Isotopic analysis by nuclear magnetic resonance


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 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 to control the naturality 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 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 for analysis of vinegar.

History

History of SNIF-NMR

Discovery

  • 1981: Invention, as RMN-FINS, the acronym for fractionnement isotopique naturel spécifique par résonance magnétique nucléaire, by Gerard Martin and Maryvonne Martin and their team at the University of Nantes/CNRS.
  • 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.

Organisational recognition

Principle

Isotopic distribution

]
]
The atoms hydrogen, oxygen, and carbon co-exist naturally in specific proportions with their stable isotopes, 2H, 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 conditions, and
  • Chemical or biochemical processes, primary metabolism, photosynthetic metabolism in plants, etc.
A phenomenon known as natural isotopic fractionation 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.

Principles underlying specific methods

SNIF-NMR

SNIF-NMR is built on the principle of fractionation of carbon isotopes in oxygenic photosynthesis (isotope 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 actual abundance ratios of 13C at each C site in a molecule.
    Steps of the method
]
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:
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 are performed on each sample.
Advantages of the method
]
The isotopic ratios of a molecule can also be determined by isotope ratio mass spectrometry, 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 online 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 of sugars or ethanol, and the official CEN and OIV method for the 18O/16O in water.
The SNIF-NMR method 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, the three ratios CH3, CH2 and can be obtained.
An example 2H-SNIF-NMR Spectrum
]
Ethanol molecules obtained after complete fermentation of the sugar coexists with 3 naturally monodeuterated isotopomers. 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 CH3 and CH2 are calculated by comparison with an Internal standard, tetramethylurea, with a certified value.
Interpretation of SNIF-NMR isotopic values
]
The figure summarizes the principles of interpretation applied:
  • Results measured by IRMS, which enable discrimination of plants according to their CO2 photosynthetic metabolism ;
  • Results measured by SNIF-NMR that can differentiate the botanical origin of sugars within the same metabolic group.
Values obtained on a test sample are then compared with the values of authentic, databased 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 to detect addition of C3 sugar.
Authenticity of wines
]
SNIF-NMR is the official method of the OIV to determine the authentication of wine origin, and as of this date, is the only method to detect C3 sugar addition.
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 concerning wines of all European members. The database contains several thousand entries for European wines, 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 the wine, can be checked.
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 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.
Vanillin
As of this date, 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 ex-bean can well be distinguished from the other sources.
Additionally, this method is the only one to discriminate between natural and biosynthetic sources of vanillin.
Other odorants
The naturality 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.
Other applications
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, natural and semisynthetic. Other applications have also been published, including for raspberry ketone, heliotropine, etc.

13C-SNIF-NMR

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 and some crassulacean acid metabolism plants like pineapple or agave.
This method can also be applied to tequila products, where it can differentiate authentic 100% agave tequila, misto tequila, and products made from a larger proportion of cane or maize sugar and therefore not complying with the legal definition of tequila.