3-108-MK-PRE-0117442 - edt. 04-2015 -Version: 1

Protection and Control of

Geographical Indications -

Tools for detecting IPR

violations and food fraud

Freddy THOMAS

1st October 2015

EXPO Milano

Food Authenticity testing in Eurofins :

the use of isotopic and NMR methods to

control the origin and the traceability of

food

1981 Invention of the SNIF-NMR® method (Site-specific Natural Isotopic

Fractionation studied by deuterium Nuclear Magnetic Resonance)

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 to food testing

FINS is the French acronym for SNIF

Eurofins laboratories: the origin...

…Today: Eurofins Analytics France laboratory based in Nantes (France)

is the group Competence Centre for authenticity

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Eurofins Analytics France

Group authenticity competence centre

Nantes, France

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Verify raw materials

Protect brand image

Protect consumer’s health

Why is it necessary to check with analytical means?

Authenticity

Is a natural product or ingredient actually from its

reputed source?

… and avoid headlines like ….

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Article UFC Que Choisir, 540, October 2015

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1994

2014

FAIM – Food Authenticity: Issues and Methodologies

European Project coordinated by Eurofins about

available analytical solutions to check food

authenticity

20 years later, after many EU projects

(MEDEO, COFAWS, PURE JUICE, WINE DB,

TRACE….)

Eurofins Analytics France: more than 25 years of experience in

food authenticity testing

www.foodintegrity.eu

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Chemical composition methods (e.g. chromatography)

Identification & quantification of defined compounds

Stable isotopes

Verification of the origin of molecular species from

the measurement of heavy/light stable isotope ratios

Molecular Biology

Identification of species, varieties, etc. through the

DNA profile of the food product

Profiling methods

Investigation of the spectroscopic or

chromatographic fingerprint of a whole food matrix

Analytical tools for authenticity testing

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Stable isotopes: a powerful authentication tool

The content of... Is influenced by…

Botanical origin Addition of sugars

from other plants

And can reveal…

13C, 2H Fruit juices

Examples

Origin of water Dilution with tap

water

Synthetic pathway Addition of artificial

substances

Geographic origin Mislabeling of origin

18O, 2H

13C, 2H

13C, 18O, 15N

2H, etc.

Wines, NFC juices

Flavours :

vanillin...

All products

Diet Feeding regime 13C, 15N Animal products

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Stable isotope analyses

Two main techniques for measuring stable isotope ratios

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 GC unit

SNIF-NMR

Measures non-statistical

deuterium distribution within a

given molecule

Method pioneered by Eurofins

Official isotopic methods for food testing

Method Product Fraction Technique Isotope ratios

AOAC Official method 995.17 fruit juice Ethanol (from fermentation) SNIF-NMR (D/H)I , (D/H)II ,R

AOAC Official method 998.12 honey honey & proteins IRMS 13C/

12C

AOAC Official method 2000.19 maple syrup Ethanol (from fermentation) SNIF-NMR (D/H)I , (D/H)II ,R

AOAC Official method 2004.01fruit juice &

maple syrupEthanol (from fermentation) IRMS 13

C/12

C

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

CEN (TC174 N108, ENV 12140) fruit juice Sugars IRMS 13C/

12C

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

16O

OIV-MA-AS311-05, OIV/OENO 381/2009 wine & spirits Ethanol SNIF-NMR (D/H)I , (D/H)II ,R

OIV-MA-AS312-06, OIV/OENO 381/2009 wine & spirits Ethanol IRMS 13C/

12C

OIV-MA-AS2-12, OIV/OENO 381/2009 wine & spirits Water IRMS 18O/

16O

CEN, EN 16466-1:2012 vinegar Acetic acid SNIF-NMR (D/H)CH3

CEN, EN 16466-2:2012 vinegar Acetic acid IRMS 13C/

12C

CEN, EN 16466-3:2012 vinegar Water IRMS 18O/

16O

AOAC = Association of Official Analytical Chemists (USA)

CEN = European Committee for Standardization (EU)

OIV = International Organisation of Vine and Wine

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ORIGIN

Agreement with declared botanical origin of alcohol

Absence of unauthorised, exogenous ingredients

Varietal compliance

Agreement with declared provenance

Authenticity of Wine and Spirits

Stable isotope analyses

Isotope ratios +

other parameters

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WINE

COMPONENTS

Ethanol

Distillation

CH2CH3OH

IRMS 13C/12C

2H/1H SNIF-NMR®

Water

IRMS

18O/16O 2H/1H

Authenticity of Wine and Spirits

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Official methods (AOAC) / limits (AIJN CoP)

100.0 105.0 110.0 115.0 120.0 125.0 130.0

Orange Beet

Cane, maize

- 35.0

- 30.0

- 25.0

- 20.0

- 15.0

- 10.0

- 5.0

85.0 90.0 95.0

(D/H) I ( ppm )

d 13 C (‰)

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

Beet

Cane, maize

- 35.0

- 30.0

- 25.0

- 20.0

- 15.0

- 10.0

- 5.0

85.0 90.0 95.0

(D/H) I ( ppm )

d 13 C (‰)

Beet

Cane, maize

- 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 )

d 13 C (‰)

AOAC 995.17

AOAC 2004.01

Orange Orange

-28 - -25

103 - 107

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1995 1996

1997 From C. Guillou, BEVABS

JRC Ispra, Italie

Wine official control: EC 2729/2000 (isotopic database)

1) Chaptalisation

2) Watering

3) Origin

4) Vintage

Wine official control: a challenge for Europe

WINE Data Base project

Commercial wines

Grapes

Micro-vinification

acc. EC reg. 2729/2000

Reference wines

Physico-chemical analyses

Stable isotopes Elemental analysis

SNIF-NMR

IRMS

ICP-MS

«classical »

analysis

Statistics

Exploratory analysis

Within a country

Discrimination analysis

HPLC, GC

Chemistry

Between countries

Commercial wines

Grapes

Micro-vinification

acc. EC reg. 2729/2000

Reference wines

Grapes

Micro-vinification

acc. EC reg. 2729/2000

Reference wines

Physico-chemical analyses

Stable isotopes Elemental analysis

SNIF-NMR

IRMS

ICP-MS

«classical »

analysis

Statistics

Exploratory analysis

Within a country

Discrimination analysis

HPLC, GC

Chemistry

Between countries

IRMS

18O/16O 13C/12C

2H NMR

(D/H)II(D/H)I

WINE

Distillation

ETHANOL

CO2

Equilibration

Combustion

WATER

IRMS

18O/16O 13C/12C

2H NMR

(D/H)II(D/H)I

WINE

Distillation

ETHANOL

CO2

Equilibration

Combustion

WATER

•BfR, Germany (coordinator) •FERA (ex CSL), United Kingdom •Eurofins Scientific Analytics, France •VUB, Belgium, •BEVABS, JRC Ispra, Italy, •UGOA, Italy, •Technische Universität Berlin, Germany •Universitatea de Stiinte Agricole si Medicina Veterinaria, Romania, •National Institude for Wine Qualification, Hungary, •Ministry of Finance, Czech Republic

Establishing of a Wine Data Bank for Analytical Parameters for Wines from Third countries Contract N° : G6RD-CT-2001-00646

Project funded by the European Community under the ‘Competitive and Sustainable Growth’ Programme (1998-2002), Measurement and Testing Activity

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All commercial wines

Model 1 (8 var.)

Parameter = 0.128

Hungary

Czech Republic

Romania

South Africa-12 -10 -8 -6 -4 -2 0 2 4 6 8

Root 1

-7

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

Ro

ot

2

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2H, 18O

15N, 34S

13C, 15N

Local climatic conditions

Agricultural practices, local « pollution »

Influence of diet in animals

Applications to other products: traceability

studies

Confirmation of specifications for

PDO, PGI labels

Examples: Mozzarella, Extra Virgin Olive Oil, balsamic vinegar, Iberico ham….

Chemical composition methods (e.g. chromatography)

Identification & quantification of defined compounds

Stable isotopes

Verification of the origin of molecular species from

the measurement of heavy/light stable isotope ratios

Molecular Biology

Identification of species, varieties, etc. through the

DNA profile of the food product

Profiling methods

Investigation of the spectroscopic or

chromatographic fingerprint of a whole food matrix

Analytical tools for authenticity testing

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Official control today

The future

Need for new analytical approach : untargeted approach

R&D project 2012-2017

coordinated by Eurofins

Observation domain: H-containing compounds, ppm to %

High Resolution 1H Nuclear Magnetic Resonance

__ Lab 1 __ Lab 2

Key Strengths:

Large linearity range

Short Turn-Around Time

High Reproducibility

Discrimination power

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The 1H-NMR spectrum is obtained on the whole matrix

Detects markers and spectral effects of adulteration

Allows to quantify key-quality parameters

Allows to confirm botanical origins of mono-floral honey

Benefits of a non-targeted method

Unique spectral fingerprint

Alternative to

conventional methods

Authenticity proof

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Wine analysis by untargeted 1H-NMR

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Country

Appelation

Classification

Variety

Vintage

Another example: caviar authenticity

Protection of the new PGI “Caviar d’Aquitaine”

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Even if these methods are not yet official, some food sectors

use the 1H-NMR untargeted approach for their daily controls in

our lab:

Fruit Juice (supported by SGF)

Honey (method presented at Apimondia / IHC workshop)

Coffee (tested by several coffee associations)

Milk (batch comparison, adulteration and degradation)

Spices (adulteration saffron, curcumin…)

Wine (discussion with OIV)

Caviar (French trade association)

State of the art

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Conclusion: our best offer to fight against food

adulteration: stable isotopes and NMR

Stable isotope analysis constitutes a powerful tool for product authentication

But isotopic techniques also have limitations...

• The sample preparation is limited by the need for large amounts of pure

compounds to be isolated without isotopic fractionation

• Need for large and up-to-date databases

Untargeted analysis : from R&D to official control

• Consolidation of database

• Standardisation of the statistical approach

• Need for officialisation

• A number of routine applications exist (wines, spirits, fruit juices, honey,

flavours,…)

• Recognized as official methods by EU / CEN / AOAC / OIV / AIJN ….

• Used by public control laboratories

Thanks for your attention

We see only what we want to see…

or what we can see at the moment…

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Freddy THOMAS