*3.2.3. 18O and 2H pyrolysis CF-IRMS*

The abundance of the stable isotopes oxygen-18 (18O) and deuterium (2H) are particularly interesting isotopic probes for both botanical and geographical identification of a variety of different food products. The primary source of all organic hydrogen and oxygen is the hydrosphere. The meteoric water that has passed through the meteorological cycle of evaporation, condensation and precipitation finally constitutes the groundwater and exhibits a systematic geographical isotope variation. Decreasing temperatures cause a progressive heavy-isotope depletion of the precipitation when the water vapour from oceans in equatorial regions moves to higher latitudes and altitudes. Evaporation of water from the oceans is a fractionating process that decreases the concentration of the heavy isotopomers of water (1H2H16O, 1H1H18O) in the clouds compared to the sea. As the clouds move inland and gain altitude further evaporation, condensation and precipitation events occur decreasing the concentration of deuterium and oxygen-18.

Consequently, the ground water reflects this isotopic gradient from the coast to inland areas. For land plants, a further pre-assimilation affects the isotopic composition of the water substrate. The hydrogen and oxygen present in plant material originates from the water taken up by the roots. The water is transported through the plants xylem system. The isotopic composition of the xylem water is the same as that of water taken in by the roots, and the water is taken into the leaves without a change in isotopic composition. Evapotranspiration of water through the leaf enriches the remaining water in the heavier isotopomers. Therefore, it is expected that growing regions with relatively low humidity, where the rate of evaporation from the leaf is higher, result in plant materials with relatively enriched δ2H and δ18O values.

Over the past 5 years there has been a marked increase in the use and application of 2H and 18O stable isotopes in many areas of food research. This has been facilitated by recent developments in on-line gas preparation devices that proceed by high temperature pyrolysis of organic products and the availability of commercial IRMS analysers capable of measuring 2H/1H ratios in the presence of a helium carrier gas. These innovations have, to a large extent, overcome the difficulties associated with offline gas preparation for DI-MS and greatly increased the applicability of this measurement. It is now possible to routinely measure 2H and 18O abundances in organic samples by Pyrolysis-Continuous Flow-Isotope Ratio Mass Spectrometry (Py-CF-IRMS).

Therefore, measurements of stable isotope ratios of the light elements (H, C ,N ,O, S and bioelements) and of the heavy element stronzium, in natural cycles, have provided geographical fingerprints (Roßmann *et al.,* 2000).

Preliminary investigations into the application of 18O-pyrolysis continuous-flow IRMS to obtain information about the geographical origin of olive oil samples has been conducted by Angerosa *et al*. (1999). They measured the δ13C and δ18O values of whole olive oil, sterols and aliphatic alcohol fractions from fruits of *Olea europaea* L. produced in Greece, Italy, Morocco, Spain, Tunisia, and Turkey. The results permitted provincial classification of the oils. However, there were some misclassifications observed for oil samples coming from neighbouring countries with similar climates.

A secure geographical classification of an olive oil, in order to ensure that the consumer is not defrauded and that the honest trader is not disadvantaged by having their PDO oils misrepresented by inferior products, can be achieved by performing heavy isotope ratios (e.g. 88Sr/86Sr) and multi-element analysis.

### *3.2.4. Nuclear magnetic resonance spectroscopy*

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

13C abundance of oleic acid compared to linoleic acid and palmitic acid.

occur decreasing the concentration of deuterium and oxygen-18.

Harwood and Sánchez, 1999).

data derived from vegetable oils.

enriched δ2H and δ18O values.

*3.2.3. 18O and 2H pyrolysis CF-IRMS* 

Royer *et al.,* (1999) examined 188 olive oils produced mainly in Greece during 1993 to 1996. The concentration and δ13C value of individual fatty acids present in the olive oils were determined by gas chromatography and GC-C-IRMS respectively. The results were examined in terms of geographical, temporal, and botanical factors. French and Italian olive oils were securely classified at the 99.9% confidence interval using the δ13C values of the principal fatty acids palmitic (C16:0), oleic (C18:1) and linoleic (C18:2). Regional classifications for the Greek olive oils were also achieved on the basis of differences in the

Glycerol is a primary metabolite in plants. It is nominally present in its ester form as glycerolipids in fats and oils (Kiritsakis and Christie, 1999). Glycerol is bio-synthesised relatively early in the lipidic metabolic pathway compared to fatty acids (Weber *et al*., 1997;

Consequently, it may be expected that the isotopic distribution in glycerol is a better indicator of the botanical and environmental influences on any given plant. A number of compound specific IRMS studies on glycerol have been performed, some of which include

The abundance of the stable isotopes oxygen-18 (18O) and deuterium (2H) are particularly interesting isotopic probes for both botanical and geographical identification of a variety of different food products. The primary source of all organic hydrogen and oxygen is the hydrosphere. The meteoric water that has passed through the meteorological cycle of evaporation, condensation and precipitation finally constitutes the groundwater and exhibits a systematic geographical isotope variation. Decreasing temperatures cause a progressive heavy-isotope depletion of the precipitation when the water vapour from oceans in equatorial regions moves to higher latitudes and altitudes. Evaporation of water from the oceans is a fractionating process that decreases the concentration of the heavy isotopomers of water (1H2H16O, 1H1H18O) in the clouds compared to the sea. As the clouds move inland and gain altitude further evaporation, condensation and precipitation events

Consequently, the ground water reflects this isotopic gradient from the coast to inland areas. For land plants, a further pre-assimilation affects the isotopic composition of the water substrate. The hydrogen and oxygen present in plant material originates from the water taken up by the roots. The water is transported through the plants xylem system. The isotopic composition of the xylem water is the same as that of water taken in by the roots, and the water is taken into the leaves without a change in isotopic composition. Evapotranspiration of water through the leaf enriches the remaining water in the heavier isotopomers. Therefore, it is expected that growing regions with relatively low humidity, where the rate of evaporation from the leaf is higher, result in plant materials with relatively During the last ten years, nuclear magnetic resonance spectroscopy (NMR) (Del Coco *et al*., 2012; Mannina & Segre, 2002), has played an ever-increasing role in the study olive oil characterization and autentication. In particular, it has been shown that high-resolution NMR together with statistical analysis constitutes a powerful tool for the geographical characterization of olive oils on Mediterranean, national, regional and PDO scales. On this regard, innovative techniques like NMR spectroscopy seem to be able to distinguish olive oils on the basis of their geographical origin, whereas the conventional analyses suitable for the determination of quality and genuineness seem not to be so appropriate for this type of discrimination (Frankel, 2010; Guillen & Ruiz, 2001).

Important information on the fatty acid distribution on the glycerol moiety can be obtained by 13C NMR (Rezzi *et al*., 2005; Petrakis *et al*., 2008; Alonso-Salces *et al.,* 2010b; Mannina *et al.,* 2010; Alonso-Salces *et al.,* 2011b). Two groups of resonances are observed in the carbonyl region of the 13C NMR spectrum of an olive oil: one group is due to fatty chains in position sn-1,3 of the glycerol moiety, the other one is due to fatty acids in position sn-2.

It must be noted that although gas chromatographic methods give the full composition of fatty chains, no information is given about the fatty chains distribution on glycerol. Thus in this case, gas chromatography and 13C NMR methodologies must be considered complementary and not alternative. 13C NMR spectroscopy together with discriminant analysis has been proposed by Mavromoustakos et *al.*, to detect the presence of soybean oil, cottonseed oil, corn oil and sunflower seed oil in virgin olive oils. Only double bonds signals have been used for the analysis. Fatty acids composition and fatty chain positional distribution on glycerol moiety determined by 13C NMR spectroscopy allow, in combination with the multivariate statistical procedure, the classification of olive oils according to their variety (Marini *et al*., 2004, 2006).

Olive Oil Traceability 275

Extra virgin olive oils sampled in three harvesting years and coming from different Italian regions (Tuscany, Lazio, Lake of Garda) have been analyzed by 1H methodology. The statistical elaboration applied on the intensity of b-sitosterol, aldehydes and some other volatile compounds has allowed the classification of the olive oils according to the

1H-NMR fingerprinting of olive oil is a valuable analytical tool for the traceability of virgin olive oils from different points of view, i.e. food authentication and food quality. As described before, 1H and 13C NMR techniques provide different information: 1H NMR spectrum allows the measurement of minor components of olive oils such as β-sitosterol, hexanal, 2-(E)-hexenal, formaldehyde, squalene, cycloartenol and linolenic acid; the 13C NMR spectrum detects major components such as glycerol tri-esters of olive oils, defining also acyl composition and positional distribution on the glycerol moiety. The main sensitivity concern about 1H NMR analysis is the dynamic range, which makes the detection of signals below the threshold imposed by the ADC (analogue to digital converter) impossible. Therefore, in a standard extra virgin oil spectrum, trace component detection is limited to signals more intense than 10−5 times the intensity of the fatty acid CH2 signals. Weak signals above the dynamic range threshold are in any case affected by severe baseline distortion. Selective pulses coupled to gradient spin-echo refocusing such as DPFGSE (69), in NMR modern instruments, allow these limitations to be circumvented, paving the way for the easy detection of minor components, especially those presenting resonances away

The power of these new sequences could be demonstrated by the detection of aldehydes, carotenoids or other specific minor components whose resonances fall in a relatively free region. It was performed a specific DPFGSE analysis on the aldehydic components of some extra virgin olive oils, mostly responsible of the sensorial properties of extra virgin olive oil. Comparison with standard 1HNMR spectra show tremendous improvement of quantitative

Fingerprinting techniques such as NMR, NIR (Galtier *et a*l., 2007; Mignani *et al*., 2011), MIR (Reid *et al*., 2006), fluorescence (Kunz *et al*., 2011), FT-IR, FT-MIR and FT-Raman (Baeten *et al*., 2005; Lopez-Diez *et al*., 2003; Yang *et al.,* 2005) spectroscopies, have been used for the

These types of techniques are particularly attractive since they are non selective, require little or no sample pre-treatment; use small amounts of organic solvents or reagents; and the

The food crisis situation seen in last years and the controversy about genetically modified organisms (GMO), with a sharp increase in basic food prices, highlights the extreme susceptibility of the current agricultural and food model and the need for more strict food quality control, which should include determination of the origin of the product and the raw

and qualitative information with drastic instrumental time reduction.

determination of food authenticity (Reid *et al.,* 2006).

**3.3. Molecular markers for the traceability of olive oil** 

analysis takes only a few minutes per sample.

geographical regions.

from other interfering resonances.

31P NMR spectroscopy has been employed for the detection and quantification of the minor compounds mono- and diacylglycerols, polyphenols, sterols, phospholipids and for the determination of free acidity and moisture (Petrakis *et al*., 2008). This analytical technique requires the derivatization of the labile hydrogens of the hydroxyl and carboxyl groups of the olive oil constituents using the phosphorous reagent 2-chloro-4,4,5,5- tetramethyl dioxaphospholane and the use of 31P chemical shifts of the phosphitylated compounds to identify the labile centers. The main phospholipids found in olive oil were phosphatidic acid, lyso-phosphatidic acid and phosphatidylinosotol, although very small amounts of other phospholipids were detected as well. In this way, the content of free fatty acids has been determined showing a good correlation with official titration method. Besides, using the same derivatization method, sn-1,2, sn-1,3-diacylglycerides and monoacylglycerides have been quantified and have showed a characteristic trend during olive oil storage.

The composition of diacylglycerols (sn-1,2- 1,3-diacylglycerols and total) determined by 31P NMR derivatization method has been used to characterize olive oils from different Greek areas (Petrakis *et al*., 2008). Some preliminary correlations between the diacylglycerols content and the geographical regions have been observed.

The geographical characterization of monovarietal virgin olive oils from three regions of Southern Greece, namely Peloponnesus, Crete and Zakynthos, has been performed applying both 1H and 31P NMR (Petrakis *et al*., 2008). The correct geographical prediction at the level of three regions, based on discriminant analysis, has been rather high (87%). Phenolic compounds and free acidity determined by 31P NMR spectroscopy have enabled to classify Koroneiki, Athinolia and Kolovi monovarietal Greek extra virgin olive oils using ANOVA and discriminant analysis.

1H NMR spectrum of an olive oil does not provide this information directly; however, it is possible to have an indirect measurement of acidity using the measurable amount of diglycerides and monoglycerides.

Another study carried out by using 1H NMR involved the characterization of the phenolic fraction of olive oil from three different areas of the Apulia region (Sacco *et al.,* 2000). Statistical evaluation has showed discrimination between Coast, Hinterland and North samples.

Extra virgin olive oils sampled in three harvesting years and coming from different Italian regions (Tuscany, Lazio, Lake of Garda) have been analyzed by 1H methodology. The statistical elaboration applied on the intensity of b-sitosterol, aldehydes and some other volatile compounds has allowed the classification of the olive oils according to the geographical regions.

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

variety (Marini *et al*., 2004, 2006).

and discriminant analysis.

samples.

diglycerides and monoglycerides.

It must be noted that although gas chromatographic methods give the full composition of fatty chains, no information is given about the fatty chains distribution on glycerol. Thus in this case, gas chromatography and 13C NMR methodologies must be considered complementary and not alternative. 13C NMR spectroscopy together with discriminant analysis has been proposed by Mavromoustakos et *al.*, to detect the presence of soybean oil, cottonseed oil, corn oil and sunflower seed oil in virgin olive oils. Only double bonds signals have been used for the analysis. Fatty acids composition and fatty chain positional distribution on glycerol moiety determined by 13C NMR spectroscopy allow, in combination with the multivariate statistical procedure, the classification of olive oils according to their

31P NMR spectroscopy has been employed for the detection and quantification of the minor compounds mono- and diacylglycerols, polyphenols, sterols, phospholipids and for the determination of free acidity and moisture (Petrakis *et al*., 2008). This analytical technique requires the derivatization of the labile hydrogens of the hydroxyl and carboxyl groups of the olive oil constituents using the phosphorous reagent 2-chloro-4,4,5,5- tetramethyl dioxaphospholane and the use of 31P chemical shifts of the phosphitylated compounds to identify the labile centers. The main phospholipids found in olive oil were phosphatidic acid, lyso-phosphatidic acid and phosphatidylinosotol, although very small amounts of other phospholipids were detected as well. In this way, the content of free fatty acids has been determined showing a good correlation with official titration method. Besides, using the same derivatization method, sn-1,2, sn-1,3-diacylglycerides and monoacylglycerides

have been quantified and have showed a characteristic trend during olive oil storage.

content and the geographical regions have been observed.

The composition of diacylglycerols (sn-1,2- 1,3-diacylglycerols and total) determined by 31P NMR derivatization method has been used to characterize olive oils from different Greek areas (Petrakis *et al*., 2008). Some preliminary correlations between the diacylglycerols

The geographical characterization of monovarietal virgin olive oils from three regions of Southern Greece, namely Peloponnesus, Crete and Zakynthos, has been performed applying both 1H and 31P NMR (Petrakis *et al*., 2008). The correct geographical prediction at the level of three regions, based on discriminant analysis, has been rather high (87%). Phenolic compounds and free acidity determined by 31P NMR spectroscopy have enabled to classify Koroneiki, Athinolia and Kolovi monovarietal Greek extra virgin olive oils using ANOVA

1H NMR spectrum of an olive oil does not provide this information directly; however, it is possible to have an indirect measurement of acidity using the measurable amount of

Another study carried out by using 1H NMR involved the characterization of the phenolic fraction of olive oil from three different areas of the Apulia region (Sacco *et al.,* 2000). Statistical evaluation has showed discrimination between Coast, Hinterland and North 1H-NMR fingerprinting of olive oil is a valuable analytical tool for the traceability of virgin olive oils from different points of view, i.e. food authentication and food quality. As described before, 1H and 13C NMR techniques provide different information: 1H NMR spectrum allows the measurement of minor components of olive oils such as β-sitosterol, hexanal, 2-(E)-hexenal, formaldehyde, squalene, cycloartenol and linolenic acid; the 13C NMR spectrum detects major components such as glycerol tri-esters of olive oils, defining also acyl composition and positional distribution on the glycerol moiety. The main sensitivity concern about 1H NMR analysis is the dynamic range, which makes the detection of signals below the threshold imposed by the ADC (analogue to digital converter) impossible. Therefore, in a standard extra virgin oil spectrum, trace component detection is limited to signals more intense than 10−5 times the intensity of the fatty acid CH2 signals. Weak signals above the dynamic range threshold are in any case affected by severe baseline distortion. Selective pulses coupled to gradient spin-echo refocusing such as DPFGSE (69), in NMR modern instruments, allow these limitations to be circumvented, paving the way for the easy detection of minor components, especially those presenting resonances away from other interfering resonances.

The power of these new sequences could be demonstrated by the detection of aldehydes, carotenoids or other specific minor components whose resonances fall in a relatively free region. It was performed a specific DPFGSE analysis on the aldehydic components of some extra virgin olive oils, mostly responsible of the sensorial properties of extra virgin olive oil. Comparison with standard 1HNMR spectra show tremendous improvement of quantitative and qualitative information with drastic instrumental time reduction.

Fingerprinting techniques such as NMR, NIR (Galtier *et a*l., 2007; Mignani *et al*., 2011), MIR (Reid *et al*., 2006), fluorescence (Kunz *et al*., 2011), FT-IR, FT-MIR and FT-Raman (Baeten *et al*., 2005; Lopez-Diez *et al*., 2003; Yang *et al.,* 2005) spectroscopies, have been used for the determination of food authenticity (Reid *et al.,* 2006).

These types of techniques are particularly attractive since they are non selective, require little or no sample pre-treatment; use small amounts of organic solvents or reagents; and the analysis takes only a few minutes per sample.
