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Recent Applications of Stable Isotopes Techniques
to check the authenticity of food products
Eric Jamin
Authenticity Analytical Unit Manager
Eurofins Scientific, Nantes (France)
[email protected]
Phone # : (33) 2 51 83 21 00
7 – 8 October 2008
www.eurofins.com
Eurofins laboratories: the origin...
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
Nantes (France): Competence Center for authenticity
and isotopic measurements
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Authenticity
Why Is it necessary to check ?
 Verify raw materials
 Check new suppliers
… and avoid headlines like ….
 Protect brand image
THE INDEPENDENT, February 1991
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A wide range of adulteration issues
ORANGE cases
2%
2%
3%
3%
3%
22%
sugar addition
8%
other fruit addition
untypical flavour profile
multiple adulteration
water addition
bad quality
12%
Mislabelling
ascorbic acid addition
16%
low brix
low vitamin C
overdilution
13%
16%
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How to check? The analytical tools available
 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
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Stable isotopes at natural abundance
Food products are composed of 4 main elements C, H, O and N.
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13
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18
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These elements are naturally present in
several isotopic forms (same atomic
number, different weights), following a
distribution influenced by natural
phenomena and human processes.
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Stable Isotope Analyses
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
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Stable Isotope Analyses
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
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Isotopic methods recognition
year Method
Product
Fraction
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
13
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1993 CEN (TC174 N108, ENV 12140)
fruit juice
Sucre
IRMS
13
12
1995 AOAC Official method 995.17
fruit juice
Ethanol (from
fermentation)
1996 OIV resolution ENO 2/96
wine
water
IRMS
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1997 EC regulation 822/97
wine
water
IRMS
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1997 CEN (TC174 N109, ENV 12141)
fruit juice
water
IRMS
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2000 AOAC Official method 2000.19
maple syrup
Ethanol (from
fermentation)
2001 OIV resolution ENO 17/2001
wine
Ethanol
IRMS
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2003 EC regulation 440/ 2003, annex 2
wine
Ethanol
IRMS
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fruit juice &
maple syrup
Vanillin
Ethanol (from
fermentation)
Vanillin
IRMS
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2004 AOAC method 2004.01
2006 AOAC method 2006.05
Techniques Isotope ratios
C/ C
C/ C
SNIF-NMR (D/H)I , (D/H)II ,R
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O/ O
O/16O
O/16O
SNIF-NMR (D/H)I , (D/H)II ,R
SNIF-NMR
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C/ C
C/12C
12
C/ C
(D/H)i
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Stable Isotope Analyses
H2O
DHO
H2O
DHO
H2O
H2O
H2O
DHO
DHO
DHO
H2O
H2O
H2O
DHO
H2O
DHO
H2O
DHO
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Influence of geographical origin
on stable isotope ratios
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Carbon metabolism
Where does the sugar in a plant
come from :
Carbon dioxide
CO2
(-8 ‰)
C3
most plants
values around -25‰
C4
Gramineae family e.g. sugarcane,
maize, values around -10‰
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What are the latest improvement
of isotopic methods for fruit juices?
Initially: bulk fractions, single isotopes
Use of multi-component profiles to increase the
sensitivity (narrower ranges of variation)
Use of multi-element profiles and site-specific methods
to discriminate more sources
Use of GC-IRMS to analyse flavour compounds
at low concentrations
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Isotopic analyses in direct fruit juice:
Improved detection of sugar addition
NFC JUICE
COMPONENTS
Water
Sugars
2H/1H
Fermentation  Ethanol
18O/16O
2H/1H
18O/16O
CH2CH3OH
13C/12C
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 sugar addition in fruit juices
d13C
(‰)
-5.0
Cane, corn
Pineapple
-10.0
-15 - -12 (AIJN)
Ethanol
From -15.0
sugars
107 - 112
-20.0
-25.0
Beet
-30.0
-35.0
85.0
Orange
90.0
95.0
100.0
105.0
110.0
(D/H)I (ppm)
115.0
120.0
125.0
130.0
Ethanol from sugars
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NEW application of 13C SNIF-NMR to pineapple:
improved detection of sugar addition
CANE
Added C4 sugar
MAIZE
BEET
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d C of ethanol CH3 (‰)
PINEAPPLE
test
d13C of ethanol CH2 (‰)
J. Agric. Food Chem. 2010, 58, 11580–11585
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Isotopic analyses in direct fruit juice:
Improved detection of water addition
NFC JUICE
COMPONENTS
Water
Sugars
2H/1H
Fermentation  Ethanol
18O/16O
2H/1H
18O/16O
CH2CH3OH
13C/12C
J. Agric. Food Chem. (2003) 51, 18, 5202-5206 ; J. Agric. Food Chem. (2006), Vol. 54, No. 2, 279-284
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Practical application:
NFC juice authentication
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10
9
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Lab-squeezed
samples
18
d O water (‰)
6
5
4
3
2
Water addition
Market NFC
juices
1
Market FC
juices
0
-1
-2
Max (95% c.i.)
-3
-4
-5
-6
-7
Authentic orange from a
specific origin
-8
Min (95% c.i.)
AIJN limit
-9
-10
d18O ethanol (‰)
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
 Industrial ascorbic acid is derived from sugar sources
 Global 13C only detects C4 sources
13C-IRMS analysis:
Site-specific
Figure 2:
for
1
of
CO
COH
COH
HC
CHOH
O
Ce(SO4)2+ H2SO4,
20°, 240 min
CO2 from Carbon 1
CH 2OH
developed in the Pure Juice Project*
*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
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Detection of added ascorbic acid
(acerola juice and dietary supplements, etc.)
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13
D d C C1 - global ascorbic acid (‰)
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3
2
1
0
artificial sources
Orange
blackcurrant
acerola fruit
market acerola
-1
-2
-3
addition of artificial ascorbic acid
-4
-5
-6
-7
-8
-9
-10
-25
-24
-23
-22
-21
-20
-19
-18
-17
-16
-15
-14
-13
-12
-11
-10
-9
-8
d13C ascorbic acid (‰)
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Fruit processing, July-August 2009, pp 170-175
Commercial sources of vanillin
Vanilla Fragrans
Vanilla Planifolia
Market: 11 000 T/year
H
Ca.11 $ / Kg
O
1200-4000 $ / Kg
Chemical Industry
Tropical
Agriculture
20-50 T/year
(Guaiacol)
OCH3
OH
Paper Industry (Lignin)
VANILLIN
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
Ex-ferulic acid
1.6
1.2
Ex-lignin
0.8
0.4
0.0
Ex-eugenol
Synthetic
-0.4
-0.8
-1.2
Ex-beans
-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
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Geographical origin of plant product:
Worldwide origins of RICE
Combines:
13C
15N
18O
Measurements
Also available for cereals, fruits, wine, coffee, olive oil, meat, honey, etc
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Salmon / Sea bass / sea bream / Cod etc.:
Wild or farmed?
d
15
N Choline (‰)
O18 Salmon oil vs. N15 Choline
Mislabeled commercial samples:
Authentic farmed samples
-Labeled « wild »,
Commercial
farmed
samples
Authentic wild
samples
-Found « farmed »
Commercial wild samples
Differentiation of wild and
farmed fish by isotopic methods
d
18
O Salmon oil (‰)
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Animal or vegetal source?
example : Squalane and squalene
squalene
squalane
mixture
-29‰
-27‰
-22‰
d13C (/VPDB)
-19‰
Determination of origin can be performed
using EA-IRMS on pure compounds or GC-C-IRMS on cosmetics
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,
should you need more information
Nantes (France) laboratories:
[email protected]
Tel. (33) 2 51 83 21 00
www.eurofins.com
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