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)
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
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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
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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
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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
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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
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H2O
DHODHO
DHODHO
H2O
H2OH2O
H2O
H2O
H2O
DHODHOH2O
H2O
H2O
DHODHO
Stable Isotope Analyses
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Influence of geographical origin on stable isotope ratios
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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‰
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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)
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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 sugar addition
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
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
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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
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-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
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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: [email protected]. (33) 2 51 83 21 00
www.eurofins.comwww.eurofins.com