J. Agric. Food Chem.2010, 58, 5991–6006 5991 DOI:10.1021/jf1007677 Chemistry of Extra Virgin Olive Oil: Adulteration, Oxidative Stability, and Antioxidants EDWIN N. FRANKEL* Department of Food Science and Technology, University of California, Davis, California 95616 Much analytical work has been published on the chemistry of extra virgin olive oil (EVOO) as a basis for the detection and quantitative analyses of the type and amount of adulteration with cheaper vegetable oils and deodorized olive oils. The analysis and authentication of EVOO represent very challenging analytical chemical problems. A significant amount of literature on EVOO adulteration has depended on sophisticated statistical approaches that require analyses of large numbers of samples. More effort is needed to exploit reliable chemical and instrumental methods that may not require so much statistical interpretation. Large assortments of methods have been used to determine lipid oxidation and oxidative stability and to evaluate the activity of the complex mixtures of phenolic antioxidants found in EVOO. More reliable chemical methods are required in this field to obviate excessive dependence on rapid antiradical methods that provide no information on the protective properties of antioxidants. The extensive literature on olive oil sensory tests, using many descriptors varying in different countries, should be supplemented by more precise gas chromato- graphic analyses of volatile compounds influencing the odor and flavors of EVOO. KEYWORDS: Extra virgin olive oil; virgin olive oil and olive oil; methods; foods; adulteration; oxidative stability; volatiles; antioxidants; statistics; vegetable oils INTRODUCTION and linolenic acids and traces of myristic, arachidic, heptadeca- Extra virgin olive oils (EVOO) prepared by cold-pressing of noic, and eicosanoic acids (1, 2). Other constituents include olive flesh are important edible oils in the Mediterranean diet and partial glycerides [diacylglycerols (DAGs), monoacylglycerols are now recognized for their potential health benefits. Adultera- (MAGs)], hydrocarbons (squalene, β-carotene, diterpenes, iso- tion of the highly desirable and costly EVOO with cheaper prenes, n-paraffins), tocopherols (R, β, γ, δ), pigments (chloro- vegetable oils and refined or processed olive oils has attracted phylls and pheophytins, carotenoids), sterols, alcohols, triterpene much attention worldwide for both economic and health con- acids, volatile compounds affecting aroma and flavors (3-8), siderations. Detection of olive oil adulteration is a difficult and phenolic compounds contributing to antioxidant activity and challenging analytical problem because EVOO consists of com- potential health effects (9), phospholipids, and proteins. plex mixtures of triacylglycerols (TAGs), partial glycerides, Much analytical work has been published in the past two hydrocarbons, tocopherols, pigments, sterols, alcohols, triterpene decades together with extensive statistical interpretation for the acids, volatile compounds, phenolic compounds, phospholipids, detection and quantitative analyses of the type and amount of and proteins (1). Several international regulations have been adulteration of EVOO. The analysis and authentication of EVOO developed to protect consumers with uniform definitions, label- represents one of the most challenging analytical problems to ing, and a multiplicity of analytical techniques to identify genuine detect and determine its adulteration with cheaper vegetable oils quality standards in many countries. Although a large number of and refined and not refined olive residue oils. Quality parameters analytical methods have been developed in the past decades to defining olive oil categories, defects, identity characteristics, and determine adulteration of EVOO, the literature in this field is still fatty acid and sterols composition have been established by controversial and confusing. different international organizations. A recent book on olive Many authors in this field are not fully exploiting the available oil (9) contains a chapter that includes five figures of unpublished powerful analytical methods to determine the authenticity of data on high-resolution GC (HRGC) of DAG and non-glyceride EVOO samples and rely too much on complex statistical methods components and HPLC of TAG profiles of a mixture of olive oil requiring the analyses of a very large number of samples to obtain with 20% rapeseed oil. A GC method is described (2) that requires usually only qualitative or semiquantitative results. a high temperature of 350 °C to detect as little as 4% adulteration of EVOO with soybean oil by measuring trilinolein, which is not DISCUSSION present in olive oil. References selected from the extensive literature (Table 1) Adulteration with Cheaper Vegetable Oils. The TAGs of EVOO illustrate a wide assortment of analytical methods aimed at contain mixtures of palmitic, palmitoleic, stearic, oleic, linoleic, evaluating the authenticity and the presence of adulterants that can devalue EVOO. A rapid reversed-phase HPLC/differential *E-mail:
[email protected]. refractometric detection system was used to determine an © 2010 American Chemical Society Published on Web 04/30/2010 pubs.acs.org/JAFC sterols EVOO MeOH/H2O HPLC total ion intensity qualitative SDA 22 positive APCI 99% prediction ESI-MS polar phenolics FAs edible oils MeOH/H2O extracts fingerprints qualitative PCA 23 GC-MS sterols hazelnut oil dimethyl-sterols sterol fraction <4% standard (24) RP-HPLC. 37 4000-650 cm-1 . hazelnut acetone/CH3CN mass spectral ANOVA. DA 17 ATR sunflower. PLS. ANN 19 TAGs extraction HPLC 1 H NMR unsat and acyl edible oils CDCl3 solution 299. pH 8.6-13. 28 fluorescence 20-120 nm vegetable oils 315-400 nm 2. 5 cultivars cold pressing acidity. TAGs vegetable oils reverse phase HPLC ELSD FID rel %: 5-10% CAT-PCA 25 ELSD SPME-MDGC filbertone hazelnut oil steam distillation GC-MS up to 7% standard 26 synchronous 270-720 nm sunflower oil hexane solutions 270-720 nm 3.. HPLC FA.46 MHz 5 DA 12 HPLC-APCI MS TAGs tocopherol. pure vegetable oils hazelnut þ ambient 1400-900 cm-1 2% sunflower oil. KNN. ZnSe. UV. PLSR 29 þ squalene CEC tocopherols vegetable oils 99:1 MeOH/ 205 and 295 nm LOD: 1.30 qualitative 30 tocotrienols aqueous buffer. SIMCA. v/v 13 C NMR polar chromatogr seed oils.8% GC-MS FAMEs edible oils hexane solutions areas of 23 FA 88-91% detection SIMCA.50-2. pfficial EU methods EVOO. Agric. TAGs. PCA 13 sterols 64:36.4% PLSR 27. DAGs.5992 J. GC. Food Chem. Extra Virgin Olive Oil (EVOO) Adulteration methods determinations adulterants exptl conditions detection threshold % data processing refs RP-HPLC TAGs LO-rich vegetable acetone/acetonitrile differential 1 standard 10 oils refractometer NP-HPLC tocopherols palm oil hexane THF fluorometric-photo 1-2 standard statistics 11 solutions diode RP-HPLC tocotrienols grapeseed oil 13 C NMR unsaturated FA seed oils CDCl3 solution 75. LDA.0 ESI-MS rel peak intensities olive oils PrOH/MeOH þ rel peak intensities 5-11% prediction LDA 31 40 mM KOH 1 H 31P NMR spectral analyses geogr origin EtOH/water extracts 87% prediction CDA 32 Raman spectral data vegetable oils neat normailzed spectra 5% by vol standard 33 spectroscopy GC. No. 25% hazenut oil vegetable oils visible and spectral data sunflower neat samples 1100-2498 nm 0. correct classification PCA. CDCl3 solution 75. 58.46 MHz qualitative 97-98% stepwise DA 14-16 fractions OO pomace oils validation FT-IR. TAGs profiles vegetable oils IOOC global method ECN R = rECN42/rECN44 Codex/IOOC 34 HPLC/APCI-MS TAGs plant oils CH3CN/2-propanol/ statistical evaluation 1% sunflower oil PCA 35 hexane FT-IR spectra spectral data vegetable oils neat samples absorbance 5% PLS-DA 36. sterols. 2010 Frankel a Table 1. 10. Vol.862 MHz IV qualitative standard 20 groups SPE-RP-HPLC polar fraction hazelnut oil no treatment UV 5% standard 21 RP-HPLC-MS. HCA.8% SDE PCA. 18 near-IR PLS GC. capillary electrophoresis. HCA. wise discriminant function analysis (SDFA) to select the variables cation of olive oil and vegetable oils. 13C NMR (SPE). 10. elenolic acid. monoacylglycerols. ELSD. headspace mass spectroscopy. CZE. This method together with discriminant analysis (DA) without chemical derivatization and purification by using step- and partial least-squares analysis (PLS) allowed correct classifi. “authenticity” factor based on the equivalent carbon number standard error of 0. thiobarbituric acid reactive substances. principal component analysis. 58. DHPEA. and C18:3 acids in linoleic acid (1. SPE. Polar components isolated by solid-phase extraction ing to their TAG composition. PRESS. GC-CIMS. Agric. authors claimed qualitative detection of adulteration above 91 scopy of 138 samples of EVOO adulterated with 1 and 5% and 88% identification of the type of adulterant (sunflower. 1H NMR. but relatively high levels were found in palm and triene absorptivity at 270 nm (19). different vegetable oil samples (20). K nearest neighbors. linear discriminant analysis. normal phase high-performance liquid chromatography. headspace-sorptive extraction. MS. 2010 5993 Table 1. thin layer chromatography. CI-MS. DA 38 diffusion NMR 30% hazelnut oils 1 H NMR unsaponifiable VOOs. high-oleic.0 crimination between authentic EVOO and its adulterated mix- for olive oil (10). and conjugated soybean oils. conjugateed dienes. HS-MS. samples. linoleate hydroperoxides.7%]. THF. low-density lipoprotein. Vol. categorical principal component analysis. were used to spectra were analyzed from 104 oils and mixtures from different detect adulteration of EVOO with pressed hazelnut oil (21). (chemical names and products) CD. mass spectrometry. LDA. oxygen stability index. FT-IR. analysis of variance. FAAES. PV. MDGC. sitostanol. SDA. ROPO. equivalent chain length. GC-electron ionization MS. discriminant analysis. Correct classification and 99% prediction rate 2% for sunflower oil. trilinolein. GC-IT-MS. CEP. diode array detection. ranging from 22. stepwise discriminant function analysis. electrospray interface.1% of the samples by using stepwise discriminant analysis 4. stepwise discrimination analysis. FAMEs. ES-MS. fatty acide methyl esters. chemical ionization MS. correct identification of all samples required components (13). were obtained with samples from three Italian olive cultivars. conjugated trienes. high-performance size exclusion chromatography. C16:1. high-oleic safflower oil. megahertz. reverse phase high-performance liquid chromatography. The Another study using visible and near-IR transflectance spectro. C16. GC-chemical ionization MS. TAGs. SPME. OPO. HOSO. ATR. Fourier transform NMR. Successful dis- linoleic acid (soybean. The method was based on sure chemical ionization-mass spectrometry (HPLC-APCI-MS) the area of signals of spectra that is proportional to the number of was used to determine adulteration of EVOO with 10-50% hydrogen atoms and different acyl groups for each type of hazelnut oil based on TAG composition and non-glyceride sample. predicted residual error sum of squares. electron spin resonance. electron paramagnetic resonance. Discriminant analysis (DA) showed that hazel. hierarchical cluster analysis. direct normal phase high-performance liquid chromato. NP-HPLC. The authors concluded that 10 grapeseed oils.44 ppm geogr origin pattern recognition (39) fingerprint matter oils hydrocarbon. GC-EIMS. LDL. discriminant component analysis. PCA. gas chromatography-ion trap-mass spectrometry. In another independent modeling of class analogy (SIMCA) and partial study. MDA. capillary zone electrophoresis. ECN. regression analyses.4 to 24. HPLC. ANOVA. sterol. solid phase extraction. and vegetable HPLC-MS with direct injection and positive APCI detection oils (17). triacylglycerols. high-resolution GC. with rapid detection level of and LDA (22). and Threshold levels as low as 5% were obtained with an accuracy high-linoleic acid oils (14-16). capillary electrochromato- graphy. refined olive pomace oil. FT-NMR. DA. soy. HS-SE. This method could detect 1% palm oil and 2% chemical indices (acidity. MHz. attenuated total reflectance. multidimensional gas chromatography. HPSEC. These relatively advanced sta- graphy (NP-HPLC) and reverse phase (RP)-HPLC/amperometric tistical methods apparently required the analyses of a very large detection without saponification were used to obtain 97 and number of samples. good precision [relative standard deviation (RSD) = for 97. C18:2. PLS. MAGs. corn. hazelnut. No. HPLC-chemiluminescence. EDA. OSI. liquid-liquid extraction. and corn oils) compared to 1. Another study using 153 samples of EVOO 102% recoveries of R-tocotrienols and γ-tocotrienols (11). ESI. malonaldehyde. evaporative light scattering detector. (LDA). adulteration. but only 25% and higher for hazelnut oil. least-squares regression (PLSR). C18:3. flame ionization detection. .Perspective J. ESR. olive pomace oi. A semiquantitative stigmasterol. linolenate hydroperoxides. DHS-HR-GC.5-130 ppm) obtain the correct classification by linear discriminant analysis was reported to detect the presence of seed oils (cottonseed. lipoxygenase. atmospheric pressure chemical ionization. FA alkyl esters. sunflower. high-performance liquid chromatography. In another study. HPLC. vegetable alcohol. data on different cultivars and authenticity of the origin of nut oil and mixtures with olive oil were clearly separated accord. SIMCA. 0-5. liquid chromatography-MS. DTD. FID. hazelnut oils examined. RP-HPLC.9 for vegetable oils high in spectral data between 1100 and 2498 nm (18). tetrahydrofuran. extra virgin olive oil. sunflower. DPPH. However. soft independent modeling of class analogy. KNN. sunflower oil allowed complete classification accuracy with a peanut. C18:1. EVOO. Food Chem. LC-MS. HR-GC. tocopherol a Abbreviations: (analytical methods and instruments) APCI.1) and determine the proportions of C18:1. and Δ7-stigmasterol) are sufficient to method using 13C NMR in the olefinic region (127. sunflower. EPR. grapeseed oil in virgin and refined olive oils.. PLSR. GC-RI/MS. and corn oils) in EVOO. LOX. static headspace. CDA. 66 samples of edible oils and of different acyl groups in 17 High-performance liquid chromatography-atmospheric pres. C18. diphenyl-picrylhydrazyl. GC-refractive index/MS. DCA. SDFA. proton nuclear magnetic resonance. CAT-PCA. which affected NMR spectroscopy was a useful tool to simultaneously the intensities of 12 peaks and the R/β ratios of oleic acid (1. electron spin MS. and coconut oils). LOOH. CEC. dynamic headspace-high resolution GC. partial least-squares regression. dihydroxyphenyletanol or hydroxytyrosol. Fourier transform infrared (FT-IR) was used to determine Correct discrimination of EVOO samples were obtained by adulteration of EVOO with hazelnut. geographical origins to distinguish among VOOs. and linearity (R2 = 0. Fourier transform infrared. TLC. Correct validation was achieved of 90%. No from 5 Italian cultivars was based on Official Analytical Methods tocotrienols were detected in olive. SHS. 1 sunflower seed. TBARS. LLE. canonical discriminant analysis. quantification of adulteration was pre- and FFA) from the same oils allowed better than 98% correct vented by the large variability of marker components in the validation by SDA. LnOOH. Determinations of minor components (DAGs. DAD. followed by RP-HPLC with UV detection. artificial neural network.998) in the range of 5-40% (SDA). direct thermal desorption. Detection of adulteration with as little as 1% of tures was achieved at levels as low as 1% by using soft vegetable oils was claimed possible by this approach.8% by using the first-derivative treatment of (ECN) 42. MAGs. solid phase microextraction. and of fatty acid composition. However. sterols. (statistical procedures) ANN. peroxide value.5) (12). C18:2. soybean. Continued methods determinations adulterants exptl conditions detection threshold % data processing refs gradient TAGs plant oils neat samples m/z 50-1200 10% sunflower. CT. and ECN42 oxidized soybean oil showing more additional ions than the fresh were obtained and the theoretical ECN42 and ECN44 were oil. sunflower. determined to detect adulteration of EVOO for rapid screening tion was quantified by using the PLSR model at a level of 3. rapeseed. more effort is needed in this field to use reliable with 5-11% average errors. and cheaper olive oils that have sensory defects and are referred to Canonical discriminant analysis (CDA) showed that 87% of the as “Lampante” oils (Table 2). corn. detection limit of adulteration was 5% for corn-sunflower ponent analysis (CAT-PCA). were used to determine the authenticity of oil from vegetable oils. Correct validation of 100% was claimed for EVOO samples and 720 nm. with different concentrations of sunflower oil. The samples. scattering detector (ELSD) (25). In another study. The extent of adultera. and coconut phical characterization of VOOs from Spain. Agric. Common according to their contents of fatty acids. to detect aging and adulteration of olive oils (33). Adulteration of virgin olive oils (VOOs) by hazelnut oil at calculated. entiated olive oil from the other refined vegetable oils and experimental ECN48. sunflower. and Syria. Tunisia. Olive oil powerful analytical methods are not exploited to provide more quality grade was predicted on the basis of ratios of peak precise and accurate chemical information.40 -dimethyl. In another study. Electrospray ionization MS lists many statistical approaches that require special training and was also used to predict olive oil quality according to European a multitude of samples. 58. To remove undesirable flavor geographic source could be predicted. using the statistical program categorical principal com. ECN46. A method was developed to detect filber. for sunflower and soybean oils and 30% for hazelnut and peanut and walnut oils by varying the excitation wavelength between 250 oils. soybean. On the basis of the results of TAGs. given in the form of two- (ESI-MS) was used to differentiate qualitatively unrefined olive dimensional charts. Vol. binary mixtures and cottonseed and rapeseed oils. ECN44. Hazelnut oils con. Excellent evaluations requiring large numbers of samples.90 for calibration and validation. corn. identified.. these oils are generally sity ratios of the cis (dC-H) and cis (CdC) bonds normalized subjected to mild deodorization at lower temperatures than . the inten. (MLR) models were used to determine binary mixtures as low as The potential application of total synchronous fluorescence 5% of EVOO with other vegetable oils. ECN42/ECN 44 (34).6-13. composition of binary mixtures of EVOO with cheaper oils tone and establish adulteration of up to 7% with virgin hazelnut (sunflower. Absorbance oils. TAG profiles were determined in variate data analysis enabled identification of adulterated oils vegetable oils by HPLC separation using an evaporative light from 1% of added sunflower oil. soybean. such as soybean oil. Multiple linear regression filbertone. canola. rapeseed oil. and classification of olive oils. Too many authors appear to depend Union (EU) marketing standards (30). Plant oils were tained 86-91% 4-dimethylsterols compared to 51-57% in authenticated by using model samples of olive oil adulterated VOOs. 93 oils including one olive oil composed of 355 relative proportions in sterol fractions (24). corn. Samples diluted in an excessively on sophisticated statistical methods to determine alkaline mixture of propanol/methanol were infused directly into degrees of adulteration of EVOO with cheaper oils. This method can distinguish more precisely and vegetable oils by analyzing the polar components extracted with easily olive oils from the mixtures of olive oils containing g5% by methanol/water (1:1) from different oils and mixtures (23). No. samples and obviate too much dependence on statistical inter- 1 H and 31P NMR spectroscopy were used to characterize pretations. The corre. 2010 Frankel -1 Direct infusion electrospray ionization mass spectrometry by the band at 1441 cm (CH2). Binary includes 16 different specialized statistical methods that were used mixtures of EVOO/VOO and EVOO/ROPO could be predicted in the analyses. adulteration practices consist of blending EVOO with low-quality total free sterols. TAGs by HPLC-MS were evaluated by PCA (35). The model Different varieties and geographical origins of mixtures of based on PLS analysis developed to detect adulteration was VOOs with 10-20% hazelnut oils were analyzed by solid phase limited to 10%. whereas olive oils contained 32-40% of 4. Food Chem. identification. Italy. This table >0. even though the electrospray ionization source of an ion trap MS. Chemometric models used were corresponding non-glyceride fractions together with statistical based on soft independent modeling of class analogy (SIMCA) pattern recognition techniques (39). and quantified on the basis of the relative [partial least-squares (PLS) and PCA model] were used to detect peak areas of 8 vegetable oils including olive oil in a total of 52 and quantify adulteration of EVOO with edible oils (36). based on NMR profiles of bulk oils and the quantitation by GC-MS (29). followed by LDA. or corn oil. and cottonseed). peanut. 10. Although Table 1 abundance of free fatty acids (FFAs). phenolics. diacylglycerols. The 1H NMR could be detected at levels of 2. 2.5 min. spectra was combined with multivariate analysis to assess adul. The values for R and ΔECN were proposed to identify levels of <4% was determined by GC-MS on the basis of adulteration. The same technique was also used (28) to differentiate Changes in D values could be detected with adulteration of 10% VOOs from olive pomace. A total of 15 peaks were Mid-IR and FT-IR spectroscopy combined with chemometrics separated. and free acidity and iodine number (32). In another study FT-IR was used to classify oil microextraction and multidimensional gas chromatography samples according to botanical origin and to determine the (SPME-MD-GC) (26). Adulteration with the different vegetable oils in VOO randomly adulterated with cheaper vegetable oils. oils was determined by sequential detection. cipal component analysis (PCA) was used to differentiate unique sunflower seed oil. and Turkey. giving results precision was obtained with PLSR as shown by R2 values of expressed often in ranges that are not always precise. this method could not be applied with some peak areas were normalized within the FT-IR spectra as pre- refined hazelnut oils containing very low concentrations of dictors of botanical origin by LDA. ECN50. and hazelnut oils) (37). authenticity. lated on the basis ECN42 (equivalent carbon number) and R = sponding ESI-MS fingerprints in the negative mode also differ. Prin. In characteristic dimethylsterols observed in significantly different another study.5994 J. corn. High-power gradient NMR diffusion coefficients (D) were teration of VOO with sunflower oil (27).4% in of adulteration of olive oils with cheaper vegetable oils (38).8%. monovarietal VOOs from 3three regions of southern Greece Adulteration with Refined and Deodorized Olive Oils. Statistical multi- sterols. by multiple linear regression and chemical methods that would not require a large number of PLSR (31). volume of other edible oils. major diagnostic ions of olive oil from other vegetable oils Olive oils were classified by the fatty acids and TAGs calcu- (soybean. and K nearest neighbors (KNN) able to predict >91% for Many studies in Table 1 are very much dependent on statistical adulterants and to identify the adulterants by 88%. fingerprint of the unsaponifiable fraction was used for geogra- EVOO adulteration with sunflower. volatiles derived from lipid oxidation. However. PV. GC analyses of 60 samples of Spanish olive oils silica gel column was used followed by size exclusion chroma. In another study. and TAGs produced when olive its chlorophyll and tocopherol constituents had been stripped by fruits are stored before milling are readily converted into alkyl column chromatography (48).67-7. In another study.5-diene produced by thermal dehydration of low-quality olive oil that has been subjected to soft deodorization. oxidation due to its relatively high levels of oleic acid. most bottles of imported EVOO Ch 5-91). size exclusion HPLC 40 <150-170 °C deodorization.12-18:2 diene isomer would Oxidative Stability. been apparently little or no control of shelf life in many retail lysis (AOCS Standard method for specific extension in the UV: markets. deodorization temperatures. thermal treatments used were deodorization. 190 °C. GC. volatiles. including tocopherols. tocopherols. DAGs. sterols esterification of FFA with low fatty acid alkyl esters (FAAEs) silica SPE cartridge GC 45 MW alcohols microwave. artificial conditions of microwave and conventional heating tify one conjugated diene 9. To improve GC analyses. polyphenols. HPSEC.11-18:2 fatty acid ester. hydrocarbons. Both conjugated isomers can be readily deter. but the results obtained under very included the use advanced GC-MS and GC-MS/MS to iden. respectively. sterols. 2 h conjugated dienes GC-MS. 10. samples were first separated by titratable acidity of the oil was maintained below 5% after storage SEC into nonpolar compounds containing hydrocarbons. found in many groceries are often not dated and generally stored A soft purification process for Lampante olive oils containing for prolonged periods without controlling their shelf life at FFAs (3. 14 at 8 °C and to a maximum PV of 8 at 5 °C. temperature. The tillates.11-18:2 fatty esters produced at the eleva. oven heating FA alkyl esters. Unfortunately. total polar compounds. 90 min. Quantification of specific compounds oxidative stability of commercial EVOO stored at 60 °C in the in fractions was carried out by GC using internal standards. For these reasons.5-diene colum chromatography. formation may be questionable. and phenolics 93%. stigmasta-3.2%) and oxidation products (PV 26-52 mequiv/ ambient temperatures. there has mined quantitatively by standard spectrophotometric UV ana. Agric. the presence of tion and oxidative stability of olive oils using a large assortment of refined olive oil in EVOO was determined on the basis of the methods and their limitations. physical refining. Food Chem. No. alkyl at 5 °C. The level of dimer solid phase cartridge and analyzed by GC equipped with a TAGs and stigmastadiene formed during deodorization was programmed temperature vaporizer injector using a polar capil- determined in olive oil and vegetable oils (40). The presence of was used to determine the effects of deodorization and physical fatty acid esters can therefore be considered a good marker of refining. 58. to form micronic aggregates removed by microfiltration (43). A statistical study of processing parameters (N2 determine admixtures of mildly deodorized olive oil with EVOO flow. FA. 2010 5995 a Table 2. dimer TAGs.. sensory 43 UV absorption deodorizer distillates TAGS. the fatty acids. 46 FA composition OFA. GC 44 alkyl esters. partial glycerides. OSI a Abbreviations: see Table 1 footnote. Another approach was based on the determina. of the corresponding conjugated 10. the corresponding formation of dimer TAGs starting at 90 °C and increasing at esters ranged from 29 to 193 ppm and from 42 to 3636 ppm. and oil load) was used to determine apparent by chromatographic and spectroscopic methods (46). The quality of olives and olive oil complex volatile and nonvolatile compounds in deodorizer dis.Perspective J. DAGs. phenolic compounds . showed wide ranges of FA methyl esters (5-23 ppm) and ethyl tography (SEC) with a refractive index detector to show the esters (3-40 ppm). FFA. A cleanup short lary column. β-sitosterol was detected and quantified in refined oils in The effects of hydrolysis and oxidation were also used to EVOO (41). and sterols (44). To simulate kinetic constants for the formation of stigma-3. dark and under fluorescent light was compared before and after The FFAs. Other The resulting methyl and ethyl esters can be isolated by a silica gel minor components. component that accelerated photooxidation of EVOO. microfiltration FFA. steryl esters. when these olive oils kg) was based on deacidification with H3PO4 and oil conditioning oxidize. and polar compounds including MAGs and DAGs. GC-MS/MS 42 neutralization. MAGs. stigmastadiene short silica column. GC. based on microwave and conventional heating at 180 °C for tion of conjugated 9. The producing objectionable and undesirable flavors. Up to 800 mg/kg fatty ethyl esters was produced The determination of non-glyceride components in olive oils after soft deodorization for 4 h at 98 and 150 °C. Vol. Although this study EVOO were compared. was evaluated by storage stability and sensory tests (47). Adulteration with Refined and Deodorized Olive Oils (“Lampante” Oils) treatment determinations analyses ref steam-washing deodorization. Different mixtures of thermally stressed olive oils with ted temperatures of deodorization (42). TLC. sterol components Table 3 summarizes selected studies that evaluate lipid oxida- 36-50%.5-diene during home cooking or food catering. polymers. silica SPE cartridge. process lowered monoglycerides up to 78%. HRGC 41 bleaching heating 160. Chlorophyll was an important esters by microbial esterification with methanol and ethanol (45). The initial PV of 4 increased sharply to a maximum PV of esters TAGs. conventionally practiced with vegetable oils. they eventually develop relatively high levels of rancidity. Because EVOO is relatively stable to be expected. FA composition. In the Lampante olive oils. Stigma-3. acidity. OSI at 100. FA. tocopherols. chlorophylls. increase in PV. squalene. 58. hexanal. calorimetric analyses at 3. and bitter taste values. The conclusion was that this simple model R-tocopherol concentration correlated with increases in PV. Vol. HOSO.. Other parameters on oxidative after 24 months of storage in the dark. 60 °C HPLC. These differences in stability were attributed to the radical by EPR and the contents of polyphenols and tocopherols. No. TBARS oxidized at 60 °C. 40. SO. tocopherols. HPLC ambient storage. total sterols by capillary GC-MS. lutein. influenced the oxidative and loss of R-tocopherol. natural vs stripped EVOO TBARS is questionable 48 dark. effect of R-tocopherol on PUFA hydroperoxides. three samples of EVOO duction time at 70 °C (53). could predict oil listed in Table 3. OH tyrosol. oligomers PV. LLE of phenolic questionable OSI 59 FA composition phenolic extract compounds decrease in OSI stability a Abbreviations: see Table 1 footnote. room temperature storage for R-tocopherol. in this study and several oleic acid content. of predicting the future stability of EVOO. and compared with storage tests (50). FA questionable OSI stability 57 120 °C. CD. HPLC analyses (49). lutein. Rancimat 15 samples of EVOO tested by EPR polyphenols and tocopherols questionable Rancimat stability 53 correlated with Rancimat PV. CD. β-carotene. EVOO profiles. oxidized at 60 °C. Carotenoids and chlorophylls showed stability in the dark. Most Using a different approach. FFA. 25. dimers. the decrease in sensory characteristics. 50. complex effect of high temperature 50 PV. storage at 60 °C. CD olive oil stability polyphenols. CT. fotal phenolics. OSI at 100 °C tocopherol. TG monomers. 98 °C sterol composition and identification questionable OSI stability 55 model systems. and oxidative at 120 °C. Rancimat (OSI) is questionable 49 60 °C. Food Chem. oxidized TAG oxidative stability. In samples stored for 24 months in the dark at ambient temperatures another study (52). FFA. 40. times required to questionable Rancimat stability 58 FA profile in PUFAs. squalene o-diphenols. 2010 Frankel a Table 3. PV stability OSI. K270. melting thermograms of EVOO. changes in storage stability 56 R-tocopherol 21 months OH tyrosol and tyrosol complexes OSI. CD. screening stability/instability by EU model developed on 10 samples: PV. and 88 days and similar oxidative stability data based on the Rancimat at 110 °C and with trends by the OSI stability test at 100 °C and development of CD radical scavenging activity toward another artificial galvinoxyl (K232) (51). Rancimat β-carotene. squalene PV. decrease rate constants. CT. PV. questionable DPPH. unsaponifiables. total aglycon. based only on months. chlorophyll. K232. CD at K232 UV index. increase in PV. questionable DPPH antiradical test 52 DPPH sensory methods CD. tocopherol. Significant increases in CD and PV were observed at a three parameters may not be justified. K232. minor polar content. As expected. HPLC. CD. Arrhenius plots reach EU standards at 25. 5 and 8 °C quality and stability of fruits and oils sensory evaluations 47 PV. total phenols. polyphenols. pheo R-tocopherol. the contents of o-diphenols. fluorescent light PV. GC. 40 and OSI. Rancimat 54 DPPH Rancimat 60 °C liquid fraction. In another study. and carotenoids in stripped olive oil. and K270 measurements and other showed that a combination of only three parameters. the oxidative stability of EVOO of the R-tocopherol was lost after 72 h at 37 °C in thin layer and was evaluated by electron paramagnetic resonance (EPR) and after 96 h at 75 °C in bulk phase. and lipid oxidation status EPR. R-tocopherol. changes in oleic/linoleic acid ratio. Agric. Rancimat (OSI) is questionable 51 R-tocopherol. UV. tocopherol vs on tocopherol stability polyphenols PV. causing significant losses of R-tocopherol of 21-50% status of fatty acids on 10 samples. CD. K270. threshold concentration of R-tocopherol of 60-70 mg/kg.5996 J. PV. peanut oil used as model systems PV. This study provides another example of using an expensive . K232. total phenolics. polar phenols. OSI at 110. viscosity. stability storage: ambient. total polar. UV. 120 °C. Methods To Evaluate Lipid Oxidation and Oxidative Stability of Olive Oils methods conditions results limitations refs acidity. tyrosol. spin trapping with N-tert-butyl-R-phenylnitrone to measure in- Under accelerated storage at 60 °C. GC-MS oxidative stability. 46. containing complex and complex polyphenols after storage at room temperature for 7 mixtures of fatty acids and phenolic compounds. whereas squalene showed much higher stability. changes in VOO similar trends. the status included cis-trans and trans-trans-CD. phenolics. 10. The EPR results correlated with exhibited induction periods of 40. the authors used the questionable Rancimat test stability expressed by the PV. Unfortunately. multivariate statistical analyses of EVOO were followed by PV. 110. ES-MS. conjugated triene thermal oxidation of EVOO in bulk and in thin layer was K270. spin trapping. K270. In another stability study. total phenols. whereas the increase of K270 followed pathways of hydroperoxide decomposition by homolytic clea- apparent pseudo-first-order kinetics according to the linear Ar. on O2 pressures because the solubility of O2 decreases and the O2 Although the addition of polyphenol extracts and EVOO to concentration becomes a significant limiting factor that increases refined peanut oil significantly increased the OSI stability. 8). like and oxidation products were used to predict oxidative stabi. at elevated temperatures. 60 °C.5 and 5. are more relevant to rancidity and flavor deterioration. In another study using accelerated storage of EVOO at phase microextraction (SPME) and supercritical fluid extraction. 40.8-10%. In VOO.Perspective J.57-59) in Table 3. and temperature OSI conditions. PV and increase of K232 followed apparent carbonyls. Other effects of temperature (25-80 °C) have (Figure 1). Agric. These results provide further evidence that the Volatile Compounds. “quite good” for solid tions. changes in R-tocopherol from 12 to 23% and of total phenols Polymerization and cyclization of PUFA that become important from 43 to 73% after storage at room temperature for 21 at elevated temperatures are not significant at room tempera- months (56). sophisticated method based on measuring the tendency to form antioxidant levels followed by an increase in oxidized pro- radicals during early stages of oxidation. In oxidative stability tests. Although the PV did not exceed the upper limit of tures. vage into aroma volatiles included C5 alcohols. many others in the literature on the oxidative stability of olive oil. headspace and stable isotope dilution. Food Chem. the Rancimat and OSI stability tests can be especially misleading ture. 190 °C (62) or at 150 °C (63) and microwave heating for extended pletely depleted after 25 h at 100 °C.1-3. and rhenius equation. the addition of its phenolic extract resulted in a decrease of “good” for distillation-extraction. including artificial and severe thermal stress with the depletion of R-tocopherol under questionable high. Phenolic compounds in extra virgin olive oil and typical average values from 116 oil samples (78)..51. alcohols. and after times (64). esters. In a further stability study (57). 2010 5997 Figure 1. Although the results of ducts (59). this rate becomes dependent 110 °C with crude and refined peanut oil model systems (55). Two comprehensive reviews of VOO aromas analyzed by GC- A kinetic study of EVOO oxidation was carried out at 25. 58. and hydrocarbons (7. without added phenols. 150. and C8 alcohols and (PUFA). and 60 °C was attributed to the increase in mat or OSI tests that cannot be used reliably to predict storage concentration of unsaturated TAGs and decrease in polyphenol shelf life at room temperature (60). Some of the phenolic compounds were The EPR method (60) measures free radicals formed by initial also modified into secoiridoids and oxidized products. and “poor” for both static . 4 h at 120 °C. including products of lipid oxidation rather than breakdown products that carboxylic acid derivatives and loss of phenolic and acidic groups. and linolenate contents decreased by 2. which correlated with increase of K232. alcohols. The physical state of EVOO based on storage from 3 to 60 °C A majority of the studies (49. antioxidants would be destroyed. the with the degree of oxidation. MS listed approximately 180 compounds composed of volatile 60 °C in the dark (58). the formation of been reported on autoxidation and changes in antioxidant levels oxidation products from sunflower and olive oils was compared of olive oils (61).53-55. Additional questionable temperatures at which they are run. the polyphenolic antioxidants in EVOO are known to be in EVOO supplemented with phenols and after 2 weeks in EVOO decomposed significantly at the high temperature used in this test. after 14 h at 110 °C. therefore important to use several storage temperatures and in a Another oxidative stability study of VOOs reported significant range as low as practical and preferably at or below 60 °C (60). Unfortunately. Considerable attention has been devoted oxidative stability of EVOO cannot be tested reliably under the to the interpretation of sensory data of olive oils by capillary GC elevated temperatures of the OSI test because the phenolic of volatile compounds that influence odor and flavors of EVOO. These tests are not reliable content. aldehyde. Hydroxytyrosol and tyrosol levels increased linearly in most in evaluating the effectiveness of antioxidants (see section E on samples by the hydrolysis of the corresponding secoiridoids Antioxidants). they are not relevant to normal storage conditions. Because volatile acids that are measured by the Rancimat 20 mequiv/kg during this storage period. these authors used the questionable because the mechanism of lipid oxidation changes at the elevated DPPH antiradical and Rancimat tests. The results of Phenolic compounds were not very stable even at room tempera. an accelerated ketones. The deviation of zero-order rate constants for PV employed unfortunately questionable high-temperature Ranci- increases at 3 25. Important pseudo-zero-order kinetics. the respective linoleate and OSI methods are produced only at elevated temperatures. No. it is amounts of sterols added did not correlate with OSI stability. Although at ambient tem- stability tests were reported of the influence of total sterols on peratures. and esters. Vol. lity (54). From the loss of polyunsaturated fatty acids ketone. conditions used to monitor oxidation by heating at 100. the rate of lipid oxidation is independent of O2 antioxidant activity of EVOO based on OSI testing at 98 and pressure. These changes could be used as quality markers for EVOO. R-tocopherol was com. C6 aldehydes. The increase in quality parameters (K232 and K270) this method correlated with the questionable stability Rancimat corresponded with a decrease in tocopherols levels after 3 weeks test. 10. The most common techniques for quantitative analysis stability test was set up at temperatures below 60 °C to estimate of olive oil volatiles were evaluated as “very good” for dynamic the potential shelf life under normal storage temperature condi. The static HS (run at 110 °C and equilibrated for sensory effects of ethanol and ethyl acetate were described as 120 min) was considered to be not sufficiently sensitive and “winey. nonanal showed the highest rate of increment during oxi- The relative sensory impact of these volatiles was calculated by dation of VOOs and was considered to be the most suitable index their flavor dilution (FD) factors. “winey-vinegary” to acetic attributes by PCA and a “sensory wheel. 2010 Frankel a Table 4. Another (2.2-nonenal 91 16 1. and ethyl acetate. Flavor-significant potent volatiles of evaluations. the results vary with different unsaturated fied by “odor activity values” (OAV) based on the ratio between vegetable oils. and hexanol. The most potent aldehydes in of degree of oxidation of olive oils (70. too acid.” attributed to 1-octene-3-ol. and 2-undecenal linolenate hydroperoxides (6855 μg/kg) than from linoleate were the most abundant volatiles in oxidized olive oils (73).E)-2. identifying. Spain. position of hydroperoxides (60). pound that characterized the oil samples (72). Table 4 summarizes a list of potent aldehydes. The (DTD) (69).98) was the varieties analyzed. GC volatiles sible for the pleasant sensory characteristics of the oil and 51 were characterized by sensory attributes determined by olfacto- compounds as possibly responsible for off-flavors (67). a much lower temperature (40 °C) is Basic evaluations of the quality of VOO on the basis of flavor required for equilibration by HS-GC to avoid excessive decom- profiles were developed early by isolating.4-decadienal 422 16 10 18:2-OOH 3 samples.” and “moldy” and “earthy” defects were attributed to unsuitable to characterize olive oil volatile compounds. the GC volatile peaks were correlated to 52 sensory and propanoic and butanoic acids. 2-decenal. decomposition of the corresponding 11-. Another of 39 varieties from 8 countries cultivated together under the same study identified 60 volatiles in EVOOs by dynamic headspace agronomic conditions were characterized by 64 volatile com- GC-olfactometry and characterized 9 compounds to be respon. Virgin olive oils difficult to reproduce by panels from different countries. The MS signal octanal 382 32 6. because linolenate is aldehydes were shown previously (60) to be derived by homolytic known to oxidize twice as quickly as linoleate (60).5998 J. These hydroperoxides (2948 μg/kg). pounds analyzed by dynamic headspace GC (75). (Z)-3-hexenal (3).94) and were analyzed by SPME.8-21%) than those of linolenate (0. At New Tunisian olive cultivars were extracted. more precisely any adulterants with fewer samples without using 18:1-OOH. compound concn (μg/kg) Italy OAV (rn) originb rel FD 1995). and “rancid” to several many attributes represented vague descriptors that may be saturated and unsaturated aldehydes and acids. headspace solid phase headspace and direct injection GC. Other volatile com- nonadienal (2).. By using a GC-sniffing method to assess aroma humidity.E)-2.4. As a marker of oxidative referred to as “odorants. Although the max- FD factor imum limit for PV of <20 is accepted by EC regulation (EC. significance is not clearly established.E)-2. hexanal (2).” Unfortunately.9%) in olive oils (9). linolenate. hexanal 1770 8 24 18:2-OOH 2 Volatile compounds were also analyzed by headspace mass (E)-3-hexenal 36 16 30 18:3-OOH 3 spectroscopy (HS-MS) in olive oils mixed with different propor- (E)-2-hexenal 6770 32 26 18:3-OOH 4 tions of sunflower and olive pomace oil (68). R = 0.” from VOOs derived by lipid oxidation. 10-. 71). This study showed a wide variability in the chemical and heating at 100 °C and purging with O2 for 55 h. but their flavor (Z)-2-nonenal. as expected.E)-2. suggested as an appropriate method to detect initial oxidation.Z)-2. nonanal. (E. However. OAV. to evaluate oxidized lipids. including static headspace (SHS). 58. based on their FD factor. An early study conducted detailed By using dynamic headspace high-resolution GC-olfactome- analyses by dynamic headspace GC of 65 volatile compounds try.4-decadienal. octanal (4). The level of rancidity was microextraction (HS-SPME). precursors.4 18:2-OOH 3 were compared between nonadulterated and adulterated olive (E. 9-.4. (E)-2-hexenal. Correct classification of 100% and prediction of adulteration (E. Potent Odorants in Different Virgin Olive Oil Samples oxidized and good-quality VOO samples. 10. (E. odor activity values = ratio of concentration to odor threshold. study using HS-GC at a relatively lower temperature of 50 °C for higher relative concentrations of aldehydes are derived from 30 min showed that octanal. No. Vol. the early sensory characteristics of the VOOs as expected by the diversity of formation of nonanal (regression coefficient. (E. During storage over several months. 18:2-OOH. analyzing. status. 18:3-OOH = hydroperoxides of oleate. and 8-hydro- Table 5 summarizes selected methods used to evaluate volatile peroxides produced by oleate oxidation. and direct thermal desorption attributed to 2-heptenal or the ratio of hexanal/nonanal. 2-hexenol. Agric. 2-hexenal Although the concentrations of linoleate are much higher decreased and C6 alcohols and C5 ketones increased. no useful chemical information of un- a ique volatile compounds was reported to identify and quantify From ref 65. pounds identified included mainly hexanal. Several headspace techniques were compared with French VOOs. and (Z)-2-nonenal (4).8 18:1-OOH 4 (Z)-2-nonenal 28 32 47 18:2-OOH 4 intensities of all the ions from total volatiles without separation (E). 3-methylbutanol. Although the HS-GC approach is more precise than VOOs from Italy. After metry. sensory methods. the most prominent volatile compounds responsible for the from VOOs that were related to sensory attributes by 5 European main sensory defects of olive oils (74) included “mustiness- panels (66). compounds in olive oils. Lipid the origin of volatile oxidation products and individual flavor oxidation derived aldehydes of high OAVs included propanal.Z)-2.Z)-2. FD. Another study based Table 4. antioxidants. decadienal (3). benzaldehyde. (Z)-3-hexenal. and Morocco were isolated and quanti. (E)-2-nonenal (3). showing 46 compounds characterized the ratio of hexanal/nonanal were used to differentiate between by GC-MS (76). and the oil volatiles later stages of oxidation. Headspace their concentrations and their nasally and retronasally odor analysis by the SPME method provides useful information on threshold values when added to a bland sunflower oil. “fusty” to ethyl butanoate notes. b From ref 60. the sensory panel rejected the oxidized oil when the PV was <4. 3-hexenol. Total and individual volatiles can be correlated hexanal. and LDA. and between oxidation time and flavor scores. and metal chelators. include in decreasing order of on headspace SPME showed that 2-hexenal was the main com- flavor impact (E.4-decadienal (1). and evaluating flavor-significant volatiles by high-resolution gas The analysis of oxidation products in VOOs by GLC can chromatography and gas chromatography-olfactometry of provide useful markers to compare with sensory panel of flavor headspace samples (65).4-nonadienal 49 8 <1 18:3-OOH 2 oils. New previously unreported volatiles included . linoleate.4-1. the formation of hexanal (R = 0. flavor dilution factor = extent of dilution required for detection. Unfortunately. Food Chem.4-decadienal 255 <8 13 18:2-OOH 1 were claimed by using LDA chemometric techniques with 121 (E. (E)-2-hexenal (4). compounds (79).9-4. Rancimat and DPPH. DTD >60 compounds identified by SHS not sensitive enough SPME and HSSE were more 69 GC-RI. UV. 2010 5999 a Table 5. and β-selinene. MVA statistical and effect hydroperoxide precursors and analyses volatiles dynamic HS-HR-GC-FID relationship between volatiles dynamic HS involves high-temp systematic comparison GC 74 olfactometry and sensory defects desorption olfactomtry and sensory dynamic HS-GC. GC-MS successful than SHS SPME-MS-FID best fiber coating (DVB-CAR. No. HS-SPME. climate and environment. 72 representing 85.19 mg/kg) were reported.9 mg/kg) and carotenoids rich in oleic acid (56-84%) and linoleic acid (3-21%) (9. swept GC sniffing of 65 compounds. Commer- Antioxidants. sensory. correct classification and 68 of olive oils with sunflower and qualitative threshold prediction by LDA chemometric olive pomace oil. statistical 66 with N2. drupe freshness of Tunisian olive oils (77). 78). ABTS total polyphenols and SAT/ 77 sensory hexanal/(E)-2-hexenal is antiradical methods PUFA-18:1/18:2 ratio = major important indicator of degree parameters for antioxidant of oxidation stability a Abbreviations: see Table 1 footnote. Ratios of total polyphenols ripening. Although claims were extraction and processing conditions on the prevalent classes of made that these oils provide a rich source of natural antioxidants.68-4. acid but also due to the abundance of its natural phenolic “Oleaster” virgin olive oils were thus regarded as distinctly antioxidants. (E)-2-hexen-1-ol. and (1. Food Chem. 58. evaluate antioxidants in olive oils and their limitations. phenols and o-diphenols.3-92. Several reviews on VOO included the effect of different from European and Tunisian oils. by the questionable high. 64 volatiles analyzed in 39 origin of samples determined by comparison GC-olfactometry 75 olfactometry varieties of VOO for volatiles statistical SLA and sensory for VOOs SPME. and and saturated to PUFAs (and/or ratio of oleic to linoleic acid) storage and oil processing techniques. 121 samples techniques SHS. 10. Agric. time of harvesting. trocosane. the their oxidative stability and antioxidant activity of phenolic hexanal/(E)-2-hexenal ratio was considered to be a very impor. Olive oil has been generally considered to be cial olive oil samples were evaluated to determine the factors nutritionally desirable for its health properties and oxidative influencing their oxidative stability (80). GC-EIMS. capillary GC DHS-GC. >100 compounds necessary analyses of VOO samples identified SPME. SPME-MS relationship between FA profiles correlation data are not = cause good relationship between 73 and volatiles. PUFA loss HS-SPME volatiles compared pentanal from 13-LOOH coeluted determination of oxidation 71 with standard methods with 3-pentanone reactions HS-SPME GC-RI/MS 41 compounds isolated olive quality limited to (E)-hex-2-enal decreased. their special fatty acid composition only levels of chlorophylls (1. HSSE.7% of (E)-hex-2-enal C6 alcohols and C5 ketones total increased on storage GC. PV.6 °C identification of genetic new 76 characterized by GC-MS = varieties of olives and VOOs 85-98% of total amount SPME. Tenax trap. cold pressing of the olive paste.Perspective J. GC-CIMS. Vol. GC-MS. (E)-3-hexen-1-ol. identifications limitations attributes refs DHS-HR-GC samples heated at 40 °C. 60 volatiles identified by excessive thermal oxidation samples evaluated by 4 trained 67 HRGC-olfactometry HRGC-olfactometry fresh and assessors initially and after oxidized at 100 °C for 55 h aging volatile methodology. Methods To Evaluate Volatile Compounds in Olive Oils methods conditions. Different trends in stability not only because of its relatively high content of oleic antioxidant activity were observed when tested at 60 °C according . unfortunately. temperature OSI test and artificial antiradical DPPH and ABTS Table 6 presents selected studies based on methods used to tests. The highest concentration were shown statistically to be the major parameters in oil of these compounds is found in EVOO obtained from the first antioxidant stability. arbitrary and vague. desorbed 9 unidentified sensory wheel at 220 °C. determination of response factors qualitative and quantitative 70 PDMS).. A variety of minor constituents found in the tant indicator to estimate the degree of oxidation and of the unsaponifiable fraction of olive oil depends on cultivars. LOX static HS compared with more emphasis on lipoxygenase improved sensitivity of static and 4 pathways enrichment step dynamic HS than chemical pathways dynamic headspace GC HS-MS volatile compounds from mixtures excessive heating 120 °C. In another study. hydrophilic phenols found. GC-MS 45 compounds isolated and Rancimat used at 101. radical scavenging. tyrosol. 10. interference by redox EVOOs metþH2O2 agents GC-MS extraction with MeOH/water claim of di-OH-phenylacetic acid as several new minor phenols (0.. oxidation products identified by MW oleuropein and ligstroside aglycons 88 oxidized products only and oxidation products. ABTS/ room temperature. ABTS assays in use of Rancimat at 80 °C. acids. diacetoxyoleuropein oxidation aglycon. FRAP.5 101 (80:20 v/v) initial oxidation product mg/kg) a Abbreviations: see Table 1 footnote. LLE-SPE-HPLC. and tyrosyl C4. RP-HPLC polar fraction oxid stability at 80 °C. No. 58. flavonoids polyphenols from olive different phenolic extracts tested by limited polyphenol composition tested limited polyphenol composition of 92 leaves HPLC tested in bulk oil and emulsions olive leaves simple. 84 R-tocopherol PV. OSI OH tyrosol. shaking relatively slow oxidation phenol extract. Food Chem. 85 RV-HPLC phenols.6000 J. LDL questionable TBARS and DPPH tests OH tyrosol. standard AOCS PLS regression analyses R-tocopherol. oleuropein/aglycon SPE and RP. incomplete separation by HPLC ohenyl alcohols. effect of storage at 37 and 75 °C on incomplete recoveries R-tocopherol. R-tocopherol fractions coumaric. Vol. hydroxytyrosol. hexanal oxidized at 60 °C. UV. lignans. Methods To Evaluate Antioxidants in Olive Oils methods conditions. . C18:1. OH tyrosol. oleuropein (MeLo) aglycon HPLC-DAD LDL oxidation HPLC separation and identification of MDA is not reliable end point of LDL OH tyrosol. TMS ethers. tyrosol. ligstroside aglycon minor polar compounds.2-1. FFA FA profile storage in diffuse and dark light. syringic. 80 R-tocopherol SPE. phenolic acids 180 °C stability derivatives. 94 storage shelf life oxidation. o-diphenols. C18:2 96 MeOH antiradical tests 3. 89 antioxidant activities activity in liposome and Me linoleate MeLo are questionable dialdehyde derivatives. vanillic. secoiridoids. o-diphenols. C16. OH tyrosol. OH tyrosol. 2010 Frankel a Table 6. elenolic ac. 91 fluorometric detection NMR identification oleuropein. p-coumaric. RV-HPLC of reference compounds. tyrosol. complex phenols. partially quantified tyrosol. OSI are questionable simple phenols. O2 flow total phenols. C18. in bulk oil. 99 acids optimized by SPE ferulic. C12. analyses before and after 12 months bleaching of β-carotene. 12 months linoleic acid. and vanillic acids β-carotene-linoleate β-carotene bleaching. secoiridoid derivative 87 extraction lignans SPE-GC-MS. and 83. emulsions 97 to yield the dialdehyde form method and LDL phenolic profile by HPLC. OH tyrosol. oleuropein 93 lignans. 86 electrophoretic methods phenolic fractions aglycons HPLC. CZE liquid-liquid and solid-liquid DPPH. oleocanthal direct injection HPLC. storage at not specific test with emulsified crude phenolic extracts of Italian 100 bleaching.HPLC Rancimat at 100 °C Rancimat is questionable o-diphenols. elenolic acid 90 OO phenolics. HPLC-UV/MS CE-UV heating at excessive thermal conditions. identifications limitations antioxidants refs PV. HPLC-DAD. HPLC-MS. tyrosol. inhibition of LDL oxidation derivs. antiradical total and individual phenols 98 LC-MS of pure standards ofstorage ABTS CE-ESI-MS polar fraction containing phenolic focus limited only to phenolic acids hydroxyphenyl acetic. antiradical DPPH and antioxidant antiradical DPPH test and use of flavonoids. Agric. HPLC phenolic extracts. 81 tyrosol esters APCI-MS methanolic extracts crude olive oil qualitative. OH tyrosol. complex phenols. OO samples from 95 method: tocopherols France and Spain synthetic tyrosol esters Rancimat. OH tyrosol acetate. 82 ligstroside. TBARS. total incomplete characterization of HPLC tyrosol.4-DHPEA-EDA Bis-Me acetal of oleuropein acidified use of questionable antiradical DPPH strong antioxidant in oil. elenolic ac derivs. DPPH oleuropein derivs LC-MS tocopherols total phenols. CE. expected to decompose significantly at the elevated temperature Unfortunately. and complex phenols were studied during storage olive leaf to obtain the same stability as VOO (93).4-DHPEA-EDA and p-HPEA-EDA (9) (Figure 1). the remaining levels of these (61. hydroxyl-oleuropein. lignan tyrosol. and their antioxidant potency deacetoxyl-oleuropein aglycons.4-DHPEA-EA. ate. R.4 for the RBD tion of phenols decreased with ripeness of olive fruits. oleuropein aglycon. and oleuropein aglycon (25. The but was more effective than the other phenolic antioxidants in radical scavenging and antioxidant activities of phenolic com- inhibiting hexanal formation in RBD olive oil. followed by RP-HPLC and colorimetric deter. hydroxytyrosol. and osols. Vol. a secoiridoid derivative (diacetoxy oleuropein ized (RBD) olive oil (8 ppm GAE) used as control. The antioxidant activity of VOO was mainly due to the Virgin olive oil was separated into fractions by SPE and dialdehyde of elenolic acid linked to hydroxytyrosol (3. 10. the effects of depleted. correlated with their total polar compounds. oleocanthal. Oxidation products from the aldehydic and dialdehydic forms of Tocopherol behaved as a prooxidant at high concentrations elenolic acid and of ligstroside and oleuropein aglycons were (>250 ppm GAE) based on PV for hydroperoxide formation. but better aldehydic and dialdehydic forms of elenolic acid linked to tyrosol antioxidant activity was observed at 100 and 200 ppm GAE on and hydroxytyrosol (Figure 1) as the most abundant compounds. SPE cartridge and analyzed by GC-MS as trimethylsilyl (TMS) p-coumaric acid. At the same milli. and the loss of tempering influence of steam produced by the moisture in total phenols was much lower than that of R-tocopherol. the hydrophilic phenols from VOO were analyzed molar concentrations. Phenolic compounds extracted from VOOs increased nately. by direct injection HPLC using a fluorescence detector and and different o-diphenols showed similar oxidative stability by the compared with traditional LLE followed by HPLC (92). ligstroside and oleuropein agly. Three HPLC fractions by HPLC with diode array detection into tyrosol. The foods (60). ligstroside aglycon. In structure of the ligstroside aglycon (83. oleuropein aglycon. In another study. cons. cinnamic acid. the basis of hexanal formation by static headspace GC.9%). deacetoxyl-ligstroside and (MDA) and conjugated dienes. their oxidative aglycon). A different study (94) aimed at the R-tocopherol was decomposed by 39-45% after 6 months and thermal decomposition of EVOO at frying temperatures showed total phenols by 50-62% after 12 months. and one was tentatively identified as an ester of opein aglycon. and decarboxymethyl oleuropein aglycon. decarboxymethyl oleuropein agly- compounds ranged between 50 and 73% under diffused light and con (28. tyrosol. hydroxytyrosol.Perspective J. factors including metal concentration. and R-tocopherol ether derivatives (88). and luteolin (90). R-tocopherol and complex polyphenols on the oxidative stability and the lignan aglycon (3%) were more thermally resistant. diacetoxy-oleur- syringic acid. hydroxytyr- were identified as mixtures of hydroxytyrosol. 58. and deodor. hydroxytyrosol. Depletion of R-tocopherol peratures is known to cause excessive thermal damage without the showed an inverse correlation with increase in CD. including catalyzed oxidation of human LDL based on malonaldehyde tyrosol. The effects of changes in the concentrations of R-tocopherol. secoiridoid derivative. Other MDA is a notoriously unreliable marker of lipid oxidation in studies separated VOO phenols quantitatively into flavones and foods and biological systems (60). The further claim that the lignans by SPE.5%). the use in this study of the antiradical method DPPH and the oxidative stability when added to the control RBD olive oil. vanillic acid.3%) were rapidly between 40 and 62% in the dark. elenolic acid (56%). The phenolic extract had the best hydroxytyrosol. Although the samples of VOOs contained higher levels of total In another study using HPLC and capillary zone electrophor- phenolic compounds. 2010 6001 to whether measurements were made of either PV for hydroper. tyrosyl and hydroxytyrosol acetate. complex phenolics were more active antioxidants in liposomes tions and the oxidative stability of olive oils and (b) the fact that and aglycons in bulk lipids. Unfortu- olive oil. elenolic acid. and time of year Complex phenols were the least stable. DHPEA-EDA). than did the refined. Better questionable Rancimat test at 100 °C and were more stable than efficiency and quantitation were obtained for phenyl alcohols and R-tocopherol and tyrosol. separation of many phenolic components analyzed. ranging were detected as the main components in EVOO. Refined olive oil was supplemented with polyphenol extracts of total phenols. After 4 months of storage under diffused light. tions analyzed for total phenols and o-diphenols were tested for Phenolic compounds from two Italian VOOs were separated antioxidant activity at 80 °C and O2 flow. R. vanillic acid). but lower efficiency and quantitation were questionable because the polyphenolic antioxidants would be found for 3. Concentrations of tyrosol and 5-hydroxytyrosol . These results of oxidative stability are 3. the HPLC chromatograms showed incomplete used for the Rancimat test. of EVOO were compared at room temperature in glass bottles Unfortunately. and two lignans (pinoresinol and acetoxypinoresinol) stability was decreased by their relatively high initial PVs. Identification of 21 compounds included added to the RBD olive oil. oil extracted from leaves was evaluated for oxidative stability and tocopherol was decomposed by 79% and the phenols by <45%. of the collection of leaves. cultivar. heating olive oils without foods at frying tem- and at 37 and 75 °C as thin layer (86).4- emphasized (a) the need to measure at least two oxidation dihydroxy moiety linked to an aromatic ring (89). This study pounds from olive pulp and olive oils were due mainly to a 3. Frac. ranging from 63 to 534 ppm gallic acid esis (CZE) (87). The oil extracts inhibited the Cu- APCI-MS in methanolic extracts of crude olive oil. The concentra- from 11 to 33 mequiv/kg compared to a PV of 0. and isomers of oleuropein (9). and equivalents (GAE). detected and their structures confirmed by HPLC-APCI-MS. phenolic profiles were identified by derivative. elenolic acid and derivative. another study. simple phenols (tyrosol. The antioxidant activity of lignans the antioxidant effectiveness of phenolic compounds in VOOs can was attributed to their chelating properties toward copper acet- be diminished in oxidized oils. 84). The antioxidant activity of a mixture of phenolic compounds Phenolic compounds in Spanish VOO were extracted with an extracted from VOO was compared with that of pure caffeic acid. Italian olive oils influenced biological activities is unsupported by mination of o-diphenols and by NMR to confirm the aldehydic most recent studies on absorption and bioavailability (91). After storage in the dark. Refined olive of VOO (85). Agric. but tyrosol (19. antioxidant activity test OSI is questionable (79).4- analyzed by RP-HPLC before and after hydrolysis (81). Unfortunately. oxide formation or hexanal for hydroperoxide decomposition. various phenolic compounds. hydroxytyrosol acetate (5%).3%). and antioxidant activity at 50 ppm GAE on the basis of PV. When the olive oil that after heating at 180 °C for 30 min the levels of hydroxytyrosol reached a PV of 20 mequiv/kg. A stability study of bottled EVOO reported changes phenolic fraction isolated by HPLC from VOO was extracted by in minor polar compounds after storage for 18 months at 18 °C in liquid-liquid (LLE) and SPE and analyzed by MS as mixtures of the dark (95). squalene. bleached. tyrosol.. In another study (82). Food Chem. tyrosol. No. Glucosides and parameters to better evaluate antioxidants at different concentra. 6002 J. The β-carotene-linoleate showed induction periods varying from 40 to 88 days at 60 °C. The activation energy of lipid oxidation is higher in the A paper on phenolic extracts from 29 monocultivar olive oil presence of antioxidants (∼20-25 kcal/mol) than in their absence samples reported that French olive oils had lower total phenol (∼18 kcal/mol). powerful antioxidant than hydroxytyrosol (102). which. 3. after the same storage period.53. spectra of complex hydroxytyrosol derivatives (98). carotenoids artificial ABTS [(2. respectively. 3.2 radical cation and in the lipid phase by the β-carotene-linoleate mg/kg) (9. to evaluate the antioxidant activity of olive oil phenolic com- pounds after storage for 18 months at 18 °C in the dark (95). pounds. Many studies on the adulteration of EVOO antioxidants for their inhibition of lipid oxidation (79).026 mg/kg). form of elenolic acid or R-tocopherol. information on olive oils that may obviate too much dependence droxy-2. on statistics. 0. ABTS/metmyoglobin þ H2O2 monitors Several potential problems become apparent from the exten- the decay of ABTS radical. The antioxidant activity in susceptible to oxidation due to their PUFAs (5-9%) and minor the aqueous phase was tested by the radical scavenging of the constituents including chlorophylls (9-20 mg/kg). bleaching test measuring the loss of β-carotene in an emulsified with PV ranging from 6 to 37 mequiv/kg after ambient storage aqueous system in the presence of O2 at 50 °C is nonspecific and between 11 and 24 months (79). The authors concluded that the phenolic compounds choice of methods and conditions to evaluate oxidative stability were not able to scavenge free radicals. 79. Although the phenolic content was strongly widely because their initial PV can range between 0.54. Food Chem.4-dihydroxyphenylacetic acid For the adulteration of EVOO with cheaper soybean and (0. weight phenolic compounds by distillation and the continuous In another advanced structural study by 1H and 13C NMR stream of air bubbled through the oil at high temperatures. 58. coumaric acids (99). Italy. any BHT and R-tocopherol (97). oxidative stability. trans-isoeugenol now available to provide more precise and accurate chemical [trans-2-methoxy-4-(1-propenyl)phenol] (0. and antioxidants is extensive and (CE-ESI-MS) was used to identify and determine seven selected often difficult to interpret. A wide variation in test methods is antioxidants (cinnamic and benzoic acids) and three isomeric used to determine adulteration. Although olive oils are A different approach was aimed at determining chemical generally stable to oxidation because of their relatively high oleic changes in Italian EVOO samples during storage at room acid content and natural phenolic antioxidants. oxidation based on capillary electrophoresis-electrospray interface-MS products. linoleic acid substrate (60). and vanillic acids For many oxidative stability studies of EVOO. TheresultsofmanystudieslistedinTables3(49.17 mg/kg).005 mg/kg). 10. the activities of these natural antioxidants are most concentrated polyphenols in olive leaves including the bis. hydroxyphenyl acetic. the end-point of lipid oxidation.51. using Trolox as reference. Powerful analytical methods are 4-hydroxyphenylacetaldehyde (0. A “new” analytical method The literature reviewed on olive oil adulteration. the evidence presented referred more decreased between 12 and 18 months of storage. In contrast. Unfortunately. For these reasons the results are often widely diverging have a much stronger antiradical activity by the questionable when antioxidants are compared at temperatures below and antiradical DPPH test (60) than the corresponding dialdehyde above 60 °C (91). An Arrhenius plot compounds by LC-MS except for lower pinoresinol in the French of log induction period versus the reciprocal of absolute tem- oil (96).6-dimethoxybenzene (0. However.550). ferulic. Therefore..59) tion measured by the questionable TBARS test and antiradical and 6 (83.58. including effectiveness of natural or added antioxidants. the specifically to a naturally occurring oxidation product of hydroxy- secoiridoids and diacetoxy-oleuropein aglycone were depleted by tyrosol.4 and 33 correlated with β-carotene bleaching test (R = 0. 2010 Frankel were relatively stable for the first 12 months and then rapidly in olive oils. Interactions between minor components in correlated with the scavenging activity by the ABTS radical cation EVOO and trace metals can produce prooxidant effects. Storage under different condi- influenced by the micelle properties of an inappropriate emulsified tions resulted in losses of polar phenolic antioxidants of 18-38%. No. 87. proved to be a more 34-55 and 38-61%. and does not test kinds of olive oils. markedly influenced by the effect of elevated oxidation tempera- methylacetal of oleuropein aglycone secoiridoid (9) was shown to tures. it was weakly mequiv/kg (80). p-coumaric.4-dihy. measures sive literature published in the past several decades on different reactivity toward the artificial ABTS radical. zyl alcohol (0. and 3. In another study. Vol. more sensitive and reliable methods (60) idants in olive oils include the loss of volatile low molecular were needed to evaluate antioxidant activities of the tyrosyl esters.839). 84. without a suitable lipid substrate (60).023 mg/kg).20 -azinobis(3-ethylbenzothiazoline-6-sulfonate)] (>10 mg/kg).5-3. The presence in substantial amounts of and oxidative stability and to evaluate phenolic antioxidants (9). are often used to accelerate oxidation. new synthetic temperature decreases (60). 0. Agric. The authors suggested that these compounds could canola oils that are most available in the United States and be used to characterize olive oils. the hydroxytyrosol testing for oxidative stability at lower temperatures requires esters were more active than hydroxytyrosol in methanol solution testing at several different temperatures. 97) confirm the misleading application of DPPH test. 1. they are still temperature for 12 months (100). because antioxidants lower the rates of oxidation content than Spanish samples but similar individual phenolic by increasing the overall energy of activation. 94. The quality of stored commercial EVOOs can vary bleaching method. and Spain invalid tests for antioxidant activity. including samples (footnote of Table 1). Other problems of the by the FRAP and ABTS methods used without a suitable lipid frequent use of the Rancimat and OSI tests to evaluate antiox- substrate. Similarly. hydroxytyrosol and may indicate initial autoxidation processes Linolenic acid ranging from 7 to 10% in soybean and canola oils . like other o-diphenols. one of the Furthermore.4-Dihydroxybenzylacetic acid Canada. Unfortunately.034 mg/kg).4-dihydroxyben. Cu. The (R = 0. The temperature coefficients are lipophilic esters of tyrosol were less active by the Rancimat test different for a fat according to the relative concentration and run at 80 °C than hydroxytyrosol and its analogues.001-0. Oxidative more example of a doubtful conclusion made on the basis of two stability evaluations of EVOOs from Greece. with cheaper vegetable oils were based on advanced sophisticated Several new minor phenolic compounds were detected in statistical methods that require the analyses of large numbers of methanol/water extracts of 34 VOOs by GC-MS (101). A stability the Rancimat and OSI tests for oxidative stability and especially study of bottled EVOO reported changes in minor polar com. and metal impurities (Fe. “antioxidant” activity was based on LDL oxida. This study represents one and antioxidants is therefore critical (60. 91).79). Amounts of R-tocopherol were generally different among perature shows that the effectiveness of antioxidants increases as the five cultivars examined. many potential problems may be caused by high- was reported as a naturally occurring oxidation product of temperature GC to analyze olive oil directly for adulteration. drastic conditions was confirmed in Spanish VOO and EVOO samples. H. M. Casals. 2002. L. These antioxidants would not be destroyed by using (10) El-Hamdy.. The use of the tion by measuring its authenticity factor using reversed-phase high- antiradical methods DPPH and ABTS to determine antioxidant performance liquid chromatography... M. 2nd ed. J. Technol. S. pp 173-190... Carrilho. the gas chromatograms. 1078. radicals by using completely artificial scavengers in the absence of (11) Dionisi. Detection of pressed hazelnut oil in LITERATURE CITED virgin olive oil by analysis of polar components: improvement and (1) Angerosa. Gómez. A.. T.. Soc.. Gordon. authentication. Aparicio. L. A.. (20) Guillén.. R. M. N.. Agric. 2nd ed.Perspective J.. analysis of soybean oil in olive oil by high-temperature capillary gas Chromatogr. 105. P... (13) Parcerisa. J. J. Galli. Review. F. Magri. Hidalgo. Hidalgo. L. Technol. D. No. Technol. Soc. 84. Soc. Marini. Oil Chem.. 75. Assessment of olive oils suitable lipid substrates (79). Prodolliet. 688–696. I. K. A. Zervou. sunflower oil adulteration in extra virgin olive oils from the Eastern Many claims have been made regarding the nutritional benefits Mediterranean by visible and near-infrared spectroscopy. J. Am. K. Aspen Publishing: Gaithersburg. The quantitation of olive oil samples adulterated with these (5) Kiritsakis.. Tagliaferri. J. Detecting and quantifying precisely the adulteration of EVOO with vegetable oils. Agozzino. more detailed 267–272. Magri. R. Bongiorno. 50. K. 2002. J... A. Chromatogr. Eur. Boatella. D. 5520–5525. Dobarganes. J.. Servili.. R. MD. 2002. Blekas. Chromatogr. Selvaggini. P. M..resolution 13C NMR spectroscopy. however. R.. Am. McIntyre.. benefits of natural antioxidants in EVOOs.. fractionation as a preliminary step in the characterization of senting vague descriptors that may be difficult to reproduce by vegetable oils by high. Campestre. J. Handbook of Olive Oil. proton NMR of unsaturation and composition of acyl groups in vegetable oils.. Cabrini. 2006. This subject will be 50. Lipid Sci. J. A 1995. changes. Avellone. G. 1054. 2002. F. F. S. Cunha. Use of high- the kinetics of lipid oxidation and do not evaluate breakdown resolution 13 C nuclear magnetic resonance spectroscopy for decomposition products causing rancidity in olive and other oils. Food Chem. and unsupported by the most recent (19) Bucci. J. Such claims may be Food Chem. Oil Chem. Kolokouris. Eur. Another questionable and relatively expensive method used to Analysis of olive oil and hazelnut oil mixtures by high-performance measure lipid oxidation is based on measuring the formation of liquid chromatography-atmospheric pressure chemical ionization- free radicals during the early stages of oxidation by EPR or ESR mass spectrometry of non-saponifiable compounds (tocopherols using artificial free radical scavengers (60). C. H. Chapter 7: Analysis and validation of the method. 72. 405–411. High-performance liquid chromatography-mass spec- (2) Antoniosi Filho.. J. C. In Olive Oil. L. Useful information of unique volatile using FT-IR spectroscopy. (21) Zabaras. C. Soc. Codony. R. A 2005. Glasante. More reliable and quantitative quality evaluated by analytical approaches and sensory panel. Lipid Sci. 2002. and the ABTS/metmyo- Petrakis. methods available for TAGs and minor constituents of vegetable (8) Angerosa. E. 73. 708. (6) Velasco. analytical results may be achieved by exploiting many HPLC J. J.. J. 90–97. N. Oil Chem. A. IL. chromatography. G.. Influence of volatile compounds on virgin olive oil nents by thermal decomposition. 475–483. K. N. Haddad. H. 79.. 104. F.. J. Ed. 661–676. Esposto.. A Tables 3 and 6. Agric. Boskou. 628–638. Characterization of vegetable and Properties. K. Lipid Sci. Am. 2006. A wide variety Chemical authentication of extra virgin olive oil varieties by of biological protocols have been used to evaluate the nutritional supervised chemometric procedures. 104. Volatile compounds in virgin olive oil: Questionable methods were used in several studies listed in occurrence and their relationship with quality. Rapid simultaneous determination by gical properties of EVOOs and their antioxidants. 78. A. Marini. 2010 6003 may undergo excessive thermal decomposition at the elevated (4) Kiritsakis. A novel method to detect adulteration of virgin olive globin þ H2O2. E. B. Davies.. R. Bogani.... J. activity of EVOO are also questionable because they measure free 351–355. D. temperatures (>350 °C) used for GC separation of TAGs (103). of using olive oils in the Mediterranean diet. compounds could also be exploited to identify and quantify more (18) Downey... the screening of virgin olive oil. Rafecas. In Rancimat and OSI tests the phenolic antioxidants in EVOO are Olive Oil. Chapter 8: Healthful properties of olive oil minor components. D.. carotene-linoleate bleaching test.. L.. D. G.. Soc. G. Mauer. 1993. ABTS radical. R. F. Eds. et al. F. Langas. Alba. Kanavouras. Analysis Sawaya. G. D. The use of high-temperature GC may Eur. chemical information may be expected by supplementing the (16) Zamora. F. J. Eur. (23) Catharino. El-Fizga. K. N.. AOCS Press: Champaign. Oil Chem. 74) graphically obtained oil fractions. information is needed to relate volatile profiles with sensory (17) Ozen. M. measuring the loss of β-car. Ruiz. Chemistry and Technology. studies on absorption and bioavailability (91).. 673–681.. AOCS significantly destroyed and the mechanism of lipid oxidation Press: Champaign.. 17–31. panels from different countries.. Much literature on olive oil sensory tests has been based on (15) Zamora.. 261–266. Flavor components of olive oil ... R. 2000. D. Food Chem. Agric. without testing for inhibition of lipid oxidation. B. 2003. 149–158.. (12) Mavromoustakos. F. F. Ed. reviewed in a separate publication on the nutritional and biolo. Eberlin. These methods use and sterols). J. 89–93. quality vegetable oils would also be compromised because the TAGs control and packaging issues of olive oil.. Technol. 2001.. A. F. 1998. A. J. 58. Visioli. Gómez. including the Rancimat and OSI tests for 2004. Chapter 5: Polar phenolic for antioxidant activity (79). oils by electrospray ionization mass spectrometry fingerprinting: . C. L. A. Lipid Sci. Am. G. completely artificial synthetic free radical scavengers that perturb (14) Zamora. F. P. J. I. Boskou. Dobarganes. 2002. Hidalgo. 2002. 881. J. M. raphy/amperometric detection of tocopherols and tocotrienols.. A 2000.. Oil Chem. Oxidative stability of virgin olive oil. G.. More precise and diagnostic Oil Chem. Vékey. oils. J. R. J. Bonas. Gómez. Tsimidou. containing linolenic acid are distributed among several peaks in 2002. 70. Vol. Am. 10. Soc. Oil panels trained to recognize and evaluate many attributes repre. Am. A. pp 113-172. pp 73-92. monitoring the reactivity toward the artificial oil by other oils.. (7) Angerosa. adulteration by reverse-phase high-performance liquid chromatog- Other invalid methods used for olive oils include the β. Detection of hazelnut oil adulteration attributes and defects. Am. have been used to characterize samples of EVOOs. 3898– 3907. Oil Chem. IL. M.. 1505–1511. Food Chem. 1995. J.. Chemical analysis. A. 50.. Agric. 2000. Soc. oils by high-resolution 13C NMR spectroscopy using chromato- Although some detailed GC-/olfactometry methods (66. D.. G. R. 1051–1053. exaggerated. 79. C. Montedoro.. 413–418.a review. 639–660. Chromatogr. M. Ceraulo. Detection of olive oil adultera- oxidative stability tests run at 60 °C (80) or lower. oxidative stability and the antiradical methods DPPH and ABTS (9) Boscou. also influence the quantitative analyses of non-glyceride compo. Classification of vegetable sensory tests with capillary GC analyses of olive oil volatiles. E. M. J. 77. J.. Taticchi. 104. (3) Harwood. (22) Nagy. Quantitative trometry based chemometric characterization of olive oils.. otene in an emulsified aqueous system. J. V. P... Chemistry and Technology. G. 2004. R. At the elevated temperatures of the compounds.. Food Chem. J. J. Ezzatpanah. 2005. Agric. A. C. G. Velasco. V. 2006. E. 969–976. (40) Gertz. 50. A.. A. P.... Lipid Sci. J. J. F. Pirisi. F.. Food Chem. A C. Food Chem. 2007. A. J.. J. 55. Food Lipids 1999. 2009. 36–42. Guo. J... of stigmasta-3.. Sotiroudis. M.. T. Chem.-L. Cert. J. 717–725. Technol. Martı́n-Polvillo. M. Constante. (30) Lerma-Garcı́a. Agric. L. F.... L.. 118. M. P. (28) Poulli. to estimate virgin olive oil potential shelf life. 107. 66–76... Evolution of major and minor components and oxida- Fourier-transform infrared spectroscopy.. Synchronous Moreda. Chaniotakis. Salvador. 103. Formation accelerated storage treatment. D. J. E.. F.. Vol. ripening and olive oil quality. Reniero.. J. 2008. Ramis-Ramos. 2010.-H.. Dobarganes. 28.. 3200–3207. G. 2008. A. H.. L. Bendini.. Valli. A. V. E. 85. Herraiz. Ramis-Ramos. Geographical characterization of Greek virgin olive oils (51) Hrncirik.. Dutta. V. V. (27) Poulli. 2002. extraction and multidimensional gas chromatography. Héberger. (52) Zanoni. Chim. Food Chem. A. Anal.. Sterol fraction s in level in thermally stressed olive oils by GC coupled to acetonitile CI- hazelnut and virgin olive oils and 4. 2005. J. 2007. 1502–1507. by-step) detection. M. 518–524.. C. W. E. X. (48) Khan. Agric. Food Chem. G.5-diene during deodorization and/or physical refin. (56) Gómez-Alonso.. Food Chem.6004 J. Cert-Trujillo. Lipid Oxidation. Ghavami. M. tion mass spectrometry data. N. K... L. Sovrano. 465–471. Food Chem. Oxidative stability and H.. S. xidation studies. P. (47) Garcı́a. Tsimidou. M. Manzocco. olive oil adulteration. Effects of membrane-based soft (25) Cunha. A.. Mariani.. Stavyiannoudaki. Rapid oxidation of commercial extra Herrero-Martı́nez. J. M. Jiménez. K. K.. J. Oliveira. Pioch. S. D. A new analytical procedure to differ. Acta 86. Cerretani.. Eur. Food Chem.. Agric... J.. spectroscopy for the rapid assessment of extra-virgin olive oil C. de Castro. Am. S. J. Food 613. 331–339. 6740–6744. L.. 742-749. Mancebo-Campos. Zhang. under oxidative stability index conditions. I. 386. Christophoridou. accelerated storage conditions. A. P.. Bioanal. 6. Influence of triacylglycerol composition in plant oils based on high-performance crystallization on the oxidative stability of extra virgin olive oil. 10055–10062. P. E. 607– by triacylglycerols evaluation of profile using HPLC/ELSD. distillates from an edible oil refining process. in virgin olive oils with different phenolic contents during an (41) León-Gamachio. Cerretani. A. Technol. Shahidi. tion indeces of virgin olive oil during 21 months storage at room 78–83.. 264– graphic profiles. M.. (29) Capote. Agric. 529–535. Food Chem. Food Chem. N. J. Lipid Sci. Rapid authentication of olive oil preliminary approach to predictive modeling of extra virgin olive oil adulteration by Raman spectrometry. Bandini.. M. Ghasemi. H. Lipid Sci. Alkyl esters of fatty acids a useful tool to detect soft fluorescence spectroscopy for quantitative determination of virgin deodorized olive oils. 2009. 97.. A. Agric. J. Food Chem. F. Holland. R. V. Chiavaro. B.. J. J. Bertuccioli. 2000. Liu. 2010. 57. paramagnetic resonance spectroscopy study. G.... G. Oil deodorized olive oil. M. Lercker. M. Xenakis. Salvador. Eur. Rovellini.. Safafar. Oily Press: Bridgwater. P. Ozen. Food Chem. Fregapane.. S. 4342–4346. 2005. K.. C. J. Chem. 1571–1575. Stability of virgin olive oil. J. C. del C.. M.K. 827–833.. 57. 110. gies and their influence on the oil oxidative stability.. Mattei. Food Chem. Salvadori. 2103–2110. 95. 2010. Discrimination of vegetable oils purification process on olive oil quality. 10. 58. 336-342. identification and quantitation of extra virgin Morilla. acid profiles by direct infusion electrospray ionization mass spec. Agric. 153–158. M. Castellano... Food Chem. Nicoli. R. D. 4867–4872. Spyros. Identification of 9(E). 329–336. 2007.. 82. 267. Chem.. 118. Chem.. A. Klostermann. Dong.. I. Mousdis. G. 116. C. J. 369–375. Chem. Bioanal. Food (32) Petrakis. Food Chem.. 92. B. F. Agiomyrgianaki. 6001–6006. M. M.... Rojas.. M. M. F. Dais. A. Soc. 2009. U. 4128–4135. P. stability. M. M..11(E)-18:2 fatty acid methyl ester at trace (24) Damirichi.. (44) Ruiz-Méndez. 7429–7433.. Agric. Sci. 110. and aging. Mazzini. oxidant system and the quality of extra virgin olive oil under nical discriminant analysis and classification binary trees. 102. Dessi. G. 105. V. M. J. Perdiguero. S. F. Formation of oxidation compounds in sunflower and olive oils adulteration.. 2006. F. G. 2008. A. H. . C. 7834–7840.. High-power gradient diffusion NMR (57) Márquez-Ruiz. Ramis-Ramos. liquid chromatography-atmospheric pressure chemical ioniza. T. J.. a possible marker for adulteration by addition of markers for detection of adulteration of virgin olive oil. 2005. F. G. 54. Simó-Alfonso. A. Georgiou.. Anal. Rodrı́guez-Estrada. 2005.. Oil Chem. Food Chem. E. G... A. Food Chem. Simó-Alfonso. L. 50. Bohac. S. A. Bellan. Sequential (step. Serrano. Food Chem. 227– (60) Frankel.. (43) Hafidi. M. Chiavaro. D. 1492–1498.. 77. 2009. P... A. A. Arab. Fritsche. M. Sofikiti.. Raffaelli. 519–525. 2005.. E. Food Chem. C. Y. E. G. M. X. (58) Mancebo-Campos. quality. M. 2007. 2006. ing using nitrogen as stripping gas. G. Agric. 453–458. Herrero-Martı́nez. Albi. S. M. Piccolo. 53. J.. 1996. Qi. M. J. olive oils: importance of R-tocopheol concentration. 2010 Frankel classification. Study of chemical changes produced Eur. 573-574. A. Kinetic Multivariate analysis of NMR fingerprint of the unsaponifiable study for the development of an accelerated oxidative stability test fraction of olive oils for authentication purposes. Influence of storage temperature on fruit olive oil adulteration by chemometric treatment of chromato. No. Mousdis. oils by CEC using methacrylate ester-based monolithic columns. Rosa. J. Z. H. P.-F. Agric. A. R. 232.. (59) Lerma-Garcı́a.. J. Guillou. 2005.-W. M. (35) Lisa. 388. Herrero-Martinez. Auto- (31) Lerma-Garcı́a. (55) Cercaci. Novel approach to study oxidative stability of extra virgin trometry. 57. Agric. Passalacqua. J. Food Agric. P. G. J. 2nd ed. G. (34) Piravi-Vanak. (42) Saba. Evaluation of the quality of olive oil using fatty (50) Deiana. Simo-Alfonso. Authentication of extra virgin olive oils by gapane. Ajana... deodorized olive oil: focus on the hydrolytic and oxidative stress. Food Chem. E. Evaluation of authenticity of Iranian olive oil by radical scavenging activity of extra virgin olive oils: an electron fatty acid and triacylglycerol profiles. 53. 1859–1865. M.. 56... (38) Smejkalov a. G. 2007. Combination of chro- (26) Flores. 118. Dobarganes. C. M. Determination of tocopherols in vegetable virgin olive oil stored under fluorescent light. B. Cao. S.. 44. Food Chem.... Lercker. Chem. Statistical evaluation of (54) Calligaris. Mattei. 2006. Agric. S. G. Poerio.. M.. Savage.. (37) Lerma-Garcı́a. 56. R. Grasas Aceites 2004. A. J. M. N. G. 1307–1313. F. Marotta. (39) Alonso-Salces. Agric. Anal. M.. Gutiérrez. Ma. Detection of adulteration of extra-virgin extra virgin olive oils obtained with different extraction technolo- olive oil by chemometric analysis of mid-infrared spectral data. Holcapek. R. 57..-Q. M. (45) Pérez-Camino. Food Chem. A.. 2002. J. Food Chem. Electrophoresis 2007. M. C. matographic techniques for the analysis of complex deodoriser Detection of the adulteration of olive oils by solid phase micro. 2005..40 -dimethylsterols as possible MS and CI-MS. 956–965. entiate virgin or non-refined from refined vegetable fats and oils. Fre- Simó-Alfonso. 2008. del Castillo. Am.. M.. Soc. 6888–6898. M. A. F.. A. (49) Psomiadou. C. D. (53) Papadimitriou. X. Lercker. 716–721. Composition of total sterols (4-desmethyl-sterols) in (36) Gurdeniz. J. Segura.. 2008. adulteration. V. (33) Zou. Rapid synchronous Study of the effects of heating of virgin olive oil blended with mildly fluorescence method for virgin olive oil adulteration assessment. P. Georgiou. M. Relation between the endogenous anti- (cv. Wang. G.. 2009. Food Chem. A. B. (46) Bendini. Technol. Moreno.. G.. Koroneiki) using 1H and 31P NMR fingerprinting with cano. Blanch. G. 100. temperature. 2009. Anal.. E... Heinonen. Lipids 2009. Comparison of static headspace. F. S. Vol. J. N. F. 2003. Chem. 2004.. Fernandez. and Chiavaro.. in extra virgin olive oils as a function of the ripening degree of olive tion techniques on chemicals composition of French olive oils.-F. 2007. 169–174. M. J.Perspective J. methods to evaluate natural antioxidants... Maestri. Ieri. M. 2008. Temime. A.. K. Cert.. Chromatogr.. R.. Hammami. Food Chem. Toschi. R. Food Chem. 56. J.. 2006. Grosch. 56. M. 2002. M.. (89) Morelló. M. J. ionization-mass spectrometry (APCI-MS).. Food Chem. Montedoro. N. L. Mechri. A.. Food Agric. J. (78) Servili. Oily Press: Bridgwater. Effect on chemical oxidative stability indices and mance liquid chromatography with diode array ultraviolet detection. H. 602– C. Food Chem. J. L. G. J. 2002. T. Cerretani. oxidante evaluation with hydroxytyrosol esters. (76) Baccouri. Brahmi. Motilva. 1996. U. M. R. E.. Food Chem. 1077–1080. 1182–1185. Campeol. López-Tamames. Agric. J. L. L. J. Characterization of 39 (93) Paiva-Martins.. A 2003. M. N. od for evaluation of phenolic compounds in virgin olive oil and tography with mass spectrometric and flame ionization detection. A. Pinto. J. Agric. J. Gutiérrez-Rosales. 53. Morales. S. 2008. S. F. Composition and stability of olive oil follow. (73) Torres. Gil. J. I. Tasioula-Margari. M. J. 850–856.. Cioni. Temperature dependence of the auto. 2002–2008. Lizzani-Cuvelier.. S. . N.. (69) Cavalli. Félix. Evaluation of phenolic compounds in virgin 82. Espartero. 6571. N. A.. 5181–5187. 98. 2001. P. Antioxidant activity of olive pulp and olive oil phenolic compounds volatile fraction: modifications induced by oxidation and suitable of the Arbequina cultivar.. Soc. E.. Food Chem.K. 2007. Mulinacci. I. Food Res. Food capacity during storage of bottled extra virgin olive oil. Solid-phase extraction López-Tamames. Romero.-J. J. volatile compounds and sensory attributes of olive oils by the 7170–7175. 1315–1320... J. M.. (97) Mateos... 104. R.. Am. Martı́nez.. Application of solid-phase microextraction to the tero. 2009. Volatile composition of compounds and R-tocopherol of virgin olive oil during storage. Gallina. J. Food Chem.. A. J. 102.. M.... A... Technol. M. 51. F. M. A. Anti- profiles. Chromatogr. Bergmans.. S. Socaciu. L. Castellote. Cantini. 239–246. J. J.... 8194–8201. Evolution of minor polar compounds and antioxidant stability.. Bendini. J. J. flavones. A 2002. 50. R. M. Agric. 48. New lipophilic tyrosol esters.. Agric. Soc. R. P. N. Lizzani-Cuvelier. and deodorized olive oil. oxidante activity of extra-virgin olive oils. Correlations of the phenolic compounds and the 613. A. E. Antioxidants in Food and Biology. and direct thermal desorp. Contribution of phenolic compounds (96) Andjelkovic. 1998. D. Agric.. Taticchi. 1997. Food Chem. Lipid Sci. 46. 2007. L.. R.. L.. J. Morales. Posthuus. E. electrophoretic methods. L. M. Conte. M. Trujillo. No..-W. Bendini. Food Res. 56. Mousdis. Pavón.. Discrimination (95) Romani.. M. 2832–2838. Buxaderas. olive oil by direct injection in high-performance liquid chromatog- (74) Morales. Agric. Urgeghe. Coinu. A. G. Technol.-M. Espartero. volatiles compounds and chemometric analysis. 2007.. G. Eur. ing partial crystallization.. assessment. 1093. Cerretani. 1995. Agric. 57. Loiseau. A multivariate 2008. (63) Jansen. B. Pereira-Caro. Loiseau. 2754–2763. (79) Frankel. Food Chem. 49. Montedoro. M. H. J. M. A.. Agric. Galli. 2006. G. J. Lapucci. 221–230. A.. 10960–10966. M. Soc. León-Camacho. Liquid-liquid and solid-phase extractions of phenols Cordero. 54.. 16..... R. 985. G. Daoud. Finley. B.. Choe. virgin olive oils by HS-SPME: identification of quality-freshness (91) Frankel. Food Chem. P. Boskou. 4771–4780... J. Rı́os. components in olive and walnut oils. 2005. 2007. (83) Mateos.. Relationship between minor components in virgin olive oil.. M.. F. Qualitative and semiquantitative analysis of phenolic compounds traction. Bendini. M. B. Alcudia. J. structural confirmation of oleuropein and ligstroside aglycons and Chromatogr. 2666–2673.. T.-M. S. H. Y. S. J.. S.. Agric. 45. T... 2185–2192. J.. Domı́nguez. phenolic content in some Spanish and French olive oils. (88) Rı́os. Food Chem. 2009. Agric. S. Vittadini. D. M.... 2000. Agric. to virgin olive oil quality. J. Cert. (68) Marcos-Lorenzo. Food Chem.. E. Urbani.. E. F. from olive leaves. Food Chem.-P. markers of oxidation status. Pedra. Odorants of virgin olive oils with different (84) Mateos. Lee. E.. G. M. G. Solid-phase microextraction in the analysis of virgin olive oil M.. phenolic compounds. Carta. Int. (85) Okogeri. fruits by different analytical techniques.. Food Chem.-R. 2009... G.. F. H. Biguzzi. J. J. Agric. Comparative study of olive raphy with fluorescente dectection. 2005.. Fernandez.. 243–252. Birch. 226. Linssen. Antioxidant activity of two virgin olive (72) Cavalli. Ferreira. G. 2008. J. S. 115. J. 105–110. M. S.. E. Food Chem. new hydroxytyrosol derivative from olive (Olea europaea) leaves. C. Vuorela.. 2004. Food Chem. J.. 55. 425–433.... 91.. Chehab. 51. L. Bonoli. M. T. T. Renders.. (71) Vichi. E. G. (70) Vichi. Lercker. J. F. A... How to standardize the multiplicity of markers. L. 3121–3125. 56. Rodrı́guez-Estrada. A 2003. Zarrouk. 55. 826–831. J.. Aparicio.. Microwave heating of different commercial categories lignans in virgin olive oils by solid-phase extraction and high-perfor- of olive oil: Part I. M.. Verhé. J. F. 72. J..... J. Schols. Am.. M. Aparicio. (94) Carrasco-Pancorbo.. T. Oil Chem. Effects of enrichment of refined olive oil with phenolic compounds 2006. R. Mulinacci.... Coireira. (86) Bendini. Facts and Fiction. Agric. virgin olive oil during oxidation: flavors and off-flavors.. Food Chem. of some Tunisian olive oil varieties according to their oxidative Visioli. from virgin olive oil and their separation by chromatographic and mass spectroscopy. (98) Paiva-Martins. Colombo. Alonso. Segura-Carre- Zarrouk... Luna. 945. Food Chem. C. 1253–1264. M. Agric. G. J. B.. 1131–1137. opein aglycon in olive oil by atmospheric pressure chemicals mally stressed edible oils. oil sensory defects. F. A. sunflower. Georgiou. F. Ieri. W. J. B. X. J. Chromatogr. 5582–5588. Detection of adulterants in olive oil by headspace. Laespada.. 1997. Cerretani. Trujillo.. (82) Caruso. Pizzale.. F. headspace sorptive extraction. 293–301. 6564– (90) Franconi. 88. Madrona. L.. Esposto. Agric. Lercker. R. Food Chem. Isolation and characterization of a (81) Litridou. Analysis of virgin olive oil volatile compounds gas chromatography-ion trap-mass spectrometry qualitative meth- by headspace solid-phase microextraction coupled to gas chroma. L. W. Agric. 4139–4143. D. (80) Satué. M. A.. A. Food Chem. Kaouther.. 54. 74.. varietal virgin olive oils by their volatile compositions. J. Chantzos. Food Chem. Conte. Aouni. Oil Chem. M. C. 151–157.. Pinto.. M. Ben. Cert. Y. Eur. M. Determination of phenols. D. S. and olive oil. L. Buxaderas. Food Chem.. J. F.. V. (65) Reiners. Effect of natural Espartero.. I. E. 2003. M.. Romani. 983. Servili.. R. Aparicio. Food Chem. R-tocopherol. Comparative anti- antioxidants in virgin olive oil on oxidative stability of refined. 4901–4908. Tsimidou. 55. Agric. 42. fractionation by solid phase extraction and antioxidant activity xidation and antioxidants of soybean. A.. 2003. M. 2010 6005 (61) Lee.. Gordon.-F. Evaluation of the influence of analysis of volatile compounds in virgin olive oils from five new thermal oxidation on the phenolic composition and on the anti- cultivars. Oil Chem. J. Agric. O. their oxidation products. M. 58. Gutiérrez. T. Lercker. T. and other (66) Aparicio. C. Rı́os. 51. R. evaluation of phenolic component profile and analysis of oleur- Synchronous fluorescence spectroscopy: tool for monitoring ther. study of the relationship between fatty acids and volatile flavor (92) Selvaggini. Food bleached. Huang. F. J.. 7709–7716. J. G. Rapid (62) Poulli. J. Am. 164–186.. Food Chem. Phenolic compounds in virgin olive oils: Agric.. (75) Luna. L. M. Patelli.. 73.. V. Agric. X. Agric. S. F. 7026–7032. Frankel. 1381–1388. P. L. (64) Cerretani. Pizzale. M. (87) Bonoli. Van Camp. 2005. oxidant effect of phenolic compounds. 52.. Changes occurring in phenolic (67) Morales. (77) Issaoui. headspace solid phase microex... 19–33.. oils depends on the concentration and composition of minor polar Characterization of volatile compounds of French and Spanish compounds. G. M.. J. K. A 2005. Giavarini. Sci. R. 2007. 10. sensory wheel. Food Chem. 167–176. A. M. A. 112.. Food Chem. La Notte.. C. 10.. V. G. 79.. Revised manuscript received 2009. purified hydroxytyrosol and 3. J. J. R. Mares. 542. H. Received for review February 26. Minor compounds in the phenolic fraction of virgin olive oils. A. G. 93. G. C. (103) Resanka.. G. Accepted April 22. No. April 12. Determination of plant triacylglycerols using Lamacchia.. Martı́nez. 525–532. Robledo... M.. F. 58. Food Sci. I. L. Terracone. Gambacorta. Food Chem. 1238–1246. 197–204.. 2010. alternative to synthetic antioxidants. Chromatogr. 2009. (101) Saitta. New CE-ESI-MS analytrical method for ther separation. Di Bella. Food Chem.4-dihydroxyphenyl acetic acid from cation of seven phenolic acids including three isomer compounds in olive mill wastewater for the stabilization of refined oils: a potential virgin olive oil. 2005. 74. Vol... M. high-performance liquid chromatogra- antioxidante activity of Italian extra-virgin olive oils during sto. A. J. 2010. C. The use of polyphenolic extract. La Torre. J.. C177–C183.. V. Agric. 145–159.. Sayadi. rage. Changes in phenolic content and capillary gas chromatography. G. (100) Baiano. 2010.. E.6006 J. Allouche. phy and mass spectrometry. 2010 Frankel (99) Nevado. B. Previtali. Penalvo. 1991. N. I. (102) Fki. Dugo. . Salvo. identifi. S. P. Talanta 2009.