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.

A mixture of closely related streptomyces fermentation products, antimycin A, is separated, and the components are identified by using reversed-phase high-performance liquid chromatography with directly linked 400-MHz proton nuclear magnetic resonance detection. Analyses of mixtures of three amino acids, alanine, glycine, and valine, are used to determine optimal measurement conditions. Sensitivity increases of as much as a factor of 3 are achieved, at the expense of some loss in chromatographic resolution, by use of an 80-microL NMR cell, instead of a smaller 14-microL cell. Analysis of the antimycin A mixture, using the optimal analytical high-performance liquid chromatography/nuclear magnetic resonance conditions, reveals it to consist of at least 10 closely related components.

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