Physics:Isotopic analysis by nuclear magnetic resonance

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Isotopic analysis by nuclear magnetic resonance refers to an overarching set of methodologies to precisely quantify differences in isotopic content at each atom of a molecule, and thus to measure the specific natural isotope fractionation for each site of the molecule. One such method, SNIF-NMR—the corresponding English of the original French acronym, which abbreviates site-specific natural isotopic fractionation-nuclear magnetic resonance[1][2][3]—is an analytical method developed to detect the over-sugaring of wine and enrichment of grape musts.[not verified in body] its main use has been to check the authenticity of foodstuffs such as wines, spirits,[not verified in body] fruit juice, honey, sugar and odorant molecules such as vanillin, benzaldehyde, raspberry ketone, and anethole.[not verified in body] The SNIF-NMR method in particular has been adopted by the International Or the [[European Union as an official method for wine analysis, by the Association of Official Agricultural Chemists (AOAC) as an official method for analyzing fruit juices, maple syrup, vanillin, and by the European Committee for Standardization (CEN) for analyzing vinegar.[not verified in body]

History

History of SNIF-NMR

Discovery

  • 1981: Invention, as RMN-FINS, the acronym for fractionnement isotopique naturel spécifique par résonance magnétique nucléaire (see following), by Gerard Martin, Maryvonne Martin, and their team at the University of Nantes/CNRS.[according to whom?][4] * 1987-1990: Eurofins Laboratories applied the SNIF-NMR method to the analysis of fruit juices and certain natural flavors.[citation needed]
  • 1990-1992: the method is tested on aromatic molecules.[citation needed]

Organisational recognition

Principle

Isotopic distribution

Natural abundances of hydrogen, carbon and oxygen. [From Eurofins Analytics France]
Isotopic Fractionation Sources. [From Eurofins Analytics France]

The atoms hydrogen, oxygen, and carbon co-exist naturally in specific proportions with their stable isotopes, 2H (or deuterium), 18O, and 13C, respectively, as shown in the figure.[citation needed]

The amount and distribution of the different isotopes in a molecule are natural products influenced by:[7]

  • Environmental (climatic and geographical) conditions, and
  • Chemical or biochemical processes, primary metabolism, photosynthetic metabolism in plants, etc.

A phenomenon known as natural isotopic fractionation (see figure) means that an isotopic fingerprint composed of ratios of isotopes at each atom of a molecule can be determined in order to provide information on the origin—botanical, synthetic, geographical of the molecule or product.[citation needed]

Principles underlying specific methods

SNIF-NMR

SNIF-NMR is based on the principle of fractionation of carbon isotopes in oxygenic photosynthesis (isotope fractionation).[8][9][10] NMR of two nuclei are routinely used for assessing food authenticity:

  • Hydrogen nuclei: the 2H-SNIF-NMR method, which was the original application of SNIF-NMR, measures the ratio of deuterium to hydrogen of the hydrogen atoms ini a sample molecule;[11][12] and
  • Carbon nuclei: the 13C-SNIF-NMR method has made for new applications of SNIF-NMR, where the method determines the ratio of 13C (carbon-13) to 12C of the carbon atoms in a sample molecule.[13][14]
Steps of the method
Steps in the SNIF-NMR of ethanol. [From Eurofins Analytics France]

The SNIF-NMR method is applied to purified molecules; therefore, preparative steps are required before instrumental analysis. For example, for the SNIF-NMR of ethanol, according to official methods, preparative steps include:

  • fermentation (for fruit juices);[clarification needed]
  • quantitative extraction of ethanol by distillation; and
  • standardized preparation of NMR samples, followed by NMR acquisition, interpretation of the results, and a report regarding sample authenticity.

At each step of the SNIF-NMR sample preparation and analysis, efforts are made to avoid parasitic isotopic fractionation. Control measurements, such as determining the alcoholic strength of the intermediate products of the analysis (fermented juice or distillate), are performed on each sample.

Advantages of the method
Principle of the IRMS. [From Eurofins Analytics France]

The isotopic ratios of a molecule can also be determined by isotope ratio mass spectrometry (IRMS). The sample quantity required for IRMS is much lower than that for NMR, and it is possible to couple the mass spectrometer to a chromatographic system to enable online purification or analysis of multiple components in a complex mixture. However, the sample is burnt after a physical transformation such as combustion or pyrolysis. Therefore, it merely gives the mean concentration of the studied isotope across all sites of the molecule. IRMS is the official AOAC technique used for the average ratio 13C/12C (or δ13C) of sugars or ethanol, and the official CEN and OIV method for the 18O/16O in water.

The SNIF-NMR method can determine, with high accuracy, the isotopic ratios at each site of the molecule, enabling better discrimination. For example, for ethanol (CH3CH2OH), the three ratios ((D/H)CH3, (D/H)CH2, and (D/H)OH) can be obtained.

An example 2H-SNIF-NMR Spectrum
2H (Deuterium) NMR spectrum of ethanol. [From Eurofins Analytics France]

Ethanol molecules obtained after complete fermentation of a sugar coexist with 3 naturally monodeuterated isotopomers (CH2D-CH2-OH, CH3-CHDOH, and-CH3-CH2OD). Their presence can then be quantified with relative precision.[15] In the presented 2H-NMR spectrum, peaks correspond to one of the three observed isotopomers of ethanol.

In the official method of the AOAC, the ratios of deuterium (D)/hydrogen (H) of CH3 and the D/H of CH2 are calculated by comparison with an internal standard, tetramethylurea (TMU), with a certified (D/H) value.[16]

Interpretation of SNIF-NMR isotopic values
The adulteration triangle: Re-partition of isotopic ratios on ethanol molecules. [From Eurofins Analytics France]

The figure summarizes the principles of interpretation applied:[citation needed]

  • Results measured by IRMS (isotopic deviation of δ 13C), which enable discrimination of plants according to their CO2 photosynthetic metabolism (C4, as in corn or maize, versus C3, as in beet, orange, or grape);
  • Results measured by SNIF-NMR that can differentiate the botanical origin of sugars within the same metabolic group (e.g., beet versus orange or grape).

Values obtained on a test sample are then compared with the values of certifiably authentic sample data.[citation needed]

Applications

Of SNIF-NMR

2H-SNIF-NMR

Isotope method recognition for food application, 2013. [From Eurofins Analytics France]
Fruit juice and maple syrup

AOAC Official Method for detecting the addition of sugar in a fruit juice[6] or in maple syrup. It is the only reliable method for detecting the addition of C3 sugar (e.g., beet sugar).

Authenticity of wines
Applications of SNIF-NMR and IRMS to wine authenticity. [From Eurofins Analytics France]

SNIF-NMR is the official method of the OIV for determining the authenticity of wine origin,[citation needed] and, as of this date,[when?] is the only method to detect C3 sugar addition (such as beet sugar).[citation needed]

The isotopic parameters of both water and ethanol are related to the humidity and temperature of the growing region of the plant. Therefore, consideration of the region's meteorological data and the year helps make a diagnosis. In the case of wine and fruit, the isotopic parameters of ethanol have been shown to respond even to subtle environmental variations and efficiently characterize the region of production.[15][17]

Since 1991, an isotopic data bank has been built at the Joint Research Center of the European Commission (EC-JRC) for wines from all European member states. The database contains several thousand entries for European wines,[18][full citation needed] and is maintained and updated every year.[citation needed] This database is accessible for all official public laboratories. Private companies involved in food and beverage controls have also collected authentic samples and built up specific data banks.[19]

Thus, by comparing the specific natural isotope fractionation corresponding to each site of a molecule of ethanol in wine with that of a molecule known and referenced in a database. The geographical origin, botany, and method of production of the ethanol molecule, and thus the authenticity of the wine, can be checked.[20]

Acetic acid in vinegar
Application to vinegar. [From Eurofins Analytics France]

The origins of vinegars obtained by bacterial or chemical oxidation of ethanol resulting from the fermentation of various sugars can be identified by the 2H-SNIF-NMR. It allows control of vinegar quality and determination of whether it comes from sugar cane, wine, malt, cider, or alcohol, or from chemical synthesis.[21]{{page needed|date=March 2025}

Vanillin

As of 2019, 2H-SNIF-NMR is the official AOAC method for determining the natural vanillin.[22]

The abundance of five monodeuterated isotopomers for vanillin can be measured by 2H-SNIF-NMR.[citation needed] Data for vanillin are shown in the figure; all observable sites for which the site-specific deuterium concentrations can be measured are referenced with a number.[citation needed]

As for wine or fruit, the interpretation of results regarding origin is done by comparison of the isotopic parameters of the sample analyzed with those from a group of referenced molecules of known origin.[citation needed] Origins of vanillin are well discriminated using 2H-NMR data; in particular, vanillin from the ex-bean can be well distinguished from the other sources (see next figure).[citation needed]

Additionally, this method is the only one to discriminate between natural and biosynthetic sources of vanillin.[23]

Other odorants

The naturality[clarification needed] of different aromas can also be checked using SNIF-NMR: for example, for anethole, abundance of only six monodeuterated isotopomers can be measured by 2H-SNIF-NMR that allows differentiating the botanical origins fennel, star anise, or pine.[24][full citation needed]

Other applications

SNIF-NMR applied to benzaldehyde can detect adulteration in bitter almond and cinnamon oils. It is demonstrated that the site specific deuterium contents of benzaldehyde allow the determination of the origin of the molecule: synthetic (ex-toluene and ex-benzal chloride), natural (ex-kernels from apricots, peaches, cherries and ex-bitter almond) and semisynthetic (ex-cinnamaldehyde extracted from cinnamon).[25][full citation needed] Other applications have also been published, including for raspberry ketone,[26][verification needed] heliotropine,[citation needed] etc.

13C-SNIF-NMR

Use of 13C-SNIF-NMR to distinguish between C3, C4, and CAM-types of plant metabolism; see text for explanation and citations.

Optimization of technique parameters has enabled reaching better accuracy for the 13C NMR measurements.[27]

The 13C-SNIF-NMR method is called the "new frontier" because it is the first analytical method that can differentiate sugars coming from C4-metabolism plants (cane, maize, etc.) and some crassulacean acid metabolism plants (CAM-metabolism) like pineapple or agave.[28]

This method can also be applied to tequila products, where it can differentiate authentic 100% agave tequila, misto tequila (made from at least 51% agave), and products made from a larger proportion of cane or maize sugar and therefore not complying with the legal definition of tequila.[28]

Further reading

References

  1. ↑ Eurofins Staff (2023). How Eurofins Got Its Name (short form informational video). LinkedIn Posts. Event occurs at 0:00-0:22. Retrieved 20 March 2025.
  2. ↑ Viskić M; Bandić LM; Korenika AJ; Jeromel A (8 January 2021). "NMR in the Service of Wine Differentiation". Foods 10 (1): 120. doi:10.3390/foods10010120. PMID 33429968. 
  3. ↑ Ogrinc, N; Kosir, IJ; Spangenberg, JE & Kidric, J (June 2003). "The Application of NMR and MS Methods for Detection of Adulteration of Wine, Fruit Juices, and Olive Oil. A Review.". Anal. Bioanal. Chem. 376 (4): 424–430. doi:10.1007/s00216-003-1804-6. PMID 12819845. 
  4. ↑ INPI Staff (6 January 2014) "Bases de Données Marques". Courbevoie, France: Institut National de la Propriété Industrielle. Accessed 6 January 2014.
  5. ↑ Commission Regulation of the European Communities, 1990. (EEC) n° 000/90: "Determining Community Methods for the Analysis of Wine". Brussels, Official Journal of the European Communities, p.64-73.
  6. ↑ 6.0 6.1 AOAC Official Method 995.17, Beet Sugar in Fruit Juices, SNIF-NMR, AOAC International 1996
  7. ↑ Akoka, Serge; Remaud, Gérald (October–December 2020). "NMR-based isotopic and isotopomic analysis". Progress in Nuclear Magnetic Resonance Spectroscopy 120-121: 1–24. doi:10.1016/j.pnmrs.2020.07.001. PMID 33198965. Bibcode: 2020PNMRS.120....1A. 
  8. ↑ Galimov, Ė M. (1985). The biological fractionation of isotopes. Orlando: Academic Press. ISBN 978-0-12-273970-5. 
  9. ↑ Drake, Brandon L (2014). "Using Models of Carbon Isotope Fractionation during Photosynthesis to Understand the Natural Fractionation Ratio" (in en). Radiocarbon 56 (1): 29–38. doi:10.2458/56.16155. ISSN 0033-8222. Bibcode: 2014Radcb..56...29D. https://www.cambridge.org/core/product/identifier/S0033822200049092/type/journal_article. 
  10. ↑ O'Leary, M. H.; Madhavan, S.; Paneth, P. (December 1992). "Physical and chemical basis of carbon isotope fractionation in plants" (in en). Plant, Cell & Environment 15 (9): 1099–1104. doi:10.1111/j.1365-3040.1992.tb01660.x. ISSN 0140-7791. Bibcode: 1992PCEnv..15.1099O. https://onlinelibrary.wiley.com/doi/10.1111/j.1365-3040.1992.tb01660.x. 
  11. ↑ Meier, Urs C. (February 2023). "Forensic analysis of the deuterium/hydrogen isotopic ratios of the nerve agent sarin, its reaction by-product diisopropyl methylphosphonate and their precursors by 2H SNIF-NMR" (in en). Talanta 253. doi:10.1016/j.talanta.2022.123890. PMID 36116239. https://linkinghub.elsevier.com/retrieve/pii/S0039914022006865. 
  12. ↑ Ciepielowski, Grzegorz; Krassowski, Jakub; Albrecht, Łukasz; Pacholczyk-Sienicka, Barbara (January 2025). "Identifying the botanical origin of alcohol using 2H SNIF NMR: A case study of "polish vodka" PGI" (in en). Food Chemistry 463 (Pt 2). doi:10.1016/j.foodchem.2024.141218. PMID 39276548. 
  13. ↑ Perini, M.; Pianezze, S.; Bontempo, L. (2024). "Stable Isotope Ratio Mass Spectrometry and Site-Specific Natural Isotope Fractionation-Nuclear Magnetic Resonance applications to discriminate between synthetic and natural analogs: A review" (in en). TrAC: Trends in Analytical Chemistry 180. doi:10.1016/j.trac.2024.117966. https://linkinghub.elsevier.com/retrieve/pii/S0165993624004497. 
  14. ↑ Portaluri, Vincent; Thomas, Freddy; Jamin, Eric; Akoka, Serge; Remaud, Gérald S. (2021-08-20). "Authentication of Agave Products through Isotopic Intramolecular 13C Content of Ethanol: Optimization and Validation of 13C Quantitative NMR Methodology". ACS Food Science & Technology 1 (7): 1316–1322. doi:10.1021/acsfoodscitech.1c00177. Bibcode: 2021AFST....1.1316P. 
  15. ↑ 15.0 15.1 Martin, G; Martin, ML. Modern Methods of Plant Analysis: "The Site-Specific Natural Isotope Fractionation-NMR Method Applied to the Study of Wines", edition: HF Linskens and JF Jackson, Springer Verlag, Berlin, 1988, p. 258-275
  16. ↑ Martin, G. G.; Hanote, V.; Lees, M. (1996). "Interpretation of combined 2H SNIF/NMR and 13C SIRA/MS analyses of fruit juices to detect added sugar". Journal of AOAC International 79: 62–72. doi:10.1093/jaoac/79.1.62. PMID 8620113. https://academic.oup.com/jaoac/article-abstract/79/1/62/5684571. Retrieved 1 January 2026. 
  17. ↑ Martin GJ, Guillou C, Martin ML, Cabanis MT, Tep Y, Aerny J. J. Agric. Food Chem. 1988;36:316–22
  18. ↑ Official Journal of the European Communities. Off. J. Eur. Commun. 1991;L214:39–43.[full citation needed]
  19. ↑ Guillou C, Jamin E, Martin GJ, Reniero F, Wittkowski R, Wood R. Bulletin OIV. 2001;74:26–36
  20. ↑ Martin, G; C. Guillou, C; Martin, YL. Natural Factors of Isotope Fractionation and the characterization of Wines", Journal of agricultural and food chemistry, n°36, 1988, p. 316-322
  21. ↑ Vallet, C; Arendt, M; Martin, G. (1988) "Site specific isotope fractionation of Hydrogen in the oxidation of ethanol into acetic acid. Application to vinegars", Biotechnology Techniques, 2:2.
  22. ↑ Sophie, Guyader; Freddy, Thomas; Eric, Jamin; Mathilde, Grand; Serge, Akoka; Virginie, Silvestre; S., Remaud, Gérald (2019). "Combination of 13C and 2H SNIF-NMR isotopic fingerprints of vanillin to control its precursors" (in en). Flavour and Fragrance Journal 34 (2). ISSN 0882-5734. https://agris.fao.org/search/en/providers/122535/records/65df8fec0f3e94b9e5d9e83c. 
  23. ↑ AOAC Official Method 2006.05, Site-Specific Deuterium/Hydrogen (D/H) Ratios in Vanillin, AOAC International 2007
  24. ↑ Martin, G; Martin, M; Mabon, F & Bricout, J. (1983) "La Résonance Magnétique Nucléaire du Deutérium en Abondance Naturelle, une nouvelle méthode d'identification de l'origine de produits alimentaires appliquée à la reconnaissance des Anétholes et des Estragoles", Sciences des Aliments.[full citation needed]
  25. ↑ Remaud, G; Debon, A; Martin, Y & Martin, G. (1997) "Authentication of Bitter Almond Oil and Cinnamon Oil: Application of the SNIF-NMR Method to Benzaldehyde", Journal of Agricultural and Food Chemistry, 45.[full citation needed]
  26. ↑ Casabianca, H; Graff, J-B; Jame, P; Perrucchietti, C & Chastrette, M (May 1995). "Application of Hyphenated Techniques to the Chromatographic Authentication of Flavors in Food Products and Perfumes". Journal of High Resolution Chromatography 18 (5): 279–285. doi:10.1002/jhrc.1240180503. https://journals.scholarsportal.info/details/09356304/v18i0005/279_aohtttfifpap.xml. Retrieved 20 March 2025. 
  27. ↑ Tenailleau, E & Akoka, S. (2007) "Adiabatic 1H decoupling scheme for very accurate intensity measurements in 13C NMR, Journal of Magnetic Resonance", n°185, p. 50-58.
  28. ↑ 28.0 28.1 Thomas, F; Randet, C; Gilbert, A; Silvestre, V; Jamin, E; et al. (2010) "Improved Characterization of the Botanical Origin of Sugar by Carbon-13 SNIF-NMR Applied to Ethanol", Journal of Agricultural and Food Chemistry, n° 58, pp. 11580-11585.

See also



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