1H ΝΜR spectroscopyOlive oil adulteration with vegetable oils

0.760.800.840.880.920.961.001.041.081.12

(ppm)

Olive oil

Soya oil

Linolenic ester

CH3

COO~

NMR spectroscopy and Chemometrics

• Quality Control of olive oil (composition, age)

• Geographical and votanical classification

• Year of production variations

• Detection of adulteration with vegetable oils and low quality olive oils

Food Chemistry and Analysis

Chromatographic techniques for lipid research

� GC-MS

� LC-MS

� SEC

Low LOD (limit of detection) but time consuming and require standards

NMR spectroscopyNMR spectroscopy

• Non-destructive

• Fast (15-20 min)

• Quantitative

• Does not require standards

144.4 144.8 145.2 145.6 146.0 146.4 146.8 147.2 147.6 148.0 148.4 148.8

(ppm)

144.4 144.8 145.2 145.6 146.0 146.4 146.8 147.2 147.6 148.0 148.4 148.8

(ppm)

1-MG

144.4 144.8 145.2 145.6 146.0 146.4 146.8 147.2 147.6 148.0 148.4 148.8

(ppm)

1-MG

2-MG

1,2-DG

1,3-DG

CH

CH2OCOR

CHOCOR

CH2OH

CH2OCOR

CHOH

CH2OCOR

SterolsAcidity

CH2OCOR

CHOH

CH2OH

CH2OH

CHOCOR

CH2OH

O

OPR-OH + HCl +

O

OPOR

I II

31Ρ NMR spectrum of olive oilGlyceride composition

A. Spyros, P. Dais, J. Agric. Food. Chem., 48, 802 (2000)

DGsin virgin olive oils

• Virgin oils contain 1-3% total DGs

• Fresh virgin oils contain mainly 1,2-DGs

D=1,2-DGs/DGs > 0.9

• D is further reduced during storage, thus it is a good index of aging

DGsin low quality olive oils

• Refined oils: 4-5 % total DGs

• Deodorized oils: 5-10% total DGs

• Low quality oils contain mainly 1,3-DGs

D=1,2-DGs/DGs ~ 0.33

• D can be used for authentication of virgin oils

Ratio D for ~140 olive oils

Determination of olive oil age

• There exists no official method

• Olive oil age is important for the consumers, olive oil bottling companies and the international olive oil trade in general, because the organolepticcharacteristics of olive oil are affected by prolonged storage.

• Can we use DGs for that ?

Kinetics of diglyceride, DG, isomerizationin olive oil

1,2-DGs 1,3-DGsk

k'

CH2OCOR

CHOCOR

CH2OCOR

CH2OCOR

CHOCOR

CH2OH

CH2OCOR

CHOH

CH2OCOR

RCOOH

TGs

H

1,2-DGs

1,3-DGs

1,2-

DG

s [µ

mol

/g]

5D

10

20

30

40

50

AD

10

20

30

40

50

Pilion

AL

10

20

30

40

50

0 6 12 18

Lesvos0 6 12 18

Sitia0 6 12 18

Iraklion0 6 12 18

time [months]

1,3-

DG

s [µ

mol

/g]

5D

-5

10

25

40

55

AD

-5

10

25

40

55

Pilion

AL

-5

10

25

40

55

0 6 12 18

Lesvos0 6 12 18

Sitia0 6 12 18

Iraklion0 6 12 18

Evolution of 1,2-DGs and1,3-DGs with aging

Evolution of total DGs and acidityΗ with aging

time [months]

Fre

e ac

idity

[µm

ol/g

]

5D

0

10

20

30

40

50

AD

0

10

20

30

40

50

Pilion

AL

0

10

20

30

40

50

0 6 12 18

Lesvos0 6 12 18

Sitia0 6 12 18

Iraklion0 6 12 18

tota

l DG

s [µ

mol

/g]

5D

01020304050607080

AD

01020304050607080

Pilion

AL

01020304050607080

0 6 12 18

Lesvos0 6 12 18

Sitia0 6 12 18

Iraklion0 6 12 18

tHH 0 hk+=

tkh+[DGs]=[DGs]0

Kinetics of diglyceride, DG, isomerizationin olive oil

1,2-DGs + H 1,3-DGs + Hk

k'

CH2OCOR

CHOCOR

CH2OCOR

CH2OCOR

CHOCOR

CH2OH

CH2OCOR

CHOH

CH2OCOR

RCOOH

TGs

H

1,2-DGs

1,3-DGsTGs

kh

tHH 0 hk+=

tk[DGs]DGs h0 +=

0 5 10 15 20

10

20

30

40

50

60

70

80

90

a

1,3-DGs

1,2-DGs

H

DGs

µmol/g

time [months]

0

10

20

30

40

50

60

70

kh

0 0.2 0.5 1

µmol/g

Evolution of DGs andΗ with time

A. Spyros, A. Filippidis, P. Dais, J.Agric. Food. Chem., 52, 157 (2004)

0 5 10 15 20

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 0.2 0.5 1

b

kh

D

time [months]

DGs

DGs2,1D

−=

tkke

kk

kk

kk

kD

H])[H'(2

1

0

00 0

])[DGs]'(

DGs][1,3'DGs],21[[)

'

'(

++−

⋅+

−−−+

+=

[µmol * month/g]

H*t

D

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

-100 100 300 500 700 900

PilionLesvosSitiaIraklion

[µmol * month/g]

H t

D/D

0

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 100 200 300 400 500

PilionLesvosSitiaIraklion

k+k’= 17.4 x 10-3 g mol-1 month-1

k+k’= 1.4 x 10-3 g mol-1 month-1

0 2 4 6 8 10 12 14 16 18 20

0

2

4

6

8

10

12

14

16

18

20

age[months]

time[months]

set A set B set C

Storage at room temperature

H*0165.0

)62.0

32.0Dln(

age

−−

=

Application to commercial extra virgin olive oils

• Eight (8) virgin olive oils were obtained from supermarkets. Four (4) of them were found to contain olive oil from the previous crop season, against labeling information.

• “Minerva”, the biggest olive oil bottler in Greeceused for two years this methodology to verify the freshness of the olive oil supplied by local agricultural cooperatives from all over the country.

1H NMR spectrum of olive oil

P. Fronimaki, A. Spyros, S. Christoforidou, P. Dais, J. Agric. Food. Chem., 50, 2207 (2002)

Fatty acid composition

Oleic acid

Linoleic acid

COOH

COOH

Linolenic acid

COOH2

3

4

5

6

7

8

9

1011

12

1314

15

16

17

18

1

COOH

COOH Palmitic acid

Stearic acid

O

OCH2

CH

CH2

O

O

O

OTripalmitin

Oleic acid

Linoleic acid

COOH

COOH

Linolenic acid

COOH2

3

4

5

6

7

8

9

1011

12

1314

15

16

17

18

1

COOH

COOH Palmitic acid

Stearic acid

O

OCH2

CH

CH2

O

O

O

OTripalmitin

Oil composition in:•saturated•monounsaturated•di-unsaturated•polyunsaturated fatty acids•sterols•Iodine Value

ChemometricsApplication of multivariate statistical methodsin the analysis of large volumes of data

What to do with all those data?

x~500 oil samples

>6000 experimental data

1,3-DGs

Total DGs

Ratio D

Acidity

Sterols

1,2-DGs

31PNMR

1,3-DGs

Total DGs

Ratio D

Acidity

Sterols

1,2-DGs

31PNMR

Iodinenumber

SFA Oleic

Linoleic

Linolenic

1Η

NMR

Iodinenumber

SFA Oleic

Linoleic

Linolenic

1Η

NMR

Geographical classification of Greek virgin olive oils

Κρήτη

Πελοπόννησος

Λέσβος

Πήλιο

Root 1

Roo

t 2

-7

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

-6 -4 -2 0 2 4 6 8 10 12 14 16

Crete

Peloponese

LesvosLesvos

Pelion

Classification of Greek virgin olive oils according to olive fruit variety

Κορωνέικη

Κολοβή

Πηλίου

Root 1

Roo

t 2

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

-6 -4 -2 0 2 4 6 8 10 12 14 16

Koroneiki

Kolovi

Pilioy

Classification of Greek virgin olive oils according to production year

2000-2001

2001-2002

2002-2003

Root 1

Roo

t 2

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

-8 -6 -4 -2 0 2 4 6

Classification of 13 types of vegetable oils

-40 -20 0 20 40 60 80-20

-15

-10

-5

0

5

10

15

20

25

30

virgin olive soybean hazelnut corn sesame groundnut sunflower almond safflower palm coconut walnut rapeseed

Roo

t 2

Root 1

Adulteration of olive oil with vegetable oils

Root 1

Roo

t 2

-15

-10

-5

0

5

10

15

20

25

-30 -20 -10 0 10 20 30

virgin olive

hazelnut

sunflower

soybean

corn

mhazelnut

msun

msoybean

mcorn

G. Vigli, A. Filippidis, A. Spyros, P. Dais, J.Agric. Food. Chem., 51, 5715 (2003)

Adulteration of virgin olive oil with low quality olive oils

G. Fragkaki, A. Spyros, G. Siragakis, A. Salivaras, P. Dais, J.Agric. Food. Chem., 53, 2810 (2005)