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—is an analytical method developed to detect the over-sugaring of wine and enrichment of grape musts. As of this date, its main use has been to check the authenticity of foodstuffs such as wines, spirits, fruit juice, honey, sugar, and vinegar, and of flavorant and odorant molecules such as vanillin, benzaldehyde, raspberry ketone, and anethole. The SNIF-NMR method in particular has been adopted by the International Organisation of Vine and Wine (OIV) and 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.
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.INPI Staff (6 January 2014) "Bases de Données Marques". Courbevoie, France: Institut National de la Propriété Industrielle. Accessed 6 January 2014. 1987-1990: Eurofins Laboratories applied the SNIF-NMR method to the analysis of fruit juices and certain natural flavors. 1990-1992: the method is tested on aromatic molecules.
Organisational recognition 1990: The SNIF-NMR method is recognized by the European Union as an official method for the analysis of wines.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. 1996: The SNIF-NMR method is recognized in the United States by the Association of Official Agricultural Chemists (AOAC) for fruit juices.AOAC Official Method 995.17, Beet Sugar in Fruit Juices, SNIF-NMR, AOAC International 1996 2001: The SNIF-NMR method is recognized by the AOAC for vanillin. 2013: The SNIF-NMR method is recognized by the European Committee for Standardization for acetic acid. The International Organisation of Vine and Wine (OIV) adopts it as an official method.
Principle
Isotopic distribution ] ]
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.
The amount and distribution of the different isotopes in a molecule are natural products influenced by: 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.
Principles underlying specific methods
SNIF-NMR SNIF-NMR is based on the principle of fractionation of carbon isotopes in oxygenic photosynthesis (isotope fractionation). 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; 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.
Steps of the method ]
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); 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 ]
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 ]
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. 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 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.
Interpretation of SNIF-NMR isotopic values ]
The figure summarizes the principles of interpretation applied:
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.
Applications
Of SNIF-NMR 2H-SNIF-NMR ]
Fruit juice and maple syrup AOAC Official Method for detecting the addition of sugar in a fruit juice or in maple syrup. It is the only reliable method for detecting the addition of C3 sugar (e.g., beet sugar).
Authenticity of wines ] SNIF-NMR is the official method of the OIV for determining the authenticity of wine origin, and, as of this date, is the only method to detect C3 sugar addition (such as beet sugar).
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.Martin GJ, Guillou C, Martin ML, Cabanis MT, Tep Y, Aerny J. J. Agric. Food Chem. 1988;36:316–22
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,Official Journal of the European Communities. Off. J. Eur. Commun. 1991;L214:39–43. and is maintained and updated every year. 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.Guillou C, Jamin E, Martin GJ, Reniero F, Wittkowski R, Wood R. Bulletin OIV. 2001;74:26–36
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.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
Acetic acid in vinegar ] 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.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.
Vanillin As of 2019, 2H-SNIF-NMR is the official AOAC method for determining the natural vanillin.
The abundance of five monodeuterated isotopomers for vanillin can be measured by 2H-SNIF-NMR. 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.
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. 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).
Additionally, this method is the only one to discriminate between natural and biosynthetic sources of vanillin.AOAC Official Method 2006.05, Site-Specific Deuterium/Hydrogen (D/H) Ratios in Vanillin, AOAC International 2007
Other odorants The naturality 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.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.
Other applications