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Food Research International 45 (2012) 103 – 108 Contents lists available at SciVerse ScienceDirect Food Research

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

journal homepage: www.elsevier.com/locate/foodres Chemical changes of thermoxidized virgin olive oil

Chemical changes of thermoxidized virgin olive oil determined by excitation emission uorescence spectroscopy (EEFS)

Noelia Tena, Ramón Aparicio, Diego L. García-González

Instituto de la Grasa (CSIC), Padre García Tejero 4, E-41012 Sevilla, Spain

article info

Article history:

Received 5 August 2011 Accepted 12 October 2011

Keywords:

Excitation emission matrix (EEM) PARAFAC Thermoxidation Virgin olive oil Phenols Oxidation products

abstract

The uorophores present in virgin olive oil evolve dur ing any thermoxidation process and thereby uores- cence spectroscopy can be used as a straightforwa rd approach to study the ox idative degree of oils. Samples of heated virgin olive oils collected from a fryer every two hours up to 94 h were analyzed to study their excitation emission matrix (EEM) and the evolution of the uorescent composition during the thermoxidation process. The potential usefulness of EEM to study thermoxidized virgin olive oils has been explored. For this purpose, parallel factor analysis (PARAFAC) was used to interpret the results. The emission pro le obtained from three-factor PARAFAC model calculated for 47 samples of thermoxidized oil in the range of λ ex =250 298 nm and λ em = 300 700 nm allows the decomposition of the uorescence landscape in speci c information about phenols, pigments and oxidation products respectively. The chemical interpretation from the PARAFAC model was checked with chemical analysis of phenols, tocoph- erols and chlorophyll pigments.

© 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The ability of virgin olive oil to emit uorescent radiation is due to the presence of uorescent compounds as chlorophylls, pheophytins, tocopherols, Vitamin E and oxidative compounds ( Galano, Durán, Correa, Roldán, & Rodriguez, 2003; Sikorska et al., 2004). Previous studies based on uorescence spectroscopy of olive oil have associat- ed several emission spectrum regions with these compounds present in olive oil. The region between 300 and 400 nm is attributed to the tocopherols and phenols ( Sikorska, Górecki, Khmelinskii, Sokorski, & Koziol, 2005; Zandomeneghi, Carbonaro, & Caffarata, 2005), the region of 440445 nm shows good correlation with the K232 and K270 ( Kyriakidis & Skarkalis, 2000), the derivatives of vitamin E are associated with the region 400600 nm ( Dupuy et al., 2005), and the bands in the region of 600700 nm are associated with the chlorophyll and pheophytin pigments ( Dupuy et al., 2005). Different approaches based on uorescence spectroscopy have been implemented for the characterization of edible oils. The differ- ences between the uorescence emission spectra of olive oil and other edible oils (Sikorska et al., 2005 ) and between different catego- ries of olive oil ( Nicoletti, 1990) have been previously described. The uorescence spectra have also been used to detect adulterations, as the presence of hazelnut oil in virgin olive oil (Sayago, Morales, & Aparicio, 2004), and even to determine the degree of oil oxidation

Corresponding author. Tel.: +34 954 61 15 50; fax: +34 954 61 67 90. E-mail address: dluisg@cica.es (D.L. García-González).

0963-9969/$ see front matter © 2011 Elsevier Ltd. All rights reserved. doi: 10.1016/j.foodres.2011.10.015

and thermal deterioration in virgin olive oils ( Poulli, Chantzos, Mousdis, & Georgiou, 2009; Tena, García-González, & Aparicio, 2009). On the other hand, the uorescence excitation spectra with emission at 330 and 450 nm have been used to control the evolution of phenols, vitamin E and degradation of hydroperoxides during heating (Cheikhousman et al., 2005). Furthermore, applications with total synchronous uorescence spectroscopy and excitationemission uorescence spectroscopy (EEFS) have been developed to monitor changes in the olive oil during storage in different conditions ( Sikorska et al., 2008 ). A technique selected to ensure the safety and quality of any food product should be rapid and easy to use ( Belton et al., 1995). Fluores- cence spectroscopy, being a rapid technique, provides valuable infor- mation on thermolabile compounds (e.g. phenols, vitamin E, pigments), and therefore, this technique has the advantage over other spectroscopic methodologies of providing specic information on oxidation. In consequence, it can be proposed as a suitable tool for monitoring the deterioration degree of frying olive oils. Synchro- nous uorescence and excitationemission uorescence spectros- copies are preferable for the analysis of complex multicomponent samples such as edible oils, compared with conventional uorescence spectroscopy ( Poulli, Chantzos, Mousdis, & Georgiou, 2009; Poulli, Mousdis, & Georgiou, 2009). In particular the excitation emission uorescence spectroscopy (EEFS) involves the simultaneous acquisition of a range of excitation wavelengths (λ ex ) and emission wavelengths (λ em ). As a result, a total uorescence prole of the sample over the scanned range is obtained. This uorescence landscape is called the excitationemission matrix (EEM) and it has the ability of obtaining

104

N. Tena et al. / Food Research International 45 (2012) 103108

simultaneous information of different compounds in the sample. This procedure improves the selectivity of uorescence spectroscopy and it has proved to be useful in discerning olive oils by slight differences in their composition. The statistical tools to interpret the EEFS results require dividing the information provided by the three-dimensional matrix (EEM) into two-dimensional arrays. The parallel factor analysis (PARAFAC) has the advantage of working with three-dimensional arrays providing a limited number of factors that represent the whole matrix. The model obtained with PARAFAC simplies the interpretation of the excitationemission proles and it provides a unique solution, regardless of the type of rotation of the factors (Harshman, 1970). The objective of this work is to propose the excitation emission uorescence spectroscopy combined with PARAFAC algorithm as a tool to monitor the changes in the uorescence composition of virgin olive oil during thermoxidation. For this purpose, the information obtained from the EEM was processed to determine when the oil had to be discarded for human consumption by correlating the most signicative spectral bands with the percentage of total polar compounds. The chemical assignment of the spectral bands was done according to the work reported by other authors and supported by the chemical analysis of phenols, tocopherols, and chlorophyll pigments.

2. Materials and methods

2.1. Sample preparation

The study was carried out with forty-seven samples of ther- moxidized virgin olive oils. These samples were obtained through a thermoxidation process carried out in a 4 L domestic fryer with automat- ic temperature controller (Heidolph EKT 3001, Schwabach, Germany). The fryer was set at 190 °C for 94 h in cycles of 8 h per day. A sample of 40 mL was collected every 2 h until the end of the heating process. Samples were kept in brown glass vials at 4 °C until further chemical and spectroscopic analyses.

2.2. Analysis of total polar compounds

The percentage of total polar compounds (% PC) was determined gravimetrically according to the IUPAC Standard Method No. 2.507. Nonpolar and polar fractions were separated from 1 g of oil by silica gel column chromatography (20 g silica adjusted to a water content of 5%, w/w) hexane/diethyl ether (90:10, v/v) and 150 mL diethyl ether as elution systems, respectively. The nonpolar fraction was eluted with 150 mL of n-hexane/diethyl ether (90:10, v/v), while the polar fraction was eluted with 150 mL of diethyl ether. Efciency of the separation was conrmed by TLC using n-hexane/diethyl ether/ acetic acid (80:20:1, v/v/v) and visualized with iodine vapor. The percentage of polar fraction was calculated by weighing both fractions after the evaporation of the solvents.

2.3. Excitationemission uorescence spectroscopy (EEFS)

Spectrouorimetric measurements were performed with a Cary Eclipse (Varian Ibérica, Madrid, Spain). This instrument was equipped with a continuous xenon lamp, excitation and emission monochroma- tors, and a photomultiplier. A 10 mm×10 mm quartz cell (3 mL) was used for right-angleRA uorometry. The wavelength range for the emission monochromator was 300700 nm, and for the excitation monochromator was 250 nm at 298 nm, intervals for each dimension being 1 and 4 nm respectively. The polarizer operated in automatic mode. The excitation and emission slits were both of 5 nm. The photo- multiplier voltage was set at 600 V to avoid saturation. The time taken to acquire a spectrum was of 10 min (intermediate speed). All data were exported to an Excel spreadsheet and were subsequently treated with the softwares Matlab v7.8 (The MathWorks, Natick, MA) and

Statistica v7.0 (Statsoft, Tulsa, OK). The experimental conditions allowed getting spectra of satisfactory intensity, resolution and signal-to-noise ratio. After each sample the cell was cleaned using detergent, followed by Milli-Q hot water and acetone to dry it and eliminate possible rests of samples. Each sample was analyzed in duplicated.

2.4. Determination of tocopherols

Tocopherols were analyzed by following the IUPAC Standard Method No. 2.432 . A solution of oil in hexane (10 mg/mL) was analyzed by HPLC (Agilent Technologies 1200) on a silica gel column (Merck, Superspher Si60, particle size 4 μm, 250 mm×4 mm i.d.), elut- ing with hexane/2-propanol (99:1, v/v) at a ow rate of 1 mL/min. The injection volume was of 20 μL. A uorescence detector (Agilent Technol- ogies 1100) with the excitation wavelength set at 290 nm and emission wavelength at 330 nm was used. The quantication was carried out by using an external calibration with a solution of α-tocopherol/Hexane (0.55.0 μg/mL) as standard.

2.5. Determination of phenols

A standard solution (0.5 mL) made of p -hydroxyphenylacetic (0.12 mg/mL) and o -coumaric acids (0.01 mg/mL) in methanol was added to a sample of ltered virgin olive oil (2.5 g). A rotary evapora- tor at 40 °C under vacuum was used to evaporate the solvent and the oily residue was dissolved in 6 mL of hexane. The diol-bonded phase cartridge was conditioned according to Mateos et al. (2001) . After the sample loading, the nal residue was extracted with 500 μL of methanol water (1:1 v/v) at 40 °C and a l- trated aliquot (20 μL) of the nal colorless solution was injected onto the HPLC system (LaChrom Elite Tokio, Japan), equipped with a diode array detector. The column was a Lichrospher 100RP-18 column (4.0 mm i.d.×250 mm; 5 μm, particle size) (Darmstadt, Germany) maintained at 30 °C. The gradient elution, at a ow rate of 1.0 mL/min, was achieved by using the following mobile phases: a mixture of water/phosphoric acid (95.5:0.5 v/v) (solvent A) and methanol acetonitrile (50:50 v/v) (solvent B). The change of solvent gradient was programmed as follows: from 95% (A)-5% (B) to 70% (A)-30% (B) in 25 min; 62% (A)-38% (B) in 10 min; 62% (A)-38% (B) in 5 min; 55% (A)-45% (B) in 5 min; 47.5% (A)-52.5%(B) in 5 min and 100% (B) in 5 min, followed by 5 min of maintenance. The chro- matographic signals were obtained at 235, 280, and 335 nm. Quantication of phenols cinnamic acid and lignans was carried out at 280 nm by using p-hydroxyphenylacetic acid as internal standard. Quantication of avones was done at 335 nm by using o-coumaric acid as internal standard. The response factors and recoveries were based on the procedure carried out by Mateos et al. (2001).

2.6. Determination of chlorophyll pigments

The determination of chlorophyll pigments was carried out by implementing the method proposed by Gertz and Fiebig (2006) to quantify the pirofeotina a. A sample of 300 mL was diluted in 1 mL of hexane. Separation of the pigments (pheophytins, pyropheophytin a and chlorophylls) was carried out by using a miniaturized column chromatography on a silica gel column. After loading the sample, the eluate of chlorophyll pigments were collected by passing 5 mL of acetone and it was analyzed by HPLC (Agilent technologies 1100, Santa Clara, California) equipped with a C18-RF Spherisorb ODS-2 (25 cm × 4 mm id; 3 μm particle size) (Waters, Saint Quentin- Yvelines, France) and a diode array detector. It worked in isocratic regime, using H 2 O/MeOH/acetone as mobile phase (4:36:60 v/v/v) with a ow of 1 mL/min. The chromatographic signals were obtained at 410 nm. The compounds identied and quantied were: pheophor- bide, chlorophyll, pheophytins a, a, b, and b, and pyropheophytin a.

N. Tena et al. / Food Research International 45 (2012) 103108

105

40 100 total phenols alfa-tocopherol 90 PC total chlorophyll pigments 80 30 70 60 20
40
100
total phenols
alfa-tocopherol
90
PC
total chlorophyll pigments
80
30
70
60
20
50
40
30
10
20
10
0
0
0
5
10
15
20
25
30
35
40
Residual content (%)
Polar compounds (%)

Thermoxidation time (h)

Fig. 1. Evolution of residual percentage of total phenols, α-tocopherol and total chlorophyll pigments and the percentage of total polar compounds (% PC) with the thermoxidation time. The dotted line indicates the legal limit to discard the frying oils (25% PC).

calculated by residual analysis indicated that three was the optimal number of factors. Nonnegative constraints were applied as they are advisable when working with spectral data set ( Bro, 1997 ). The

processing of spectroscopic data with PARAFAC algorithm has been extensively described elsewhere ( Andersen & Bro, 2003; Bro, 1997 ). The multiway model PARAFAC allows an easy interpretation of three dimensional matrix because the model decomposes three-

way uorescence data into spectral excitation and emission pro les of uorophores. Thus, the total matrix was decomposed into three matrices (excitation, emission and samples), each one having three factors. The relationship between factors and the chemical

variables (e.g. vitamin E, phenols) was studied by multiple linear regression analysis (MLRA). In order to avoid hyperoptimistic results MLRA was carried out under the strictest conditions (Fisher-distribution table at p=0.05). The arrangement of data and PARAFAC analysis was

carried out with Matlab v7.8 (The MathWorks, Natick, MA), while Statistica 7.0 (StatSoft, Tulsa, OK) was used for carrying out the rest of statistical analyses.

3. Results and discussion

The quantication was carried out by using an external calibration line with a solution of chlorophyll a/acetone (0.20.5 mg/kg).

2.7. Statistical analysis

Prior to examining the whole set of uorescence data, the EEMs of the 47 samples were arranged in a three-dimensional structure

of

size 47 × 400 × 13 (samples × λ em × λ ex ). The array was decom-

posed by PARAFAC algorithm. The explained variance (93.991%)

The thermoxidation process of vegetable oils during frying induces a series of changes in its chemical composition that affects the quality and the safety of the frying oil and food ( Matthäus, 2006). The most established method to regulate the use of frying oils is the determination of the percentage of the total polar com- pounds (% PC), 25% being the maximum limit to discard the frying oils in most of the European countries (Stier, 2001 ). Thus, the % PC was determined in the samples to know the alteration degree and their suitability for consumption. Fig. 1 shows the evolution of % PC of the samples heated up to 40 h. The oil reached 25% PC after 30 h

Table 1 Evolution of the concentration (mg/kg) of individual uorophores present in the olive oil samples during 40 h of thermoxidation.

Time

% PC a

Chlorophyll pigments

 

Phenols

α -Tocopherol

(h)

Pheoph b

Pheo c

Pyroph d

HyT e

Ac-HyT f

Ty g

Pinoresinol

p-HPEA-EA h

0

3.45

2.66

2.07

1.62

17.71

7.17

16.82

3.85

tr

i

228.38

2

3.94

1.06

0.23

4.11

10.60

6.31

13.20

3.42

tr

i

220.49

4

4.73

0.36

0.14

2.32

8.30

4.12

12.62

3.36

tr

i

177.85

6

5.10

0.15

0.12

2.12

7.03

4.53

11.52

3.26

tr

i

162.15

8

5.67

4.83

3.09

10.50

2.80

tr

i

152.38

10

7.11

2.25

1.97

9.78

2.50

3.67

127.89

12

8.66

0.06

0.06

0.57

1.22

1.14

9.55

2.30

3.76

119.56

14

9.07

0.84

0.94

8.52

2.18

4.49

106.60

16

9.40

0.04

tr i

0.40

0.22

0.93

7.80

2.10

4.90

80.04

18

12.60

0.23

0.35

7.29

2.05

5.20

78.01

20

13.02

0.11

0.18

6.11

1.70

5.42

59.06

22

13.94

0.12

0.12

5.09

1.42

4.82

56.27

24

14.47

0.03

tr i

0.13

0.08

0.11

4.97

1.23

4.63

4.56

26

18.56

0.08

0.10

4.31

0.94

4.32

28

20.05

0.07

0.08

4.08

0.78

4.24

29

20.22

0.09

0.09

3.99

0.56

4.08

30

20.76

tr

i

tr i

tr

i

0.07

0.07

4.48

0.52

3.81

tr

i

31

26.91

0.08

0.06

4.05

0.50

3.49

 

32

27.52

0.06

0.05

4.10

0.49

3.47

33

27.60

0.06

0.05

4.00

0.48

3.43

34

29.03

0.07

0.04

3.99

0.47

3.37

35

30.95

0.07

0.03

3.31

0.46

3.33

36

31.03

tr

i

0.02

2.75

0.42

2.90

38

36.40

tr

i

tr i

tr

i

tr

i

tr i

2.23

0.40

2.50

tr

i

a Percentage of total polar compounds.

b Pheophorbide.

c Pheophytin b.

d Pyropheophytin.

e Hydroxytyrosol.

f Hydroxytyrosol acetate.

g Tyrosol.

h Ligstroside aglycone, oxidized aldehyde and hydroxylic form.

i Trace levels.

106

N. Tena et al. / Food Research International 45 (2012) 103108

of thermoxidation (dotted line), which points out the maximum time that this oil can be used as frying oil for that temperature and frying conditions. Fig. 1 also shows the evolution of the residual contents (%) of total phenols and chlorophyll pigments, both of them having uorescent properties. The chlorophyll pigments were the uorophores whose concentrations decrease faster during the thermoxidation process. The concentration decay of these compounds was slower after reach- ing 7.1% PC and they reduced their concentration at trace levels when the oil had 25% PC. The evolution of the concentration of the individ- ual chlorophyll pigments ( Table 1) shows that all of them decreased during the rst hours of thermoxidation, except for pyropheophytin, which increased its concentration fourfold in the rst two hours of thermoxidation and it degraded up to trace levels after 30 h. The concentration of total phenols and α-tocopherol (Fig. 1) shrunk at the same rate and they reached a plateau when the oil had 25% PC. The individual study of the phenols shows that the simple o-diphenols, hydroxytyrosol and hydroxytyrosol acetate (Table 1) were the phenols with more variation in their concentrations during the thermoxidation process. All these compounds reached trace levels after 20 h of thermo- xidation, the oil containing 20% PC (Table 1). On the contrary tyrosol and pinoresinol, together with some secoiridoid oxidized derivatives as p- HPEA-EA (Table 1), remained in the oil after 38 thermoxidation hours. These results partially match with those presented by Brenes, García, Dobarganes, Velasco, and Romero (2002). Once the chemical analyses revealed the chemical changes on the main uorophores in the oil, the evolution of these compounds was studied by excitation emission uorescence spectroscopy (EEFS). The 47 excitationemission matrices were analyzed by PARAFAC

16 Oxidation compounds 12 8 4 0 300 350 400 450 500 550 600 650
16
Oxidation
compounds
12
8
4
0
300 350
400
450
500
550
600
650
700
Factor 1
20 16 Chlorophyll pigments 12 8 4 0 -4 300 350 400 450 500 550
20
16
Chlorophyll
pigments
12
8
4
0
-4
300 350
400
450
500
550
600
650
700
Factor 2
8 Tocopherols and phenols 6 4 2 0 300 350 400 450 500 550 600
8
Tocopherols and phenols
6
4
2
0
300 350
400
450
500
550
600
650
700
Factor 3

Wavelenght (nm)

Fig. 2. Spectral pro les obtained from the three-factor calculated for the emission wavelengths from the PARAFAC model.

algorithm. Fig. 2 shows the emission proles obtained from the three-factor PARAFAC model. The three plots one for each factor showed a band with a maximum at 675 nm ( λ em ), which is associated with the presence of chlorophyll pigments in the samples ( Dupuy et al., 2005). The rest of the emission prole depended on the individ- ual factor. Thus, the emission prole of factor 1 showed a band with a maximum at 525 nm, assigned to oxidation compounds ( Dupuy et al., 2005). This band (λ em = 450650 nm) is absent in the emission pro- le of factor 2, and it appears at less intensity in the emission prole of factor 3. The emission prole of factor 2 was characterized with the ab- sence of bands except for that assigned to the chlorophyll pigments in the range λ em =650700 nm. Finally, the emission prole of factor 3 showed a characteristic band with a maximum at 350 nm (λ em ), which was absent in the proles of the other two factors and it was as- sociated with the presence of tocopherols and phenols (Sikorska et al., 2005; Zandomeneghi et al., 2005). In general, taking into account the range 300650 nm (omitting the λ em range of pigments), the emission prole of factor 3 was similar to the spectra of the samples at the beginning of the thermoxidation process when they were acquired at a single excitation wavelength (270 nm) (Tena et al., 2009). On the contrary, the emission prole of factor 1 was related to the spectra of samples at the end of the process, which include an intensive band in the range 450650 mm (λ em ) assigned to oxidation products (Tena et al., 2009). These results showed that the data modelling carried out by PARAFAC allows obtaining uorescence proles that can be used as ngerprints of specic compounds. Fig. 3 shows the score plot for the excitation wavelengths ( λ ex = 250298 nm) by representing the factors 2 and 3. The plot of these two factors pointed out a clear division of the excitation wave- lengths into two groups, establishing the boundary between both at 280 nm. The excitation wavelengths above this value (280 300 nm) include the maximum for phenols and tocopherols ( Sikorska et al., 2004; Zandomeneghi et al., 2005), which partly justies the distinc- tion between these two groups. By contrast, an excitation wavelength of 260270 nm (value 0.3 on the factor 2) generates information on oxidation compounds ( COI/T.15/NC no. 3/Rev. 3, 2009; Kyriakidis & Skarkalis, 2000). This results show that the optimum excitation wavelength to study the thermoxidized olive oils by conventional uorescence spectroscopy would be next to 280 nm since it would allow acquiring information from both phenols and oxidation products. Regarding the sample scores, Fig. 4 shows the projection plot of the PARAFAC model using the factors 1 and 2. This information was used to carry out a variance analysis of thermoxidized oils. Fig. 4 shows that the initial sample of olive oil, nonheated (3.5%PC), clearly differs from the other samples. According to the percentage of total polar compounds ( Fig. 1), the rest of the samples can be divided

0.9 298 0.8 0.7 0.6 0.5 294 0.4 0.3 290 0.2 286 278 0.1 266
0.9
298
0.8
0.7
0.6
0.5
294
0.4
0.3
290
0.2
286
278
0.1
266
282
274 270
254
262
258
250
0.0
0.1
0.2
0.3
0.4
0.5
Factor 3

Factor 2

Fig. 3. Projection plot of factors 2 and 3 calculated for the excitation wavelengths from the PARAFAC model. The numbers on the gure correspond to the λ ex in nm.

N. Tena et al. / Food Research International 45 (2012) 103108

107

0.5 3.5 3.9 0.4 4.7 < 8% PC 5.1 0.3 5.7 7.1 0.2 8.7 9.1
0.5
3.5
3.9
0.4
4.7
< 8% PC
5.1
0.3
5.7
7.1
0.2
8.7
9.1
9.4
0.1
8-36% PC
12.6
13.0
13.9
> 36% PC
14.5
0.0
67.9
18.6
67.7
66.0
65.7
65.3
20.1
64.8
64.0
63.863.0
20.2 20.8
62.4
61.961.3
27.627.5
26.9
55.2
60.5
54.5
30.9
29.0
31.0
53.9
-0.1
46.247.850.6
52.2
36.036.4
42.9
37.136.8
45.6
37.9
-0.2
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Factor2

Factor1

4. Conclusions

Excitationemission uorescence spectroscopy combined with

the PARAFAC algorithm allows getting individualized information

about series of uorescence compounds. The information obtained

by this procedure may serve for a better interpretation of the spectra

collected with conventional uorescence spectroscopy ( Tena et al.,

2009). On the other hand, the emission proles obtained from the

three-factor PARAFAC model minimize the intensity of chlorophyll

pigment, which means an advantage in the study of other series of

uorophores that show lower absorption intensity. As a result, the

spectra obtained for each factor permit an individualized study of oxidation products, tocopherols and phenols.

References

Fig. 4. Sample projection plot of the rst two factors from the PARAFAC model. The numbers on the gure correspond to the percentage of total polar compounds (% PC).

into three different groups as it is shown in Fig. 4: oils with less than 8% PC, this moment of the experiment matching with the decrease of the degradation rate of the chlorophyll pigments (Fig. 1); oils in the range from 8% to 36% of total polar compounds; and oils with more than 36% of total polar compounds. The latter percentage (36% PC) was achieved at approximately 38 h of thermoxidation when the minimum concen- tration of phenols and trace levels of pigments were quantied (Fig. 1). The low concentrations of these compounds at that moment and thereafter provide a chemical justication for the separation of sam- ples with more and less than 36% PC. The three factors that explain the variance of the samples were used as variables in a regression model relating these factors to the percent- age of total polar compounds and the concentration of phenols, tocoph- erols and chlorophyll pigments. The objective was to evaluate the relationship between the spectral information processed by PARAFAC and the chemical data from chromatographic analyses. Table 2 summa- rizes the results obtained with multiple linear regression analysis (MLRA). In all cases the adjusted quadratic regression coefcients (R 2 adj ) are higher than 0.90. The standardized coefcients (beta) (Table 2) allow comparing the relative contribution of each factor to the prediction of the concentration of those compounds. Considering these factors, factor 1 is the largest contributor to predict the percentage of total polar compounds as its standardized coefcient corresponded to the higher absolute value (Table 2). Factor 2, in particular, has a higher relative contribution in the regression model to predict the concentra- tion of phenols and tocopherols, while factor 3 further explains the concentration of chlorophyll pigments. These results demonstrate the power of PARAFAC to analyze complex mixtures of compounds, such as virgin olive oil, and allow studying individual chemical series by means of the decomposition of spectral data. Furthermore, these regression results explain the distribution of the samples in Fig. 4, since the percentage of total polar compounds and the concentration of phenols are explained by factors 1 and 2 respectively.

Table 2 R 2 -adjusted (R 2 -adj) and standardized coef cients of multiple linear regression analy- sis (MLRA) between the three PARAFAC factors calculated for the samples and several series of uorophores.

Parameters

R 2 - adj

Standardized coef cient (beta)

 

Factor 1

Factor 2

Factor 3

Polar compounds

0.90

0.72

0.32

0.19

Chlorophyll pigments

0.92

3.66

1.80

4.51

Total phenols

0.96

0.02

0.97

0.01

α -Tocopherol

0.95

0.20

1.18

0.32

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