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Food Chemistry 134 (2012) 22832290

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Food Chemistry
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Analytical Methods

Blends of olive oil and sunower oil: Characterisation and olive oil quantication using fatty acid composition and chemometric tools
M. Monfreda a,b,, L. Gobbi b, A. Grippa c
a

Central Directorate for Chemical Analysis and Development of Laboratories, Italian Customs Agency, via M. Carucci 71, 00143 Rome, Italy Department of Management, Sapienza University of Rome, via del Castro Laurenziano 9, 00161 Rome, Italy c Department of Business and Law, Roma Tre University, via Silvio DAmico 77, 00154 Rome, Italy
b

a r t i c l e

i n f o

a b s t r a c t
A method capable of recognising the percentage of olive oil in a blend is required to verify whether its labelling complies with the statements set out by the Commission Regulation (EC) No. 1019/2002. In this study an analytical methodology was developed in order to dene blends of olive oil and sunower oil, which contain 50% of olive oil, compared to blends with 40% and 60% of it, respectively. Methyl esters of fatty acids were analysed by GCFID and processed through chemometric tools (PCA, TFA, SIMCA and PLS). A strong differentiation of blends according to the amount of olive oil contained and a quantication model with a standard error of prediction of 1.51% were obtained. As this issue represents a signicant analytical challenge, variability associated with the fatty acid composition of olive oil was rst studied. 2012 Elsevier Ltd. All rights reserved.

Article history: Received 30 December 2011 Received in revised form 29 February 2012 Accepted 15 March 2012 Available online 4 April 2012 Keywords: Olive oil Blend Principal component analysis Target factor analysis Soft independent models of class analogy Partial least squares

1. Introduction Olive oils have been extensively studied; in 1991 an EEC Commission Regulation (No. 2568) was issued, which regulates not only the characteristics of olive oil and olive-residue oil but also the relevant methods of analysis for establishing any oils conformity to the relevant characteristics established for each category. The olive oil control eld is in constant evolution; as a result, the above regulation has been continuously amended, most recently with the introduction of fatty acid methyl esters and fatty acid ethyl esters method of analysis in Commission Regulation (EU) No. 61/2011. In Italy, the olive-growing industry is worth an estimated 3.2bn and is made up of over 775,000 businesses that use over 1.1 million hectares of land and produce around 550,000 tonnes a year (DellOrece, 2011). With exports and imports booming, strict control that aims to combat illegal attempts to boost prots is of absolute importance. European concern about commercial fraud on olive oils has always been signicant: a typical case is the unlawful addition of oilseeds in oils which should have been obtained exclusively from
Corresponding author at: Central Directorate for Chemical Analysis and Development of Laboratories, Italian Customs Agency, via M. Carucci 71, 00143 Rome, Italy. Tel.: +39 3396093528; fax: +39 0650244115. E-mail address: maria.monfreda@gmail.com (M. Monfreda).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.03.122

olive pressing. This fraud can be revealed by means of several chemical analyses provided for in EEC Regulation No. 2568/91 and its subsequent modications and supplements. However, no ofcial method currently exists for quantifying olive oil and/or an oilseed in a blend. Several studies have been carried out on this subject, using various analytical methods followed by appropriate chemometric tools, with the aim of accurately measuring the relative quantities of olive oil and oilseeds in ad hoc blends. It is noteworthy to observe that these studies focused their attention on the detection and quantication limits of seed oils in samples where olive oil is the main ingredient (Aparicio & Aparicio-Ruz, 2000; Cristopoulou, Lazaraki, Komaitis, & Kaselimis, 2004; Fauhl, Reniero, & Guillou, 2000; Gurdeniz & Ozen, 2009; Papadopoulos, Triantis, Tzikis, Nikokavoura, & Dimotikali, 2002; Parcerisa, Casals, Boatella, Codony, & Rafecas, 2000; Pea, Crdenas, Gallego, & Valcrcel, 2005; Poulli, Mousdis, & Georgiou, 2007; Priego Capote, Ruiz Jimnez, & Luque de Castro, 2007; Vigli, Philippidis, Spyros, & Dais, 2003; Vlachos et al., 2006). This approach appears reasonable when considering that blends have always been associated with the battle against commercial fraud regarding oils that should have been obtained exclusively by olive pressing. Consequently, even if the concentration range of binary blends has been completely explored (0100%), these studies have focused, however, on their evaluation at a low concentration of seed oils, instead of examining blends in the strict sense of the word. It should also be noted that in several cases, a relatively small number of olive oil samples has been used

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for preparing blends with seed oils: in other words, the response variability range of olive oil towards analytical methods could be more fully detailed. The subject of blends of olive oils and other vegetable oils is handled by Commission Regulation (EC) No. 182/2009, amending Commission Regulation (EC) No. 1019/2002 on marketing standards for olive oil. In these regulations, the marketing of blends of olive oils and other vegetable oils is also included. Regulation No. 1019/2002 sets the following trade description for blends whose labelling highlights the presence of olive oil elsewhere than in the list of ingredients, using words, images or graphics: Blend of vegetable oils (or the specic names of the vegetable oils concerned) and olive oil, directly followed by the percentage of olive oil in the blend. It is also stipulated that the presence of olive oil may be highlighted by images or graphics on the label of a blend, only where it accounts for more than 50% of the blend concerned. Commission Regulation (EC) No. 182/2009 declares, moreover, that Member States may prohibit the production in their territory of blends of olive oil and other vegetable oils for internal consumption. However, they may not prohibit the marketing on their territory of such blends originating from other countries and they may not prohibit the production on their territory of such blends for marketing in another Member State or for export. This regulation, in fact, laid the foundations for the boom in the blends market. In light of what is stated in Regulation No. 182/2009, an analytical method capable of quantifying the percentage of olive oil in a mixed blend of olive oil and other vegetable oils will soon be necessary, in order to prevent marketing fraud. The percentage of 50% of olive oil is the most important discriminator, with particular reference to the labelling. De la Mata-Espinosa, Bosque-Sendra, Bro, and Cuadros-Rodrguez (2011) suggested an interesting approach to the problem of olive oil quantication in a blend, using high performance liquid chromatography (HPLC) analysis of triacylglycerols and subsequent multivariate regression. The fatty acid composition, evaluated by means of methyl esters of fatty acids, is a noteworthy parameter for the characterisation of an olive oil ngerprint. Limit values for the percentage of methyl esters of fatty acids in olive oils are established in the EEC Regulation No. 2568/91 and its subsequent modications and supplements. It can be seen that such limits are consistent for each category of olive oil (except for the trans isomers), because they depend on the type of oil. The method of analysis is also set out in the Commission Regulation (Annex XA and XB). The methyl esters limits set by the EEC Regulation No. 2568/91 and its subsequent modications and supplements are shown in Table 1, from which it can be seen that the percentages of some fatty acids (palmitic, palmitoleic, stearic, oleic and linoleic) are

expected to vary over quite a large range, whilst for the other acids, found at amounts lower than 1% mass/mass (m/m), only the upper limits are established. By contrast, the fatty acid composition of seed oils is not set out in any rule (neither in European law, nor in any single Member State law, but some references can be found in literature (Amelotti, 1990)). In Table 2, the fatty acid compositions of some seed oils (corn, peanut, grape seed, sunower and rice) are shown. In light of the high variability found in the fatty acid composition of olive oil, it can be deduced that mixing olive oil with seed oil will produce some blends that have even more variable fatty acid composition. For this reason, the identication and the quantication of the olive oil percentage in a blend is an analytical challenge. This issue may be studied with the help of chemometric procedures, such as principal component analysis (PCA), target factor analysis (TFA), soft independent models of class analogy (SIMCA) and partial least squares (PLS). PCA, SIMCA and PLS have already been employed for the study of blends. PCA has also been used for the evaluation of virgin olive oil evolution with time (Alonso-Salces, Holland, & Guillou, 2011). PCA and SIMCA have also been widely used for olive oil discrimination in relation to geographical origin (Alonso-Salces et al., 2010; Longobardi et al., 2012; Mannina, Marini, Gobbino, Sobolev, & Capitani, 2010; Marini, Magr, Bucci, Balestrieri, & Marini, 2006; Papadia et al., 2011; Pizarro, Rodrguez-Tecedor, Prez-delNotario, & Gonzlez-Siz, 2011), or the olive fruit variety (Bucci, Magr, Magr, Marini, & Marini, 2002; Diaz, Mers, Casas, & Franco, 2005; Pouliarekou et al., 2011). TFA has been employed in order to verify the presence or the absence of specic compounds in matrices which, as a result of some instrumental analyses, gave a notable overlapping of signals (Lohnes, Guy, & Wentzell, 1999; Miao, Cai, & Shao, 2011; Shao, Roske, & Grifths, 2010). The aim of this study was to verify whether it is possible to recognise, rstly in blends of olive oil and sunower oil, the percentage of 50% of olive oil (because of the discrimination power of this gure in blend labelling), comparing such blends with ones containing 40% and 60% of olive oil, and by applying the ofcial method for the analysis of methyl esters of fatty acids by GCFID, followed by chemometric tools. 2. Materials and methods 2.1. Samples First of all, 12 olive oil samples with signicant variability in their fatty acid composition were selected, in order to obtain results covering the full variability that exists among olive oils. For each olive oil sample, three blends with a sunower oil sample were prepared, with a total volume of 10 mL and containing 40%, 50% and 60% respectively in olive oil volume. Volumes were measured and transferred with a 10-mL micropipette and the blends were vigorously shaken. Finally, 36 samples (spread in three categories related to the percentage of olive oil) were prepared and analysed. 2.2. Sample preparation and analysis by GCFID Sample (0.1 g) was dissolved in 2 mL of heptane and shaken. The sample was then trans-esteried with 0.2 mL of 2 N methanolic potassium hydroxide solution and vigorously shaken. The upper layer of the solution was analysed by GCFID (Commission Regulation (EEC) No.2568/91). GC was performed on a 60 m 0.2 mm i.d. 0.25 lm lm HP88 capillary column using an Agilent 6890 gas chromatograph connected to a ame ionisation detector (FID). The GC conditions used were as follows: injection volume 1 lL, split injection 50:1 at

Table 1 Fatty acids methyl esters limits set by the Commission Regulation(EEC) No. 2568/91 for olive oils. Fatty acids methyl esters Myristic Palmitic Palmitoleic Margaric Margaroleic Stearic Oleic Linoleic Arachidic Linolenic Eicosenoic Behenic Lignoceric Limits (% m/m) 60.05 7.520.0 0.33.5 60.3 60.3 0.55.0 55.083.0 3.521.0 60.6 61.0 60.4 60.2 60.2

M. Monfreda et al. / Food Chemistry 134 (2012) 22832290 Table 2 Some examples of fatty acid composition of seed oils. Corn (% m/m) Myristic Palmitic Palmitoleic Stearic Oleic Linoleic Arachidic Linolenic Eicosenoic Behenic Lignoceric Max 0.1 10.015.0 Max 0.5 1.53.0 23.041.0 41.063.0 0.20.7 0.61.1 0.20.5 Max 0.2 Peanut (% m/m) Max 0.1 8.013.5 Max 0.5 2.05.0 35.070.0 15.048.0 1.02.5 Max 0.2 0.91.5 2.04.0 1.02.5 Grape seed (% m/m) Max 0.2 6.08.0 Max 0.1 3.06.0 12.025.0 60.076.0 Max 0.5 Max 0.5 Max 0.2 Max 0.2 Sunower (% m/m) Max 0.1 5.08.0 Max 0.5 2.57.0 15.040.0 40.074.0 Max 0.5 Max 0.3 Max 0.5 0.51.0

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Rice (% m/m) 0.10.7 17.022.0 0.10.4 1.02.5 30.045.0 35.050.0 0.10.5 1.02.0 0.20.3 tr0.3

240 C; oven temperature set at 170 C for 30 min, then ramped to 230 C at 5 C min1 (held for 3 min), giving a total run time of 45 min. Helium gas carrier was held at a constant ow rate of 1 mL min1, whilst the detector was set at a temperature of 300 C. A typical fatty acid methyl esters chromatogram of a blend containing 50% of olive oil is shown in Fig. 1. 2.3. Preliminary statistical tests The fatty acid variance associated with the 12 blends containing 50% of olive oil was compared with the variance associated with 10 blends containing 50% of the same olive oil by a one-tailed F-test at 0.05 signicance level. Furthermore, a one-way Anova was performed on the whole data set in order to compare, for each variable, the variance within any category with the variance between categories. 2.4. Multivariate statistical analysis First of all, an unsupervised tool, namely principal component analysis (PCA), was applied in order to have a general overview of the data. PCA was applied in order to highlight a natural grouping of samples depending on their olive oil percentage and to gain some useful information about how variable loadings are linked to a potential separation between groups. Target factor analysis (TFA) is an extremely useful technique for studying mixtures of pure samples. It is strongly linked to PCA, from which the p number of signicant factors is achieved, corresponding to the number of pure samples used to produce the

mixtures. If the composition of some pure samples is known, TFA tests if they are possible constituents of the mixture by evaluating the distance of each pure sample in the data base, as the residual variance from the hyperplane dened by the p signicant components. Among supervised pattern recognition procedures, class modelling tools show some advantages compared to classication methods as they build a model for each category instead of a simple delimiter between classes. The modelling rule discriminates between the studied category and the rest of the universe. As a consequence, each sample can be assigned to a single category, to more than one category (if more than one class is modelled) or, alternatively, considered as an outlier if it falls outside the model. SIMCA (Soft independent models of class analogy) builds a mathematical model of the category with its principal components and a sample is accepted by the specic category if its distance to the model is not signicantly different from the class residual standard deviation. SIMCA was applied, considering a 95% condence level, to dene the class space and an unweighted augmented distance (Wold & Sjostrom, 1977). Multivariate regression was nally applied, using PLS algorithm which looks for the direction of maximum variance among the variables, but weighing the variables upon their higher or lower correlation with the response variable. The model builds latent variables, which are a combination of original variables. The number of latent variables used for calculating the closed form was obtained from the minimum value of the standard deviation of the error of prediction (SDEP), as a function of the number of latent variables.

Fig. 1. Typical fatty acids methyl esters chromatogram of a blend containing 50% of olive oil: myristic (1), palmitic (2), palmitoleic (3), margaric (4), margaroleic (5), stearic (6), oleic (7) trans-linoleic (80 ), linoleic (8), arachidic (9), linolenic (10), eicosenoic (11), behenic (12) and lignoceric (13).

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M. Monfreda et al. / Food Chemistry 134 (2012) 22832290 Table 4 Fatty acid composition of the sunower oil sample used in preparing the blends. Sunower oil (% m/m)

All the computations were performed using V-PARVUS (Forina, Lanteri, Armanino, Cerrato-Oliveiros, & Casolino, 2010) and SPSS (IBM Statistics Computer Program, 2010). 3. Results and discussion The fatty acid composition of 241 olive oil samples, analysed over the last 2 years (20102011) was evaluated in order to select 12 samples with an acidic prole variable over as wide a range as possible. These data are shown in Table 3 where, for each fatty acid, the mean value, the standard deviation, the minimum value, the maximum value and the range of variability (the difference between the maximum and minimum value) are indicated. From this table, for instance, a range of variability of 24.58% for oleic acid (with a minimum value of 55.30% and a maximum of 79.88%) and a range of 15.59% for linoleic acid (with a minimum value of 4.38% and a maximum of 19.97%) can be noted. The fatty acid composition of the sunower oil sample used in preparing the aforementioned blends is shown in Table 4, whilst the fatty acid proles of the 12 olive oil samples selected for this study are presented in Table 5. 3.1. Preliminary statistical tests A preliminary statistical test was carried out in order to check whether a single analysis for each blend might be adequate for this study; in other words, whether the variability associated with the measurements repeatability in one sample would be signicantly lower than the variability among samples belonging to the same category (variability within category), considering that each category is made up of blends of a sunower oil with widely varying olive oils. This comparison (made by a one-tailed F-test) showed that variance within category is signicantly higher than the variance due to the measurements repeatability (the olive oil sample labelled number 4 in Table 5 was chosen for this evaluation). Therefore, it seemed adequate for the purpose of this study to perform only one analysis for each sample, with vigorous homogenisation of each blend before analysis. One-way ANOVA was performed on the whole data set, in order to select, for further statistical analysis, the more signicant fatty acids for discrimination between categories. Myristic, margaroleic, oleic, linoleic, arachidic, linolenic, behenic and lignoceric acids have a between-category variability signicantly higher than the within-category variability (for lignoceric acid the discrimination power was veried only between categories containing 50% and 60% of olive oil, because the preliminary assumption of homogeneity of variance, was veried only between these two categories).

Myristic Palmitic Palmitoleic Margaric Margaroleic Stearic Oleic Linoleic Arachidic Linolenic Eicosenoic Behenic Lignoceric

0.07 6.55 0.11 0.04 0.03 3.67 32.00 56.03 0.23 0.04 0.13 0.72 0.38

Palmitic, palmitoleic, margaric, stearic and eicosenoic acids do not have discrimination power because the between-category variability is not signicantly different from the within-category variability. 3.2. Multivariate statistical analysis Multivariate statistical analysis was performed on a data set of 36 samples and eight variables selected by ANOVA (corresponding to % m/m of myristic, margaroleic, oleic, linoleic, arachidic, linolenic, behenic and lignoceric acids) in order to:  highlight the natural variability existing within groups containing 40%, 50% and 60% of olive oil by PCA;  identify the ngerprint of pure oils used for preparing the blends (olive and sunower) by TFA;  nd a decision rule for evaluating whether an unknown blend could be associated to one of the analysed groups by means of SIMCA;  build a quantitative model which would allow one to nd the percentage of olive oil in a blend of olive oil and sunower oil, using PLS. 3.2.1. Principal component analysis (PCA) Variables were preprocessed by column autoscaling: yiv = (xiv xvm)/sv, where xiv is the value of the variable v relative to the sample i, xvm is the mean value of the variable v, and sv is the standard deviation of the variable v. Two PCs were extracted, with eigenvalues >1; they explain 79.7% of the total variance. The bi-plot of PC2 versus PC1 is shown in Fig. 2. A well-dened separation of samples in accordance with the percentage of olive oil was achieved on PC1: the blends containing 40% olive oil cluster in the negative values of PC1, samples with 50% olive oil are grouped around the origin, whilst blends with 60% of olive oil have the highest positive scores on PC1. From the bi-plot, it can be seen that variables with positive loadings on PC1 are linolenic, oleic, arachidic and margaroleic acids, whilst variables with negative loadings on PC1 are behenic, linoleic, myristic and lignoceric acids. Such loading distribution is strongly related to the fatty acid composition of pure oils; in fact, olive oil has a signicantly higher content of oleic, linolenic, arachidic and margaroleic acids compared to sunower oil, whilst the latter has a signicantly higher concentration of behenic, linoleic, myristic and lignoceric acids compared to olive oil (see Tables 2 and 3). This explains why blends that are more similar to olive oil than sunower oil (with 60% of olive oil) cluster toward positive values of PC1, whilst blends that are more similar to sunower oil than olive oil

Table 3 Summary of fatty acid composition of 241 olive oils: mean value (% m/m), standard deviation, minimum value, maximum value and range of variability (difference between the maximum and minimum value) for each fatty acid are shown. Mean Myristic Palmitic Palmitoleic Margaric Margaroleic Stearic Oleic Linoleic Arachidic Linolenic Eicosenoic Behenic Lignoceric 0.010 12.09 1.15 0.05 0.10 3.01 72.77 9.47 0.36 0.60 0.23 0.11 0.05 St. Dev. 0.004 2.12 0.44 0.02 0.03 0.53 5.94 3.90 0.03 0.05 0.04 0.02 0.02 Min 0.000 9.49 0.58 0.00 0.06 1.61 55.30 4.38 0.23 0.24 0.09 0.03 0.00 Max 0.038 18.98 2.54 0.12 0.21 4.15 79.88 19.97 0.45 0.71 0.42 0.22 0.16 Range 0.04 9.50 1.96 0.12 0.15 2.54 24.58 15.59 0.22 0.47 0.33 0.19 0.16

M. Monfreda et al. / Food Chemistry 134 (2012) 22832290 Table 5 Fatty acid composition of the 12 olive oil samples selected for this study (% m/m). Sample 1 Myristic Palmitic Palmitoleic Margaric Margaroleic Stearic Oleic Linoleic Arachidic Linolenic Eicosenoic Behenic Lignoceric Others 0.01 11.85 1.04 0.05 0.11 2.97 74.49 8.13 0.34 0.58 0.24 0.13 0.04 0.02 Sample 2 0.01 13.22 0.74 0.06 0.09 2.86 66.73 14.67 0.42 0.71 0.33 0.11 0.05 0.00 Sample 3 0.01 13.32 1.38 0.04 0.09 2.62 67.69 13.55 0.36 0.59 0.25 0.03 0.07 0.00 Sample 4 0.01 13.41 1.37 0.04 0.09 2.63 67.29 13.83 0.36 0.58 0.22 0.11 0.06 0.00 Sample 5 0.01 17.33 2.08 0.03 0.07 2.42 58.44 18.21 0.37 0.67 0.19 0.12 0.06 0.00 Sample 6 0.01 10.35 0.86 0.06 0.10 3.57 76.66 7.04 0.36 0.61 0.22 0.11 0.05 0.00 Sample 7 0.01 18.06 2.42 0.03 0.08 2.26 57.16 18.59 0.37 0.67 0.17 0.11 0.07 0.00 Sample 8 0.01 10.36 0.87 0.04 0.08 3.81 77.72 5.78 0.36 0.60 0.21 0.11 0.05 0.00 Sample 9 0.01 10.4 0.85 0.04 0.08 3.74 77.62 5.92 0.36 0.61 0.21 0.11 0.05 0.00 Sample 10 0.01 13.79 1.40 0.07 0.15 2.19 70.18 10.8 0.36 0.60 0.25 0.12 0.08 0.00 Sample 11 0.01 9.51 0.75 0.09 0.15 3.54 77.28 7.16 0.37 0.66 0.24 0.11 0.13 0.00

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Sample 12 0.00 16.79 2.16 0.03 0.08 2.20 59.54 17.76 0.36 0.64 0.17 0.11 0.16 0.00

Fig. 2. Bi-plot of PC1 versus PC2 obtained from the data set relative to 36 samples and eight variables.

(with 40% of olive oil) are grouped toward negative values of PC1. Finally, blends containing 50% of olive oil cluster in the middle of the plot.

Table 6 Fatty acid composition of six pure oils used in target factor analysis (% m/m). Olive Myristic Palmitic Palmitoleic Margaric Margaroleic Stearic Oleic Linoleic Arachidic Linolenic Eicosenoic Behenic Lignoceric Others 0.01 12.09 1.15 0.05 0.10 3.01 72.77 9.47 0.36 0.60 0.23 0.11 0.05 0.00 Corn 0.03 9.97 0.11 0.06 0.03 2.02 28.02 57.98 0.32 0.90 0.17 0.19 0.20 0.00 Peanut 0.04 11.82 0.07 0.06 0.03 3.38 39.97 37.75 1.45 0.10 0.90 2.85 1.46 0.12 Grape seed 0.04 6.58 0.10 0.05 0.03 3.84 19.11 69.65 0.15 0.23 0.15 0.02 0.05 0.00 Sunower 0.1 8.01 0.12 0.00 0.00 3.21 27.52 60.11 0.18 0.05 0.11 0.45 0.14 0.00 Rice 0.36 19.5 0.24 0.03 0.00 1.97 42.19 32.55 0.99 0.92 0.46 0.29 0.47 0.03

3.2.2. Target factor analysis Target factor analysis was applied in order to check whether the ngerprints of olive oil and sunower oil would have been recognised in the 36 blends, forming the original data matrix. For this purpose, a test set of six pure objects, corresponding to the fatty acid composition of the same number of pure oils, was added to the aforementioned data matrix. The pure oils fatty acid composition is reported in Table 6 where for the olive oil composition a mean value was chosen among the 241 olive oils already mentioned, whilst for the sunower oil a different sample was chosen from the one used for preparing blends. For the purpose of this study, corn, peanut, rice and grape seed oil were also chosen, as shown in Table 6. Factors with eigenvalues >1 were chosen as signicant. TFA was applied to a data set consisting of 36 samples, six pure objects and eight variables (the same variables, pre-processed by column autoscaling: yiv = (xiv xvm)/sv, used for PCA). As only two signicant factors, namely two factors with eigenvalues >1, were extracted with PCA, TFA has to identify the same number of target

factors, in other words two types of oil used to produce blends. Indeed, the rst and the second target factor (ordered according to the residual variance) were the chromatographic proles of olive oil and sunower oil, respectively. It must be noted that, despite the sunower oil used as a pure sample having a different

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composition from the one used for preparing blends, and although the olive oil used as a pure sample had a mean composition among 241 samples (most of them were not included in this data set), the ngerprints of olive oil and sunower oil were still recognised among six different kinds of vegetable oils. Such a result is noteworthy because, in real cases, where the exact composition of pure oils used for preparing blends might not be available, it would be possible to check whether a blend actually contains the oils listed on the label. This result is also important because it demonstrates (for blends of olive oil with sunower oil) that just a single analysis of fatty acids, in spite of the variability existing in pure samples, could be enough for a qualitative control of the blend composition stated on the label. 3.2.3. SIMCA SIMCA was applied to the same data matrix used for PCA and TFA, performing a cross validation with six cancellation groups and using seven components to build the mathematical model of each class. Classication ability (modelling rate) and prediction ability (prediction rate) of 100% in both were obtained. The same parameters are normally provided by all the classication methods too but the performance of a class-modelling tool can also be evaluated by sensitivity and specicity. The sensitivity (the percentage of objects belonging to the category which are correctly identied by the mathematical model) and specicity (the percentage of objects from other categories which are classied as foreign) were both equal to 100%. From Coomans plots (Coomans et al., 1984), representing the distance of each sample from a specic category, all samples are classied in their own class, there are no outliers, nor are there any samples assigned to both classes. Fig. 3 is relative to classes 1 (blends with 40% of olive oil) and 2 (blends with 50% of olive oil), whilst Fig. 4 is relative to classes 2 and 3 (blends with 60% of olive oil). Coomans plot relative to classes 1 and 3 is not displayed as it is less relevant for the purpose of this study than the discrimination between class 2 and each of the other classes. 3.2.4. PLS Twelve samples were extracted from the data set in order to construct the external calibration set. Variables were centred, eight cancellation groups were used for model validation, obtaining a

Fig. 4. Coomans plot for classes 2 and 3.

prediction ability that was less optimistic but more realistic than the one given by the leave-one-out application. Performance of the model was evaluated through the mean of SDEP (because this parameter was calculated for each cancellation group), whilst model stability was evaluated by the standard deviation of SDEP in cancellation groups and the standard deviation of the mean of SDEP in cancellation groups. External evaluation of the model was quantied by the rootmean-squared error of prediction (RMSEP) (Kowalski & Seasholtz, 1991; Massart et al., 1997). The best prediction was obtained with ve latent variables; therefore, the closed form was calculated with a complexity of ve. The prediction ability, evaluated by the mean value of SDEP in cancellation groups, is extremely satisfying, considering that equals 1.51% of olive oil in blends. Model stability (that is, performance variability among cancellation groups) was also good, because of the low values of the standard deviation of SDEP in cancellation groups (equals 0.68%) and the standard deviation of the mean of SDEP in cancellation groups (equals 0.24%). The RMSEP obtained for the external evaluation set was 2.10%. In Fig. 5, the predicted (5a) and computed percentages (5b) of olive oil versus the actual ones are shown. 4. Conclusions Multivariate statistical analysis, applied to blends of olive oil (12 samples were used) with sunower oil (1 sample) gave outstanding results. PCA allowed the highlighting of the differences existing between groups containing 40%, 50% and 60% of olive oil. Such groups are, in fact, well separated on the rst principal component, even though the data set was studied to represent the existing variability in the fatty acid composition of olive oil in the widest way possible. PCA also gave some useful information about variable loadings in the separation between groups. With TFA, it was possible to recognise the ngerprints of both oils used for preparing blends, in spite of the sunower and olive oil targets having a different fatty acid composition from that of the samples used for preparing blends. Such a procedure might be applied, in real cases, in order to check whether a blend is really obtained from the oils listed on the label, where only the type of pure oils and not their fatty acid

Fig. 3. Coomans plot for classes 1 and 2.

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Fig. 5. Predicted (a) and computed (b) percentages of olive oil versus actual ones.

composition is supposed to be indicated. Moreover, TFA indicated that just a single analysis of fatty acids, in spite of the existing variability in the fatty acid composition of pure samples, might well be enough for an (qualitative) identication of pure oils forming a blend. SIMCA allowed the construction of class models without outliers or overlapping areas between classes (sensitivity and specicity both of 100%). It can be concluded that blends containing 50% of olive oil were perfectly distinguishable from the others, even though there was a great variability in the olive oils used for preparing them. Finally, with PLS a good quantitative model was constructed, having a standard error of prediction of 1.51% and a standard error of prediction on the external evaluation set of 2.10%. This study provides an initial evaluation of how natural variability in the olive oil might affect the blends with a specic seed oil (sunower) and suggests also a methodological approach, be it qualitative or quantitative, for verifying the composition of a binary blend of olive oil and sunower oil, in compliance with the

rules established by Commission Regulation (EC) No. 1019/2002. This method provides some advantages because it involves the application of only one analysis (against the several analyses required by the EEC Regulation No. 2568/91 for the classication of a pure olive oil) using an ofcial method (relatively quick and already set for the characterisation of olive oils) and is subject only to the analyses of some blends that have a known composition. The model proposed by this work will be extended to also include the variability associated with different types of seed oils, in order to build wider decision rules that could be applied to blends of vegetable oils to identify the percentage of olive oil, and/or the threshold value for the use of olive imagery or graphics on blended oil labelling. References
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