Multivariate Characterization of Olive Oilsusing 1H-NMR and 13C-IRMS Techniques

Rosa Maria Alonso Salces, Fabiano Reniero, Jose Manuel Moreno Rojas, Claude Guillou,

Károly Héberger†

Institute for Health and Consumer Protection, Physical and Chemical Exposure Unit, BEVABS T.P. 281, I-21020 Ispra (VA), Italy

† Chemical Research Center, Hungarian Academy of Sciences, P.O. Box 17, H-1525, Budapest, Hungary

Geographical characterization of olive oilsby 1H-NMR and 13C-IRMS

1. Trace 2005*

2. Italian olive oils 2005 (regions)

3. Olive oils 2005 (countries)

4. Unsaponifiable fraction of olive oils

(countries)

* TRACE project is funded by the EU through the Sixth Framework Programme under the Food Quality and Safety Priority (http://www.trace.eu.org). WP2: Fingerprinting and Profiling Methods.

1. Trace 2005

Variables:

175 buckets of 1H-NMR spectra

Objective:

Classification of olive oils as Ligurian or non-Ligurian

Origin samples ClassesItaly - Liguria 63 LigurianItaly - other regions 162 Non-LigurianSpain 42 Non-LigurianGreece 26 Non-LigurianTurkey 14 Non-LigurianFrance 9 Non-Ligurian

ITALY: 226(162 PDO + 63 EV)

SPAIN: 42(22 PDO + 20 EV)

GREECE: 25

TURKEY: 14(EV)

FRANCE: 9(PDO)

316

ITALY: 225

SPAIN: 42(22 PDO + 20 EV) (14 PDO + 12 EV)

GREECE: 26

TURKEY: 14(EV)

FRANCE: 9(PDO)

316

Samples:

316 olive oils 63 Ligurian

253 non-Ligurian

TRACE (http://www.trace.eu.org) is project funded by the EU through the Sixth Framework Programme under the Food Quality and Safety Priority, which aims to develop cost effective analytical methods, that should allow the determination of authenticity and detection of fraud in several food products, olive oil being one of them.

1.Trace 2005Principal Component Analysis: Ligurian olive oils are grouped,

but overlapping with non-Ligurian class.LigurianNon-Ligurian

-3 -2 -1 0 1 2 3 4

PC 2 (14.4 % of total variance)

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PC 3

(9.5

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PC 1 (31.6% of total variance)

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316c x 175v

1. Trace 2005Comparison of supervised pattern recognition techniques: PLS DA and LDA

Cross-validation (x3):

- 2/3 of samples in training set

- 1/3 of samples in test set

LDA:

- Variable selection, 175v:

Modified best-subset and forward stepwise

- 6 selected variables:

6.90, 6.02, 5.38, 4.70, 1.62, 0.90 ppm

PLS DA

175v, no variable selection

3500v, no variable selection,

Uncertainty test (Martens)

1. Trace 2005

316c x 175v

316c x 175v

1H-NMR spectra contains useful information for the classification of olive oils according to their origin: Ligurian or non-Ligurian olive oils.

The supervised pattern recognition techniques PLS DA and LDA achieve complementary results, allowing the correct classification (>98% of hits) of both classes.13C-IRMS does not containsignificant information for the classification of olive oils in Ligurianor non-Ligurian.

1. Trace 2005Supervised pattern recognition techniques: PLS DA and LDA

Abilities (%) PLS DA LDA

Recognition 98.6 95.3Ligurian 99.4 82.3Non-Ligurian 95.2 98.4

Prediction 96.5 95.3Ligurian 98.4 82.3Non-Ligurian 88.7 98.4

Classification 97.9 95.3Ligurian 99.1 82.3Non-Ligurian 93.0 98.4

316c x 175v

Concepts, IdeasPercent Correct 0 p=0.830601 1 p=0.16939999.671 303 195.161 3 5998.907 306 60

366c x 3500vCV-LOO

366c x 3500v

2. ITALIAN OLIVE OILS 2005

Liguria Centre South Garda

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PC 1 (28.6% of total variance)

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Principal Component Analysis:

Samples of each class are grouped, but groups are overlapping.

Objective:

Classification of Italian olive oils by regions

Samples: 225 olive oils

Origin SamplesLiguria 62Garda 18Centre 73

Lazio 29Umbria 18Abruzzo 6Molise 13Campania 7

South 72Puglia 28Calabria 13Sicilia 31

Variables:175 buckets 1H-NMR spectra

+13C-IRMS

Supervised pattern recognition technique: LDA

Crossvalidation (x3):

- 2/3 of samples in training set

- 1/3 of samples in test set

LDA:

- Variable selection:

Modified best-subset and forward stepwise

- 8 selected variables:

6.66, 5.10, 4.70, 4.62, 4.22, 2.82, 2.14, 2.10 ppm

Abilities (%) Recognition Prediction Classification

82.4 79.1 81.3Liguria 96.0 91.9 94.6Centre 80.1 75.3 78.5South 74.3 73.6 74.1Garda 77.8 72.2 75.9

LDA results using 9 variables:

8 NMR

+13C-IRMS

2. ITALIAN OLIVE OILS 2005

1H-NMR spectra and 13C-IRMS data contain useful informationfor the classification of Italian olive oils according to their origin: Liguria, Garda, Centre and South of Italy.

LDA achieves a satisfactory classification for Ligurian olive oils, although better classification results are expected by increasing and equilibrating the number of samples.

Supervised pattern recognition technique: LDA

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Liguria Centre South Garda

LDA: 8 NMR variables + 13C-IRMS

2. ITALIAN OLIVE OILS 2005

Objective:Classification of olive oils by country of origin.

Samples: 364 olive oils

Origin SamplesItaly 163Spain 72Greece 44Turkey 14France 9Liguria 62

* 15 samples were selected randomly,the rest was used for external validation

**

*

*

Variables:175 buckets 1H-NMR spectra

+13C-IRMS

Principal Component Analysis:

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PC 1 (30.1% of total variance)

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(11.

6 %

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otal

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ianc

e)Italy Spain Greece France Turkey Liguria

3. OLIVE OILS 2005

3. OLIVE OILS 2005

Supervised pattern recognition techniques: PLS DA and LDA

Crossvalidation (x3):

- 2/3 of samples in training set

- 1/3 of samples in test set

LDA:

- Variable selection:

Modified best-subset and forward stepwise

- 7 selected NMR variables:

6.62, 5.18, 4.66, 4.26, 2.58, 2.18, 1.34 ppm

LDA: 7 NMR variables + 13C-IRMS

Italy Spain Greece France Turkey Liguria

Root 1

Root 2

Roo

t 3

3. OLIVE OILS 2005Supervised pattern recognition techniques: PLS DA and LDA

1H-NMR spectra and C13-IRMS contains useful information for the classification of olive oils according to their country of origin.

LDA achieves satisfactory classification of Greek oils (96%), and 78-89% for the other oil origins.

Better classification are expected by increasing and equilibrating the number of samples.

CROSS-VALIDATION EXTERNAL VALIDATION

Abilities (%) PLS LDA PLS LDARecognition 77.7 89.6Italy 66.7 93.3Spain 80.0 83.3Greece 80.0 96.7France 50.0 93.8Turkey 89.3 82.1Liguria 100.0 90.0

Prediction 51.9 80.5Italy 66.7 80.0 54.3 66.9Spain 40.0 66.7 36.3 52.6Greece 53.3 93.3 63.1 84.5France 0.0 75.0Turkey 71.4 78.6Liguria 80.0 86.7 64.5 75.2

Classification 69.1 86.6Italy 66.7 88.9Spain 66.7 77.8Greece 71.1 95.6France 33.3 87.5Turkey 83.3 81.0Liguria 93.3 88.9

Objective:

Classification of olive oils by country of origin.

Variables:209 buckets of 1H-NMR spectra

Samples:54 olive oil unsaponifiable fractions

Origin SamplesItaly 22Spain 10Greece 15Turkey 7

Principal Component Analysis:

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PC 1 (40.8% of total variance)

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Italia SpainGreece Turkey

4. UNSAPONIFIABLE FRACTION OF OLIVE OILS

4. UNSAPONIFIABLE FRACTION OF OLIVE OILSSupervised pattern recognition techniques: LDA

Crossvalidation (x3):

- 2/3 of samples in training set

- 1/3 of samples in test set

LDA:

- Variable selection: Modified best-subset and forward stepwise

- 5 selected variables: 5.28, 4.76, 3.72, 0.88, 0.60 ppm

This preliminary results of the analysis of the unsaponifiablefraction of olive oils by 1H-NMR spectra indicate potential, although better classification results are expected by increasing the number of samples.

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Italia Spain

Turkey

Abilities (%) LDA

Recognition 83.3Italia 97.7Spain 85.0Greece 56.7Turkey 92.9

Prediction 81.3Italia 90.9Spain 80.0Greece 66.7Turkey 85.7

Classification 82.7Italia 95.5Spain 83.3Greece 60.0Turkey 90.5

1H-NMR spectra of olive oils and olive oil unsaponifiable fractions contain useful information for the classification of olive oils according to their geographical origin.13C-IRMS data of olive oils can provide ‘complementary’ information to NMR data for the geographical characterization of olive oils.

Both analytical techniques, NMR and IRMS, together with multivariate data analysis have a ‘potential’ for the differentiation of olive oils from different geographical origins.

Better classification results are expected by increasing the number of samples, and equilibrating the number of samples in each class.

CONCLUSIONS

AcknowledgementsRosa Maria Alonso Salces, Fabiano Reniero, Jose Manuel Moreno Rojas,Claude Guillou

JRC Institute for Health and Consumer Protection, Physical and Chemical Exposure UnitChemical Research Center, Hungarian Academy of Sciences

TRACE project (http://www.trace.eu.org)