www.eurofins.com7 – 8 October 2008

Recent Applications of Stable Isotopes Techniques to check the authenticity of food products

Eric Jamin

Authenticity Analytical Unit ManagerEurofins Scientific, Nantes (France)

ericjamin@eurofins.com

Phone # : (33) 2 51 83 21 00

2

1981 Invention of the SNIF-NMR® method allowing the determination

of the botanical origin of alcohol (Pr. G and M Martin, Nantes

University)

1987 Creation of Eurofins Laboratory, specializing in the application

of isotopic methods

1997 Introduction in Paris stock exchange

2010 Network of 150 laboratories present in 30 countries, >8000

staff, 40,000 methods

Eurofins laboratories: the origin...

EUROFINSEUROFINS

Nantes (France): Competence Center for authenticity

and isotopic measurements

3

Verify raw materials

Check new suppliers

Protect brand image

Why Is it necessary to check ?

Authenticity

… and avoid headlines like ….

THE INDEPENDENT, February 1991

4

ORANGE cases

22%

16%

16%

13%

12%

8%

3%3%

3%

2%

2%

sugar addition

other fruit addition

untypical flavour profile

multiple adulteration

water addition

bad quality

Mislabelling

ascorbic acid addition

low brix

low vitamin C

overdilution

A wide range of adulteration issues

5

Compositional analysis

Identification and quantification of major and minor components

Stable isotope analysis

Origin of the components

Molecular biology and immunology

plant and animal species identification

Profiling methods

Spectroscopic patterns of global matrices applied for screening

How to check? The analytical tools available

6

Stable isotopes at natural abundance

Food products are composed of 4 main elements C, H, O and N.

These elements are naturally present in

several isotopic forms (same atomic

number, different weights), following a

distribution influenced by natural

phenomena and human processes.OC12

13

18

16

17

NND

H12

14

15

OC

7

IRMS : Isotopic Ratio Mass Spectrometry

Access to overall isotope ratios of a product or constituent

Combustion or Pyrolysis of the organic product

Can be coupled to Gas Chromatography unit

Stable Isotope Analyses

8

SNIF-NMR

measures non-statistical deuterium distribution within a given molecule

method pioneered by Eurofins Scientific

Site-specific Natural Isotopic Fractionation studied by deuterium Nuclear Magnetic Resonance

Stable Isotope Analyses

9

Isotopic methods recognition

year Method Product Fraction Techniques Isotope ratios

1987 OIV, recueil des méthodes d'analyse wine Ethanol SNIF-NMR (D/H)I , (D/H)II ,R

1990 EC regulation 2676/90, annex 8 wine Ethanol SNIF-NMR (D/H)I , (D/H)II ,R

1991 AOAC method 998.12 honey honey & proteins IRMS 13C/12C

1993 CEN (TC174 N108, ENV 12140) fruit juice Sucre IRMS 13C/12C

1995 AOAC Official method 995.17 fruit juiceEthanol (from fermentation)

SNIF-NMR (D/H)I , (D/H)II ,R

1996 OIV resolution ENO 2/96 wine water IRMS 18O/16O

1997 EC regulation 822/97 wine water IRMS 18O/16O

1997 CEN (TC174 N109, ENV 12141) fruit juice water IRMS 18O/16O

2000 AOAC Official method 2000.19 maple syrupEthanol (from fermentation)

SNIF-NMR (D/H)I , (D/H)II ,R

2001 OIV resolution ENO 17/2001 wine Ethanol IRMS 13C/12C

2003 EC regulation 440/ 2003, annex 2 wine Ethanol IRMS 13C/12C

2004 AOAC method 2004.01fruit juice & maple syrup

Ethanol (from fermentation)

IRMS 13C/12C

2006 AOAC method 2006.05 Vanillin Vanillin SNIF-NMR (D/H)i

10

H2O

DHODHO

DHODHO

H2O

H2OH2O

H2O

H2O

H2O

DHODHOH2O

H2O

H2O

DHODHO

Stable Isotope Analyses

11

Influence of geographical origin on stable isotope ratios

12

Where does the sugar in a plant come from :

Carbon dioxide

C3 C4

CO2

(-8 ‰)

Carbon metabolism

most plantsvalues around -25‰

Gramineae family e.g. sugarcane, maize, values

around -10‰

13

What are the latest improvement of isotopic methods for fruit juices?

Use of multi-element profiles and site-specific methods

to discriminate more sources

Use of GC-IRMS to analyse flavour compounds

at low concentrations

Initially: bulk fractions, single isotopes

Use of multi-component profiles to increase the sensitivity (narrower ranges of variation)

14

13C/12C

NFC JUICE

Sugars

COMPONENTS

Fermentation Ethanol

2H/1H

CH2CH3OH18O/16O 2H/1H

Water

18O/16O

Isotopic analyses in direct fruit juice:Improved detection of sugar addition

J. Agric. Food Chem. (2003) 51, 18, 5202-5206 ; J. Agric. Food Chem. (2006), Vol. 54, No. 2, 279-284

15

Detection of sugar addition in fruit juices

100.0 105.0 110.0 115.0 120.0 125.0 130.0

OrangeBeet

-35.0

-30.0

-25.0

-20.0

-15.0

-10.0

-5.0

85.0 90.0 95.0

(D/H)I (ppm)

13C(‰)

100.0 105.0 110.0 115.0 120.0 125.0 130.0100.0 105.0 110.0 115.0 120.0 125.0 130.0

Beet

-35.0

-30.0

-25.0

-20.0

-15.0

-10.0

-5.0

85.0 90.0 95.0

(D/H)I (ppm )

13C(‰)

Beet

Cane, corn

-35.0

-30.0

-25.0

-20.0

-15.0

-10.0

-5.0

-35.0

-30.0

-25.0

-20.0

-15.0

-10.0

-5.0

85.0 90.0 95.0

(D/H)I (ppm )

13C(‰)

Ethanol from sugars

EthanolFromsugars

Pineapple-15 - -12 (AIJN)

107 - 112

16

NEW application of 13C SNIF-NMR to pineapple:improved detection of sugar addition

13C of ethanol CH2 (‰)

13 C

of

eth

ano

l C

H3

(‰)

PINEAPPLE

CANE

MAIZE

BEET

test

Added C4 sugar

J. Agric. Food Chem. 2010, 58, 11580–11585

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13C/12C

NFC JUICE

Sugars

COMPONENTS

Fermentation Ethanol

2H/1H

CH2CH3OH18O/16O 2H/1H

Water

18O/16O

Isotopic analyses in direct fruit juice:Improved detection of water addition

J. Agric. Food Chem. (2003) 51, 18, 5202-5206 ; J. Agric. Food Chem. (2006), Vol. 54, No. 2, 279-284

18

-10

-9

-8

-7

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

18O ethanol (‰)

18O

wat

er

(‰)

Lab-squeezedsamples

Market NFCjuices

Market FCjuices

Max (95% c.i.)

Min (95% c.i.)

AIJN limit

Water addition

Authentic orange from a specific origin

Practical application: NFC juice authentication

J. Agric. Food Chem. (2003) 51, 18, 5202-5206 ; J. Agric. Food Chem. (2006), Vol. 54, No. 2, 279-284

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Detection of added ascorbic acid

*PURE JUICE is a shared-cost RTD project funded under the Fifth Framework Programme of the European Community, within the Competitive and Sustainable Growth Programme, Measurement and Testing Activity. Contract N° : G6RD-CT-2002-00760. Coordination: SGF

developed in the Pure Juice Project*

Figure 2: for of Site-specific 13C-IRMS analysis:

COH

COH

CH

CHOH

CH2OH

O

CO

CO2 from Carbon 1Ce(SO 4)2+ H2SO4,20°, 240 min

1

Industrial ascorbic acid is derived from sugar sources Global 13C only detects C4 sources

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Detection of added ascorbic acid(acerola juice and dietary supplements, etc.)

-10

-9

-8

-7

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

-25 -24 -23 -22 -21 -20 -19 -18 -17 -16 -15 -14 -13 -12 -11 -10 -9 -8

13C ascorbic acid (‰)

D 1

3 C C

1 -

glo

bal

asc

orb

ic a

cid

(‰

)

artificial sourcesOrangeblackcurrantacerola fruitmarket acerola

addition of artificial ascorbic acid

Fruit processing, July-August 2009, pp 170-175

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Commercial sources of vanillin

Paper Industry

TropicalAgriculture

VANILLIN

Vanilla Fragrans

Chemical Industry1200-4000$/ Kg

Vanilla Planifolia

Market: 11 000T/year

20-50 T/year

Ca.11$/ Kg

OCH3

OH

OH

(Guaiacol)

(Lignin)

Biotechnology(new sources)

Paper Industry

TropicalAgriculture

VANILLIN

Vanilla Fragrans

Chemical Industry1200-4000$/ Kg

Vanilla Planifolia

Market: 11 000T/year

20-50 T/year

Ca.11$/ Kg

OCH3

OH

OH

(Guaiacol)

(Lignin)

Biotechnology(new sources)

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Principal Component Analysis of (D/H)i and 13C/12C ratios of vanillin from various sources

-2.0

-1.6

-1.2

-0.8

-0.4

0.0

0.4

0.8

1.2

1.6

2.0

-2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5

Ex-beans

Ex-lignin

Ex-ferulic acid

Ex-eugenolSynthetic

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Geographical origin of plant product:Worldwide origins of RICE

Also available for cereals, fruits, wine, coffee, olive oil, meat, honey, etc

Combines:13C15N18OMeasurements

24

Salmon / Sea bass / sea bream / Cod etc.: Wild or farmed?

O18 Salmon oil vs. N15 Choline

18 O Salmon oil (‰)

1

5N

Ch

olin

e (‰

)

Authentic farmed samplesAuthentic wild samplesCommercial farmed samplesCommercial wild samples

Mislabeled commercial samples:

-Labeled « wild »,-Found « farmed »

Differentiation of wild and farmed fish by isotopic methods

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Animal or vegetal source?example : Squalane and squalene

Determination of origin can be performed

using EA-IRMS on pure compounds or GC-C-IRMS on cosmetics

-29‰ -22‰-27‰ -19‰

mixture

13C (/VPDB)

squalane

squalene

Rapid Commun. Mass Spectrom 2010, 24, 1810-1814; SOFW-Journal | 136 | 1/2-2010

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Conclusion: the analytical challenge

Adulteration is a moving issue due to changes in prices, regulation etc. and remains a constant challenge for the analyst

A number of routine applications recognized as official methods exist (wines, spirits, fruit juices, honey, flavours,…)

Further analytical developments are needed to establish always more efficient methods

Customised projects can be set up to solve problems of product origin, suspected counterfeits, brand protection, etc.

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Thank you for your attention!

And feel free to contact us, And feel free to contact us, should you need more informationshould you need more information

Nantes (France) laboratories: Nantes (France) laboratories: eurofinsfr@eurofins.comTel. (33) 2 51 83 21 00

www.eurofins.comwww.eurofins.com