**3. Analytical methods for the traceability of extra virgin olive oil**

The increase in the demand for high-quality *olive oil*s has led to the appearance in the market of *olive oil*s elaborated with specific characteristics. They include oils of certain regions possessing well-known characteristics, that is, *olive oil*s with a denomination of origin, or with specific olive variety composition, that is, coupage or monovarietal *olive oil*s. Monovarietal *olive oil*s have certain specific characteristics related to the olive variety from which they are elaborated (Montealegre *et al*., 2010). However, coupage *olive oil*s are obtained from several olive varieties to achieve a special flavor or aroma.

The appearance of denominations and protected indications of origin has promoted the existence of oils labeled according to these criteria. Regulation 2081/92, to promote and protect food products, created the systems known as:

Protected Designation of Origin (PDO);

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

safety in the human and animal food chains.

**2. Traceability in regulation (EC) 178/2002** 

Regulation (CE) 178/2002.

products that are not involved.

identify two inverse processes:

the same system.

same process of transformation.

path.

and at every stage of the journey;

"General Laws for food safety" and was not applied only to food safety but also: to introduce the concept of traceability where food companies (producers, manufacturers and importers) need to guarantee to be able to demonstrate the traceability of every food, animal feed and ingredient, showing the chain from producer to consumer; create the " European Food Safety Authority" (EFSA) to unify the various commities and to make public the scientific process and risks; to strenghthen the ealry warning system adopted by European governments and the European Commission to enable rapid interventions in cases of food

In this chapter, we will only consider the "traceability" as a process that allows us to track "from downstream to upstream" the informations distributed along the olive-oil production chain, from producer to consumer (from farm to fork), and to include a evaluation of analytical methods useful in ascertaining what is claimed on the label, in according to the

Since 1 January 2005, a rule has required that EU countries implement labelling and identification procedures for products sold by farmers, producers and first importers to the EU to enable and facilitate their traceability when they are put on the market. The main purpose is to be able to initiate a withdrawal and/or recall procedure for products in the event of a food crisis. The quality of traceability will enable targeted and precise withdrawals. It will also limit the extent of recalls and ensure the removal of holds on

The law (EC Regulation No. 178/2002) defines traceability as: "The ability to reconstruct and follow a food, feed, a food-producing animal or substance intended to be, or to join a food or feed, through all stages of production, processing and distribution" (Article 3, paragraph 15).

Many use the terms tracking and traceability synonymously. In reality, these two terms

tracking identifies the location of a product from upstream to downstream in the chain

 traceability or tracing is the inverse process, which allow us to gather the information previously issued. It is evident that the two processes are strongly related and based on

Traceability does not refer to the production of a generic good. It makes each unit of production physically identifiable, managing production processes that are determined by "lots", and manage traceability means identifying each group of products and following the

It is necessary to record information relating to inputs (products and companies), processing (product lots, which lots and what end products) and outflows (products/companies /clients). The key is to define the composition of a set of products that have undergone the


An *olive oil* with a PDO denomination requires meeting precise definition of several parameters such as cultivar, geographical origin, agronomic practice, production technology, and organoleptic qualities (Giménez *et al*., 2010), and all of these parameters have to be investigated to study its traceability and to certify its quality. The introduction of certifications of origin and quality for virgin *olive oil* as PDO makes necessary the implementation of traceability procedures.

Any research dealing with *olive oil* traceability is focused on investigating the botanical or geographical origin. In both cases, the selection of the markers (compounds with discriminating power) to be studied is complicated because the composition of extra virgin *olive oil*s is the result of complex interactions among olive variety, environmental conditions, fruit ripening, and oil extraction technology (Araghipour *et al*., 2006). The verification of the

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

Olive Oil Traceability 269

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 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)

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).

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

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

(MS) overcomes this drawback (Cavaliere *et al.,* 2007; Lazzez *et al.,* 2008).

detectors, has also had a great expansion into the field of food analysis.

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

mass spectrometry (IRMS) have been applied to the authentication of foods.

on the fractionation of the elements in groundwater.

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

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

In recent years, there has been increasing legislation to ensure consumer confidence and to protect the rights of both the consumer and honest producers.

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 (Giménez *et al*., 2010).
