Isotopic analysis by nuclear magnetic resonance


Isotopic analysis by nuclear magnetic resonance allows the user to quantify with great precision the differences of isotopic contents on each site of a molecule and thus to measure the specific natural isotope fractionation for each site of this molecule. The SNIF-NMR analytical method was developed to detect the sugaring of wine and enrichment of grape musts, and is mainly used to check the authenticity of foodstuffs and to control the naturality of some aromatic molecules. The SNIF-NMR method has been adopted by the International Organisation of Vine and Wine and the European Union as an official method for wine analysis. It is also an official method adopted by the Association Of Analytical Chemists for analysis of fruit juices, maple syrup, vanillin, and by the European Committee for Standardization for vinegar.

Background

The OIV adopts it as an official method
→ Implementation of the SNIF-NMR method for official laboratories in Europe
→ Implementation of the SNIF-NMR< method for official laboratories in US
→ Implementation of the SNIF-NMR method for official laboratories in Asia

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 2 below.
The amount and distribution of the different isotopes in a molecule is influenced by:
This phenomenon is known as natural isotopic fractionation. The resulting isotopic fingerprint can provide information on the origin - botanical, synthetic, geographical - of the molecule or product.

General principle

The Principle of the SNIF-NMR is built on: “The Natural Isotopic Fractionation”. Routinely for food authenticity, two nuclei are used:
The SNIF-NMR is applied on pure molecules. Therefore, some preparation steps may be required in the lab before analysis.
For example, for the SNIF-NMR of ethanol, according to official methods:
At each step of the SNIF-NMR analysis, efforts should be 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 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 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.

Examples of applications of 2H-SNIF-NMR

Application for 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.

NMR Spectrum (example of the 2H-SNIF-NMR)

Ethanol molecules obtained after complete fermentation of the sugar coexists with 3 naturally monodeuterated isotopomers. Their presence can then be quantified with relative precision.
On the following 2H-NMR spectrum, a peak corresponds 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 following Figure 9 summarizes the principle of interpretation of:
Values obtained on a test sample are then compared with the values of authentic samples.

Application for authenticity of wines

SNIF-NMR is the official method of the OIV to determine the authentication of wine origin. It 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 wine can be checked.

Application for vinegar and acetic acid

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.

Application for vanillin

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. The vanillin molecule is represented in figure 11, all observable sites for which the site specific deuterium concentrations can be measured are referenced with a number.
As for the wine or the fruit, the interpretation of results in terms of origin is done by comparison of the isotopic parameters of the sample analyzed with those from a group of referenced molecules of known origin.
It appears that all the origins of vanillin are well discriminated using 2H-NMR data. Particularly, vanillin ex-bean can well be distinguished from the other sources, as we can see in figure 12 below.
Additionally, this method is the only one to discriminate between natural and biosynthetic sources of vanillin.

Application for other aromas

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: raspberry ketone), heliotropine,…

13C-SNIF-NMR

As described in the work of E. Tenailleau and S. Akoka, an optimization of the technique parameters have enabled to reach a 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.
This method will certainly have further applications in future, in the field of food and beverage analysis authenticity.