Detection of hazelnut oil in virgin olive oil by assessment of free sterols and triacylglycerols

Free sterols were evaluated as factors for discriminating between genuine virgin olive oil and hazelnut-mixed virgin olive oil. Numeric analyses of the results amplified the differences between groups. The application of this method to virgin olive oil samples and their mixtures with 10% hazelnut oil distinguished between genuine and nongenuine virgin olive oil with statistical certainty. Triacylglycerol analysis was tested for the same purpose by using parameter deltaECN42, but although it possessed a discriminating capacity, it alone could not distinguish the aforementioned groups with sufficient certainty. Free delta7-sterols data were combined with deltaECN42 data into a single discriminating function to improve differentiation and bring more ruggedness, and for detection of low amounts (10%) of hazelnut oil in virgin olive oil. In fact, the values obtained by addition of delta7-sterol data and deltaECN42 data showed a higher discriminating capacity than single parameters. In a single operation the method produced all the oil fractions necessary for analysis of free sterols and triacylglycerols with ECN42. Solid-phase extraction was applied in substitution of traditional chromatography on a silica column.     

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.     

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.     

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.     

Novel solid-phase extraction method to separate 4-desmethyl-, 4-monomethyl-, and 4,4'-dimethylsterols in vegetable oils

Conventional methods for sterol fractions separation by TLC have some drawbacks such as low recovery and time consuming. A new solid-phase extraction (SPE) method was developed with stepwise elution by increasing the polarity of solvents mixture: n-hexane and diethyl ether. This method was applied to separate sterol fractions of hazelnut and virgin olive oils, and our results were compared with those of TLC method. The recovery of spiked authentic sample of 4-desmethylsterols in oil was higher with the SPE method (94%) compared with the TLC method (62%). The amount of 4,4'-dimethylsterols and 4-desmethylsterols separated with SPE in both hazelnut and virgin olive oil samples were at least 75% and 35%, respectively, higher than that of TLC. Generally, both methods obtained similar results for 4-monomethylsterols of the two oils. This new SPE method to separate phytosterol fractions was less time consuming, simpler and can be used instead of preparative TLC to detect adulteration of virgin olive oil with hazelnut oil.     

Quantification of free and esterified sterols in Portuguese olive oils by solid-phase extraction and gas chromatography-mass spectrometry

A simple and accurate method based on solid-phase extraction (SPE), transesterification and gas chromatography-mass spectrometry (GC-MS) was developed for the quantitative analysis of free and esterified sterols of olive oil. In order to achieve better separation of esterified and free sterols, silica and alumina SPE adsorbents were tested. Separations by silica provided more reproducible results. The transesterification of both sterol fractions was found to be more user friendly than saponification as a method to liberate the sterols from the respective esters. The free sterols were then silylated with N,O-bis-trimethylsilyltrifluoroacetamide (BSTFA) with 1% of trimethylchlorosilane (TMCS). The most favourable conditions for exploitation of this reagent were established. The optimized methodology was suitable for evaluation of free and esterified sterols in Protected Designation of Origin (PDO) olive oils and monovarietal olive oils with different maturation indices. The prevailing phytosterols in all olive oils were beta-sitosterol and campesterol. The free sterols predominated, although they seemed to decrease with the maturation of the olive fruits.     

The detection and quantification of adulteration in olive oil by near-infrared spectroscopy and chemometrics.

A new procedure has been developed for the classification and quantification of the adulteration of pure olive oil by soya oil, sun flower oil, corn oil, walnut oil and hazelnut oil. The study was based on a chemometric analysis of the near-infrared (NIR) spectra of olive-oil mixtures containing different adulterants. The adulteration of olive oil was carefully carried out gravimetrically in a 4 mm quartz cuvette, starting with pure olive oil in the cuvette first. NIR spectra of the 525 adulterated mixtures were measured in the region of 12,000-4000 cm(-1). The spectra were subjected batch wise to multiplicative signal correction (MSC) before calculating the principal component (PCA) models. The MSC-corrected data were subjected to Savitzky-Golay smoothing and a mean normalization procedure before developing partial least-squares calibration (PLS) models. The results revealed that the models predicted the adulterants, corn oil, sun flower oil, soya oil, walnut oil and hazelnut oil involved in olive oil with error limits +/-0.57, +/-1.32, +/-0.96, +/-0.56 and +/-0.57% weight/weight, respectively. Furthermore, the PCA developed models were able to classify unknown adulterated olive oil mixtures with almost 100% certainty. Quantification of the adulterants was carried out using their respective PLS models within the same error limits as mentioned above.     

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.     

Analysis of free and esterified sterols in vegetable oil methyl esters by capillary GC

The introduction of quality standards for vegetable oil methyl esters is gaining in importance due to their increased use as diesel fuel substitutes and as technical products. Free and esterified sterols, the main constituents of the unsaponifiable matter in vegetable oils, are recovered in vegetable oil methyl esters and may influence the technical properties of vegetable oil methyl ester products. A rapid gas chromatographic method for the qualitative and quantitative determination of free and esterified sterols in vegetable oil methyl esters has therefore been developed. The concentration of the free sterols as well as their qualitative and quantitative composition and the concentration of the sterol esters have been determined in rape seed oil methyl ester samples by GC–FID. Prior to analysis, the free sterols were silylated with N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane; betulinol was used as an internal standard. Calibration was performed by analysis of standard solutions containing β-sitosterol, cholesteryl stearate, and betulinol. The reproducibility of the quantitative results has been evaluated by repeated injections of the same test solution and by repeated complete analysis of the same sample.     

Optimization and application of methods of triacylglycerol evaluation for characterization of olive oil adulteration by soybean oil with HPLC-APCI-MS-MS

Triacylglycerols (TAGs) are the main constituents of vegetable oils where they occur in complex mixtures with characteristic distributions. Mass spectrometry using an atmospheric pressure chemical ionization interface (APCI-MS) run in positive mode and an Ion Trap mass analyser were applied in the study of olive and soybean oils and their mixtures. Direct injections of soybean and olive oil solutions allowed the identification of ions derived from the main TAGs of both oils. This procedure showed to be a simple and powerful tool to evaluate mixtures or addition of soybean to olive oil. TAG separation was optimized by high performance liquid chromatography (HPLC) using an octadecylsilica LiChrospher column (250 mm × 3 mm; 5 μm) and a gradient composed of acetonitrile and 2-propanol allowed the separation of the main TAGs of the studied oils. APCI vaporization temperature was optimized and best signals were obtained at 370 °C. Multiple reaction monitoring (MRM) employing the transition of the protonated TAG molecules ([M+H]⁺) to the protonated diacylglycerol fragments ([M+H−R]⁺) improved the selectivity of TAG detection and was used in quantitative studies. Different strategies were developed to evaluate oil composition following TAG analysis by MRM. The external standard calibration and standard additions methods were compared for triolein quantification but the former showed to be biased. Further quantitative studies were based on the estimates of soybean and olive oil proportions in mixtures by comparison of TAG areas found in mixtures of known and unknown composition of both oils. Good agreement with expected or labeled values was found for a commercial blend containing 15% (w/w) of olive oil in soybean oil and to a 1:1 mixture of both oils, showing the potential of this method in characterizing oil mixtures and estimating oil proportions. Olive oils of different origins were also evaluated by mass spectra data obtained after direct injections of oil solutions and principal component analysis (PCA). Argentinean olive oils were clustered in a different area of the principal components plot (PC2 × PC1) in comparison with European olive oils. The commercial blend containing 15% (w/w) of olive oil in soybean oil appeared in a completely different area of the graphic, showing the potential of this method to screen out for olive oil adulterations.     

Differentiation of vegetable oils by mass spectrometry combined with statistical analysis

The main triacylglycerol (TAG) composition of different plant oils (almond, avocado, corn germ, grape seed, linseed, mustard seed, olive, peanut, pumpkin seed, sesame seed, soybean, sunflower, walnut and wheat germ) were analyzed using two different mass spectrometric techniques: HPLC/APCI-MS (high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry) and MALDI-TOFMS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry).Linear discriminant analysis (LDA) as a multivariate mathematical statistical method was successfully used to distinguish different plant oils based on their relative TAG composition. With LDA analysis of either APCI-MS or MALDI-MS data, the classification among the almond, avocado, grape seed, linseed, mustard seed, olive, sesame seed and soybean oil samples was 100% correct. In both cases only 6 different oil samples from a total of 73 were not classified correctly.     

Comprehensive two-dimensional liquid chromatography x gas chromatography: evaluation of the applicability for the analysis of edible oils and fats

Edible fats and oils are complex mixtures containing a wide range of (classes of) compounds. The most important group of compounds are the triglycerides (triacylglycerides, TAGs). Because of the large number of possible fatty acid combinations, an enormous number of TAGs is possible. In the present feasibility study, the applicability of different modes of comprehensive two-dimensional LCXGC for detailed oil and fat analysis is evaluated. Comprehensive LCXGC was found to be an extremely powerful analytical method for the analysis of complex TAG samples. Using the new comprehensive set-ups, TAGs can be separated according to two independent parameters: carbon number vs. number of double bonds, or fatty acid composition vs. number of double bonds. The information content of comprehensive separations by far exceeds that of the current generation of analytical methods. The quantitative results of the separations show a good agreement with data obtained from standard analytical methods. The comprehensive methods studied can also be used for fingerprinting of oil samples, as well as for the analysis of target compounds or compound groups. Highly detailed separations of olive oil samples were obtained. Zooming in on one region of the chromatogram allowed reliable analysis of wax esters without interferences of sterol esters.     

Comprehensive two-dimensional liquid chromatography×gas chromatography: evaluation of the applicability for the analysis of edible oils and fats

Edible fats and oils are complex mixtures containing a wide range of (classes of) compounds. The most important group of compounds are the triglycerides (triacylglycerides, TAGs). Because of the large number of possible fatty acid combinations, an enormous number of TAGs is possible. In the present feasibility study, the applicability of different modes of comprehensive two-dimensional LC×GC for detailed oil and fat analysis is evaluated. Comprehensive LC×GC was found to be an extremely powerful analytical method for the analysis of complex TAG samples. Using the new comprehensive set-ups, TAGs can be separated according to two independent parameters: carbon number vs. number of double bonds, or fatty acid composition vs. number of double bonds. The information content of comprehensive separations by far exceeds that of the current generation of analytical methods. The quantitative results of the separations show a good agreement with data obtained from standard analytical methods. The comprehensive methods studied can also be used for fingerprinting of oil samples, as well as for the analysis of target compounds or compound groups. Highly detailed separations of olive oil samples were obtained. Zooming in on one region of the chromatogram allowed reliable analysis of wax esters without interferences of sterol esters.     

Optimization of a technique for the estimation of the composition of edible oil blends

The fatty acid, sterol and tocopherol contents of edible oils were used to determine the formulation of blends of oils, which are themselves complex mixtures, by means of a weighted least-squares estimator with backwards elimination. A t-test was used to determine whether an oil should be eliminated from the blend. Initially, the minimum t-value was set at 2.7, which corresponds to a probability of ca. 0.01 if normal distribution is assumed. Some compounds are present in one oil but cannot be detected in others and are valuable in differentiating between various oils. Assumptions about levels for the undetected compounds and their corresponding weightings were made earlier in order to implement the weighted least-squares technique. A simplex optimization procedure was applied to avoid the use of assumed values. An optimum for the t-value as well as for the levels and the weightings of the undetected compounds was established. Analytical results for 378 pure oils were used to develop the models. The optimized technique is slightly better for assessing the composition of unknown blends than the original technique, when tested on 93 samples which were blends of known amounts of sunflower-seed oil, groundnut oil, cottonseed oil, maize oil, olive oil and palm oil.     

Combining chromatography and chemometrics for the characterization and authentication of fats and oils from triacylglycerol compositional data—A review

The characterization and authentication of fats and oils is a subject of great importance for market and health aspects. Identification and quantification of triacylglycerols in fats and oils can be excellent tools for detecting changes in their composition due to the mixtures of these products. Most of the triacylglycerol species present in either fats or oils could be analyzed and identified by chromatographic methods. However, the natural variability of these samples and the possible presence of adulterants require the application of chemometric pattern recognition methods to facilitate the interpretation of the obtained data. In view of the growing interest in this topic, this paper reviews the literature of the application of exploratory and unsupervised/supervised chemometric methods on chromatographic data, using triacylglycerol composition for the characterization and authentication of several foodstuffs such as olive oil, vegetable oils, animal fats, fish oils, milk and dairy products, cocoa and coffee.     

Biosensor immunoassay for traces of hazelnut protein in olive oil

The fraudulent addition of hazelnut oil to more expensive olive oil not only causes economical loss but may also result in problems for allergic individuals as they may inadvertently be exposed to potentially allergenic hazelnut proteins. To improve consumer safety, a rapid and sensitive direct biosensor immunoassay, based on a highly specific monoclonal antibody, was developed to detect the presence of hazelnut proteins in olive oils. The sample preparation was easy (extraction with buffer); the assay time was fast (4.5 min only) and the limit of detection was low (0.08 μg/g of hazelnut proteins in olive oil). Recoveries obtained with an olive oil mixed with different amounts of a hazelnut protein containing hazelnut oil varied between 93% and 109%. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of phytosterols in extra-virgin olive oil by nano-liquid chromatography

In this work the applicability of nano-liquid chromatography (nano-LC) was evaluated for the determination of phytosterols in extra-virgin olive oil samples. These compounds represent a minor part of lipids in vegetable oils, but their quantification can be useful to establish oil origin and to reveal intentional adulterations. The analysis of five main sterols, specifically brassicasterol, stigmasterol, campesterol, cholesterol and β-sitosterol, was performed in a laboratory-assembled nano-LC system coupled with a UV detector. The separation of all compounds was obtained in about 20 min, employing a capillary column packed with a C18-RP (sub-2 μm particles) stationary phase for 15 cm. Methanol only was used as mobile phase. The simple method developed and optimized was validated in terms of repeatability, linearity, limit of detection and limit of quantification (0.78 and 1.56 μg/mL, respectively) achieving good results. After this, it was applied to the determination of phytosterols in extra-virgin olive oil samples. Isolation of phytosterols was obtained by solid-phase extraction, after saponification and liquid–liquid extraction of the unsaponified fraction with diethyl ether. Recovery tests were performed and values between 90% and 103%, with RSDs within 5%, were obtained. Moreover the nano-LC system was coupled with a mass spectrometer for an accurate identification of phytosterols.     

Analysis of virgin olive oil volatile compounds by headspace solid-phase microextraction coupled to gas chromatography with mass spectrometric and flame ionization detection

The efficiency of headspace solid-phase microextraction (SPME) was evaluated for the qualitative and semi-quantitative analysis of virgin olive oil volatile compounds. The behaviour of four fibre coatings was compared for sensitivity, repeatability and linearity of response. A divinylbenzene-Carboxen-polydimethylsiloxane fibre coating was found to be the most suitable for the analysis of virgin olive oil volatiles. Sampling and chromatographic conditions were examined and the SPME method, coupled to GC with MS and flame ionization detection, was applied to virgin olive oil samples. More than 100 compounds were isolated and characterised. The presence of some of these compounds in virgin olive oil has not previously been reported. The main volatile compounds present in the oil samples were determined quantitatively.     

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.     

Determination of pesticide residues by GC-MS using analyte protectants to counteract the matrix effect.

An analytical method was developed to determine pesticides of various chemical classes in soil, juice and honey using analyte protectants to counteract the enhancement of the chromatographic response produced by the presence of matrix components (matrix effect). This effect was more pronounced for soil and honey samples than for juice samples; regarding the pesticide chemical class, organochlorine pesticides were less affected by the presence of matrix components than triazines and organophosphorus pesticides. Several analyte protectants (2,3-butanediol, L-gulonic acid gamma-lactone, corn oil and olive oil) were tested for counteracting the observed matrix effect. L-Gulonic acid gamma-lactone was an effective protecting agent for most of the pesticides studied in soil and honey samples, whereas olive oil was very effective for juice samples. The combination of these two protectants was found to be an effective analyte protectant for all compounds in soil and honey samples.