Stable Isotope Ratio Analysis provides the methods of choice for food authentication

Michèle Lees

Independent Expert on Food Integrity, previously Director of Collaborative Research, Eurofins, France

Context

Stable Isotope Ratio Analysis (SIRA)

now widely deployed to authenticate food and beverages

a number have gained official recognition and have been successfully used in legal court cases

What has led to this broad acceptance ?

good practices have ensured that the measuring systems and their results are robust and fit for purpose

the tools developed often provide the only solution to real problems of food fraud

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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 2004.01 Fruit juice & maple syrup Ethanol (from fermentation) IRMS 13C/12C

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 N108, ENV 12141) Fruit juice Water IRMS 18O/16O

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

OIV-MA-AS312-06, OIV/OENO 381/2009 Wines & Spirits Ethanol IRMS 13C/12C

OIV-MA-AS2-12, OIV/OENO 381/2009 Wines & 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

Officially-recognised methods for food authentication

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FIT - Food analyses using

Isotopic Techniques

Contract N° : SMT4 - CT95 - 7500

An early initiative to develop and promote stable isotope analyses applied to food authentication

Industry

Retail Sector

State Control Bodies

Consumers APPLICATIONS

NEEDS

Stable Isotope

Analyses

Universities Research Centres

Official Control labs Private specialised labs

A helping hand from the European Commission

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BELGIUM P. de Bièvre & P. Taylor , J. Pauwels & G.N Kramer ,

IRMM

FRANCE G.J. Martin, CEAIS J. Koziet, Pernod Ricard A. Le Leuch, DGCCRF M.H. Merle & S. Giraudon, DGCCRF C. Lamoureux, DGDDI E. Jamin, M. Lees, G.G. Martin, Y.L. Martin & G.Remaud, Eurofins Scientific G.J. Martin, N. Naulet & R. Robins, U. de Nantes

IRELAND K. Mac Namara, Irish Distillers

UNITED KINGDOM J. Dennis, S. Kelly, I. Parker & M. Sharman, CSL S. Brookes & A. Bradley, Europa Scientific G. Goldwin, MAFF London R. Wood, MAFF Norwich P. Turner & F. Fourel, Micromass

SPAIN A.I. Blanch-Cortes, Min. de Agricultura, Pesca y Alimentacion

PORTUGAL M. Curto, A. Teixeira & L. Santos, INETI J. Lopes, Instituto da Vinho e do Vinho Paulo Barros , Instituto do Vinho do Porto

FINLAND

P. Lehtonen , Alko Group

ITALY G. Versini, D. Depentori & A. Monetti, Ist. Agrario di San Michele all’Adige A. Ciambotti & P. Bosia, Ist. Sper. per l’Enologia di Asti G. Serrini, C. Guillou & F.Reniero, JRC Ispra

AUSTRIA F. Pichlmayer, IAEA G. Haberhauer , Austrian Research Centre

GREECE G. Bonas, T .Mavromoustakos & E. Zervou , NHRF

GERMANY R. Wittkowski , BgVV A. Hermann , Chem. Unters. Speyer. H. Förstel , Forschungszentrum Jülich N. Christoph , LUA Nordbayern A. Mosandl & U. Hener, U. Frankfurt

H.L. Schmidt & A. Rossmann , T.U. München

SWEDEN G. Fuchs & S. Wretling ,

National Food Administration

The FIT Thematic Network 1996-1998

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Using stable isotope techniques as official control methods

Key requirements

Applications that target areas of concern for food control laboratories

Interpretation of results: extensive reference data base

Reliability: robust method performance, quality control

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3 Case studies

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Developing a method to differentiate between wild and farmed salmon

Solving the problem of adulterated palm sugar

Keeping up with clever fraudsters: the case of vanilla authentication

Case study N°1: Developing a method to differentiate between wild and farmed salmon

Background (www.fao.org/fishery/culturedspecies/Salmo_salar/en)

Considerable increase in global production of farmed salmon

Farmed Atlantic salmon >90 percent of the farmed salmon market, and >50 percent of the total global salmon market

Commission Regulation 2065/2001/EC

labelling and traceability requirements for aquaculture products, including:

• the production method (farmed or caught at sea or in freshwater)

EU-funded project G6RD-CT-2001-00512 COFAWS (Confirmation of the Origin of Farmed and Wild Salmon)

Muscle

Water

extraction

Fatty Acid Methyl Esters (FAMEs)

saponification Fatty Acids

Glycerol (+Choline)

Feed

transesterification

Seawater

extraction

Muscle

extraction

saponification

transesterification

extraction 13C-IRMS 15N-IRMS

13C-IRMS 18O-IRMS

Feed Oil

Triacylglycerides

+ PC 13C-IRMS

2H-SNIF-NMR (Triacetin) 13C-IRMS, 18O-IRMS

15N-IRMS, %N GC

2H-SNIF-NMR 13C GC-C-IRMS

2H-SNIF-NMR 18O-IRMS

2H-SNIF-NMR 18O-IRMS

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Database requirements – the COFAWS project

Geographical origin

Authentic

Wild

Farmed

Slow/fast growth

Season

Type of feed Market As labelled

Sample collection Norway, Scotland, Ireland, Faroes, Iceland, Chile, Canada …

Database requirements – the COFAWS project

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Analysis of Variance (ANOVA) : wild vs. farmed

0

20

40

60

80

100

120

140

O18oil

N15chol

(D/H

)1T

CLU

3

C18:2

n-6

C16:1

n-7

C14:0

CLU

1

(D/H

)2T

C13A

c

C18:0

C22:6

n-3

O18G

ly

C13R

P

CLU

9

CLU

6

C18:1

n-7

C13G

ly

C20:5

n-3

CLU

8

C16:0

C18:1

n-9

C22-1

n-1

1

C20:1

n-9

CLU

5

(D/H

)3T

CLU

7

CLU

2

C22:5

n-3

CLU

4

Best discriminatory parameters

COFAWS project results

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F. Thomas et al. J. Agric. Food Chem., 2008, 56, 989-997.

d 18

O Salmon oil (‰)

d 1

5

N C

ho

line

(‰)

Authentic farmed samples Authentic wild samples Market farmed samples Market wild samples

Mislabeled market samples:

-Labeled « wild », -Found « farmed »

Differentiation of wild and farmed fish by isotopic methods

Validated method to distinguish wild and farmed salmon

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Case study N°2: Solving the problem of adulterated palm sugar

The background

Environmental Health Office UK tested samples of palm sugar (origin Thailand): d13C ~ -13‰

Palm expected to be C3 plant (d13C ~-25‰)

Sugar profile of palm sugar not distinctive

More samples (other brands) taken from UK market all shown to contain varying amounts of C4 sugar

A sample bought in France (origin Cambodia): d13C ~ -27‰

Requirement

Find out whether the palm sugar in the UK market is adulterated

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Where does the sugar in a plant come from :

Carbon dioxide

C3 C4

CO2

most plants values around -25‰

Gramineae family e.g. sugarcane, maize, values around -10‰

Authentication of palm sugar

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C3 C4

? ?

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Authentication of palm sugar

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Authentication of palm sugar

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Authentication of palm sugar

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Authentication of palm sugar

Samples of sap taken from the coconut plantation (as well as leaves, flowers)

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Authentication of palm sugar

The results : d13C‰

-26.9 -25.2 -24.8 -25.7

-20.3

-15.4

-30

-20

-10

0

Palm

leaf Palm

flower Sap

(A) Sap

(B) Palm

sugar Commercial

samples

Authentication of palm sugar

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Authentication of palm sugar

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Case study N°3: Keeping up with clever fraudsters: the case of vanilla authentication

Vanilla Planifolia – the background

Premium product

Important flavour compound for the food industry

Also used extensively in perfumes, cosmetics, etc.

Labour intensive production

High and variable costs linked to poor yields and harvesting difficulties

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Building up a database for the main vanilla production regions

Vanilla authentication: an interesting case study Geographical origin ?

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1964 1974 1984 1994 2004 2006 2008 2009

Indonesia 150 300 520 1770 3700 3700 4146 4362

Madagascar 1050 2283 2277 1320 839 2534 3055 2830

China 0 0 0 400 900 1200 1400 1382

Mexico 90 29 161 167 252 291 600 524

Tonga 0 10 16 100 131 140 199 263

French Polynesia 100 21 6 13 44 25 49 74

Comores 175 160 160 131 60 75 70 65

Uganda 10 10 10 20 61 48 52 48

Réunion 45 27 56 33 25 22 12 12

Amount of vanilla produced (tons) (FAO-STAT)

Different species : Vanilla Tahitiensis

Cyclones and political unrest in Madagascar scarcity of supply, new producers enter

market

China appears on the scene and is becoming a major producer

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Vanilla authentication: an interesting case study

Vanilla authentication: an interesting case study

A classic case of supply and demand …

Euros/tonne Tonnes

produced

High risk of adulteration!

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Main component : Vanillin

OCH3

OH

OH

Typical vanilla aromatic profile (regulatory guidelines exist to assess vanilla authenticity)

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Vanilla authentication: an interesting case study

Paper Industry

Tropical

Agriculture

VANILLIN

Vanilla Fragrans

Chemical Industry

Vanilla Planifolia

OCH3

OH

OH

(Guaiacol)

(Lignin)

Commercial sources of vanillin

2001: 400 € / tonne

2003: 750 € / tonne

Guaiacol

Lignin

12 - 15 € / tonne

Vanilla authentication: an interesting case study

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Vanilla is a CAM (Crassulacean Acid Metabolism) plant

Use of 13C/12C ratios to detect frauds

d13C Vanillin (‰ /V.PDB) > -21.2 (vanilla beans) ; ~ -30 (synthesis)

d13C (‰ /V.PDB)

for pHB > -19.2 ; for vanillic acid > -24.0 ; for pHB acid > -23.0

13C/12C ratios of other vanilla components specified in the legislation

13C enrichment during synthesis able to fool the authentication method BUT

Selective 13C enrichment is undectable when analysing whole compounds BUT

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PCA using (D/H)1, (D/H)3,

(D/H)4, (D/H)5

SNIF–NMR® ANALYSIS of vanillin

OCH3

OH

OH(D/H)1

(D/H)2

(D/H)3

(D/H)4

(D/H)5

(D/H)3

1 23 4

5

TMU

Solvent

8.4

7.5

7.0

3.9

2.8

2.0

9.8

OCH3

OH

OH(D/H)1

(D/H)2

(D/H)3

(D/H)4

(D/H)5

(D/H)3

1 23 4

5

TMU

Solvent

8.4

7.5

7.0

3.9

2.8

2.0

9.8

1 23 4

5

TMU

Solvent

8.4

7.5

7.0

3.9

2.8

2.0

9.8

8.4

7.5

7.0

3.9

2.8

2.0

9.8

Site specific isotope ratios measured using 2H SNIF-NMR

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F1

F2

ex-lignin

ex-beans

ex-guaïacol

Zoom of 2H-NMR spectrum

Presence of a small peak due to OCHD2

From Remaud et al., J. Agric. & Food Chem., 1997

10.6

0

9.6

3

8.6

5

7.6

8

6.7

0

5.7

3

4.7

5

3.7

8

2.8

0

1.8

3

ppm

1 2

3

4

5

TMU

Solvent

OCHD 2

SNIF-NMR® and detection of isotopic manipulation of vanillin BUT

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Using stable isotope techniques as official control methods

Key requirements

Applications that target areas of concern for food control laboratories

Interpretation of results: extensive reference database

Reliability: robust method performance, quality control

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Proficiency Testing

Initiated in 1994 => The ONLY one dedicated to Food analysis using Isotopic Techniques (FIT)

Complies with the ISO/IUPAC/AOAC International Harmonised Protocol for Proficiency Testing of analytical laboratories

Expert Committee reviews results

70 participants in 2016

3 rounds per year, 6 samples per round

The FIT- Proficiency Testing Scheme

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FIT- Proficiency Testing Scheme

Type of samples: Wine, spirit Fruit juice Pure alcohol Honey Olive oil Vinegar Pure organic product Flour, cereal, rice Water Aroma extract Pure aroma molecule

Techniques: EA-IRMS (Combustion,

Pyrolysis, Equilibration) GC-C-IRMS SNIF-NMR

Parameters: C13 Ethanol, Sugars, Pulp, Honey, Proteins, Acetic

Acid C13 CO2 Sparkling wine C13 Vanillin, pHB, … O18 Water H2 Water N15 Proteins, Raw Product H2-Py Proteins, Raw Product O18-Py Ethanol, Raw Product S34 Proteins, Raw Product (D/H)1 and (D/H)2 Ethanol (D/H)1 Acetic Acid (D/H)i Vanillin Contact: FreddyThomas@Eurofins.com

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Thank you for your attention!

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