Food Research International xxx (2013) xxx–xxx

FRIN-04645; No of Pages 6

Contents lists available at SciVerse ScienceDirect

Food Research International

j ourna l homepage: www.e lsev ie r .com/ locate / foodres

Putative markers of adulteration of extra virgin olive oil with refined olive oil:Prospects and limitations

Raquel Garcia ⁎, Nuno Martins, Maria João CabritaICAAM — Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7002-554 Évora, Portugal

⁎ Corresponding author. Tel.: +351 266 760 869; faxE-mail addresses: raquelg@uevora.pt, rmartagarcia@

0963-9969/$ – see front matter © 2013 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.foodres.2013.05.008

Please cite this article as: Garcia, R., et al., PutFood Research International (2013), http://dx

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 January 2013Received in revised form 8 April 2013Accepted 4 May 2013Available online xxxx

Keywords:Olive oilAdulterationsRefined olive oilExtra virgin olive oil

Authentication is becoming an issue of increasing relevance in olive oil and is generally motivated byeconomic benefits. Blending of extra virgin olive oil (EVOO) with refined olive oil (ROO) constitutes one ofthe most common types of adulteration of this top grade product. Concerning this particular topic, themost recent attempts of several research groups on the finding of some target compounds as markers of adul-teration of EVOO with ROO will be reviewed. All efforts developed until now to find markers of adulterationin blends of EVOO with ROO have contributed to increase the knowledge about this topic although it ismandatory to exploit new compounds that could be assigned as reliable adulteration markers able to detectwith high selectivity, sensitivity and accuracy this fraudulent practice.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Nowadays, olive oil authentication has emerged as an issue ofthe most relevance world-wide (Angerosa, Campestre, & Giansante,2006; Frankel, 2010). In the last years, much attention has beengiven into fraudulent practices associated with olive oil traceabilityfocused with special emphasis on the botanical origin due to therecent introduction in the market of high-quality monovarietal oliveoil. Aiming to find traceability markers several studies have beenperformed allowing the discrimination of compositional and geneti-cal markers (Montealegre, Alegre, & García-Ruiz, 2010). The guaran-tee of olive oil geographical origin is another matter of concern forthe olive oil industry and recently some researchers have proposednew methodologies based on Near Infrared (NIR) Spectroscopy(Woodcock, Downey, & O'Donnell, 2008), Nuclear Magnetic Reso-nance (NMR) Spectroscopy (Agiomyrgianaki, Petrakis, & Dais, 2012;Mannina & Sobolev, 2011) and Synchronous Fluorescence Spectros-copy (Kunz, Ottaway, Kalivas, Georgiou, & Mousdis, 2011). Sinceextra virgin olive oil (EVOO) is considered the top grade of olive oil,it could be more susceptible of economic fraud, being the mostcommon practice the addition of seed oils, such as sunflower, soybeanand hazelnut oil. Recent work by Sánchez-Hernández, Marina, andCrego (2011) studied the role of non-protein amino acids as novelmarkers for the detection of EVOO adulterated with seed oils usinga new analytical methodology based on capillary electrophoresis–mass spectrometry enabling the identification and quantificationof ornithine and alloisoleucine in EVOO adulterated with soybean

: +351 266 760 828.yahoo.com (R. Garcia).

rights reserved.

ativemarkers of adulteration o.doi.org/10.1016/j.foodres.20

oil. This methodology seems to be promising allowing the qualityevaluation of EVOO as well as its authentication. Additionally, Chenet al. (2011) have performed preliminary screening studies relatedwith the usefulness of δ-tocopherol as marker of EVOO/sunflower,EVOO/hazelnut and EVOO/peanut blends. More recently, studies de-veloped by Calvano, De Ceglie, D'Accolti, and Zambonin (2012) haveproposed phospholipids as eventual markers of EVOO adulterationwith hazelnut oil based on a methodology comprising a previousselective extraction and enrichment of phospholipids from EVOOand hazelnut oil (HO) followed by the analyses using matrix- assistedlaser desorption time of flight mass spectrometry (MALDI-TOF-MS).The results seem to indicate that this methodology allows the detec-tion of low percentages of hazelnut oil in blends of EVOO and HO.

Another possible fraudulent economic practice is the forbidden ad-dition of cheaper olive oil, namely refined olive oil (ROO) to EVOO.The detection of adulterations requires the use of analytical methodol-ogies which can easily, rapidly and accurately detect those fraudulentpractices. Recently, Torrecilla, García, García, and Rodríguez (2011)have proposed a methodology based on thermophysical properties toquantify adulterations of EVOO with ROO at relatively low concentra-tions allowing also the quantification of impurities during olive oilproduction to control on-line the quality of EVOO before bottling.These studies were also extended to quantify adulterations of EVOOwith refined olive pomace, sunflower or corn oils. Indubitably, thedetection of those illegal practices is crucial either for consumer's healthandwealth protection (García-González& Aparicio, 2010), aswell as forquality assurance. European Union and global food policies continuallydemand even stricter monitoring and control of food quality. Sinceadulterations have become more sophisticated, new methodologieswill be required with suitable sensitivity, selectivity and accuracy todetect those fraudulent practices.

f extra virgin olive oil with refined olive oil: Prospects and limitations,13.05.008

2 R. Garcia et al. / Food Research International xxx (2013) xxx–xxx

In the last years, in order to assure the authenticity of EVOO several re-search groups have been focused on the development of analytical meth-odologies comprising mainly the use of chromatographic techniques,namelyGas Chromatography (GC) andHigh Performance Liquid Chroma-tography (HPLC) and more recently on spectroscopic techniques, such asNuclear Magnetic Resonance (NMR) (Dais & Hatzakis, 2013; Fragaki,Spyros, Siragakis, Salivaras, & Dais, 2005; Ogrinc, Košir, Spangenberg, &Kidrič, 2003). Some approaches relying with electronic nose (Oliveroset al., 2002), near-infrared spectroscopy (Christy, Kasemsumran, Du, &Ozaki, 2004), fluorescence spectroscopy (Sikorska, Khmelinskii, &Sikorski, 2011), differential scanning calorimetry (Chiavaro et al., 2008),weak chemiluminescence (Papadopoulos, Triantis, Tzikis, Nikokavoura,& Dimotikali, 2002), Raman Spectroscopy (Dong, Zhang, Zhang, &Wang, 2012; Zou et al., 2009) and gas chromatograph–ionmobility spec-trometer with an ultraviolet source (Garrido-Delgado, Arce, & Valcárcel,2012; Garrido-Delgado, Mercader-Trejo, Arce, & Valcárcel, 2011;Garrido-Delgado et al., 2011) have been also introduced as analyticalmethodologies to discriminate olive oil commercial categories.

Generally, detection of olive oil adulterations with other vegetableoils has been focused on the major constituents of olive oil, namelyfatty acids (FA) and triacylglycerols (TAG) (Angerosa et al., 2006;Frankel, 2010). Since authentication of EVOO is of mandatory rele-vance many methodologies based on the identification of eventualmarkers have been explored. Triacylglycerols have been explored asa tool in the authentication and genuineness of EVOO and the resultsproved that those compounds have potential for the detectionof changes in the EVOO composition due to forbidden blending(Bosque-Sendra, Cuadros-Rodríguez, Ruiz-Samblás, & de la Mata,2012). More recently, Lukic, Lukic, Krapac, Sladonja, and Pilizota(2013) have proved that sterols and triterpene diols can be used asreliable indicators of variety and ripening degree among olive oils.Since the detection of EVOO adulteration based on fatty acid compo-sition analysis combined with traditional current analytical tech-niques constitute a very challenging task, there is a growing intereston exploring an alternative methodology based on the applicationof DNA-based detection methods (Agrimonti, Vietina, Pafundo, &Marmiroli, 2011) in order to assess the role of DNA as a tool to detectadulterations (Costa, Mafra, & Oliveira, 2012; He et al., 2013; Kumar,Kahlon, & Chaudhary, 2011; Zhang et al., 2012).

Particularly, to detect bleeding of EVOO with ROO several at-tempts have been proposed mainly based on some target compoundswhich belong to olive oil's bioactive compounds, namely chlorophylls,diacylglycerols (DGs), ester derivatives of FA, straight chain waxesters, sterol components and TAGs. Due to sample complexity,the detection of olive oil adulteration constitutes a really difficultand challenging analytical hindrance (Maggio, Cerretani, Chiavaro,Kaufman, & Bendini, 2010). Moreover, blending of EVOO with ROOdoes not produce an easily detectable modification on the chemicalcomposition of the final blend due to the mild conditions used inthe deodorization process and, consequently, the identification ofeventual adulteration is a demanding analytical task (Saba, Mazzini,Raffaelli, Mattei, & Salvadori, 2005). The finding of chemical com-pounds which could be assigned as adulteration markers in EVOOand ROO blends is considered of crucial significance and indubita-bly will contribute to detect more accurately those fraudulentpractices.

Aiming to contribute to the current knowledge about the usefulnessof some target compounds as potential candidates of adulterationmarkers of EVOO and ROO blends, the most significant attempts intro-duced in the last years on the detection of this kind of adulterationwill be focused in this paper.

2. Olive oil commercial categories

Olive oil has been reported as a primary consumption product andan essential component of healthy diet for majority of people living in

Please cite this article as: Garcia, R., et al., Putativemarkers of adulterationFood Research International (2013), http://dx.doi.org/10.1016/j.foodres.20

Mediterranean countries due to its rich nutritional value, namely highmonounsaturated fatty-acid and antioxidant properties (Bendini etal., 2007; Frankel, 2011; Visioli, Bogani, & Galli, 2006).

According to the European Union legislation (EC. Off. J. Eur.Communities, 2003), olive oil is classified into some categoriesreflecting its quality and organoleptic properties, namely extra virginolive oil (EVOO), virgin olive oil (VOO), lampante virgin olive oil(LVOO) and also refined olive oil (ROO) among others. In particular,EVOO is considered to be the oil of the highest quality since it isobtained from olive fruits solely by mechanical or other physicalmeans that do not lead to alterations of the oil, and also has not un-dergone treatment other than washing, decantation, centrifugationand filtration. Its free acidity expressed as oleic acid must be b0.8%and other characteristics are fixed for this category by the Interna-tional Olive Oil Council (IOOC) and European Community (EC) Regu-lations (IOOC, 2011; Official Journal of European Community, 2003).

The high cost of EVOO makes it prone to adulteration with oliveoils of lower categories in order to increase economic benefits.However, this practice deteriorates its quality and nutritional valueleading to major economic losses for the consumers and the loss ofconsumer confidence can also arise (Fragaki et al., 2005; Gurdeniz &Ozen, 2009; Mignani et al., 2011).

One of the most common adulteration practices consists of blend-ing EVOO with ROO (Fragaki et al., 2005; Frankel, 2010) which isobtained usually from virgin olive oil mechanically extracted fromdamaged olive fruits or from olives stored in unsuitable conditionsand using refining methods that does not lead to alterations in the ini-tial glyceridic structure. It has a free acidity, expressed as oleic acid, ofnot more than 0.3 g per 100 g (IOOC, 2011).

Particularly, the refining process can be accomplished in twoways: alkali or physical refining. In the alkali refining, the oil is treat-ed with dilute acid (the two most common degumming agents arecitric and phosphoric acid) to precipitate the phosphatides and pro-teinaceousmaterial, which are separated by settling or centrifugation.After degumming, the oil is neutralized either in a continuous orbatched system. Next, the oil obtained is bleached under vacuumwith mixtures of various adsorbents and filtered by filter pressesequipped with a solvent system for recovering oil which is thenentrained in the bleaching earth, followed by deodorization of thebleached oil. Finally, the refined oil is mixed with virgin oil in orderto improve the organoleptic attributes as well as the chemical proper-ties of the final oil. In the process of physical refining, the oil is initiallydegummed and bleached and after that, in order to remove the vola-tiles and free fatty acids (92–95%) the oil is deodorized (continuousdistillation). Usually, deodorization is finished before removal of allof the free fatty acids, and the oil is alkali refined to remove theremainder of the free fatty acids. The advantage of this procedure isthe improvement of the oil stability by hampering the formation ofoxidation products (Firestone, 2005; Petrakis, 2006).

In the current context, it is crucial to ensure EVOO authenticity inorder to guarantee the safety and consumer protection and to avoidthe image of a hypothetical uncontrolled distribution of adulteratedolive oil into the market.

3. Putative markers of EVOO adulterations with ROO

During the last years, several researchers have been devoted to thestudy of reasonable marker candidates of adulteration of EVOO withROO. It should be taken into account that most of those compoundsbelong to the major constituents of olive oil and are chosen basedon some quality indicators, such as TAG composition and sterols.More recently, new approaches have been explored based on minorcomponents of olive oil. In this section will be discussed in detailthe more significant compounds belonging to those class whichwere proposed as putative markers of EVOO adulteration with ROO.

of extra virgin olive oil with refined olive oil: Prospects and limitations,13.05.008

3R. Garcia et al. / Food Research International xxx (2013) xxx–xxx

3.1. Natural chlorophyll

Natural chlorophylls comprising chlorophyll A and B are thepigments responsible for the characteristic green color of the olivedrupe. During the ripening of the olives some chemical and physicalchanges occur leading to changes in the chlorophyll fraction profilewhich affects the color of the olives as well as the olive oil extractedfrom them (Giuliani, Cerretani, & Cichelli, 2011). Those compoundsare irreversibly converted into more stable pigments called aspheophytins, in which the central Mg2+ ion of the porphyrin ring isreplaced by two hydrogen atoms. Pyropheophytins are another classof natural chlorophylls which are also formed resulting from theremoval of the carboxy-methyl group from the pheophytin structure(Giuliani et al., 2011). Chemical structures of pheophytin A andpyropheophytin A are depicted in Fig. 1.

Earlier studies of Serani and Piacenti (1992) were related with thestudy of pheophytin A photodecomposition in EVOO aiming to assessthe influence of temperature and light intensity on the kineticconstant of this reaction. More recently, Gertz and Fiebig (2006)have developed a procedure to determine the degradation productsof chlorophyll A, namely pheophytin A and pyropheophytin A, inolive oil. However, since pyropheophytin A content increases alongEVOO shelf life only scarcely this compound can be considered amarker of adulteration.

3.2. Diacylglycerols

Diacylglycerols (DGs) are present in EVOO in two isomeric forms1,2- and 1,3-isomers, as depicted in Fig. 2, ranging from 1 to 3%(Mannina & Sobolev, 2011).

While the occurrence of 1,2 isomers of DG (1,2-DG) is attributedto the incomplete biosynthesis of TAGs, the presence of 1,3 isomers

NNH

HCH3

CH3

CH3O

NH

CH2 CH3

N

CH3H

O

O

CH3

O

O

CH3

HH

Pheophytin A

NHN

NNH

H

H

OO

O

Pyro

Fig. 1. Natural chlorophy

Please cite this article as: Garcia, R., et al., Putativemarkers of adulteration oFood Research International (2013), http://dx.doi.org/10.1016/j.foodres.20

of DG (1,3-DG) is ascribed to enzymatic or chemical hydrolysis ofTAGs which are formed before or during olive oil extraction process(Pérez-Camino, Moreda, & Cert, 2001). Then, olive oils obtainedwith poor-quality olive fruits show a significant raise of 1,3-DGwhile olive oils originating from healthy olive fruits contain almostexclusively 1,2-DG. Mild refining process of virgin oil which involvessome steps, such as neutralization, washing and deodorization of theoil at low temperatures under vacuum leads to a decrease of totalcontents of DGs and subsequent raise of 1,3-DG/1,2-DG ratio(Pérez-Camino et al., 2001). During the storage, 1,2-DG is graduallytransformed into 1,3-DG and hence 1,3-DG/1,2-DG ratio has beenconsidered as an valuable marker of olive oil's freshness and quality(Mannina & Sobolev, 2011).

For the past 20 years, the role of DGs as possible markers ofadulteration has been investigated and within this purpose severalapproaches have been explored.

One of the first studies developed by Pérez-Camino et al. (2001),aimed to assess the evolution of 1,3- and 1,2-DG isomers in oliveoils obtained from healthy olives during storage and to detectdeodorized oils in virgin olive oils (VOO) using an analytical proce-dure comprising a solid phase extraction (SPE) followed by GC analy-sis on polar columns. This approach proved to have scarce utility onthe detection of blends of mild refined oils in VOO due to the lowerDG contents. However, particularly for VOO with low acidities theusefulness of 1,3-DG/1,2-DG ratio to evaluate their genuineness andthe storage conditions as well as on the determination of VOO aginghas been claimed by the authors.

Furthermore, Mannina, Sobolev, and Segre (2003) have applied 1HNMR spectroscopy to evaluate 1,2-DG and 1,3-DG contents in EVOOand ROO. Results obtained in this study also corroborated the hypoth-esis that 1,2-DGs usually present in olive oils are transformed pro-gressively during the storage into 1,3-DGs. Thus, these compounds

CH3

CH3

CH3

CH3

O

pheophytin A

ll present in EVOO.

f extra virgin olive oil with refined olive oil: Prospects and limitations,13.05.008

CH2OOCR

CH2OH

HR'COO

CH2OOCR

CH2OOCR'

HOHC

sn-1,2- diacylglycerol

C

sn-1,3- diacylglycerol

Fig. 2. Diacylglycerol isomeric forms in olive oil.

H

HH

HH

OH OH

Stigmasta 3,5- diene

β-Sitosterol Δ5- Avenasterol

Fig. 3. Some sterols present in VOO.

4 R. Garcia et al. / Food Research International xxx (2013) xxx–xxx

could be considered as a good indicator of freshness and quality of anolive oil (Mannina et al., 2003), but seem to be not suitable as markersof EVOO adulteration with ROO.

Recently, studies performed by Fragaki and co-workers haveallowed the determination in a single experiment of 1,2-DG, 1,3-DGand total DGs' (TDGs) contents and the ratios of 1,2-DGs over TDGsusing 31P NMR spectroscopic experiments on different grades of oliveoil, namely EVOO, ROO and blended olive oil composed of ROO andEVOO (BOO) (Fragaki et al., 2005). To achieve this purpose an analyticalmethodology has been employed comprising a derivatization stepinvolving the replacement of the labile hydrogens of hydroxyl andcarboxyl groups with 2-chloro-4,4,5,5-tetramethyl dioxaphospholaneleading to the production of the correspondent phosphitylated com-pounds, followed by the subsequent 31P NMR analysis (Spyros & Dais,2000; Vigli, Philippidis, Spyros, & Dais, 2003). The results have shownthe usefulness of this methodology to detect olive oil adulterationapplying a 31P NMR method and a multivariate statistical analysisallowing the estimation of BOO sample composition. According tothese results, EVOO samples possess 1,3-DG contents much lowerthan the corresponding values in the ROO samples which corroboratethe fact that isomerization of 1,2-DGs to 1,3-DGs, which frequentlyoccurs during prolonged olive oil storage, also happens through oliveoil refinement (Fragaki et al., 2005). This finding also corroboratesthat the use of diacylglycerols isomers as markers of adulterationsmust be done carefully since it represents some limitations.

3.3. Fatty acids and wax esters

Olive oil includes on its composition a class of natural neutrallipids called fatty acid alkyl esters (FAAEs) which are formed by ester-ification of free fatty acids (FA). These compounds were detectedfirstly by Mariani and Fedeli (1986) which isolated the “nonpolarfraction” of olive oil by means of column liquid chromatography andanalyzed it by GC. Formation of FAAEs is promoted by unsuitablepractices related with olive oil extraction and scarce quality of olivefruits (Pérez-Camino, Moreda, Mateos, & Cert, 2002).

Recent studies developed by Pérez-Camino and co-workers aredevoted to the evaluation of soft deodorization on the concentrationof FAAEs and the potential utility of these compounds as adulterationmarkers on the detection of blends of mildly ROO with EVOO(Pérez-Camino, Cert, Romero-Segura, Cert-Trujillo, & Moreda, 2008).Within this purpose, the authors have quantified FAAE contentsusing a previous sample preparation method based on the isolationof those compounds with a silica gel solid phase extraction cartridgefollowed by GC analysis. The study has been applied to around onehundred of Spanish olive oils of several categories, varieties andgeographical origin allowing differentiating of EVOO from other cate-gories of olive oils that has been subjected to a mild refining process.The results shown that blending process of EVOO with refined lowquality olive oils can be assessed by the measurement of their alkylester concentrations (Pérez-Camino et al., 2008).

The profiles of fatty acids and fatty alcohol esters are related witholive oil categories thus straight chain wax esters were indicated asuseful quality and purity indicators of olive oils. Hence, the distinctionof VOO from ROO based on wax esters could be achieved since VOO

Please cite this article as: Garcia, R., et al., Putativemarkers of adulterationFood Research International (2013), http://dx.doi.org/10.1016/j.foodres.20

posses a higher content of C36 and C38 waxes than of C40, C42, C44 andC46 while ROO exhibits an inverse relation (Aparicio & Aparicio-Ruíz,2000).

Since straight chain wax esters were indicated as potentiallyuseful quality/purity markers in olive oils some attention weregiven to those compounds in order to evaluate their relevance onthe detection of EVOO and ROO blends (Biedermann, Hase-Aschoff,& Grob, 2008). Those compounds are located in the waxy surfacelayer of the olive but are poorly extracted from the olive fruit byphysical pressing operation. According to the literature, olive oils oflow quality possess these wax esters at higher concentrations.Biedermann and co-workers have optimized an LC–GC–FID methodto measure methyl and ethyl oleate contents as well as wax esters'fraction of the C26 and C28 alcohol with unsaturated C18 fatty acidsin order to clarify their significance for the evaluation of olive oil qual-ity (Biedermann et al., 2008). Results have shown that the presence ofhigh wax ester content is a consequence of degrading olives and theirformation also occurs during the storage of olive oils. Therefore,assignment of straight chain wax esters as quality markers is morecontroversial since those compounds are continuously formed duringstorage constituting a limitation for their use as adulteration markers(Aparicio & Aparicio-Ruíz, 2000; Biedermann et al., 2008).

3.4. Sterols

Sterols are present on the unsaponifiable matter of almost all fatsand oils. The analysis of the unsaponifiable fraction is being consideredas a powerful tool to detect adulteration allowing the differentiationbetween olive oils of different quality (Lerma-Garcia, 2012). Thesecompounds have been ascribed the genuineness of some vegetableoils because this fraction is more characteristic than the fatty acidprofile, which explains why this family of compounds has been widelyused in olive oil authentication (Martínez-Vidal, Garrido-Frenich,Escobar-García, & Romero-González, 2007). Further investigations onvariations of the ratio of free to total sterols in different categories ofolive oil and the usefulness of this ratio as a parameter for olive oil qual-ity have been evaluated by Pasqualone and Catalano (2000). In particu-lar, VOO possesses high levels ofβ-sitosterol andΔ5-avenasterol (Fig. 3)constituting a potential differentiation from other oils (Aparicio &Aparicio-Ruíz, 2000). In general, refining process leads to a decreasein the total content of sterols and increase esterified sterols in theoil (Jiménez de Blas & dell Valle González, 1996; Phillips, Ruggio,Toivo, Swank, & Simpkins, 2002). Concretely, during refining processof edible oils and fats a dehydration of β-sitosterol occurs yielding

of extra virgin olive oil with refined olive oil: Prospects and limitations,13.05.008

5R. Garcia et al. / Food Research International xxx (2013) xxx–xxx

stigmasta-3,5-diene (Fig. 3) (León-Camacho, Serrano, & Constante,2004). Experimentally, its quantification is performed by means ofan analytical methodology comprising the previous purification ofstigmasta-3,5-diene by preparative column chromatography followedby its analysis using gas chromatography. However, this procedure isconsidered a time-consuming and laborious work.

Kinetic studies performed by León-Camacho et al. (2004) using dif-ferent independent variables namely time, temperature, flow of strip-ping gas and the thickness of oil layer have allowed the measurementof apparent thermodynamic parameters related with the formationof stigmasta-3,5-diene through the deodorization refining process.According to these studies, the formation of stigmasta-3,5-diene fromβ-sitosterol and therefore stigmasta-3,5-diene contents are stronglydependent of the temperature used during the physical refining(León-Camacho et al., 2004). Then, stigmasta-3,5-diene has beenconsidered potentially remarkable on the detection of ROO in VOO(León-Camacho et al., 2004). However, stigmastadienes could be onlyseen as reliable indicators of olive oil adulteration when their concen-trations ranged between 0.01 and 4 mg/kg (Al-Ismail, Alsaed, Ahmad,& Al-Dabbas, 2010) limiting their use on the detection of ROO andolive oil blends.

More recently, Martínez-Vidal et al. (2007) have introduced ananalytical methodology based on liquid chromatography coupled tomass spectrometry (LC–MS) with atmospheric-pressure chemicalionization in positive ion mode which enables the determination ofthe sterol composition of different types of olive oils, namely EVOOand ROO. This methodology seems to be most advantageous since itallows the reduction of sample handling and analysis time. Accordingto these results, sterol contents can be used to distinguish betweendifferent types of oil, in particular authentic olive oil and seed oils(Martínez-Vidal et al., 2007).

3.5. TAG oligopolymers

Recently, studies of Caponio and co-workers are committed to thediscrimination between VOO and ROO based on the quantificationof some polar compounds (PC), namely triacylglycerol oligopolymers(TAGP), oxidized triacylglycerols (ox-TAG) and diacylglycerols (DAG)by means of High Performance Size-Exclusion Chromatography(HPSEC) involving a previous step comprising a purification of oliveoil samples by silica gel column chromatography. In particular,TAGP is produced during deodorization in the refining process dueto the higher temperatures achieved, being considered as a relevantindicator of the secondary oxidative degradation of olive oil sincetheir presence is independent of processing conditions. The othercompounds — ox-TAG and DAG, are linked with primary oxidationlevel of the olive oil and the hydrolytic degradation of ROO, respec-tively (Caponio et al., 2011). Ox-TAG could undertake polymerizationreactions promoting the formation of TAGP. It's assumed that TAGPwere absent or present at very low concentrations in EVOO althoughpresent in relatively high amounts in ROO and the same trend is ob-served for ox-TAG. Therefore, especially TAGP could be a valuablemark-er to discriminate olive oils complemented with HPSEC (Caponio et al.,2011). Since the formation of TAGP could be ascribe to the bleachingand especially deodorization steps during the refining process, thiscompound displays a potential role on the discrimination amongEVOO and ROO. However, the analytical methodology implementedby Caponio et al. (2011) has not been applied to more complex experi-mental designs including several blends of oils thus the usefulness ofthat methodology for the detection of EVOO adulterated with ROOrequires more detailed studies.

4. Conclusions

Adulteration of olive oil is an issue of crucial significance becauseof its impact in quality, nutritional value and safety of consumers.

Please cite this article as: Garcia, R., et al., Putativemarkers of adulteration oFood Research International (2013), http://dx.doi.org/10.1016/j.foodres.20

Due to the inherent EVOO high-cost, the adulteration of this kind ofproduct with low-quality olive oils seems to be actually one of themost common types of fraud. The development of analytical method-ologies which enable detection of adulterations is warranted since theaddition of ROO to EVOO at low percentages could be a very challeng-ing task.

In spite of all attempts developed until now, it seems to be rela-tively consensual that none of the compounds described above canbe considered as a reliable adulteration marker of EVOO and ROOblends, since their appearance is not exclusively related with therefining process used on the ROO production. Thus, the assignmentof some components intimately ascribed to the refining step couldbe the key to overcome the limitations of the methodologies pro-posed until now to detect this kind of fraudulent practice.

Nevertheless, the discovery of putative markers that could identifypossible adulteration of EVOO with ROO could be considered ofmandatory importance and will open new avenues in the field of“Food Authenticity”. Briefly, more efforts are needed to exploit newcompounds that could be assigned as reliable adulteration markersable to detect with high selectivity, sensitivity and accuracy blendsof EVOO with ROO.

Acknowledgments

This work is funded by FEDER funds through the OperationalProgramme for Competitiveness Factors — COMPETE and nationalfunds through FCT — Foundation for Science and Technology underthe Strategic Project PEst-C/AGR/UI0115/2011.

References

Agiomyrgianaki, A., Petrakis, P. V., & Dais, P. (2012). Influence of harvest year, cultivarand geographical origin on Greek extra virgin olive oils composition: A study byNMR spectroscopy and biometric analysis. Food Chemistry, 135, 2561–2568.

Agrimonti, C., Vietina, M., Pafundo, S., & Marmiroli, N. (2011). The use of food genomicsto ensure the traceability of olive oil. Trends in Food Science and Technology, 22,237–244.

Al-Ismail, K. M., Alsaed, A. K., Ahmad, R., & Al-Dabbas, M. (2010). Detection of olive oiladulteration with some plant oils by GLC analysis of sterols using polar column.Food Chemistry, 121, 1255–1259.

Angerosa, F., Campestre, C., & Giansante, L. (2006). Analysis and authentication. In D.Boskou (Ed.), Olive oil: Chemistry and technology (pp. 113–172). American OilChemists' Society Press.

Aparicio, R., & Aparicio-Ruíz, R. (2000). Authentication of vegetable oils by chromato-graphic techniques. Journal of Chromatography. A, 881, 93–104.

Bendini, A., Cerretani, L., Carrasco-Pancorbo, A., Gómez-Caravaca, A. M., Segura-Carretero,A., Fernández-Gutiérrez, A., et al. (2007). Phenolic molecules in virgin olive oils: Asurvey of their sensory properties, health effects, antioxidant activity and analyticalmethods. An overview of the last decade. Molecules, 12, 1679–1719.

Biedermann, M., Hase-Aschoff, P., & Grob, K. (2008). Wax ester fraction of edible oils:Analysis by on-line LC–GC–MS and GC × GC-FID. European Journal of Lipid Scienceand Technology, 110, 1084–1094.

Bosque-Sendra, J. M., Cuadros-Rodríguez, L., Ruiz-Samblás, C., & de la Mata, A. P.(2012). Combining chromatography and chemometrics for the characterizationand authentication of fats and oils from triacylglycerol compositional data — Areview. Analytica Chimica Acta, 724, 1–11.

Calvano, C. D., De Ceglie, C., D'Accolti, L., & Zambonin, C. G. (2012). MALDI-TOF massspectrometry detection of extra-virgin olive oil adulteration with hazelnut oil byanalysis of phospholipids using an ionic liquid as matrix and extraction solvent.Food Chemistry, 134, 1192–1198.

Caponio, F., Summo, C., Bilancia, M. T., Paradiso, V. M., Sikorska, E., & Gomes, T. (2011).High performance size-exclusion chromatography analysis of polar compoundsapplied to refined, mild deodorized, extra virgin olive oils and their blends: An ap-proach to their differentiation. LWT — Food Science and Technology, 44, 1726–1730.

Chen, H., Angiuli, M., Ferrari, C., Tombari, E., Salvetti, G., & Bramanti, E. (2011). Tocoph-erol speciation as first screening for the assessment of extra virgin olive oil qualityby reversed-phase high-performance liquid chromatography/fluorescence detec-tor. Food Chemistry, 125, 1423–1429.

Chiavaro, E., Rodriguez-Estrada, M. T., Barnaba, C., Vittadini, E., Cerretani, L., & Bendini,A. (2008). Differential scanning calorimetry: A potential tool for discrimination ofolive oil commercial categories. Analytica Chimica Acta, 625, 215–226.

Christy, A. A., Kasemsumran, S., Du, Y., & Ozaki, Y. (2004). The detection and quantifi-cation of adulteration in olive oil by near-infrared spectroscopy and chemometrics.Analytical Sciences, 20, 935–940.

Costa, J., Mafra, I., & Oliveira, M. B. P. P. (2012). Advances in vegetable oil authenticationby DNA-based markers. Trends in Food Science and Technology, 26, 43–55.

f extra virgin olive oil with refined olive oil: Prospects and limitations,13.05.008

6 R. Garcia et al. / Food Research International xxx (2013) xxx–xxx

Dais, P., & Hatzakis, E. (2013). Quality assessment and authentication of virgin olive oilby NMR spectroscopy: A critical review. Analytica Chimica Acta, 765, 1–27.

Dong, W., Zhang, Y., Zhang, B., & Wang, X. (2012). Quantitative analysis of adulterationof extra virgin olive oil using Raman spectroscopy improved by Bayesian frame-work least squares support vector machines. Analytical Methods, 4, 2772–2777.

EC, Commission Regulation (EC) No 1989/2003 of 6 November 2003 (2003). OfficialJournal of the European Communities, L295, 57–77.

Firestone, D. (2005). Olive oil. In F. Shahidi (Ed.), Bailey's industrial oil and fat products(pp. 303–331). John Wiley & Sons, Inc.

Fragaki, G., Spyros, A., Siragakis, G., Salivaras, E., & Dais, P. (2005). Detection of extravirgin olive oil adulteration with lampante olive oil and refined olive oil using nu-clear magnetic resonance spectroscopy and multivariate statistical analysis. Journalof Agricultural and Food Chemistry, 53, 2810–2816.

Frankel, E. N. (2010). Chemistry of extra virgin olive oil: Adulteration, oxidative stabil-ity, and antioxidants. Journal of Agricultural and Food Chemistry, 58, 5991–6006.

Frankel, E. N. (2011). Nutritional and biological properties of extra virgin olive oil.Journal of Agricultural and Food Chemistry, 59, 785–792.

García-González, D. L., & Aparicio, R. (2010). Research in olive oil: Challenges for thenear future. Journal of Agricultural and Food Chemistry, 58, 12569–12577.

Garrido-Delgado, R., Arce, L., & Valcárcel, M. (2012). Multi-capillary column-ion mobil-ity spectrometry: A potential screening system to differentiate virgin olive oils.Analytical and Bioanalytical Chemistry, 402, 489–498.

Garrido-Delgado, R., Mercader-Trejo, F., Arce, L., & Valcárcel, M. (2011a). Enhancingsensitivity and selectivity in the determination of aldehydes in olive oil by use ofa Tenax TA trap coupled to a UV-ion mobility spectrometer. Journal of Chromatog-raphy. A, 1218, 7543–7549.

Garrido-Delgado, R., Mercader-Trejo, F., Sielemann, S., de Bruyn, W., Arce, L., &Valcárcel, M. (2011b). Direct classification of olive oils by using two types of ionmobility spectrometers. Analytica Chimica Acta, 696, 108–115.

Gertz, C., & Fiebig, H. J. (2006). Pyropheophytin a — Determination of thermal degrada-tion products of chlorophyll a in virgin olive oil. European Journal of Lipid Scienceand Technology, 108, 1062–1065.

Giuliani, A., Cerretani, L., & Cichelli, A. (2011). Chlorophylls in olive and olive oil: Chem-istry and occurrences. Critical Reviews in Food Science and Nutrition, 51, 678–690.

Gurdeniz, G., & Ozen, B. (2009). Detection of adulteration of extra-virgin olive oils bychemometric analysis of mid-infrared spectral data. Food Chemistry, 116, 519–525.

He, J., Xu, W., Shang, Y., Zhu, P., Mei, X., Tian, W., et al. (2013). Development andoptimization of an efficient method to detect the authenticity of edible oils. FoodControl, 31, 71–79.

International Olive Oil (IOOC) Council (2011). COI/T.15/NC No.3/Rev. 6 November 2011.Trade standard applying to olive oils and olive-pomace oils, 1–17.

Jiménez de Blas, O., & dell Valle González, A. (1996). Determination of sterols by capil-lary column gas chromatography. Differentiation among different types of oliveoil: virgin, refined, and solvent-extracted. Journal of the American Oil Chemists'Society, 73(12), 1685–1689.

Kumar, S., Kahlon, T., & Chaudhary, S. (2011). A rapid screening for adulterants in oliveoil using DNA barcodes. Food Chemistry, 127, 1335–1341.

Kunz, M. R., Ottaway, J., Kalivas, J. H., Georgiou, C. A., & Mousdis, G. A. (2011). Updatinga synchronous fluorescence spectroscopic virgin olive oil adulteration calibrationto a new geographical region. Journal of Agricultural and Food Chemistry, 59,1051–1057.

León-Camacho, M., Serrano, M. A., & Constante, E. G. (2004). Formation of stigma-3,5-dienein olive oil during deodorization and/or physical refining using nitrogen as strippinggas. Grasas y Aceites, 55(3), 227–232.

Lerma-Garcia, M. J. (2012). Characterization and authentication of olive and othervegetable oils— New analytical methods. Doctoral Thesis accepted by the Universityof València, Spain, (Springer Theses). Heidelberg: Springer-Verlag.

Lukic, M., Lukic, I., Krapac, M., Sladonja, B., & Pilizota, V. (2013). Sterols and triterpenediols in olive oil as indicators of variety and degree of ripening. Food Chemistry, 136,251–258.

Maggio, R. M., Cerretani, L., Chiavaro, E., Kaufman, T. S., & Bendini, A. (2010). A novelchemometric strategy for the estimation of extra virgin olive oil adulterationwith edible oils. Food Control, 21, 890–895.

Mannina, L., & Sobolev, A. P. (2011). High resolution NMR characterization of olive oilsin terms of quality, authenticity and geographical origin. Magnetic Resonance inChemistry, 49, 3–11.

Mannina, L., Sobolev, A. P., & Segre, A. L. (2003). Olive oil as seen by NMR andchemometrics. Spectroscopy Europe, 15(3), 6–14.

Mariani, C., & Fedeli, E. (1986). Individuazione di oli di estrazione in quelli di pressione[olio di oliva]. Pt.1. Rivista Italiana delle Sostanze Grasse, 63(1), 3–17.

Please cite this article as: Garcia, R., et al., Putativemarkers of adulterationFood Research International (2013), http://dx.doi.org/10.1016/j.foodres.20

Martínez-Vidal, J. L., Garrido-Frenich, A., Escobar-García, M. A., & Romero-González, R.(2007). LC–MS determination of sterols in olive oil. Chromatographia, 65, 695–699.

Mignani, A. G., Ciaccheri, L., Ottevaere, H., Thienpont, H., Conte, L., Marega, M., et al.(2011). Visible and near-infrared absorption spectroscopy by an integrating sphereand optical fibers for quantifying and discriminating the adulteration of extra virginolive oil from Tuscany. Analytical and Bioanaytical Chemistry, 399, 1315–1324.

Montealegre, C., Alegre, M. L. M., & García-Ruiz, C. (2010). Traceability markers to thebotanical origin in olive oil. Journal of Agricultural and Food Chemistry, 58, 28–38(and references therein).

Ogrinc, N., Košir, I. J., Spangenberg, J. E., & Kidrič, J. (2003). The application of NMR andMS methods for detection of adulteration of wine, fruit juices, and olive oil. Areview. Analytical and Bioanalytical Chemistry, 376(4), 424–430.

Oliveros, M. C. C., Pavon, J. L. P., Pinto, C. G., Fernandez Laespada, M. E., Cordero, B. M., &Forina, M. (2002). Electronic nose based on metal oxide semiconductor sensors asa fast alternative for detection of adulteration of virgin olive oils. Analytica ChimicaActa, 459, 219–228.

Papadopoulos, K., Triantis, T., Tzikis, C. H., Nikokavoura, A., & Dimotikali, D. (2002).Investigations of extra virgin olive oils with seed oils using weak chemilumines-cence. Analytica Chimica Acta, 464, 135–140.

Pasqualone, A., & Catalano, M. (2000). Free and total sterols in olive oils. Effects ofneutralization. Grasas y Aceites, 51(3), 180–182.

Pérez-Camino, M. C., Cert, A., Romero-Segura, A., Cert-Trujillo, R., & Moreda, W. (2008).Alkyl esters of fatty acids a useful tool to detect soft deodorized olive oils. Journal ofAgricultural and Food Chemistry, 56, 6740–6744.

Pérez-Camino, M. C., Moreda, W., & Cert, A. (2001). Effects of olive fruit quality and oilstorage practices on the diacylglycerol content of virgin olive oils. Journal ofAgricultural and Food Chemistry, 49(2), 699–704.

Pérez-Camino, M. C., Moreda, W., Mateos, R., & Cert, A. (2002). Determination of estersof fatty acids with low molecular weight alcohols in olive oils. Journal of Agriculturaland Food Chemistry, 50, 4721–4725.

Petrakis, C. (2006). Olive oil extraction. In D. Boskou (Ed.), Olive oil: Chemistry andtechnology (pp. 191–224). American Oil Chemists' Society Press.

Phillips, K. M., Ruggio, D. M., Toivo, J. I., Swank, M. A., & Simpkins, A. H. (2002). Free andesterified sterol composition of edible oils and fats. Journal of Food Composition andAnalysis, 15, 123–142.

Saba, A., Mazzini, F., Raffaelli, A., Mattei, A., & Salvadori, P. (2005). Identification of 9(E),11(E)-18:2 fatty acid methyl ester at trace level in thermal stressed olive oils by GCcoupled to acetonitrile CI–MS and CI–MS/MS, a possible marker for adulteration byaddition of deodorized olive oil. Journal of Agricultural and Food Chemistry, 53,4867–4872.

Sánchez-Hernández, L., Marina, M. L., & Crego, A. L. (2011). A capillary electrophoresis-tandem mass spectrometry methodology for the determination of non-proteinamino acids in vegetable oils as novel markers for the detection of adulterationsin olive oils. Journal of Chromatography. A, 1218, 4944–4951.

Serani, A., & Piacenti, D. (1992). Kinetics of pheophytin — A photodecomposition inextra virgin olive oil. Journal of the American Oil Chemists' Society, 69(5), 469–470.

Sikorska, E., Khmelinskii, I., & Sikorski, M. (2011). Analysis of olive oils by fluorescencespectroscopy: Methods and applications. In D. Boskou (Ed.), Olive oil — Constituents,quality, health properties and bioconversions (pp. 63–88). InTech.

Spyros, A., & Dais, P. (2000). Application of 31P NMR spectroscopy in food analysis.Quantitative determination of the mono- and diglyceride composition of oliveoils. Journal of Agricultural and Food Chemistry, 48, 802–805.

Torrecilla, J. S., García, J., García, S., & Rodríguez, F. (2011). Quantification of adulterantagents in extra virgin olive oil by models based on its thermophysical properties.Journal of Food Engineering, 103, 211–218.

Vigli, G., Philippidis, A., Spyros, A., & Dais, P. (2003). Classification of edible oils byemploying 31P and 1H NMR spectroscopy in combination with multivariate statis-tical analysis. A proposal for detection of seed oil adulteration in virgin olive oils.Journal of Agricultural and Food Chemistry, 51, 5715–5722.

Visioli, F., Bogani, P., & Galli, C. (2006). Healthful properties of olive oil minor compo-nents. In D. Boskou (Ed.), Olive oil: Chemistry and technology (pp. 173–190).American Oil Chemists' Society Press.

Woodcock, T., Downey, G., & O'Donnell, C. P. (2008). Confirmation of declared prove-nance of European extra virgin olive oil samples by NIR spectroscopy. Journal ofAgricultural and Food Chemistry, 56, 11520–11525.

Zhang, H., Wu, Y., Li, Y., Wang, B., Han, J., Ju, X., et al. (2012). PCR-CE-SSCP used toauthenticate edible oils. Food Control, 27, 322–329.

Zou, M. Q., Zhang, X. F., Qi, X. H., Ma, H. L., Dong, Y., Liu, C. W., et al. (2009). Rapidauthentication of olive oil adulteration by Raman spectrometry. Journal of Agricul-tural and Food Chemistry, 57(14), 6001–6006.

of extra virgin olive oil with refined olive oil: Prospects and limitations,13.05.008