Chemometric classification of olive cultivars based on compositional data of oils

European Union regulations provide important guidelines for maintaining the Protected Designation of Origin (PDO) of olive oil and other foods. This includes characterization of foods based on variety (cultivar) and geographical origin, as this may be used as a criterion for determining authenticity and quality. Therefore, analytical method standards need to be established to ensure these criteria. This study describes how cultivar differences can be established between Italian oils, obtained from single varieties, based on acid, sterol, and TAG differences determined by chemometrics. TAG and FA composition provided the best basis for differentiation of olive oils among cultivars. The results were compared with those obtained using ¹³C NMR analysis, and a similar differentiation between oils of different cultivars was achieved. ¹³C NMR provides useful information on the acyl composition and on the positional distribution of the glycerol moiety and can be used for classification of cultivars based on oil composition. Furthermore, the advantages of this technique come from the rapidity with which information can be obtained and from the very simple preparation procedure required for analysis.     

Use of high-resolution 13C nuclear magnetic resonance spectroscopy for the screening of virgin olive oils

¹³C Nuclear magnetic resonance (NMR) spectra of 104 oil samples were obtained and analyzed in order to study the use of this technique for routine screening of virgin olive oils. The oils studied included the following: virgin olive oils from different cultivars and regions of Europe and north Africa, and refined olive, “lampante” olive, refined olive pomace, high-oleic sunflower, hazelnut, sunflower, corn, soybean, rapeseed, grapeseed, and peanut oils, as well as mixtures of virgin olive oils from different geographical origins and mixtures of 5–50% hazelnut oil in virgin olive oil. The analysis of the spectra allowed us to distinguish among virgin olive oils, oils with a high content of oleic acid, and oils with a high content of linoleic acid, by using stepwise discriminant analysis. This parametric method gave 97.1% correct validated classifications for the oils. In addition, it classified correctly all the hazelnut oil samples and the mixtures of hazelnut oil in virgin olive oil assayed. All of these results suggested that ¹³C NMR may be used satisfactorily for discriminating some specific groups of oils, but to obtain 100% correct classifications for the different oils and mixtures, more information than that obtained from the direct spectra of the oils is needed.     

Characterization of italian olive oils based on analytical and nuclear magnetic resonance determinations

Analytical measurements and proton nuclear magnetic resonance (¹H NMR) spectra of phenolic extracts were performed on a set of italian extra-virgin olive oils from different cultivars and geographical locations of Apulia region. Multivariate statistical analysis (principal component analysis, hierarchical clustering analysis, and discriminant analysis) was applied separately to analytical and NMR data. Analytical parameters, in particular fatty acid compositions, permit the discrimination of olive variety, while ¹H NMR data of phenolic extracts permit a classification according to the geographical origin of the samples.     

A novel analytical method to detect adulteration of virgin olive oil by other oils

The present study focuses on the olefinic region of the ¹³C nuclear magnetic resonance (¹³C NMR) spectrum of virgin olive oil which shows 12 peaks resonating between 127.5 and 130 ppm. These peaks are assigned to the most abundant unsaturated fatty acid moieties of the olive oil, oleic and linoleic acids, which are present in α and β positions of the glycerol backbone. With the use of an internal reference pyrazine, the 12 peaks were integrated and their areas were expressed in mmol/g of virgin olive oil. The intensities of the 12 observed peaks were affected when an authentic virgin olive oil was mixed with a seed oil. This observation was used to develop a semiquantitative method to detect adulteration of virgin olive oil by other oils based on ¹³C NMR spectroscopy.     

Stable isotope characterization of olive oils. I—Compositional and carbon-13 profiles of fatty acids

Nearly 200 olive oils produced in the Mediterranean basin, mainly in Greece, during 4 yr from 1993 to 1996, were studied by gas chromatography (GC) and on-line GC-isotope ratio mass spectrometry (GC-C-IRMS). The composition of the oils in the more abundant fatty acids (C_16:0, C_16:1, C_18:0, C_18:1, C_18:2, and C_18:3) was obtained by GC after transesterification of the triglycerides into methyl esters. Using the hyphenated GC-C-IRMS technique, the ¹³C contents of the three most abundant acids, C_16:0, C_18:1, and C_18:2, were measured with satisfactory accuracy. The results, analyzed in terms of geographical, temporal, and botanical factors, provide new criteria for the authentication of olive oils.     

Classification of vegetable oils by high-resolution <Superscript>13</Superscript>C NMR spectroscopy using chromatographically obtained oil fractions

¹³C NMR spectra of oil fractions obtained chromatographically from 109 vegetable oils were obtained and analyzed to evaluate the potential use of those fractions in the classification of vegetable oils and to compare the results with the NMR analysis of complete oils. The oils included the following: virgin olive oils from different cultivars and regions of Europe and north Africa; “lampante” olive, refined olive, refined olive pomace, hazelnut, rapeseed, high-oleic sunflower, corn, grapeseed, soybean, and sunflower oils; and mixtures of virgin olive oils from different geographical origins. Oils were divided into two sets of samples. The training set (98 samples) was employed to select the variables that resulted in significant discrimination among the different oil classes. By using stepwise discriminant analysis, more than 98% of correct validated assignments were obtained; these results were confirmed when applied to the test set (11 blind samples). Results suggest that the use of oil fractions considerably increases the discriminating power of NMR in the analysis of vegetable oils.     

Discrimination of Cod Liver Oil According to Wild/Farmed and Geographical Origins by GC and <Superscript>13</Superscript>C NMR

The objective of this study was to test the possibility of using lipid profiles obtained by gas chromatography (GC) and ¹³C nuclear magnetic resonance (NMR) in authentication of cod liver oils according to wild/farmed and geographical origin. GC and ¹³C-NMR data of cod liver oil from wild and farmed fish from different locations in Norway and Scotland were obtained, and analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA) to test if it was possible to differentiate oil from wild and cultured cod (Gadus morhua L.), and to further elucidate differences between fish from the different farms/catch area. Cod liver oils of wild and farmed origin were clearly separated in the PCA score plot both from GC and NMR data. From NMR data it was also possible to observe groupings based on geographical origin (farm/catch area) of the different samples. Using LDA with cross validation the wild/farmed classification rates were 97% for GC data and 100% for NMR data. In the classification of cod liver oils according to geographical origin (38 samples from six different farms/catch area), the correct classification rate was 63% for GC data and 95% for NMR data.     

Improved quantitative13C nuclear magnetic resonance criteria for determination of grades of virgin olive oils. The normal ranges for diglycerides in olive oil

A quantitative method was established to determine the presence and composition of mono-, di-, and triglycerides of olive oils of superior gradevia ¹³C NMR. The total diglyceride content and the ratio ofsn-1,2- andsn-1,3-diglycerides in extra virgin oils extracted from different olive cultivars were correlated with maturity. The correlation can be applied to identify the oils by variety. No monoglycerides were detected in the oils examined.     

Stable isotope characterization of olive oils: II-deuterium distribution in fatty acids studied by nuclear magnetic resonance (SNIF-NMR)

Site-specific isotope fractionation of hydrogen was investigated, at natural abundance, by deuterium nuclear magnetic resonance (SNIF-NMR) on nearly two hundred olive oil samples. Owing to the complexity of the ²H-NMR spectra of the mixtures of fatty acids obtained after hydrolysis of the oils, the different signals were gathered into six clusters. Knowing the contribution to the clusters of each of the four fatty acids considered (C_16:0, C_18:0, C_18:1, and C_18:2) and the composition of the fatty acids in the mixture, it is possible to compute the site-specific isotope ratios of the clusters from the molar fractions obtained from the ²H-NMR-spectra and from the total isotope ratio of the mixture, determined by isotope ratio mass spectrometry (IRMS). The results are discussed in terms of geographical (country, region and elevation) and temporal (year) parameters and they are tentatively explained on a climatic basis.     

The triglyceride composition,structure, and presence of estolides in the oils ofLesquerella and related species

Members of the genusLesquerella, native to North America, have oils containing large amounts of hydroxy fatty acids and are under investigation as potential new crops. The triglyceride structure of oils from twenty-fiveLesquerella species in the seed collection at our research center has been examined after being hydrolysis-catalyzed by reverse micellar-encapsulated lipase and alcoholysis-catalyzed by immobilized lipase. These reactions, when coupled with supercritical-fluid chromatographic analysis, provide a powerful, labor-saving method for oil triglyceride analysis. A comprehensive analysis of overall fatty acid composition of these oils has been conducted as well.Lesquerella oils (along with oils from two other Brassicaceae:Physaria floribunda andHeliophilia amplexicaulis) have been grouped into five categories: densipolic acid-rich (Class I); auricolic acid-rich (Class II); lesquerolic acid-rich (Class III); an oil containing a mixture of hydroxy acids (Class IV); and lesquerolic and erucic acid-rich (Class V). The majority of Class I and II triglycerides contain one or two monoestolides at the 1- and 3-glycerol positions and a C₁₈ polyunsaturated acyl group at the 2-position. Most Class III and IV oil triglycerides contain one or two hydroxy acids at the 1- and 3-positions and C₁₈ unsaturated acid at the 2-position. A few of the Class III oils have trace amounts of estolides. The Class V oil triglycerides are mostly pentaacyl triglycerides and contain monestolide and small amounts of diestolide. Our triglyceride structure assignments were supported by¹H nuclear magnetic resonance data and mass balances.     

Quantitative determination of castor oil in edible and heat-abused oils by13C nuclear magnetic resonance spectroscopy

Application of¹³C nuclear magnetic resonance (NMR) spectroscopy for detection of castor oil (CO) in various edible oils, such as coconut oil, palm oil, groundnut oil and mustard oil, is described. Characteristic signals observed at δ 132.4, δ 125.6, δ 71.3, δ 36.8 and δ 35.4 ppm, due to C10, C9, C12, C13 and C11 carbons of ricinoleic acid (RA) in CO, were selected for distinguishing it from edible oils. Quantitative¹³C NMR spectra of oils were recorded in CDCl₃ with a gated decoupling technique. The minimum detection limits for qualitative and quantitative analyses were 2.0 and 3.0%, respectively. The proposed method is simple, nondestructive and requires no sample pretreatment. Its application to heat-abused oils has also been demonstrated successfully without any of the interferences observed in most other methods.     

Determination of unsaturated fatty acid composition by high-resolution nuclear magnetic resonance spectroscopy

High-resolution nuclear magnetic resonance (NMR) spectroscopy provides useful data for analyzing fatty acid compositions of edible vegetable oils. Quantitation of each fatty acid was carried out by evaluation of particular peaks. According to the ¹H NMR method, terminal methyl protons, divinyl protons, and allyl protons are useful to calculate linolenic acid, linoleic acid, and oleic acid, respectively. The ω-2 carbon, divinyl carbon, and allylic carbons were used for calculation of these acids by the ¹³C NMR method. Compositional results obtained by NMR coincided well with those of the conventional gas chromatography (GC) method. Results from ¹³C NMR were in better agreement with those from GC than were the results obtained by the ¹H NMR method.     

Determination of unsaturated fatty acid composition by high-resolution nuclear magnetic resonance spectroscopy

High-resolution nuclear magnetic resonance (NMR) spectroscopy provides useful data for analyzing fatty acid compositions of edible vegetable oils. Quantitation of each fatty acid was carried out by evaluation of particular peaks. According to the ¹H NMR method, terminal methyl protons, divinyl protons, and allyl protons are useful to calculate linolenic acid, linoleic acid, and oleic acid, respectively. The ω-2 carbon, divinyl carbon, and allylic carbons were used for calculation of these acids by the ¹³C NMR method. Compositional results obtained by NMR coincided well with those of the conventional gas chromatography (GC) method. Results from ¹³C NMR were in better agreement with those from GC than were the results obtained by the ¹H NMR method.     

Conifer seed oils: Distribution of Δ5 acids between α and β chains by13C nuclear magnetic resonance spectroscopy

Nine seed oils from three different conifer families, already examined by gas chromatography and known to contain diene, triene, and tetraene C₁₈ and C₂₀Δ5 acids, have been reexamined by high-resolution¹³C nuclear magnetic resonance spectroscopy. The Δ5 acids are apparent only in the α-chains. This location is independent of chainlength, double-bond number, and the species considered and is probably a general factor of conifer seed oils. The spectra confirm the presence of oleic, linoleic, α-linolenic, and of Δ5 acids and give quantitative information about (total) n-6, n-3, and Δ5 acids that is in accord with that obtained by gas chromatography.     

Determination of Cis- and Trans-18:1 fatty acid isomers in hydrogenated vegetable oils by high-resolution carbon nuclear magnetic resonance

Carbon nuclear magnetic resonance (¹³C NMR) methods for determining the composition of cis-/trans- and positional isomers in hydrogenated vegetable oils were developed to reduce analytical time. By selecting appropriate olefinic carbon peaks and by measuring individual peak areas subsequent to the identification of isomeric peaks on the NMR spectrum, compositional results of the isomers coincided well with those obtained by conventional gas chromatography (GC). Therefore, it is highly beneficial to choose the ¹³C NMR method when analysis time is limited. Though the proposed ¹³C NMR method is promising, further development is needed. For the time being, combination with the traditional GC method is still encouraged for precise compositional analysis of cis-/trans- and positional isomers.     

An efficient method for a numerical description of virgin olive oil color with only two absorbance measurements

Four polynomial expressions are obtained that provide a good approximation and an easy, rapid calculation of the chromatic coordinates and the chroma—L ^*, a ^*, b ^*, and C—for the illuminant C and the standard observer, for a virgin or extra virgin olive oil; absorbance is measured at only 480 and 670 nm. These are as follows: L ^*=0.556458(A₄₈₀)²−2.51145A₄₈₀+0.55504(A₆₇₀)²−8.53016A₆₇₀+98.4089; a ^*=0.177372(A₄₈₀)²+2.1363A₄₈₀+1.43254(A₆₇₀)²−0.789231A₆₇₀−13.9246; b ^*=−16.0277(A₄₈₀)²+79.8932A₄₈₀−5.06558(A₆₇₀)²+3.36169A₆₇₀+31.9405; C=−15.8439(A₄₈₀)²+78.9312A₄₈₀−5.26784(A₆₇₀)²+3.56917A₆₇₀+33.3927. These give acceptable results, making the method a practical alternative to the extremely laborious Commission Internationale d’Eclairage (CIE) L ^* a ^* b ^* system, by which 391 absorbance values must be measured individually, nanometer by nanometer, before applying more complex equations. The validity of the proposed method has been confirmed by comparison, using a set of 20 sample oils different from the set of 25 oils used to generate the order of the equations. The variations between the values provided by the proposed and standard methods, respectively, had a mean of 0.00 for each of the chromatic variables—L ^* , a ^* , b ^* , and C; SD were moderate (0.71, 0.52, 1.22, and 1.22, respectively); the root mean square and the R ²-terms also confirmed the validity of the method.     

High-performance liquid chromatography and spectroscopic studies on fish oil oxidation products extracted from frozen atlantic mackerel

The formation of stable hydroxy derivatives from hydroperoxides produced during the oxidation of linoleic acid methyl ester and fish oil were studied by reverse-phase high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The oxidation products identified were mixtures of four isomeric hydroxy derivatives: 13-hydroxy-9-cis,11-trans-octadecadienoic, 13-hydroxy-9-trans,11-trans-octadecadienoic, 9-hydroxy-10-trans,12-cis-octadecadienoic, and 9-hydroxy-10-trans,12-trans-octadecadienoic acids. The presence of hydroxy compounds was confirmed by ¹³C NMR, which gave rise to a hydroxy carbon peak at 87 ppm, and by GC-MS, which showed three peaks corresponding to isomeric mixtures of trimethylsilyl ethers of the oxidized linoleic acid methyl ester. The mass spectra scans of the three peaks showed that they represent isomers of molecular weight 382 and are consistent with the molecular formula C₂₂H₄₂O₃Si. In oil extracted from stored frozen mackerel, 13-hydroxy-9-cis,11-trans-octadecadienoic acid was more prominent compared to the model lipid systems. HPLC offered a sensitive means of detection of hydroxy compounds produced both in the initiation and latter stages of oxidation. The effect of antioxidants added to the fish mince prior to storage can also be monitored by HPLC. Thus, the monitoring of lipid oxidation hydroxy derivatives by HPLC is of practical value in the efficient processing and quality control of fish, fish oils, and other fatty foodstuffs in order to enhance the acceptability, nutritional, and safety aspects.     

Identification and classification of olive oils by high-resolution13C nuclear magnetic resonance

Unsaponifiable matter from 19 olive and olive pomace oils were studied by high-resolution¹³C nuclear magnetic resonance spectroscopy. Their spectra showed characteristic peaks that corresponded to molecular substructures rather than the individual constituents present in the unsaponifiable matter. The presence of squalene and other hydrocarbons, sterols and triterpenic alcohols, in addition to other groups of minor compounds, were observed. Based on the analysis of these spectra, it was possible to distinguish among different grades of olive oils by using stepwise discriminant analysis. This direct method of analysis is suggested to be used in artificial neural networks to define oil identity and quality.     

Wax Ester Variation in Olive Oils Produced in Calabria (Southern Italy) During Olive Ripening

The wax ester composition of pressed olive oil and its variation during olive ripening were investigated by column chromatography/GC-on column technique. Six compounds were identified: C₃₆, C₃₈, C₄₀, C₄₂, C₄₄ and C₄₆ wax esters, which were grouped as total detected wax esters (TDWEs). The European Union (EU) includes C₄₀, C₄₂, C₄₄ and C₄₆ waxes (TEWEs) as a distinctive characteristic between different categories, with a maximum total content ≤250 mg/kg for an extra virgin olive oil. The International Olive Council (IOC) includes C₄₂, C₄₄ and C₄₆ waxes (TIOCWEs) as a purity parameter, with a maximum total content ≤150 mg/kg for an extra virgin olive oil. The analytical technique proposed by EU and IOC do not separate the wax esters from fatty acids esters with diterpenic alcohols (phytol and geranylgeraniol) that interfere with detected peaks. Although the examined cultivars were grown in the same geographical area and the same agricultural practices were applied to the trees, ANOVA analysis found significant differences among the oils extracted with the same machinery. The oil produced from the Itrana cultivar showed the lowest content in TEWEs (25.00–39.00 mg/kg) and in TIOCWEs (5.67–9.00 mg/kg). Wax content in Leccino and Pendolino cultivars showed a significant tendency to decrease during olive maturation, and a tendency to increase in all other cultivars from the first to the last harvest date when olive pigmentation changed from green to black.     

Quantitative triacylglycerol analysis of whole vegetable seeds by1H and13C magic angle sample spinning NMR spectroscopy

High-resolution¹³C and¹H magic angle sample spinning nuclear magnetic resonance (NMR) spectra have been obtained and used to define the relative unsaturated acyl distribution of triacylglycerols in whole oil seeds. Inverse gated proton decoupled¹³C and¹H NMR spectra permit the quantitative analysis of seeds containing simple oils,e.g., sunflower seeds containing oleyl and linoleyl unsaturates only. More sensitive¹³C NMR techniques are necessary for the analysis of specific seed classes. One such class is the rapeseed, which is especially difficult due to its low oil content (≈ 2 mg oil/seed) and complex unsaturated acyl profile of oleyl, linoleyl, linolenyl, erucyl, and eicosenoyl. The Distortionless Enhancement by Polarization Transfer technique significantly improves sensitivity to the extent that single rapeseeds can be examined within an hour of acquisition time. Furthermore, some positional (1,3- or 2-glycerol attachment) groups can be identified leading to a partial estimation of the 1,3-, 2-acyl distribution.