A New Method for Olive Oil Screening Using Multivariate Analysis of Proton NMR Spectra

A new NMR-based method for the discrimination of olive oils of any grade from seed oils and mixtures thereof was developed with the aim of allowing the verification of olive oil authenticity. Ten seed oils and seven monovarietal and blended extra virgin olive oils were utilized to develop a principal component analysis (PCA) based analysis of ¹H NMR spectra to rapidly and accurately determine the authenticity of olive oils. Another twenty-eight olive oils were utilized to test the principal component analysis (PCA) based analysis. Detection of seed oil adulteration levels as low as 5% v/v has been shown using simple one-dimensional proton spectra obtained using a 400 MHz NMR spectrometer equipped with a room temperature inverse probe. The combination of simple sample preparation, rapid sample analysis, novel processing parameters, and easily interpreted results, makes this method an easily accessible tool for olive oil fraud detection by substitution or dilution compared to other methods already published.     

Detection of refined olive oil adulteration with refined hazelnut oil by employing NMR spectroscopy and multivariate statistical analysis

NMR spectroscopy was employed for the detection of adulteration of refined olive oil with refined hazelnut oil. Fatty acids and iodine number were determined by ¹H NMR, whereas ³¹P NMR was used for the quantification of minor compounds including phenolic compounds, diacylglycerols, sterols, and free fatty acids (free acidity). Classification of the refined oils based on their fatty acids content and the concentration of their minor compounds was achieved by using the forward stepwise canonical discriminant analysis (CDA) and the classification binary trees (CBTs). Both methods provided good discrimination between the refined hazelnut and olive oils. Different admixtures of refined olive oils with refined hazelnut oils were prepared and analyzed by ¹H NMR and ³¹P NMR spectroscopy. Subsequent application of CDA to the NMR data allowed the detection of the presence of refined hazelnut oils in refined olive oils at percentages higher than 5%. Application of the non-linear classification method of the binary trees offered better possibilities of measuring adulteration of the refined olive oils at a lower limit of detection than that obtained by the CDA method.     

13C nuclear magnetic resonance spectroscopy to determine olive oil grades

¹³C nuclear magnetic resonance spectroscopy was used in a first attempt to differentiate olive oil samples by grades. High resolution ¹³C NMR Distortionless Enhancement by Polarization Transfer (DEPT) spectra of 137 olive oil samples from the four grades, extra virgin olive oils, olive oils, olive pomace oils and lampante olive oils, were measured. The data relative to the resonance intensities (variables) of the unsaturated carbons of oleate (C-9 and C-10) and linoleate (L-9, L-10 and L-12) chains attached at the 1,3- and 2-positions of triacylglycerols were analyzed by linear discriminant analysis. The 1,3- and 2- carbons of the glycerol moiety of triacylglycerols along with the C-2, C-16 and C-18 resonance intensities of saturated, oleate and linoleate chains were also analyzed by linear discriminant analysis. The three discriminanting functions, which were calculated by using a stepwise variable selection algorithm, classified in the true group by cross-validation procedure, respectively, 76.9, 70.0, 94.4 and 100% of the extra virgin, olive oil, olive pomace oil and lampante olive oil grades.     

Rapid determination of BTEXS in olives and olive oil by headspace-gas chromatography/mass spectrometry (HS-GC-MS)

In this work, a straightforward, reliable and effective automated method has been developed for the direct determination of monoaromatic volatile BTEXS group (namely benzene, toluene, ethylbenzene, o-, m- and p-xylenes, and styrene) in olives and olive oil, based on headspace technique. Separation, identification and quantitation were carried out by headspace-gas chromatography-mass spectrometry (HS-GC-MS) in selected ion monitoring (SIM) mode. Sample pretreatment or clean-up were not necessary (besides olives milling) because the olives and olive oil samples are put directly into an HS vial, automatically processed by HS and then injected in the GC-MS for chromatographic analysis. The chemical and instrumental variables were optimized using spiked olives and olive oil samples at 50 μg kg⁻¹ of each targeted species. The method was validated to ensure the quality of the results. The precision was satisfactory with relative standard deviations (RSD (%)) in the range 1.6-5.2% and 10.3-14.2% for olive oil and olives, respectively. Limits of detection were in the range 0.1-7.4 and 0.4-4.4 μg kg⁻¹ for olive oil and olives, respectively. Finally, the proposed method was applied to the analysis of real olives and olive oil samples, finding positives of the studied compounds, with overall BTEXS concentration levels in the range 23-332 μg kg⁻¹ and 4.2-87 μg kg⁻¹ for olive oil and olives, respectively.     

LC–MS Determination of Sterols in Olive Oil

Sterols in olive oils have been analyzed by liquid chromatography coupled to mass spectrometry with atmospheric-pressure chemical ionization in positive-ion mode. A simple procedure based on saponification and extraction of the compounds from olive oils was studied. Validation of the method included calibration and determination of recovery and repeatability was carried out. Good linearity was obtained up to 100 mg kg^?1 for all the sterols studied except β-sitosterol, for which linearity was obtained up to 2,000 mg kg^?1. Recovery ranged from 88 to 110%, detection limits from 0.9 to 3.1 mg kg^?1, and precision was good. The method has been successfully used for analysis of sterols in different types of oil. The predominant sterol was β-sitosterol; other minor components, for example sitostanol and cholesterol, were also detected. Total sterol content depended on the type of oil, and ranged from 687 to 2,479 mg kg^?1. Stigmasterol and the amount of erythrodiol plus uvaol can be used to distinguish between olive oil and seed oil.     

Synthesis, spectral studies and complexation properties of N,N′-bis(5-bromo-salicylidene)-2-hydroxy-1,3-propanediamine (BSHP) and iron extraction with BSHP from oils

A Schiff base was synthesized by reaction of 1,3-diamino-2-propanol and 5-bromo-2-hydroxy benzaldehyde. The Schiff base N,N′-bis(5-bromo-salicylidene)-2-hydroxy-1,3-propanediamine (BSHP) was characterized by elemental analysis, ¹H NMR, ¹³C NMR, IR, UV-Vis spectral studies. The complexation of iron with BSHP was investigated spectrophotometrically. The extraction of iron from oil phase to aqueous phase was carried out with BSHP. Extraction conditions were optimized using central composite design. Optimum extraction conditions were found to be 32°C, 2 mL/g for the ratio of the volume of the Schiff base solution to the amount of oil, and 11 min for the stirring time. The method has been validated by using oil based metal standard, obtaining satisfactory results. The limit of detection (LOD) of the improved method is 0.04 μg/g. It has been applied to analysis of different oil samples. The concentrations of iron in olive, sunflower and corn oils were found to be 0.65 ± 0.02, 0.41 ± 0.01, 0.36 ± 0.03 μg/g, respectively. A simple, cheap, rapid, efficient, sensitive and accurate alternative analytical method for the iron determination in oils has been presented in this paper.     

Olive Oil Traceability Studies Using Inorganic and Isotopic Signatures: A Review

The olive oil industry is subject to significant fraudulent practices that can lead to serious economic implications and even affect consumer health. Therefore, many analytical strategies have been developed for olive oil’s geographic authentication, including multi-elemental and isotopic analyses. In the first part of this review, the range of multi-elemental concentrations recorded in olive oil from the main olive oil-producing countries is discussed. The compiled data from the literature indicates that the concentrations of elements are in comparable ranges overall. They can be classified into three categories, with (1) Rb and Pb well below 1 µg kg⁻¹; (2) elements such as As, B, Mn, Ni, and Sr ranging on average between 10 and 100 µg kg⁻¹; and (3) elements including Cr, Fe, and Ca ranging between 100 to 10,000 µg kg⁻¹. Various sample preparations, detection techniques, and statistical data treatments were reviewed and discussed. Results obtained through the selected analytical approaches have demonstrated a strong correlation between the multi-elemental composition of the oil and that of the soil in which the plant grew. The review next focused on the limits of olive oil authentication using the multi-elemental composition method. Finally, different methods based on isotopic signatures were compiled and critically assessed. Stable isotopes of light elements have provided acceptable segregation of oils from different origins for years already. More recently, the determination of stable isotopes of strontium has proven to be a reliable tool in determining the geographical origin of food products. The ratio ⁸⁷Sr/⁸⁶Sr is stable over time and directly related to soil geology; it merits further study and is likely to become part of the standard tool kit for olive oil origin determination, along with a combination of different isotopic approaches and multi-elemental composition.     

A novel approach to the rapid assignment of 13C NMR spectra of major components of vegetable oils such as avocado,mango kernel and macadamia nut oils

Assignment of ¹³C nuclear magnetic resonance (NMR) spectra of major fatty acid components of South African produced vegetable oils was attempted using a method in which the vegetable oil was spiked with a standard triacylglycerol. This proved to be inadequate and therefore a new rapid and potentially generic graphical linear correlation method is proposed for assignment of the ¹³C NMR spectra of major fatty acid components of apricot kernel, avocado pear, grapeseed, macadamia nut, mango kernel and marula vegetable oils. In this graphical correlation method, chemical shifts of fatty acids present in a known standard triacylglycerol is plotted against the corresponding chemical shifts of fatty acids present in the vegetable oils. This new approach (under carefully defined conditions and concentrations) was found especially useful for spectrally crowded regions where significant peak overlap occurs and was validated with the well‐known ¹³C NMR spectrum of olive oil which has been extensively reported in the literature. In this way, a full assignment of the ¹³C{1H} NMR spectra of the vegetable oils, as well as tripalmitolein was readily achieved and the resonances belonging to the palmitoleic acid component of the triacylglycerols in the case of macadamia nut and avocado pear oil resonances were also assigned for the first time in the ¹³C NMR spectra of these oils. Copyright © 2009 John Wiley & Sons, Ltd.     

NMR and chemometrics in tracing European olive oils: The case study of Ligurian samples

An NMR and chemometric analytical approach to classify extra virgin olive oils according to their geographical origin was developed within the European TRACE project (FP6-2003-FOOD-2-A, contract number: 0060942). Olive oils (896 samples) of three consecutive harvesting years (2005, 2006, and 2007) coming from Mediterranean areas were analyzed by ¹H NMR spectroscopy. Olive oil samples from Liguria, an Italian region, were chosen as a case study and PLS-DA and SIMCA modeling analyses were used to build up statistical models both to discriminate between Ligurian and non-Ligurian olive oils and to define the Ligurian olive oil class to confirm the declared provenience.     

Determination of Dimethoate in Olive Oil by Adsorptive Stripping Square‐Wave Voltammetry

A method for the determination of dimethoate in olive oil by adsorptive stripping square‐wave voltammetry has been developed on the base that this pesticide is hydrolyzed in basic media giving rise to an adsorptive‐reductive peak at ?0.780 V. Extraction of dimethoate from olive oil with ethanol‐water and posterior clean‐up with C18 cartridges is carried out. Response Surface Methodology has been used for the optimization of the extraction procedure. A matrix effect is observed in olive oil extract; therefore the standard addition method must be used. The detection limit is 19.00 ng g^?1 and recoveries for three levels of fortification are ranged from 79.9% to 104.5%.     

Stable isotope ratios of carbon and hydrogen to distinguish olive oil from shark squalene‐squalane

Squalene and its hydrogenated derivate squalane are widely used in the pharmaceutical and cosmetic fields. The two compounds are mainly produced from the liver oil of deep sea sharks and from olive oil distillates. Squalene and squalane from shark cost less than the same compounds derived from olive oil, and the use of these shark‐derived compounds is unethical in cosmetic formulations. In this work we investigate whether ¹³C/¹²C and ²H/¹H ratios can distinguish olive oil from shark squalene/squalane and can detect the presence of shark derivates in olive oil based products. The ¹³C/¹²C ratios (expressed as δ¹³C values) of bulk samples and of pure compounds measured using isotope ratio mass spectrometry (IRMS) were significantly lower in authentic olive oil squalene/squalane (N: 13; ?28.4 ± 0.5‰; ?28.3 ± 0.8‰) than in shark squalene/squalane samples (N: 15; ?20.5 ± 0.7‰; ?20.4 ± 0.6‰). By defining δ¹³C threshold values of ?27.4‰ and ?26.6‰ for olive oil bulk and pure squalene/squalane, respectively, illegal addition of shark products can be identified starting from a minimum of 10%. ²H/¹H analysis is not useful for distinguishing the two different origins. δ¹³C analysis is proposed as a suitable tool for detecting the authenticity of commercial olive oil squalene and squalane samples, using IRMS interfaced to an elemental analyser if the purity is higher than 80% and IRMS interfaced to a gas chromatography/combustion system for samples with lower purity, including solutions of squalane extracted from cosmetic products. Copyright © 2010 John Wiley & Sons, Ltd.     

Fluorescence Monitoring Oxidation of Extra Virgin Olive Oil Packed in Different Containers

‘Picual’ olive oil was stored in different types of containers for 10 months and monitored via quality parameters. In combination with the mentioned analysis, non-destructive fluorescence spectroscopy was performed combined with multivariate analysis to monitor and quantify oil quality levels. Excitation emission matrices (EMMs) were analyzed using parallel factor analysis (PARAFAC). According to the quality parameters, it was observed that Transparent Crystal (TC) and Opaque Crystal (OC) samples were the ones that deteriorated faster due to their higher exposure to light in comparison with Plastic (P) and Canned (C) samples. In a fast and non-destructive manner, the fluorescence spectroscopy-based prototype successfully monitored the oxidation changes in the EVOOs. Unfolded partial least squares (U-PLS) was used to generate a regression model to quantify quality parameters. Good correlation coefficients were found for the peroxide index, K₂₃₂ and the oxidative stability index (r² between 0.90 and 0.94 for cross-validation and validation). For all of that, the results obtained confirmed the ability of fluorescence spectroscopy to monitor the quality of olive oil and EEMs combined with U-PLS can be used to analyze these parameters, eluding the classical methods.     

Determination of styrene in olive oil by an automatic purge-and-trap system coupled to gas chromatography-mass spectrometry

Summary A new gas chromatographic method using an automatic purge-and-trap system coupled to a GC with mass selective detection to analyze styrene at the parts-per-trillions (ng kg^–1) level is described. The method shows a good sensitivity and the detection limit is 10 ng kg^–1 with a relative standard deviation (RSD) of 4.7% for 164 ng kg^–1 styrene in olive oil. This analytical method has been successfully applied to the analysis of styrene in extra-virgin olive oil from the European market.     

Determination of styrene in olive oil by an automatic purge-and-trap system coupled to gas chromatography-mass spectrometry

Summary A new gas chromatographic method using an automatic purge-and-trap system coupled to a GC with mass selective detection to analyze styrene at the parts-per-trillions (ng kg⁻¹) level is described. The method shows a good sensitivity and the detection limit is 10 ng kg⁻¹ with a relative standard deviation (RSD) of 4.7 % for 164 ng kg⁻¹ styrene in olive oil. This analytical method has been successfully applied to the analysis of styrene in extra-virgin olive oil from the European market.     

The solubility of CO2 and N2O in olive oil

The solubility of CO₂ and N₂O in olive oil has been measured at temperatures of about 298, 310, and 323 K with a gravimetric microbalance under pressures up to 2 MPa. The molecular weight of olive oil has been analyzed and found to be about 882 g mol⁻¹ as a mixed oil compound. The observed solubility data have been correlated with a cubic equation of state (EOS) model. N₂O has a larger solubility than CO₂ in olive oil based on either the mole or mass fraction. The present results clarify some ambiguities from the previous N₂O solubility data in the literature.     

Territorial origin of olive oil: representing georeferenced maps of olive oils by NMR profiling

ABSTRACT Proton NMR profiling is nowadays a consolidated technique for the identification of geographical origin of food samples. The common approach consists in correlating NMR spectra of food samples to their territorial origin by multivariate classification statistical algorithms. In the present work, we illustrate an alternative perspective to exploit territorial information, contained in the NMR spectra, which is based on the implementation of a geographic information system (GIS). Nuclear magnetic resonance spectra are used to build a GIS map permitting the identification of territorial regions having strong similarities in the chemical content of the produced food (terroir units). These terroir units can, in turn, be used as input for labeling samples to be analyzed by traditional classification methods. In this work, we describe the methods and the algorithms that permit to produce GIS maps from NMR profiles and apply the described method to the analysis of the geographical distribution of olive oils in an Italian region. In particular, we analyzed by ¹H NMR up to 98 georeferenced olive oil samples produced in the Abruzzo Italian region. By using the first principal component of the NMR variables selected according to the Moran test, we produced a GIS map, in which we identified two regions incidentally corresponding to the provinces of Teramo and Pescara. We then labeled the samples according to the province of provenience and built an LDA model that provides a classification ability up to 99% . A comparison between the variables selected in the geostatistics and classification steps is finally performed. Copyright © 2016 John Wiley & Sons, Ltd.     

1H-NMR Profiling Shows as Specific Constituents Strongly Affect the International EVOO Blends Characteristics: The Case of the Italian Oil

Considering the growing number of extra virgin olive oil (EVOO) producers in the world, knowing the influence of olive oils with different geographical origins on the characteristics of the final blend becomes an interesting goal. The present work is focused on commercial organic EVOO blends obtained by mixing multiple oils from different geographical origins. These blends have been studied by ¹H-NMR spectroscopy supported by multivariate statistical analysis. Specific characteristics of commercial organic EVOO blends originated by mixing oils from Italy, Tunisia, Portugal, Spain, and Greece were found to be associated with the increasing content of the Italian component. A linear progression of the metabolic profile defined characteristics for the analysed samples—up to a plateau level—was found in relation to the content of the main constituent of the Italian oil, the monocultivar Coratina. The Italian constituent percentage appears to be correlated with the fatty acids (oleic) and the polyphenols (tyrosol, hydroxytyrosol, and derivatives) content as major and minor components respectively. These results, which highlight important economic aspects, also show the utility of ¹H-NMR associated with chemometric analysis as a powerful tool in this field. Mixing oils of different national origins, to obtain blends with specific characteristics, could be profitably controlled by this methodology.     

Determination of fenthion and fenthion-sulfoxide, in olive oil and in river water, by square-wave adsorptive-stripping voltammetry

Square-wave adsorptive-stripping voltammetry technique has been used to develop a method for the determination of fenthion in olive oil. Due to the fact that fenthion does not give any electrochemical signal at mercury electrode, the method has been based on a previous oxidation of fenthion to its metabolite, fenthion-sulfoxide, by using KMnO₄. The metabolite gives rise to a peak due to an adsorptive-reductive process at −0.786 V. Fenthion is isolated from olive oil by carrying out a solid–liquid extraction procedure using silica cartridge, followed by a liquid–liquid partitioning with acetonitrile. The detection limit in olive oil is 78.8 ng g⁻¹ and recoveries for four levels of fortification are ranged from 85% to 109%. On the other hand, it has been developed a method for the simultaneous determination of fenthion and its metabolite fenthion-sulfoxide, in river water. Pesticides are isolated from water by carrying out a liquid–liquid partitioning with trichloromethane. The detection limits are 0.41 ng g⁻¹ and 0.44 ng g⁻¹, for fenthion and fenthion-sulfoxide, respectively. Recoveries for three levels of fortification are ranged from 96% to 103% for fenthion and 94% to 104% for fenthion-sulfoxide.     

Review about olive oil characterization using high-field nuclear magnetic resonance

In the last years Nuclear Magnetic Resonance (NMR) spectroscopy has found many applications in food analysis. Here, we present a review of the most significant results we have obtained in the olive oil characterization using NMR techniques in combination with multivariate statistical methods.     

New CE-ESI-MS analytical method for the separation, identification and quantification of seven phenolic acids including three isomer compounds in virgin olive oil

A sensitive and expeditious CE-ESI-MS analytical method for the separation, identification and determination of seven selected antioxidants (cinnamic and benzoic acids), including three isomers of coumaric acid (ortho-, meta- and para-) has been developed. In order to obtain the analytical separation, capillary electrophoresis and CE-MS interface parameters (e.g., buffer pH and composition, sheath liquid and gas flow rates, sheath liquid composition, electrospray voltage, etc.) were carefully optimized.The polar fraction containing the selected phenolic acids was obtained using a previously optimized SPE pretreatment. An MS detector in order to extract structural information about the target compounds and facilitate their qualitative analysis was used in the negative ion mode. The proposed off-line SPE CE-ESI-MS method was validated by assessing its precision, LODs and LOQs, linearity range and accuracy.The optimized and validated method was used in order to quantify the selected antioxidants in various samples of virgin olive oil and extra-virgin olive oil obtained from the main olive varieties cropped in Castilla-La Mancha, Spain. Salicylic acid was used as internal standard throughout in order to ensure reproducibility in the quantitative analysis of the oil samples.The results confirmed the presence of hydroxyphenyl acetic, p-coumaric, ferulic and vanillic acids in substantial amounts (μg g⁻¹ level) in all samples.