*3.2.2. 13C compound-specific isotope analysis*

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

*3.2.1. Inductively coupled plasma mass spectrometry technique* 

organism has been exposed.

Cindric *et al*., 2007).

the soil and in precipitation.

technique is to be applied.

The elemental composition of vegetation reflects (to a certain extent) that of the soil in which it has grown (Kelly *et al*., 2002) which in turn will depend on the topography, geology and soil characteristics. Therefore, no two countries will have identical soil maps, and the concentration of elements in food product, can then be used to assign their geographical origin. As with other methods of authentication, a database of samples of known origin must be available, against which unknown samples can be compared. The most useful elements for the assignment of origin are those that are not homeostatically controlled. Elements such as K, Ca and Zn are actively absorbed by organisms and will therefore be present in samples at similar levels, regardless of the environmental conditions experienced. Elements that have no role in normal physiological processes, such as the rare earth elements (REEs) and the heavy metals, are passively absorbed and the concentration of these elements in an organism will strongly reflect the environmental levels to which the

Several studies have used multi-element concentration profiles in the determination of food authenticity, either alone or in combination with chromatographic or stable isotope ratio data. Elemental concentrations were determined by atomic absorption spectroscopy (AAS), inductively coupled plasma - atomic emission spectroscopy (ICP-AES) or inductively coupled plasma – mass spectrometry (ICP-MS) (Dugo *et al.,* 2004; Benincasa *et al.,* 2007;

Several trace elements have variable natural isotopic abundance due to the decay of radioactive isotopes. These include Li, B, Cu, Sr, Nd, Hf, Pb and U. The composition and age of the local rocks dictates the abundance of the radioactive precursors and their daughter species. Elements are taken up into plants in the same isotopic proportions as they occur in

Therefore isotope ratios in plant-derived food products depend on the geology of the region in which the source crop was grown, and are different in produce of different geographical origin. There have, however, been relatively few reports of the use of heavier stable isotope ratio measurements for the authentication of foodstuffs. For many years thermal ionization mass spectrometry (TIMS) was the only technique capable of performing isotope ratio measurements with sufficient precision to allow geographical assignment of food products based on trace element isotopic composition. Samples for TIMS analysis must be loaded in the form of the pure element, meaning that extensive sample preparation is required if this

ICP-MS is now a well established technique for isotopic trace element determinations. ICP-MS allows rapid analysis of a large range of sample types, requires minimal sample preparation and due to the ionising power of the ICP, can be applied to a wider range of elements than TIMS. The precision of isotope ratio analyses by ICP-MS has only recently matched that achievable by TIMS, through the application of double focusing mass analysers coupled to multi-collector detection arrays. The increasing availability of multiOne of the most powerful techniques to be used in food authenticity studies is stable isotope ratio mass spectrometry (SIRMS). SIRMS has found application in the authentication of a wide range of foodstuffs, especially for the detection of added cane sugar in fruit juices (Bricout and Koziet, 1987), wines (Dunbar and Schmidt, 1984), spirits (Parker *et al*., 1998), honey (White *et al*., 1998) and to detect the adulteration of flavour compounds with synthetic analogues (Culp and Noakes, 1990). The majority of the reported research has focused on bulk or global stable carbon isotope ratios (13C/12C).

Approximately 98.89% of all carbon in nature is 12C and 1.1% is 13C. The amount of 13C present in a sample is expressed as a ratio to the amount of 12C present, relative to an international standard using the δ notation. This notation has the units per mil (‰). The δ13C value of plants varies because of isotopic fractionation during physical, chemical and biological processes.

Photosynthetic fixation of CO2 by plants takes place by three different routes, depending on the nature of the plant. Most terrestrial plants photosynthesise using the Calvin or C3 pathway in which CO2 is fixed via the carbon cycle to from a three-carbon compound. Some plants, mainly tropical grasses, such as maize and sugar cane use the Hatch-Slack or C4 pathway in which the initial "Hatch-Slack" step is via a dicarboxylic acid, a four carbon compound. A third photosynthetic class of plants uses the CAM (Crassulacean acid metabolism) pathway.

Typical CAM plants are succulents and are of minor importance in the oleochemical industry. In the C3 pathway carbon dioxide is fixed via the carboxylation of ribulose-1,5 diphosphate to form phosphoglyceric acid (a 3-carbon molecule). This enzyme catalysed reaction discriminates against 13C and so proceeds with a relatively large isotope effect (O'Leary, 1981). This means that less 13CO2 is incorporated into C3 plants than into C4 plants. C3 plants have bulk δ13C values in the range -24 to -30‰ whereas C4 plants have bulk δ13C values from -9 to –14‰.

Olive oil shows a δ13C values of 28.7‰.

The most widely used technique to assess the authenticity of edible oils is to measure the fatty acid composition. However it is not always possible to detect adulteration by this technique because of the natural variation in fatty acid profiles and because blends of oils with fatty acid compositions similar to the authentic oil may be prepared relatively easily. A more sophisticated approach is to determine the fatty acid composition at the 2 position of the triacylglycerol, since this is known to differ from the overall fatty acid composition. Woodbury *et al.*, (1998) used this technique to obtain the compound-specific δ13C values of the fatty acids specifically at the 2-position of the triacylglycerol.

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

Olive Oil Traceability 273

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

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

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

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

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

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.

Ratio Mass Spectrometry (Py-CF-IRMS).

geographical fingerprints (Roßmann *et al.,* 2000).

neighbouring countries with similar climates.

(e.g. 88Sr/86Sr) and multi-element analysis.

*3.2.4. Nuclear magnetic resonance spectroscopy* 

discrimination (Frankel, 2010; Guillen & Ruiz, 2001).

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; Harwood and Sánchez, 1999).

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 data derived from vegetable oils.
