**3.1. Chromatographic techniques for the traceability of olive oil**

In the last decade, gas chromatography (GC), high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), and capillary electrophoresis (CE) have been widely used in the authentication analysis of olive oil. The fat, sterols, protein, carbohydrate or other natural compound profiles (Aparicio-Ruiz, 2000; Benincasa *et al*., 2003; Vichi et al., 2005; Lopez Ortiz et al., 2006; Temime et al., 2006; Canabate-Diaz et al., 2007; Cavaliere et al., 2007; Haddada et al., 2007; Vichi et al., 2007; Lazzez et al., 2008) have been used to provide species and geographical differentiation. In several cases, enantiomeric composition has been utilised (Rossmann et al., 2000). These methods of discrimination rely on samples of different species and/or different geographical origin having different chemical compositions. This is not always the case, samples from the same location have been found to contain different components and conversely samples from different regions may display identical chemical composition.

Detectors usually used, in combination with chromatographic techniques (GC and HPLC), may be more or less selective and sensitive, but lack information about the identity of compounds. Therefore, the coupling of chromatographic techniques and mass spectrometry (MS) overcomes this drawback (Cavaliere *et al.,* 2007; Lazzez *et al.,* 2008).

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

using anonymous or less costly cultivars (Sanz-Cortes *et al*., 2003).

olive varieties (Sanz-Cortes *et al*., 2003).

(Giménez *et al*., 2010).

may display identical chemical composition.

cultivars employed to produce an *olive oil* sample may contribute to address the oil origin. This fact may have commercial interest in the case of monovarietal *olive oil*s or *olive oil*s with PDO because these high-quality *olive oil*s may be adulterated by other oils of lower quality,

Unfortunately, morphological traits have been difficult to evaluate, are affected by subjective interpretations, and are severely influenced by the environment and plant developmental stage (Japon-Lujan *et al*., 2006). Nowadays, several efforts have been focused on the investigation of one or several compounds present in *olive oil*s usable to differentiate

Compositional markers (substances that take part of the composition of the olive oils) include major and minor components. Major components such as sterols, phenolic compounds, volatile compounds, pigments, hydrocarbons, tocopherols, fatty acids and triglycerides may provide basic information on olive cultivars. Minor components can provide more useful information and have been more widely used to differentiate the

In recent years, there has been increasing legislation to ensure consumer confidence and to

It is of great importance the development of analitycal methods to verify the correspondence between what is stated on the label and what is contemplated in the documents (EC Regulation 182/2009 and Reg. 834/2007) in relation to the production of olives and extra virgin olive from organic farming. Moreover, to enforce these laws, a measure of the authenticity of samples must be made, most often in the form of proving the presence/absence of adulterants, or verifying geographical or cultivar origin by comparison with known and reliable samples. The latter method often includes the use of multivariate statistical techniques such as principal components analysis, linear discriminant analysis, canonical variance analysis and partial least squares regression to investigate sample data

In the last decade, gas chromatography (GC), high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), and capillary electrophoresis (CE) have been widely used in the authentication analysis of olive oil. The fat, sterols, protein, carbohydrate or other natural compound profiles (Aparicio-Ruiz, 2000; Benincasa *et al*., 2003; Vichi et al., 2005; Lopez Ortiz et al., 2006; Temime et al., 2006; Canabate-Diaz et al., 2007; Cavaliere et al., 2007; Haddada et al., 2007; Vichi et al., 2007; Lazzez et al., 2008) have been used to provide species and geographical differentiation. In several cases, enantiomeric composition has been utilised (Rossmann et al., 2000). These methods of discrimination rely on samples of different species and/or different geographical origin having different chemical compositions. This is not always the case, samples from the same location have been found to contain different components and conversely samples from different regions

botanical origin of *olive oil*s (Howarth and Vlahov, 1996; Lanteri *et al*., 2002).

**3.1. Chromatographic techniques for the traceability of olive oil** 

protect the rights of both the consumer and honest producers.

MS is a sensitive and selective detector, sometimes allowing preparation steps to be avoided. GC–MS is a robust technique, used routinely in many laboratories for food analysis; for example for the determination of aroma compounds and pesticide analysis. More recently, LC coupled to quadrupoles, magnetic sectors or time-of-flight (TOF) detectors, has also had a great expansion into the field of food analysis.

The recently introduced spray methods (ESI) have fostered qualitative and quantitative analysis of medium to high polar analytes by mass spectrometry. The designing of ion source houses for ESI has also fostered the rediscovery of atmospheric pressure ionization (API) methods such APCI where the chemical ionization (CI) is achieved at atmospheric pressure. Both techniques produce soft ionization, but additional fragmentation can be achieved by performing in-source collision induced dissociation (CID) in tandem or trap instruments. MS/MS in space (tandem, sectors, quadrupoles, TOFs, etc.) and in time (traps) provide additional and unique information on the structure of analytes. ESI is useful for polar and ionic solutes ranging in molecular weight from 100 to 150103 dalton. APCI is applicable to non-polar and medium polarity molecules with a molecular weight from 100 to 2000 dalton. Although the choice of the right interface, as well as the detection polarity are based mostly on the compounds polarity and thermal stability, and the HPLC operating conditions, many classes of compounds can give good response with both ionization techniques. In certain circumstances both positive and negative ionization modes are needed, while in most of the cases the choice of only one operation mode is enough*.* The number of applications of HPLC–API-MS to food analysis has rapidly increased in recent years. ESI is much more widespread than APCI, but for both techniques the trend is towards an increase in the number of applications (Sindona et al., 1999; 2000; Di Donna et al., 2001).

### **3.2. Stable isotope techniques for the traceability of olive oil**

Stable isotope techniques enable differentiation of chemically identical substances through alterations in their isotopic fingerprint, and have been used in authenticity studies for many food products. The isotopic composition of light elements (lighter than calcium) in plant material can vary depending on location but, the dominant factor is the influence of latitude on the fractionation of the elements in groundwater.

Fractionation occurs during physical processes such as evaporation. Lighter isotopes evaporate very slightly faster than their heavier counterparts, therefore in warmer regions where the amount of evaporation is higher, the isotopes are fractionated to a greater degree. The discrimination between isotopes in such physical processes is only significant for light elements, with a high relative mass difference between the isotopes. Thus hydrogen ratios, measured by site-specific natural isotope fractionation nuclear magnetic resonance (SNIF-NMR), and carbon, nitrogen, oxygen and sulphur isotope ratios measured by isotope ratio mass spectrometry (IRMS) have been applied to the authentication of foods.

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.

Olive Oil Traceability 271

collector ICP-MS is likely to lead to wider application of heavier stable isotope ratio

One 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

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

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

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

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

measurements to the authentication of food products.

focused on bulk or global stable carbon isotope ratios (13C/12C).

*3.2.2. 13C compound-specific isotope analysis* 

biological processes.

metabolism) pathway.

bulk δ13C values from -9 to –14‰.

Olive oil shows a δ13C values of 28.7‰.

the fatty acids specifically at the 2-position of the triacylglycerol.

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 organism has been exposed.
