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
2
Officially-recognised methods for food authentication
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
AOAC Official Method 2000.19
Maple syrup
Ethanol (from fermentation)
SNIF-NMR
AOAC Official Method 2004.01
Fruit juice & maple syrup
Ethanol (from fermentation)
IRMS
AOAC Official Method 2006.05
Vanillin
Vanillin
SNIF-NMR
CEN (TC174 N108, ENV 12140)
Fruit juice
Sugars
IRMS
13
CEN (TC174 N108, ENV 12141)
Fruit juice
Water
IRMS
18
OIV-MA-AS311-05, OIV/OENO 381/2009
Wines & Spirits
Ethanol
SNIF-NMR
OIV-MA-AS312-06, OIV/OENO 381/2009
Wines & Spirits
Ethanol
IRMS
13
OIV-MA-AS2-12, OIV/OENO 381/2009
Wines & Spirits
Water
IRMS
18
CEN, EN 16466-1:2012
Vinegar
Acetic acid
SNIF-NMR
CEN, EN 16466-2:2012
Vinegar
Acetic acid
IRMS
13
CEN, EN 16466-3:2012
Vinegar
Water
IRMS
18
AOAC = Association of Official Analytical Chemists (USA); CEN = European Committee for Standardization (EU)
OIV = International Organisation of Vine and Wine
13
C/12C
(D/H)I, (D/H)II, R
13
C/12C
(D/H)i
C/12C
O/16O
(D/H)I, (D/H)II, R
C/12C
O/16O
(D/H)CH3
C/12C
O/16O
3
A helping hand from the European Commission
An early initiative to develop and promote stable isotope analyses applied to food
authentication
FIT - Food analyses using
Isotopic Techniques
Contract N° : SMT4 - CT95 - 7500
Universities
Research Centres
Official Control labs
Private specialised labs
NEEDS
Industry
Stable
Isotope
Analyses
Retail Sector
State Control Bodies
Consumers
APPLICATIONS
4
The FIT Thematic Network 1996-1998
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
FINLAND
P. Lehtonen , Alko Group
BELGIUM
P. de Bièvre & P. Taylor ,
J. Pauwels & G.N Kramer ,
IRMM
IRELAND
K. Mac Namara,
Irish Distillers
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
PORTUGAL
M. Curto, A. Teixeira
& L. Santos, INETI
J. Lopes, Instituto da
Vinho e do Vinho
Paulo Barros , Instituto do
Vinho do
Porto
SWEDEN
G. Fuchs & S. Wretling ,
National Food Administration
SPAIN
A.I. Blanch-Cortes,
Min. de Agricultura,
Pesca y Alimentacion
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
AUSTRIA
F. Pichlmayer, IAEA
G. Haberhauer , Austrian Research Centre
GREECE
G. Bonas, T .Mavromoustakos
& E. Zervou , NHRF
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
5
Using stable isotope techniques as official control
methods
Applications that target areas of
concern for food control
laboratories
Key
requirements
Interpretation of results:
extensive reference data base
Reliability: robust method
performance, quality control
6
3 Case studies
 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
7
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)
Database requirements – the COFAWS project
Feed
13C-IRMS
extraction
15N-IRMS
Feed Oil
13C-IRMS
18O-IRMS
extraction
Muscle
Triacylglycerides
+ PC
Water
2H-SNIF-NMR
18O-IRMS
Seawater
2H-SNIF-NMR
18O-IRMS
transesterification
Fatty Acid Methyl Esters
(FAMEs)
GC
2H-SNIF-NMR
13C GC-C-IRMS
saponification
Fatty Acids
13C-IRMS
Glycerol (+Choline)
2H-SNIF-NMR
(Triacetin)
13C-IRMS, 18O-IRMS
15N-IRMS, %N
9
Database requirements – the COFAWS project
Sample collection
Norway, Scotland, Ireland,
Faroes, Iceland, Chile,
Canada …
Wild
Slow/fast
growth
Farmed
Season
As labelled
Type of feed
Authentic
Geographical
origin
Market
10
COFAWS project results
Analysis of Variance (ANOVA) : wild vs. farmed
140
120
Best discriminatory parameters
100
80
60
40
20
0
CLU4
C22:5n-3
CLU2
CLU7
(D/H)3T
CLU5
C20:1n-9
C22-1n-11
C18:1n-9
C16:0
CLU8
C20:5n-3
C13Gly
C18:1n-7
CLU6
CLU9
C13RP
O18Gly
C22:6n-3
C18:0
C13Ac
(D/H)2T
CLU1
C14:0
C16:1n-7
C18:2n-6
CLU3
(D/H)1T
N15chol
O18oil
11
Authentic farmed samples
Authentic wild samples
Market farmed samples
Market wild samples
Mislabeled market samples:
-Labeled « wild »,
-Found « farmed »
d
15
N Choline (‰)
Validated method to distinguish wild and farmed salmon
Differentiation of wild and farmed fish
by isotopic methods
d
18
O Salmon oil (‰)
F. Thomas et al. J. Agric. Food Chem., 2008, 56, 989-997.
12
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
13
Authentication of palm sugar
Where does the sugar in a plant
come from :
Carbon dioxide
CO2
C3
most plants
values around -25‰
sugarcane, maize,
C4 Gramineae family e.g.values
around -10‰
14
Authentication of palm sugar
?
C3
?
C4
15
Authentication of palm sugar
16
Authentication of palm sugar
17
Authentication of palm sugar
18
Authentication of palm sugar
 Samples of sap taken from the coconut
plantation (as well as leaves, flowers)
19
Authentication of palm sugar
13C‰
d
 The results :
0
-10
-15.4
-20
-20.3
-26.9
-25.2
-24.8
-25.7
-30
Palm
leaf
Palm
flower
Sap
(A)
Sap
(B)
Palm Commercial
sugar
samples
20
Authentication of palm sugar
21
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
22
Vanilla authentication: an interesting case study
Geographical origin ?
Building up a database for the main vanilla production regions
23
Vanilla authentication: an interesting case study
1964
1974
1984
1994
2004
2006
China appears
the scene
and
520
1770 on3700
3700
Indonesia
150
300
Madagascar
1050
2283
2277
1320
839
China
0
0
0
400
Different species :
Mexico
Vanilla
Tahitiensis90
29
161
0
10
16
French Polynesia
100
21
6
Comores
175
160
160
Uganda
10
10
10
Réunion
45
27
56
2009
4146
4362
2534
3055
2830
900
1200
1400
1382
167
252
291
600
524
100
131
140
199
263
Cyclones44and political
13
25 unrest
49
74
is becoming a major producer
Tonga
2008
in Madagascar scarcity of
131
60 producers
75
70
supply, new
enter
market
65
20
61
48
52
48
33
25
22
12
12
Amount of vanilla produced (tons) (FAO-STAT)
24
Vanilla authentication: an interesting case study
A classic case of supply and demand …
Tonnes
produced
Euros/tonne
High risk of adulteration!
25
Vanilla authentication: an interesting case study
Typical vanilla aromatic profile (regulatory guidelines exist to assess vanilla authenticity)
Main component : Vanillin
H
O
OCH 3
OH
26
Vanilla authentication: an interesting case study
Commercial sources of vanillin
Vanilla Fragrans
Vanilla Planifolia
Guaiacol
H
O
Chemical Industry
(Guaiacol)
OCH 3
Tropical
Agriculture
2001: 400 € / tonne
2003: 750 € / tonne
12 - 15 € / tonne
OH
(Lignin)
VANILLIN
Paper Industry
Lignin
27
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)
13C
BUT

enrichment during synthesis able to fool the authentication method
13C/12C
ratios of other vanilla components specified in the legislation
d13C (‰ /V.PDB)
for pHB > -19.2 ; for vanillic acid > -24.0 ; for pHB acid > -23.0
BUT
Selective 13C enrichment is undectable when analysing whole compounds
28
2H SNIF-NMR
Site specific isotope ratios
measured
using
SNIF–NMR® ANALYSIS of vanillin
(D/H)1
H
O
(D/H)3
(D/H)3
(D/H)4
OCH 3
TMU
(D/H)5
OH
ex-lignin
5
(D/H)2
34
2
2.0
2.8
3.9
7.0
7.5
8.4
9.8
F2
1
Solvent
ex-guaïacol
ex-beans
PCA using (D/H)1, (D/H)3,
(D/H)4, (D/H)5
F1
29
BUT SNIF-NMR® and detection of isotopic manipulation of vanillin
Solvent
OCHD2
Zoom of 2H-NMR
spectrum
TMU
5
Presence of a small peak
due to OCHD2
3
2
1
4
1.83
2.80
3.78
4.75
5.73
6.70
7.68
8.65
9.63
10.60
ppm
From Remaud et al., J. Agric. & Food Chem., 1997
30
Using stable isotope techniques as official control methods
Applications that target areas of
concern for food control
laboratories
Key
requirements
Interpretation of results:
extensive reference database
Reliability: robust method
performance, quality control
31
Proficiency Testing
The FIT- Proficiency Testing Scheme
 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
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FIT- Proficiency Testing Scheme
Techniques:
Parameters:
Type of samples:
 EA-IRMS (Combustion,  C13 Ethanol, Sugars, Pulp, Honey, Proteins, Acetic
 Wine, spirit
Pyrolysis, Equilibration)
Acid
 Fruit juice
 GC-C-IRMS
 C13 CO2 Sparkling wine
 Pure alcohol
 SNIF-NMR
 C13 Vanillin, pHB, …
 Honey
 O18 Water
 Olive oil
 H2 Water
 Vinegar
 N15 Proteins, Raw Product
 Pure organic product
 H2-Py Proteins, Raw Product
 Flour, cereal, rice
 O18-Py Ethanol, Raw Product
 Water
 S34 Proteins, Raw Product
 Aroma extract
 (D/H)1 and (D/H)2 Ethanol
 Pure aroma molecule
 (D/H)1 Acetic Acid
Contact: [email protected]
 (D/H)i Vanillin
33
Thank you for your attention!
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