Determination of Site-Specific (Deuterium/Hydrogen) Ratios in Vanillin by 2H-NuclearMagnetic Resonance Spectrometry: Collaborative Study

Abstract

The results of collaborative study are reported for a method that determines the site-specific isotope ratios of deuterium/hydrogen (D/H)i in vanillin by deuterium-nuclear magnetic resonance (2H-NMR) spectrometry. This method allows characterization of all the main commercial sources of commercial vanillin and detection of undeclared mixtures. It is based on the fact that the amounts of deuterium at various positions in the vanillin molecule are significantly different from one source to another. Vanillin is dissolved in acetonitrile and analyzed with a high-field NMR spectrometer fitted with a deuterium probe and a fluorine lock. The proportions of isotopomers monodeuterated at each hydrogen position of the molecule are recorded, and the corresponding (D/H) ratios are determined by using a calibrated reference. Nine laboratories analyzed 5 materials supplied as blind duplicates (1 natural vanillin from vanilla beans, 2 synthetic vanillins from guaiacol, 1 semisynthetic vanillin from lignin, and a mixture of natural and synthetic vanillins). The precision of the method for measuring site-specific ratios was as follows: for (D/H)1 the within-laboratory standard deviation (sr) values ranged from 2.2 to 5.8 ppm, and the among-laboratories standard deviation (sR) values ranged from 3.6 to 5.1 ppm; for (D/H)3 the sr values ranged from 1.7 to 3.2 ppm, and the sR values ranged from 2.4 to 3.7 ppm; for (D/H)4 the sr values ranged from 2.3 to 6.2 ppm, and the sR values ranged from 2.4 to 6.4 ppm; for (D/H)5 the sr values ranged from 0.8 to 2.7 ppm, and the sR values ranged from 0.9 to 2.3 ppm. It was shown that these values allow a satisfactory discrimination between vanillin sources. Therefore, the Study Director recommends the method for adoption as a First Action Official Method by AOAC INTERNATIONAL.

Combination of 13C and 2H SNIF‐NMR isotopic fingerprints of vanillin to control its precursors

“…13 C and 2 H NMR data were processed using the automatic software Eurospec developed by Eurofins for SNIF-NMR spectra, 40 following the steps described during the inter-laboratory study for 13 C spectra and the AOAC method for 2 H spectra. 36,38 Steps for the spectral treatment were as follows: an exponential multiplication inducing a line broadening of 2 Hz, automatic phasing, and baseline correction using an adequate polynomial order.…”

“…Indeed, this routine method was developed based on the addition of an internal reference with a known (D/H) value as described in the official method. 38 Results obtained by 13 C SNIF-NMR were expressed as isotopic composition by site in ‰ (δ 13 C i ), as described during the interlaboratory study. 36 The calculation did not use an internal reference, but deduced from the total 13 C/ 12 C ratio measured by EA-IRMS as previously described.…”

A review of flavors authentication by position‐specific isotope analysis by nuclear magnetic resonance spectrometry: the example of vanillin

“…Irm‐ 2 H NMR spectrometry (often designed as the SNIF‐NMR TM experiment: Specific Natural Isotope Fractionation‐Nuclear Magnetic Resonance) is recognized as a powerful technique for verifying the origin of commercial products . However, certain limitations of the method were highlighted: (i) a long measurement time; even for a relatively simple molecular structure, obtaining a sufficiently resolved 2 H‐NMR spectrum required several hours of spectrometer time and (ii) large molecules could not be studied by 2 H‐NMR because of the complexity of the NMR spectrum.…”

Authentication of the origin of sucrose-based sugar products using quantitative natural abundance13C NMR

“…Nuclear magnetic resonance (NMR) spectroscopy serves as an alternative for the determination of isotope ratios at each position of a given molecule. The direct assessment of site‐specific isotope ratios of 2 H/ 1 H at natural abundance by IR‐NMR spectroscopy has become a well‐established analytical approach, with applications for food products, drugs and forensics . However, like IR‐MS, this approach often requires elaborate work‐up procedures …”