Direct olive oil authentication: detection of adulteration of olive oil with hazelnut oil by direct coupling of headspace and mass spectrometry, and multivariate regression techniques

Control of adulteration of olive oil, together with authentication and contamination, is one of the main aspects in the quality control of olive oil. Adulteration with hazelnut oil is one of the most difficult to detect due to the similar composition of hazelnut and olive oils; both virgin olive oil and olive oil are subjected to that kind of adulteration. The main objective of this work was to develop an analytical method able to detect adulteration of virgin olive oils and olive oils with hazelnut oil by means of its analysis by a headspace autosampler directly coupled to a mass spectrometer used as detector (ChemSensor). As no chromatographic separation of the individual components of the samples exists, a global signal of the sample is obtained and employed for its characterization by means of chemometric techniques. Four different crude hazelnut oils from Turkey were employed for the development of the method. Multivariate regression techniques (partial least squares and principal components analysis) were applied to generate adequate regression models. Good values were obtained in both techniques for the parameters employed (standard errors of prediction (SEP) and prediction residual error sum of squares (PRESS)) to evaluate its goodness. With the proposed method, minimum adulteration levels of 7 and 15% can be detected in refined and virgin olive oils, respectively. Once validated, the method was applied to the detection of such adulteration in commercial olive oil and virgin olive oil samples.     

Analytical determination of polyphenols in olive oils

The increasing popularity of olive oil is mainly attributed to its high content of oleic acid, which may affect the plasma lipid/lipoprotein profiles, and its richness in phenolic compounds, which act as natural antioxidants and may contribute to the prevention of human disease. An overview of analytical methods for the measurement of polyphenols in olive oil is presented. In principle, the analytical procedure for the determination of individual phenolic compounds in virgin olive oil involves three basic steps: extraction from the oil sample, analytical separation, and quantification. A great number of procedures for the isolation of the polar phenolic fraction of virgin olive oil, utilizing two basic extraction techniques, LLE or SPE, have been included. The reviewed techniques are those based on spectrophotometric methods, as well as analytical separation (gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE)). Many reports in the literature determine the total amount of phenolic compounds in olive oils by spectrophometric analysis and characterize their phenolic patterns by capillary gas chromatography (CGC) and, mainly, by reverse phase high-performance liquid chromatography (RP-HPLC); however, CE has recently been applied to the analysis of phenolic compound of olive oil and has opened up great expectations, especially because of the higher resolution, reduced sample volume, and analysis duration. CE might represent a good compromise between analysis time and satisfactory characterization for some classes of phenolic compounds of virgin olive oils.     

Synchronous fluorescence spectroscopy for quantitative determination of virgin olive oil adulteration with sunflower oil

Adulteration of extra virgin olive oil with sunflower oil is a major issue for the olive oil industry. In this paper, the potential of total synchronous fluorescence (TSyF) spectra to differentiate virgin olive oil from sunflower oil and synchronous fluorescence (SyF) spectra combined with multivariate analysis to assess the adulteration of virgin olive oil are demonstrated. TSyF spectra were acquired by varying the excitation wavelength in the region 270–720 nm and the wavelength interval (Δλ) in the region from 20 to 120 nm. TSyF contour plots for sunflower, in contrast to virgin olive oil, show a fluorescence region in the excitation wavelength range 325–385 nm. Fifteen different virgin olive oil samples were adulterated with sunflower oil at varying levels (0.5–95%) resulting in one hundred and thirty six mixtures. The partial least-squares regression model was used for quantification of the adulteration using wavelength intervals of 20 and 80 nm. This technique is useful for detection of sunflower oil in virgin olive oil at levels down to 3.4% (w/v) in just two and a half minutes using an 80-nm wavelength interval.     

Visible and near-infrared absorption spectroscopy by an integrating sphere and optical fibers for quantifying and discriminating the adulteration of extra virgin olive oil from Tuscany

Because of its high price, extra virgin olive oil is frequently targeted for adulteration with lower quality oils. This paper presents an innovative optical technique capable of quantifying and discriminating the adulteration of extra virgin olive oil caused by lower-grade olive oils. An original set-up for diffuse-light absorption spectroscopy in the wide 400–1,700 nm spectral range was experimented. It made use of an integrating sphere containing the oil sample and of optical fibers for illumination and detection; it provided intrinsically scattering-free absorption spectroscopy measurements. This set-up was used to collect spectroscopic fingerprints of authentic extra virgin olive oils from the Italian Tuscany region, adulterated by different concentrations of olive-pomace oil, refined olive oil, deodorized olive oil, and refined olive-pomace oil. Then, a straightforward multivariate processing of spectroscopic data based on principal component analysis and linear discriminant analysis was applied which was successfully capable of predicting the fraction of adulterant in the mixture, and of discriminating its type. The results achieved by means of optical spectroscopy were compared with the analysis of fatty acids, which was carried out by standard gas chromatography.     

Simultaneous determination of long-chain aliphatic aldehydes and waxes in olive oils

A procedure for the simultaneous determination of long-chain aliphatic aldehydes, and aliphatic and triterpenic waxes in virgin olive oils is described. A fraction containing these compounds was isolated from the oil using solid-phase extraction on silica-gel cartridges. The fraction was analyzed by capillary GC on 35%-dimethyl-65%-diphenylpolysiloxane phase using on-column injection. In extra virgin olive oils, the long-chain aliphatic aldehydes with even carbon atom numbers from C22 to C30 were identified by comparison of retention times and mass spectra with those of synthesized standards. The concentration of total aldehydes ranged from 20.2 to 108.0 mg/kg-n-hexacosanal being the most abundant aldehyde. The determination of aliphatic waxes was achieved with similar or better precision than that of the EU official methods.     

Sequential (step-by-step) detection,identification and quantitation of extra virgin olive oil adulteration by chemometric treatment of chromatographic profiles

An analytical method for the sequential detection, identification and quantitation of extra virgin olive oil adulteration with four edible vegetable oils--sunflower, corn, peanut and coconut oils--is proposed. The only data required for this method are the results obtained from an analysis of the lipid fraction by gas chromatography-mass spectrometry. A total number of 566 samples (pure oils and samples of adulterated olive oil) were used to develop the chemometric models, which were designed to accomplish, step-by-step, the three aims of the method: to detect whether an olive oil sample is adulterated, to identify the type of adulterant used in the fraud, and to determine how much aldulterant is in the sample. Qualitative analysis was carried out via two chemometric approaches--soft independent modelling of class analogy (SIMCA) and K nearest neighbours (KNN)--both approaches exhibited prediction abilities that were always higher than 91% for adulterant detection and 88% for type of adulterant identification. Quantitative analysis was based on partial least squares regression (PLSR), which yielded R2 values of >0.90 for calibration and validation sets and thus made it possible to determine adulteration with excellent precision according to the Shenk criteria.     

Investigations of the adulteration of extra virgin olive oils with seed oils using their weak chemiluminescence

A weak chemiluminescence (CL) emission was observed in commercial Greek extra virgin olive oils (Knossos, Spitiko, Ananias, Altis, Minerva, Xenia) and in refined seed oils such as sunflower oils (Marata, Sanola, Sun, Mana, Sol, Minerva) as well as in corn oils (Flora, Minerva, Marata Sun and Sol) with potassium superoxide in the aprotic solvent dimethoxyethylene.On measuring the CL of mixtures of extra virgin olive oils with the cheaper refined seed oils, calibrations were produced which can be used for the determination of the adulteration of olive oils with seed oils down to 3%. Furthermore, depending on the kind of oils, “low” authenticity-CL-factors for olive oils (0.8-2.15 μmol l⁻¹ gallic acid) and “high” for seed oils (4.5-11.2 μmol l⁻¹ gallic acid) were calculated.     

Determination of betaines in vegetable oils by capillary electrophoresis tandem mass spectrometry--application to the detection of olive oil adulteration with seed oils

A CE–tandem mass spectrometry (MS²) methodology enabling the simultaneous determination of betaines (glycine betaine, trigonelline, proline betaine and total content of carnitines) in vegetable oils was developed. Betaines were derivatized with butanol previous to their baseline separation in 10 min using a 0.1 M formic acid buffer at pH 2.0. Ion trap conditions were optimized in order to maximize the selectivity and sensitivity. Analytical characteristics of the proposed method were established by evaluating its selectivity, linearity, precision (RSDs ranged from 4.8 to 10.7% for corrected peak areas) and accuracy by means of recovery studies (from 80 to 99%) and LODs and LOQs at 0.1 ppb level. The method was applied for the determination of the selected betaines in seed oils and extra virgin olive oils. MS² experiments provided the fingerprint fragmentation for the betaines identified in vegetable oils. In extra virgin olive oils, carnitines were not detected, making it possible to propose them as a feasible novel marker for the detection of adulterations of olive oils. Application of the developed method for the analysis of different mixtures of extra virgin olive oil with seed oil (between 2 and 10%) enabled the detection and quantitation of the total content of carnitines. The results obtained show the high potential of the developed method for the authentication and quality control of olive oils.     

Use of flow injection atmospheric pressure photoionization quadrupole time-of-flight mass spectrometry for fast olive oil fingerprinting

The recently introduced technique of an atmospheric pressure photoionization (APPI) source coupled to quadrupole time-of-flight mass spectrometry (QqTOFMS) has been applied to fast olive oil fingerprinting on the basis of the accurate mass measurements obtained with this instrumentation. The key compounds can be characterized as [M+H]+ (produced by proton transfer) or as [M]+* (by charge transfer) ions in the mass spectra. [M+H]+ ions, however, show higher abundance, especially for triacylglycerols. Other ions present in APPI-MS are the acylium ion [RiCO]+ and [RiCO-H2O]+. This latter ion is absent in the electrospray ionization (ESI)-MS spectra, and this represents valuable complementary information. Several critical parameters in the APPI source were optimized such as LC eluent composition, ion spray voltage and, especially, declustering potential. APPI-QqTOFMS allows easy discrimination among different edible oils: olive, extra virgin olive, olive-pomace, hazelnut, sunflower, corn and several mixed oils, with high throughput (approximately 1 min per sample). Cluster analysis was applied to obtain the best experimental conditions for oil discrimination on the basis of declustering potential. Principal components analyses of these APPI-MS spectra show that the approach can be used for studies of olive oil adulteration with other oils, even in the case of hazelnut oil that exhibits a high chemical similarity with olive oil.     

Effects of conventional heating on the stability of major olive oil phenolic compounds by tandem mass spectrometry and isotope dilution assay

The quality of olive oils is sensorially tested by accurate and well established methods. It enables the classification of the pressed oils into the classes of extra virgin oil, virgin oil and lampant oil. Nonetheless, it would be convenient to have analytical methods for screening oils or supporting sensorial analysis using a reliable independent approach based on exploitation of mass spectrometric methodologies. A number of methods have been proposed to evaluate deficiencies of extra virgin olive oils resulting from inappropriate technological treatments, such as high or low temperature deodoration, and home cooking processes. The quality and nutraceutical value of extra virgin olive oil (EVOO) can be related to the antioxidant property of its phenolic compounds. Olive oil is a source of at least 30 phenolic compounds, such as oleuropein, oleocanthal, hydroxytyrosol, and tyrosol, all acting as strong antioxidants, radical scavengers and NSAI-like drugs. We now report the efficacy of MRM tandem mass spectrometry, assisted by the isotope dilution assay, in the evaluation of the thermal stability of selected active principles of extra virgin olive oil.     

Solid-phase extraction-thin-layer chromatography-gas chromatography method for the detection of hazelnut oil in olive oils by determination of esterified sterols

The sterol composition of extra virgin olive oil is very characteristic and, thus, has become a helpful tool to detect adulterations with other vegetable oils. Special attention has been addressed to the separate determination of the free and esterified sterol fractions, since both have different compositions and can thus provide more precise information about the actual origin of the olive oil. In the case of admixtures with small amounts of hazelnut oil, this approach can be extremely useful, because the similarity between the fatty acid compositions of both oils hampers the detection of the fraud. A hyphenated chromatographic method was developed for a sensitive and precise determination of esterified sterols in olive oils. The oil was subjected to silica solid-phase extraction (SPE) fractionation, cold saponification of the collected fraction and purification on silica TLC. The sterol band was then injected into an SPB-5 (30 m x 0.25 mm I.D., 0.25 microM film thickness) and the ratio [% campesterol x (% 7-stigmastenol)2]/(% 7-avenasterol) was calculated. The method was tested on extra virgin olive oil; good sterol recoveries and repeatability were obtained. The results were compared with another method. which has a different sample preparation sequence (silica column chromatography, hot saponification and silica TLC). Similar results were achieved with both methods; however, the SPE-cold saponification-TLC-capillary GC was faster, required less solvent and prevented sterol decomposition. The SPE-method was applied to an admixture with 10% of hazelnut oil and to a screening of 11 oils (husk oil, virgin and refined olive oils) from different Mediterranean countries.     

A capillary electrophoresis-tandem mass spectrometry methodology for the determination of non-protein amino acids in vegetable oils as novel markers for the detection of adulterations in olive oils

A new analytical methodology based on capillary electrophoresis-mass spectrometry (CE-MS(2)) is presented in this work, enabling the identification and determination of six non-protein amino acids (ornithine, β-alanine, GABA, alloisoleucine, citrulline and pyroglutamic acid) in vegetable oils. This methodology is based on a previous derivatization with butanol and subsequent separation using acidic conditions followed by on-line coupling to an ion trap analyzer for MS(2) detection established through an electrospray-coaxial sheath flow interface. The electrophoretic and interface parameters were optimized obtaining the separation of all compounds in less than 15 min and with resolutions higher than 5. The proposed method was validated by assessing its accuracy, precision (RSD<7% for corrected peak areas), LODs and LOQs (between 0.04-0.19 ng/g and 0.06-0.31 ng/g, respectively) and linearity range (R(2)>0.99), and it was used in order to identify the selected non-protein amino acids in soybean oils, sunflower oils, corn oils and extra virgin olive oils. MS(2) experiments performed the fingerprint fragmentation of these compounds allowing to corroborate ornithine and alloisoleucine in seed oils but not in olive oils. The method was applied to identify and quantify olive oil adulterations with soybean oil detecting in a single run the amino acids in mixtures up to 2% (w/w). The results showed a high potential in using these compounds as novel markers for the detection of adulterations of extra virgin olive oils with seed oils. Thus, the developed method could be considered a simple, rapid and reliable method for the quality evaluation of extra virgin olive oil permitting its authentication.     

Fluorescence spectra measurement of olive oil and other vegetable oils

Fluorescence spectra of some common vegetable oils, including olive oil, olive residue oil, refined olive oil, corn oil, soybean oil, sunflower oil, and cotton oil, were examined in their natural state, with a wavelength of 360 nm used as excitation radiation. All oils studied, except extra virgin olive oil, exhibited a strong fluorescence band at 430-450 nm. Extra virgin olive oil gave a different by interesting fluorescence spectrum, composed of 3 bands: one low intensity doublet at 440 and 455 nm, one strong at 525 nm, and one of medium intensity at 681 nm. The band at 681 nm was identified as the chlorophyll band. The band at 525 nm was at least partly derived from vitamin E. The low intensity doublet at 440 and 455 nm correlated with the absorption intensity at 232 and 270 nm of olive oil. The measurements of these fluorescence spectra were quick (about 5 min) and easy and could possibly be used for authentification of virgin olive oil.     

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.     

Linear and non linear chemometric models to quantify the adulteration of extra virgin olive oil

Two mathematical methods to quantify adulterations of extra virgin olive oil (EVOO) with refined olive oil (ROO), refined olive-pomace oil (ROPO), sunflower (SO) or corn (CO) oils have been described here. These methods are linear and non linear models based on chaotic parameters (CPs, Lyapunov exponent, autocorrelation coefficients and two fractal dimensions) which were calculated from UV-vis scans (190-900 nm wavelength) of 817 adulterated EVOO samples. By an external validation process, linear and non linear integrated CPs/UV-vis models estimate concentrations of adulterant agents with a mean correlation coefficient (estimated versus real concentration of cheaper oil) greater than 0.80 and 0.97 and a mean square error less than 1% and 0.007%, respectively. In the light of the results shown in this paper, the adulteration of EVOO with ROO, ROPO, SO and CO can be suitably detected by only one chaotic parameter integrated on a radial basis network model.     

Discriminating olive and non-olive oils using HPLC-CAD and chemometrics

This work presents a method for an efficient differentiation of olive oil and several types of vegetable oils using chemometric tools. Triacylglycerides (TAGs) profiles of 126 samples of different categories and varieties of olive oils, and types of edible oils, including corn, sunflower, peanut, soybean, rapeseed, canola, seed, sesame, grape seed, and some mixed oils, have been analyzed. High-performance liquid chromatography coupled to a charged aerosol detector was used to characterize TAGs. The complete chromatograms were evaluated by PCA, PLS-DA, and MCR in combination with suitable preprocessing. The chromatographic data show two clusters; one for olive oil samples and another for the non-olive oils. Commercial oil blends are located between the groups, depending on the concentration of olive oil in the sample. As a result, a good classification among olive oils and non-olive oils and a chemical justification of such classification was achieved.     

On testing quality and traceability of virgin olive oil by calorimetry

Extra Virgin olive oils (7 samples) originating from different areas of Tuscany, defective olive oils (5 samples), commercial edible seed oils (4 samples) and two commercial samples of olive oil (one declared ‘extra virgin olive oil’ and one ‘olive oil’) were studied by different calorimetric techniques: high sensitivity isothermal, differential scanning, and modulated scanning calorimetry. The temperature interval (–60) – (+30)°C was explored for monitoring: i) the main features of the liquid↔solid phase transitions, ii) the nucleation and growth rate of the polymorphous crystalline phases of the triacylglicerols, and iii) the melting process. This investigation was planned for verifying the utility and effectiveness of calorimetry for screening quality and origin of olive oil. To this end, the main calorimetric operation modes have been applied, the experimental results reported and their utility for developing an effective and reliable screening protocol discussed.     

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.     

Rapid and quantitative extraction method for the determination of chlorophylls and carotenoids in olive oil by high-performance liquid chromatography

Colour is an organoleptic characteristic of virgin olive oil and an important attribute that affects the consumer perception of quality. Chlorophylls and carotenoids are the main pigments responsible for the colour of virgin olive oil. A simple analytical method for the quantitative determination of chlorophylls and carotenoids in virgin olive oils has been developed. The pigments were isolated from small samples of oil (1.0 g) by solid-phase extraction using diol-phase cartridges (diol-SPE), and the extract was analysed by reverse-phase HPLC with diode-array UV detection. Chromatographic peak resolution, reproducibility (coefficient of variation (C.V.) <4.5%) and recovery (>98.4%) for each component were satisfactory. A comparative study of the proposed method was performed versus classical liquid-liquid extraction (LLE) with N,N′-dimethylformamide and solid-phase extraction using a C18 column (C18-SPE). While 96.4% of the pigments were recovered by LLE, only 51.3% were isolated by C18-SPE in comparison to diol-SPE. Likewise, a higher alteration of pigment composition was observed when such LLE and C18-SPE procedures were used. In this sense, a higher ratio of pheophytin in comparison to that isolated by the diol-SPE procedure was achieved with both extraction procedures, indicating a greater extent of the pheophytinization reaction. Therefore, quantification of pigments from virgin olive oil by diol-SPE followed by RP-HPLC was found to be rapid, simple, required only a small amount of sample, consumed only small amounts of organic solvents, and provided high recoveries, accuracy and precision.     

Preliminary study on application of mid infrared spectroscopy for the evaluation of the virgin olive oil "freshness"

The freshness of virgin olive oils (VOO) from typical cultivars of Garda regions was evaluated by attenuated total reflectance (ATR) and Fourier transform infrared (FTIR) spectroscopy, in combination with multivariate analysis. The olive oil freshness decreased during storage mainly because of oxidation processes. In this research, 91 virgin olive oils were packaged in glass bottles and stored either in the light or in the dark at room temperature for different periods. The oils were analysed, before and after storage, using both chemical methods and spectroscopic technique.Classification strategies investigated were partial least square discriminant analysis (PLS-DA), linear discriminant analysis (LDA), and soft independent modelling of class analogy (SIMCA).The results show that ATR-MIR spectroscopy is an interesting technique compared with traditional chemical index in classifying olive oil samples stored in different conditions. In fact, the FTIR PCA results allowed a better discrimination among fresh and oxidized oils, than samples separation obtained by PCA applied to chemical data. Moreover, the results obtained by the different classification techniques (PLS-DA, LDA, SIMCA) evidenced the ability of FTIR spectra to evaluate the olive oil freshness. FTIR spectroscopy results are in agreement with classical methods. The spectroscopic technique could be applied for the prediction of VOOs freshness giving information related to chemical modifications. The great advantages of this technique, compared to chemical analysis, are related to rapidity, non-destructive characteristics and low cost per sample. In conclusion, ATR-MIR represents a reliable, cheap and fast classification tool able to assess the freshness of virgin olive oils.