Detection of extra virgin olive oil adulteration with lampante olive oil and refined olive oil using nuclear magnetic resonance spectroscopy and multivariate statistical analysis

High-field 31P NMR (202.2 MHz) spectroscopy was applied to the analysis of 59 samples from three grades of olive oils, 34 extra virgin olive oils from various regions of Greece, and from different olive varieties, namely, 13 samples of refined olive oils and 12 samples of lampante olive oils. Classification of the three grades of olive oils was achieved by two multivariate statistical methods applied to five variables, the latter being determined upon analysis of the respective 31P NMR spectra and selected on the basis of one-way ANOVA. The hierarchical clustering statistical procedure was able to classify in a satisfactory manner the three olive oil groups. Subsequent application of discriminant analysis to the five selected variables of oils allowed the grouping of 59 samples according to their quality with no error. Different artificial mixtures of extra virgin olive oil-refined olive oil and extra virgin olive oil-lampante olive oil were prepared and analyzed by 31P NMR spectroscopy. Subsequent discriminant analysis of the data allowed detection of extra virgin olive oil adulteration as low as 5% w/w for refined and lampante olive oils. Further application of the classification/prediction model allowed the estimation of the percent concentration of refined olive oil in six commercial blended olive oils composed of refined and virgin olive oils purchased from supermarkets.     

Determination of the diglyceride content in greek virgin olive oils and some commercial olive oils by employing (31)P NMR spectroscopy

In this study, the diglyceride contents of 96 samples of virgin olive oils from the regions of Crete, Lesvos, Messinia, Pilion, Zakynthos, Halkidiki, and Ilia, 15 samples of commercial extra virgin and pure olive oils, and 3 samples each of refined olive oils and pomace oils were determined by a facile method introduced in a previous publication. This method is based on the phosphitylation of the free hydroxyls of the diglycerides with 2-chloro-4,4,5,5-tetramethyldioxaphospholane and the integration of the appropriate peaks in the (31)P NMR spectra. This preliminary study showed interesting trends in the diglyceride content of the virgin olive oils from the various regions of Greece that can be used as simple criteria to assess the olive oil characteristics. Analysis of variance has been carried out for the diglyceride content of each region in an attempt to detect possible differences in the diglyceride levels among the various regions. Finally, the relationship between the ratio of 1,2-diglycerides to the total amount of diglycerides and the total amount of diglycerides has been used to monitor the quality of virgin olive oils, commercial olive oils, refined olive oils, and pomace oils.     

Determination of coenzyme Q10, coenzyme Q9, and melatonin contents in virgin argan oils: comparison with other edible vegetable oils

Virgin argan oil possesses high antioxidant capacity (AC), which may be partially explained by its high content in antioxidant molecules such as polyphenols and tocopherols. However, the content in other antioxidant molecules, for example, coenzyme Q10 (CoQ(10)), coenzyme Q9 (CoQ(9)), and melatonin (Mel), which have been identified in other edible vegetable oils, have not been evaluated in virgin argan oil. Consequently, it was decided to evaluate the contents of CoQ(10), CoQ(9), and Mel in virgin argan oils and compare the results to those obtained in extra virgin olive oils and some varieties of seed oils. By the use of sensitive HPLC-EC/F methods, the results showed that virgin argan oil is a rich source of CoQ(10) and Mel, but no CoQ(9) was detected. Extra virgin olive oil showed higher levels of CoQ(10) and lower levels of Mel than virgin argan oil. Between the seed oil samples, only virgin soybean oil showed higher CoQ(10) and Mel levels than virgin argan oil. The results may be relevant for the contribution of CoQ(10) and Mel to the biological activities of virgin argan oil.     

Detection of hazelnut oil adulteration using FT-IR spectroscopy

Fourier transform infrared spectroscopy (FT-IR) was used to detect the adulteration of hazelnut oil with different types of oils and to detect the adulteration of extra-virgin olive oil with hazelnut oil. Spectra of hazelnut oil, seven other types of oils, extra-virgin olive oil, and the adulterated oils were collected with a FT-IR equipped with a ZnSe-ATR accessory and a MCTA detector. Discriminant analysis and partial least-squares analysis were used to analyze the data. Classification of hazelnut oil, olive oil, and the other types of oils was achieved successfully with FT-IR. The detection level for sunflower oil adulteration of hazelnut oil was 2%, and the correlation coefficient for the PLS model was 0.99. Adulteration of virgin olive oil with hazelnut oil could be detected only at levels of 25% and higher.     

Qualitative and semiquantitative analysis of phenolic compounds in extra virgin olive oils as a function of the ripening degree of olive fruits by different analytical techniques

Capillary electrophoresis (CE) can be effectively used as a fast screening tool to obtain qualitative and semiquantitative information about simple and complex phenolic compounds of extra virgin olive oil. Three simple phenols (tyrosol, hydroxytyrosol, and vanillic acid), a secoiridoid derivative (deacetoxy oleuropein aglycon), and two lignans (pinoresinol and acetoxypinoresinol) were detected as the main compounds in extra virgin olive oils by high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). Spectrophotometric indices, radical scavenging activity, and oxidative stability of extra virgin olive oil samples obtained from olives hand-picked at different ripening degrees were statistically correlated with the CZE and HPLC quantification. The concentration of phenols in extra virgin olive oil decreased with ripeness of olive fruits. The high correlations found between CZE and the other analytical results indicate that CE can be applied as a rapid and reliable tool to routinely determine phenolic compounds in extra virgin olive oils.     

Alkyl esters of fatty acids a useful tool to detect soft deodorized olive oils

Fatty acid alkyl esters (FAAEs) are a family of natural neutral lipids present in olive oils and formed by esterification of free fatty acids (FFAs) with low molecular alcohols. Inappropriate practices during the olive oil extraction process and bad quality of the olive fruits promote their formation. Quantification can be done by isolation with a silica gel solid phase extraction cartridge followed by analysis on a gas chromatograph equipped with a programmed temperature vaporizer injector using a polar capillary column. The application of the method to more than 100 Spanish olive oils from different categories, varieties, and geographical origin allowed for establishing the average content of FAAEs and distinguishing the Spanish protected denomination of origin (PDO) and extra virgin olive oils from other categories of olive oils. Those other categories of oils can be subjected to a mild refining process, which leads to blending with extra virgin olive oils. Studies on low quality oils subjected to mild refining showed that FAAEs remain after that process. Thereby, blends of extra virgin olive and mildly refined low quality olive oils can be detected by their alkyl ester concentrations.     

Exploitation of the chloroplast trnL (UAA) intron polymorphisms for the authentication of plant oils by means of a lab-on-a-chip capillary electrophoresis system

Methods to discriminate plant oils facilitate the detection of either deliberate or accidental adulteration. To this direction, the variability in length among plant species of the chloroplast trnL intron was exploited for the authentication of edible and cosmetic plant oils, with an extra emphasis on olive oil. The methodology was based on the combinatorial use of a PCR assay with a capillary electrophoresis system such as the lab-on-a-chip technology. Application of the assay on DNA extracted from different oil producing plant species, including olive oil and sesame oil, indicated the ability of the trnL intron to be used as an analytical target. Furthermore, this assay could be used for the detection of adulteration of olive oil with various other plant oils, with the exception of avocado and sesame oil.     

Excitation-emission fluorescence spectroscopy combined with three-way methods of analysis as a complementary technique for olive oil characterization

This paper shows the potential of excitation-emission fluorescence spectroscopy (EEFS) and three-way methods of analysis [parallel factor analysis (PARAFAC) and multiway partial least-squares (N-PLS) regression] as a complementary technique for olive oil characterization. The fluorescence excitation-emission matrices of a set of Spanish extra virgin, virgin, pure, and olive pomace oils were measured, and the relationship between them and some of the quality parameters of olive oils (peroxide value, K232, and K270) was studied. N-PLS was found to be more suitable than PARAFAC combined with multiple linear regression for correlating fluorescence and quality parameters, yielding better fits and lower prediction errors. The best results were obtained for predicting K270. EEFS allowed detection of extra virgin olive oils highly degraded at early stages (with high peroxide value) and little oxidized pure olive oils (with low K270). The proposed methodology may be used as an aid to analyze doubtful samples.     

Determination of esters of fatty acids with low molecular weight alcohols in olive oils

A simple and precise analytical method was developed for the simultaneous determination of squalene and methyl, ethyl, propyl, and butyl esters of fatty acids present in olive and olive pomace oils. A fraction containing squalene and fatty acid alkyl esters was isolated from the oil by solid phase extraction on silica gel cartridges and quantitatively analyzed by gas chromatography. A modification of the procedure allowed the isolation of squalene and esters separately. Repeatability and recovery of the method were good. The method was applied to extra and lampant virgin olive oil categories and also to oils obtained from olive pomace by second centrifugation and solvent extraction. Extra virgin olive oils contained low amounts of fatty acid methyl and ethyl esters, while oils obtained from altered olive or olive pomace showed high concentrations of fatty acid alkyl esters, mainly ethyl esters. Correlation between oil acidity and ethyl esters concentration was poor.     

Determination of nonprotein amino acids and betaines in vegetable oils by flow injection triple-quadrupole tandem mass spectrometry: a screening method for the detection of adulterations of olive oils

A novel screening method using an automated flow injection electrospray ionization tandem mass spectrometry system is proposed for the simultaneous determination of five nonprotein amino acids (β-alanine, alloisoleucine, ornithine, citrulline, pyroglutamic acid) and three betaines (glycine betaine, trigonelline, proline betaine) after derivatization with butanolic HCl. MS/MS experiments were carried out in a triple-quadrupole instrument using multiple reaction monitoring mode in <2 min. The proposed method provided high fingerprinting power to identify the presence of five of the studied compounds in different types of vegetable oils (soybean, sunflower, corn, olive) with LODs at parts per billion levels. The method was validated, and different mixtures of extra virgin olive oil with seed oils were analyzed, achieving the typification for the detection of adulterations in extra virgin olive oils up to 2% w/w. The nonprotein amino acid ornithine was confirmed as a marker for adulteration in the olive oils analyzed.     

Determination of proteins in refined and nonrefined oils

Five methods using aqueous/organic solvents for the separation of proteins from oils were compared. The extraction with acetone-hexane followed by amino acid analysis was found to be the most suitable method for isolation and quantification of proteins from oils. The detection limit of the method was 0.18 mg protein/kg oil, and the quantification limit was 0.6 mg protein/kg. The relative repeatability limit for samples containing 1-5 mg protein/kg sample was 27%. The protein recovery ranged between 68 and 133%. Using this method, the protein content of 14 refined and nonrefined oils was determined. In none of the refined oils were proteins detected, whereas the protein content of the unrefined oils ranged between undetectable in extra virgin olive oil to 11 mg/kg in rapeseed oil. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with silver staining, many protein bands were visible in the unrefined soy, olive, peanut, and rapeseed oil samples. Proteins bands were not obtained from the refined fish oil. In the other refined oil samples, a few proteins bands could be visualized. Two protein bands with apparent molecular molecular masses of 58 and 64 kDa were always observed in these oils.     

Interpretation of Fourier transform Raman spectra of the unsaponifiable matter in a selection of edible oils.

The unsaponifiable matter of edible oils is a source of information for their characterization and authentication. FT-Raman spectroscopy has been applied with success to the determination of the spectra of the unsaponifiable matter of varietal olive oils as well as other refined and crude edible oils. The spectra of the major unsaponifiable series of compounds (squalene, sterolic, and terpenic alcoholic fractions), together with beta-carotene and lutein, have been used to explain the most prominent bands found in the spectra of the unsaponifiable matter of 15 edible oil samples. The order of the scattering intensities of the varietal olive oils agrees with the results obtained by chromatography. An unsupervised multivariate statistical analysis of selected bands points out differences between olive oils and the other seed oils and also among varietal virgin olive oils.     

Comparison of volatile aldehydes present in the cooking fumes of extra virgin olive, olive, and canola oils

Emissions of low molecular weight aldehydes (LMWAs) from deep-frying of extra virgin olive oil, olive oil, and canola oil (control) were investigated at two temperatures, 180 and 240 degrees C, for 15 and 7 h, respectively. The oil fumes were collected in Tedlar bags and then analyzed by gas chromatography-mass spectrometry. Seven alkanals (C2-C7 and C9), eight 2-alkenals (C3-C10), and 2,4-heptadienal were found in the fumes of all three cooking oils. The generation rates of these aldehydes were found to be dependent on heating temperature, showing significant increases with increases in temperature. The LMWA emissions from both kinds of olive oils were very similar and were lower than those observed from canola oil under similar conditions. These results suggest that frying in any type of olive oil, independent of its commercial category, will effectively decrease the generation of volatile aldehydes in the exhaust. This fact is important because less expensive refined olive oil is usually used for deep-frying operations, whereas extra virgin olive oil is usually used as salad dressing.     

Updating a synchronous fluorescence spectroscopic virgin olive oil adulteration calibration to a new geographical region

Detecting and quantifying extra virgin olive adulteration is of great importance to the olive oil industry. Many spectroscopic methods in conjunction with multivariate analysis have been used to solve these issues. However, successes to date are limited as calibration models are built to a specific set of geographical regions, growing seasons, cultivars, and oil extraction methods (the composite primary condition). Samples from new geographical regions, growing seasons, etc. (secondary conditions) are not always correctly predicted by the primary model due to different olive oil and/or adulterant compositions stemming from secondary conditions not matching the primary conditions. Three Tikhonov regularization (TR) variants are used in this paper to allow adulterant (sunflower oil) concentration predictions in samples from geographical regions not part of the original primary calibration domain. Of the three TR variants, ridge regression with an additional 2-norm penalty provides the smallest validation sample prediction errors. Although the paper reports on using TR for model updating to predict adulterant oil concentration, the methods should also be applicable to updating models distinguishing adulterated samples from pure extra virgin olive oil. Additionally, the approaches are general and can be used with other spectroscopic methods and adulterants as well as with other agriculture products.     

A simple high-performance liquid chromatography method for the determination of throat-burning oleocanthal with probated antiinflammatory activity in extra virgin olive oils

A high-performance liquid chromatography (HPLC) method was developed to quantitatively analyze oleocanthal in extra virgin olive oils. Oleocanthal, a deacetoxy ligstroside aglycone, is known to be responsible for the back of the throat irritation of olive oils and to have probated antiinflamatory activity. Oleocanthal was isolated from small amounts of olive oil sample (1 g) by liquid-liquid extraction. Hexane-acetonitrile was found to be the best solvent system to extract oleocanthal from the oil matrix. The solvent extract was analyzed by reversed-phase HPLC with UV detection at 278 nm. Chromatogaphic separation of oleocanthal from other extracted compounds and of the two geometric isomers of oleocanthal was achieved by an elution gradient with acetonitrile and water. Both the external standard calibration curve and the internal standard calibration curve were established, and quantitation using both calibration curves gave essentially the same result. The reproducibility (RSD = 4.7%), recovery (> 95%), and limit of quantitation (< 1 microg/g) were also determined. Concentrations of oleacanthal in 10 selected throat-burning extra virgin olive oils were determined using the method (ranged from 22 to 190 microg/g) with external standard calibration.     

Classification of edible oils by employing 31P and 1H NMR spectroscopy in combination with multivariate statistical analysis. A proposal for the detection of seed oil adulteration in virgin olive oils

A combination of (1)H NMR and (31)P NMR spectroscopy and multivariate statistical analysis was used to classify 192 samples from 13 types of vegetable oils, namely, hazelnut, sunflower, corn, soybean, sesame, walnut, rapeseed, almond, palm, groundnut, safflower, coconut, and virgin olive oils from various regions of Greece. 1,2-Diglycerides, 1,3-diglycerides, the ratio of 1,2-diglycerides to total diglycerides, acidity, iodine value, and fatty acid composition determined upon analysis of the respective (1)H NMR and (31)P NMR spectra were selected as variables to establish a classification/prediction model by employing discriminant analysis. This model, obtained from the training set of 128 samples, resulted in a significant discrimination among the different classes of oils, whereas 100% of correct validated assignments for 64 samples were obtained. Different artificial mixtures of olive-hazelnut, olive-corn, olive-sunflower, and olive-soybean oils were prepared and analyzed by (1)H NMR and (31)P NMR spectroscopy. Subsequent discriminant analysis of the data allowed detection of adulteration as low as 5% w/w, provided that fresh virgin olive oil samples were used, as reflected by their high 1,2-diglycerides to total diglycerides ratio (D > or = 0.90).     

Detecting and quantifying sunflower oil adulteration in extra virgin olive oils from the eastern mediterranean by visible and near-infrared spectroscopy

One hundred and thirty-eight oil samples have been analyzed by visible and near-infrared transflectance spectroscopy. These comprised 46 pure extra virgin olive oils and the same oils adulterated with 1% (w/w) and 5% (w/w) sunflower oil. A number of multivariate mathematical approaches were investigated to detect and quantify the sunflower oil adulterant. These included hierarchical cluster analysis, soft independent modeling of class analogy (SIMCA method), and partial least squares regression (PLS). A number of wavelength ranges and data pretreatments were explored. The accuracy of these mathematical models was compared, and the most successful models were identified. Complete classification accuracy was achieved using 1st derivative spectral data in the 400-2498 nm range. Prediction of adulterant content was possible with a standard error equal to 0.8% using 1st derivative data between 1100 and 2498 nm. Spectral features and chemical literature were studied to isolate the structural basis for these models.     

Geographical characterization of italian extra virgin olive oils using high-field (1)H NMR spectroscopy.

1H high-field nuclear magnetic resonance (NMR) was used to analyze 216 extra virgin olive oils collected in three years (1996, 1997, and 1998) in different Italian areas in order to evaluate the potential contribution of this technique to the geographical characterization of olive oils. A statistical procedure performed on the intensity of selected NMR peaks has been proposed. Tree clustering analysis of NMR data performed without any a priori hypothesis showed the existence of reliable parameters able to group the olive oils according to the location of olive oil production. Linear discriminant analysis applied to selected NMR parameters of olive oils of the same year of production allowed the grouping of samples according to their geographical origin with only very few errors. Moreover, a satisfactory grouping is reached by combining the NMR data of olive oils from two different years (1996 and 1997). Operating on appropriate sampling, a careful analysis of data yielded the conclusion that the place of olive production could be singled out as a discriminating factor regardless of the cultivars from which the olive oils are derived.     

Antioxidant effect of two virgin olive oils depends on the concentration and composition of minor polar compounds

In vitro studies show that some individual minor polar phenolic compounds (MPC) present in virgin olive oil prevent oxidation of human low-density lipoproteins (LDL), but few data are available on the antioxidant effect of whole oil extract. Thus, whole virgin olive extracts were studied to determine whether they maintain the antioxidant activity and whether this last is linked to MPC composition of a single virgin oil. Using HPLC-DAD the MPC content in Taggiasca and Seggianese virgin olive oils was measured. Taggiasca oil was less rich in total MPC (208.5 mg/L) than Seggianese oil (441.9 mg/L). In addition, the major compounds of Taggiasca oil were lignan derivatives, whereas the major compounds in Seggianese oils were secoiridoid derivatives. Moreover, Taggiasca oil was practically free of 5-hydroxytyrosol and 5-hydroxytyrosol derivatives, deacetoxy-oleuropein aglycone and oleuropein aglycone. The antioxidant activity of the oils on human LDL was evaluated by measuring malondialdehyde and conjugate diene generation induced by copper ions. In both tests, the oil extracts dose-dependently reduced malondialdehyde and conjugate diene generation. Moreover, antioxidant potency correlated with total MPC; thus, Seggianese extract was more active. The two oils differed quantitatively and qualitatively, and these differences influenced their biological activities; thus clinical trials focused on studying the effects of olive oils should specify the oils used.     

Quality and stability of edible oils enriched with hydrophilic antioxidants from the olive tree: the role of enrichment extracts and lipid composition

Phenolic extracts from olive tree leaves and olive pomace were used to enrich refined oils (namely, maize, soy, high-oleic sunflower, sunflower, olive, and rapeseed oils) at two concentration levels (200 and 400 μg/mL, expressed as gallic acid). The concentration of characteristic olive phenols in these extracts together with the lipidic composition of the oils to be enriched influenced the mass transfer of the target antioxidants, which conferred additional stability and quality parameters to the oils as a result. In general, all of the oils experienced either a noticeable or dramatic improvement of their quality-stability parameters (e.g., peroxide index and Rancimat) as compared with their nonenriched counterparts. The enriched oils were also compared with extra virgin olive oil with a natural content in phenols of 400 μg/mL. The healthy properties of these phenols and the scarce or nil prices of the raw materials used can convert oils in supplemented foods or even nutraceuticals.