7 – 8 October 2008 Recent Applications of Stable Isotopes Techniques to check the authenticity of food products Eric Jamin Authenticity Analytical Unit Manager Eurofins Scientific, Nantes (France) Phone # : (33)

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... 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 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

6 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. O C N N D H O C

7 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

8 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

9 Isotopic methods recognition

10 H2OH2O DHO H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O H2OH2OH2OH2OH2OH2O Stable Isotope Analyses

11 Influence of geographical origin on stable isotope ratios

12 Where does the sugar in a plant come from : Carbon dioxide C3C3 C4C4 CO 2 (-8 ‰) Carbon metabolism most plants values around -25‰ Gramineae family e.g. sugarcane, maize, values around -10‰

13 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)

14 13 C/ 12 C NFC JUICE Sugars COMPONENTS Fermentation  Ethanol 2 H/ 1 H CH 2 CH 3 OH 18 O/ 16 O 2 H/ 1 H Water 18 O/ 16 O Isotopic analyses in direct fruit juice: Improved detection of sugar addition J. Agric. Food Chem. (2003) 51, 18, ; J. Agric. Food Chem. (2006), Vol. 54, No. 2,

15 Detection of sugar addition in fruit juices Orange Beet (D/H) I (ppm)  13 C (‰) Beet (D/H) I (ppm)  13 C (‰) Beet Cane, corn (D/H) I (ppm)  13 C (‰) Ethanol from sugars Ethanol From sugars Pineapple (AIJN)

16 NEW application of 13 C SNIF-NMR to pineapple: improved detection of sugar addition Added C4 sugar J. Agric. Food Chem. 2010, 58, 11580–11585

17 13 C/ 12 C NFC JUICE Sugars COMPONENTS Fermentation  Ethanol 2 H/ 1 H CH 2 CH 3 OH 18 O/ 16 O 2 H/ 1 H Water 18 O/ 16 O Isotopic analyses in direct fruit juice: Improved detection of water addition J. Agric. Food Chem. (2003) 51, 18, ; J. Agric. Food Chem. (2006), Vol. 54, No. 2,

18 Practical application: NFC juice authentication J. Agric. Food Chem. (2003) 51, 18, ; J. Agric. Food Chem. (2006), Vol. 54, No. 2,

19 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 Coordination: SGF developed in the Pure Juice Project* Figure 2: for of Site-specific 13 C-IRMS analysis: COH COH CH CHOH CH 2 OH O CO CO 2 from Carbon 1 Ce(SO 4 ) 2 + H 2 SO 4, 20°, 240 min 1  Industrial ascorbic acid is derived from sugar sources  Global 13 C only detects C4 sources

20 Detection of added ascorbic acid (acerola juice and dietary supplements, etc.) Fruit processing, July-August 2009, pp

21 Commercial sources of vanillin

22 Principal Component Analysis of (D/H)i and 13 C/ 12 C ratios of vanillin from various sources

23 Geographical origin of plant product: Worldwide origins of RICE Also available for cereals, fruits, wine, coffee, olive oil, meat, honey, etc Combines: 13C 15N 18O Measurements

24 Salmon / Sea bass / sea bream / Cod etc.: Wild or farmed? Commercial farmed samples Commercial wild samples Mislabeled commercial samples: -Labeled « wild », -Found « farmed » Differentiation of wild and farmed fish by isotopic methods

25 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  13 C (/VPDB) squalane squalene Rapid Commun. Mass Spectrom 2010, 24, ; SOFW-Journal | 136 | 1/2-2010

26 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.

27 Thank you for your attention! And feel free to contact us, should you need more information Nantes (France) laboratories: Tel. (33)