization of olive oil (e.g., by methoxyamine), allowing the formation of mass tags and increasing the ionization efficiency for such king of molecules (e.g., oleopentanedialdehydes) in less than one minute.

**Figure 17.** Variation of the distribution of the aroma markers with the harvesting time in the olive oils

The volatile fraction of olive oil is considered as a source of quality data either for its organoleptic properties and for its correlation to genetic, agronomic and environmental

produced from two different southern Italy cultivar.

ionization mode (-).

A similar approach was exploited in the characterization, at the molecular level, of olive oils produced from the whole fruits or from stoned olives obtained from a pilot plant.67 The absolute amount of OLP was 3÷4 times higher in stoned oils, total phenol content showed a similar behaviour, whereas the amount of α-tocopherols was higher in oils obtained by conventional milling procedures. The total phenol content should be enhanced either when more lipophilic aglycones are formed, after the removal of the hydrophilic sugar moiety, and, likely, when oleocanthal55 and its hydroxytyrosol homologue are obtained after deglycosylation and demethylation at position 11 of oleuropein.68 This effect, indirectly, resembles the action of cell-wall-degrading enzymes added to the paste to improve the olive oil quality.69

**Figure 15.** ESI-MS (-) spectra taken directly from the solution containing OLP and the water extracts of Carolea pits after 60 min. of incubation.

The main technological difference in the production of the sampled oils was represented by the presence or absence of stones during the manufacturing procedure. It was therefore checked, always by LC-MS/MS, the action of water extracts of crushed stones on pure samples of OLP was therefore planned. The extracted ion chromatogram showed, after 60 min of incubation, that elenoic acid and its open chain isomers were formed whereas hydroxytyrosol was not detected (Figure 15). This was considered an evidence of the preponderant presence of glucosidases in the stone enzyme pool. The quality of olive oils in relation to the identification and assay of the nutraceutical pool has been recently performed by the new tools of ambient ionization mass spectrometry. A low temperature plasma (LTP) ion source has allowed a rapid screening of free fatty acids, selected bioactive phenolic compounds, volatiles and adulterants in virgin olive oils (Figure 16).70

**Figure 16.** LTP-MS full scan spectrum of crude virgin olive oil in the positive (+) and negative ionization mode (-).

258 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

0.116÷0.344 ppm, respectively.

Carolea pits after 60 min. of incubation.

oil quality.69

The sensitivity and accuracy of the method allowed the discrimination of olive oil samples with very similar content of OLP. In particular filtered and not-filtered products from different southern Italian regions, located at latitude ranging between (38° 57' N) and (42° 28' N), were discriminated by the amount of OLP which was in the range of 0.093÷0.225 and

A similar approach was exploited in the characterization, at the molecular level, of olive oils produced from the whole fruits or from stoned olives obtained from a pilot plant.67 The absolute amount of OLP was 3÷4 times higher in stoned oils, total phenol content showed a similar behaviour, whereas the amount of α-tocopherols was higher in oils obtained by conventional milling procedures. The total phenol content should be enhanced either when more lipophilic aglycones are formed, after the removal of the hydrophilic sugar moiety, and, likely, when oleocanthal55 and its hydroxytyrosol homologue are obtained after deglycosylation and demethylation at position 11 of oleuropein.68 This effect, indirectly, resembles the action of cell-wall-degrading enzymes added to the paste to improve the olive

**Figure 15.** ESI-MS (-) spectra taken directly from the solution containing OLP and the water extracts of

The main technological difference in the production of the sampled oils was represented by the presence or absence of stones during the manufacturing procedure. It was therefore checked, always by LC-MS/MS, the action of water extracts of crushed stones on pure samples of OLP was therefore planned. The extracted ion chromatogram showed, after 60 min of incubation, that elenoic acid and its open chain isomers were formed whereas hydroxytyrosol was not detected (Figure 15). This was considered an evidence of the preponderant presence of glucosidases in the stone enzyme pool. The quality of olive oils in relation to the identification and assay of the nutraceutical pool has been recently performed by the new tools of ambient ionization mass spectrometry. A low temperature plasma (LTP) ion source has allowed a rapid screening of free fatty acids, selected bioactive phenolic

compounds, volatiles and adulterants in virgin olive oils (Figure 16).70

The peculiarity of the method is also represented by the possibility of analyzing each component present in the full scan spectrum with conventional MS/MS devices. Further, another ion source related to the ambient mass spectrometry techniques, named paper spray, has been used for olive oil rapid analysis. The latter, allowed for qualitative determination of several analytes as well as for their assay adding a suitable labeled internal standard. Currently, this technique is applyed also to the *is situ* derivatization of olive oil (e.g., by methoxyamine), allowing the formation of mass tags and increasing the ionization efficiency for such king of molecules (e.g., oleopentanedialdehydes) in less than one minute.

The volatile fraction of olive oil is considered as a source of quality data either for its organoleptic properties and for its correlation to genetic, agronomic and environmental

factors. Aroma components of products of plant origin are dependent on genetic, agronomic, and environmental factors.71 The identification and assay of the terminal species of the "lipoxygenase pathway" which are present in the volatile fraction of olive oils, has been performed by electron impact and/or chemical ionization mass spectrometry in a GC/MS Ion Trap apparatus. The quantitative data for each compound were subjected to principal component analysis to characterize the different cultivars of this work. PCA methodology was applied to confirm the hypothesis that the five selected markers of specific lipoxygenase oxidation could be used to differentiate the various cultivars (Figure 17).72 The same method was applied to distinguish the origin of experimental oils produced from drupes harvested in different areas of Italian Calabria region and of Tunisia. An easy discrimination was achieved from each cluster of samples. Olive oils produced from irrigated and nonirrigated farms in Tunisia were also clearly distinguishable.73 Quality and safety of oil is also associated to the presence of dangerous organic residues such as phthalates (PAEs). These compounds tend to be distributed mostly in fatty foods and this can cause the presence of remarkable amounts of PAEs in olive oil. Their determination in fatty matrices represents,therefore, a very important goal for the consumers' health and confidence. A rapid method for the analysis of phthalates in olive oil by GC-MS/MS after a GPC clean-up has been developed which exploits the capability of tandem mass spectrometry (GC-MS/MS) for the unequivocal confirmation and accurate quantification of PAEs at low limits of detection (LOD) levels in fatty matrices without the need for a liquidliquid extraction prior to GPC and for SPE clean-up following GPC.74 The interest in the identification of secondary metabolites by MS methods has been extended to different olive tissues. An extensive investigation, by means of high-resolution tandem mass spectrometry (MS/MS) has shown that some of these micro components are strongly cultivar dependent, and probably ripening dependent, while others are widespread and present in all the analyzed cultivars.75 The new secoiridoid metabolites found in drupes reveal that the key molecules produced by secondary metabolism of terpenes can be conjugated with hydroxytyrosol, a secondary metabolite of phenol biosynthesis, through the formation of differently structured glucosides. The origin of this new species could be related to transport phenomena which can be different among the various tissues of a given plant. Moreover, more stringent evidence of the biogenetic similarity of the members of different oleaceae families is provided by the discovery of metabolites typical of ligustrum and fraxinus in olive tissues. Secondary metabolites of Olea europaea leaves have been selected as markers for the discrimination of cultivars and cultivation zones by multivariate analysis; moreover, the statistical approach has been exploited to correlated the identity and relative amounts of metabolites in leaves with the harvesting period. The mean values of the concentration of each compound, detected by tandem mass spectrometry were inputted into PCA analysis. The bidimensional plot (Figure 18) shows a shifting along PC1 going from March–April to January which indicates the increase of concentration of compounds on the left of plot and the decrease for methoxytyrosol glucoside and 2-methoxyhydroxytyrosol glucoside. Moreover, position of samples of July at highest score values on PC2 means a decrease of concentration of variables verbascoside and hydroxytyrosol glucoside in leaves harvested in this period.76

Modern Methodologies to Assess the Olive Oil Quality 261

**Figure 18.** Biplot of principal component scores and loadings for leaves of Carolea cv harvested in

[1] Q.Q Wu, K.X Fan, H. Q Ruan, R. Zeng, C. H. Shieh. *Chinese Science Bulletin* 2009, 54,732-

[4] A. De Nino, F. Mazzotti, E. Perri, A. Procopio, A. Raffaelli, G. Sindona, *J. Mass Spectrom*.

[5] Di Donna L, Benabdelkamed H, Mazzotti F, Napoli A, Nardi M, Sindona G, Anal.

[7] Mazzotti F, Di Donna L, Benabdelkamel H, Gabriele B, Napoli A, Sindona, G, J. Mass

[8] J. Charrow, S. I. Goodman, E. R.G. McCabe P. Rinaldo. *Genet. Med.* 2000, *2*, 267-269.

[2] E.M. Usai , E. Gualdi , V. Solinas , E. Battistel *Bioresour. Technol.* 2010, *101* 7707–7712. [3] G. Sindona, A. Caruso, A. Cozza, S. Fiorentini, E. Marini, A. Procopio, S. Zicari, *Curr.* 

March, April, July and January (variables are indicated as m/z values).

*University of Calabria, Dept. Chemistry, Arcavacata di Rende (CS), Italy* 

Giovanni Sindona and Domenico Taverna

*Med. Chem.* 2012, *19*, 4006-4013.

Chem., 2011, 83, 6, 1990-1995.

Spectreom, 2010, 45, 4, 358-63.

[6] Cooks RG et al., Science, 2006, 311, 5767, 1566-70.

2000, *35*, 461–467.

**Author details** 

**2. References** 

737.

**Figure 18.** Biplot of principal component scores and loadings for leaves of Carolea cv harvested in March, April, July and January (variables are indicated as m/z values).
