Isotopic analysis by nuclear magnetic resonance

Isotopic analysis by nuclear magnetic resonance refers to 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.^{[not verified in body]} 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 over-sugaring of wine and enrichment of grape musts.^{[not verified in body]} As of this date,^[when?] its main use has been to check the authenticity of foodstuffs such as wines, spirits,^{[not verified in body]} fruit juice, honey, sugar, and vinegar, and to control the naturality^{[clarification needed]} of flavorant 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 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 analysis of fruit juices, maple syrup, vanillin, and by the European Committee for Standardization (CEN) for analysis of vinegar.^{[not verified in body]}

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

• 1990: The SNIF-NMR method is recognized by the European Union as an official method for the analysis of wines.
• 1996: The SNIF- NMR method is recognized in the United States by the Association of Official Agricultural Chemists (AOAC) for fruit juices.
• 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.^[when?]

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

The amount and distribution of the different isotopes in a molecule is influenced in for natural products 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 to provide information on the origin—botanical, synthetic, geographical—of the molecule or product.

SNIF-NMR is built on the principle of natural isotopic fractionation. NMR of two nuclei are routinely used for assessing for food authenticity:

• Hydrogen nuclei, ²H-SNIF-NMR method which was the original application of SNIF-NMR, measuring the ratio of deuterium/hydrogen at each atom of a sample molecule; and
• Carbon nuclei: ¹³, where the C-SNIF-NMR method has opened new applications of SNIF-NMR, where the method determines the ratios of ¹³C over ¹²C at each site of a molecule.

The SNIF-NMR is applied on 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, and interpretation of the results, and report regarding sample authenticity.

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

The isotopic ratios of a molecule can also be determined by isotope ratio mass spectrometry (IRMS), sample quantity for IRMS is much lower than for NMR, and there is the possibility of coupling the mass spectrometer to a chromatographic system to enable on-line purification or analyses of several components of a complex mixture. However the sample is burnt after a physical transformation such as combustion or pyrolysis. Therefore, it gives a mean value of the concentration of the isotope studied between 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 (Site-Specific Natural Isotope Fractionation studied by Nuclear Magnetic Resonance) is able to determine, to a high level of accuracy, the isotopic ratios for each of the sites of the molecule, which enables a better discrimination. For example, for ethanol (CH₃CH₂OH), the three ratios ((D/H)CH₃, (D/H)CH₂ and (D/H)_OH) can be obtained.

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

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

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 like can or maize versus C3 like 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 authentic, databased sample data.

AOAC Official Method for detecting the addition of sugar in a fruit juice or in maple syrup. It is the only reliable method to detect addition of C3 sugar (ex: beet sugar).

SNIF-NMR is the official method of the OIV to determine the authentication of wine origin, and as of this date,^[when?] is the only method to detect C3 sugar addition (like 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, considerations of meteorological data of the region and of the year help to make a diagnosis. In the case of wine and fruits, the isotopic parameters of ethanol have been shown to respond even to subtle environmental variations and they efficiently characterize the region of production.

Since 1991, an isotopic data bank is built in the Joint Research Centre of the European Commission (EC-JRC) concerning wines of all European members. The database contains several thousand entries for European wines,^{[full citation needed]} 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.

Thus, by comparing the specific natural isotope fractionation corresponding to each site of a molecule of ethanol of 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 wine can be checked.

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 to control the quality of vinegar and to determine if it comes from sugar cane, wine, malt, cider, and alcohol or from a chemical synthesis.^{[page needed]}

As of this date,^{[when?] 2}H-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 ex-bean can well be distinguished from the other sources (see next figure).

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

The naturality^{[clarification needed]} of different aroma 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.^{[full citation needed]}

The SNIF-NMR applied to benzaldehyde can detect adulterated 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).^{[full citation needed]} Other applications have also been published, including for raspberry ketone,^{[verification needed]} heliotropine, etc.

Optimization of technique parameters have enabled to reach better accuracy for the ¹³C NMR measurements.

The ¹³C-SNIF-NMR method is called method “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.

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.

• 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. Retrieved 20 March 2025.{{cite journal}}: CS1 maint: multiple names: authors list (link)

• 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.{{cite journal}}: CS1 maint: multiple names: authors list (link)

• Akoka, Serge; Remaud, Gérald (October–December 2020). "NMR-Based Isotopic and Isotopomic Analysis". Progress in Nuclear Magnetic Resonance Spectroscopy. 120–121: 1–24. Bibcode:2020PNMRS.120....1A. doi:10.1016/j.pnmrs.2020.07.001. PMID 33198965. Retrieved 11 February 2024.

• 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. PMC 7827514. PMID 33429968.{{cite journal}}: CS1 maint: multiple names: authors list (link)

• Nuclear magnetic resonance
• Food chemistry
• Food security