This paper describes recent progress in the isotopic analysis of the two main aromatic constituents of vanilla flavor (vanillin and p-hydroxybenzaldehyde (pHB)). Some improvements concerning the SNIF-NMR analysis of vanillin are presented. They include (i) improvement of the analytical precision by using new software for automatic phasing, baseline correction and curve fitting of the signals of the 2H-NMR spectra; (ii) significant enrichment of the database containing measurements performed on vanillin extracted from vanilla beans harvested in different countries and on synthetic vanillin; (iii) standardization of the purification process to obtain pure vanillin, in order to avoid isotopic fractionation; and (iv) improvement of the statistical tools for both proving and quantifying adulterations. All these improvements were also successfully applied to site specific deuterium NMR analysis of pHB. It is now also possible with the SNIF-NMR method to discriminate between the natural and chemical origins of pHB. New results concerning the δ13C deviation of pHB are also presented. Thus the lowest value of δ13C of pHB extracted from vanilla bean can be set at −19.5‰. We recommend the methodology presented in this paper as a standard procedure for purifying vanillin and pHB, without significant isotopic fractionation, from most matrices (for example from ice cream, yogurts, etc.), in order to perform isotopic analyses (13C IRMS and 2H-NMR). Keywords: SNIF-NMR; vanillin; pHB; δ13C; adulteration; vanilla flavor; vanilla extract; ice cream; purification

The stability over time (repeatability) for the determination of site-specific 13C/12C ratios at natural abundance by quantitative 13C NMR spectroscopy has been tested on three probes: enriched bilabeled [1,2-13C2]ethanol; ethanol at natural abundance; and vanillin at natural abundance. It is shown in all three cases that the standard deviation for a series of measurements taken every 2-3 months over periods between 9 and 13 months is equal to or smaller than the standard deviation calculated from 5-10 replicate measurements made on a single sample. The precision which can be achieved using the present analytical 13C NMR protocol is higher than the prerequisite value of 1-2 per thousand for the determination of site-specific 13C/12C ratios at natural abundance (13C-SNIF-NMR). Hence, this technique permits the discrimination of very small variations in 13C/12C ratios between carbon positions, as found in biogenic natural products. This observed stability over time in 13C NMR spectroscopy indicates that further improvements in precision will depend primarily on improved signal-to-noise ratio.

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Position-specific isotope effects (PSIEs) have been measured by isotope ratio monitoring (13)C nuclear magnetic resonance spectrometry during the evaporation of 10 liquids of different polarities under 4 evaporation modes (passive evaporation, air-vented evaporation, low pressure evaporation, distillation). The observed effects are used to assess the validity of the Craig-Gordon isotope model for organic liquids. For seven liquids the overall isotope effect (IE) includes a vapor-liquid contribution that is strongly position-specific in polar compounds but less so in apolar compounds and a diffusive IE that is not position-specific, except in the alcohols, ethanol and propan-1-ol. The diffusive IE is diminished under forced evaporation. The position-specific isotope pattern created by liquid-vapor IEs is manifest in five liquids, which have an air-side limitation for volatilization. For the alcohols, undefined processes in the liquid phase create additional PSIEs. Three other liquids with limitations on the liquid side have a lower, highly position-specific, bulk diffusive IE. It is concluded that evaporation of organic pollutants creates unique position-specific isotope patterns that may be used to assess the progress of remediation or natural attenuation of pollution and that the Craig-Gordon isotope model is valid for the volatilization of nonpolar organic liquids with air-side limitation of the volatilization rate.

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The stable carbon isotope 13 C is used as a universal tracer in plant eco-physiology and studies of carbon exchange between vegetation and atmosphere. Photosynthesis fractionates against 13 CO 2 so that source sugars (photosynthates) are on average 13 C depleted by 20‰ compared with atmospheric CO 2 . The carbon isotope distribution within sugars has been shown to be heterogeneous, with relatively C abundance in their specific precursor C-atom positions. However, the intramolecular isotope pattern in source leaf glucose and the isotope fractionation associated with key enzymes involved in sugar interconversions are currently unknown. To gain insight into these, we have analyzed the intramolecular isotope composition in source leaf transient starch, grain storage starch, and root storage sucrose and measured the site-specific isotope fractionation associated with the invertase (EC 3.2.1.26) and glucose isomerase (EC 5.3.1.5) reactions. When these data are integrated into a simple steady-state model of plant isotopic fluxes, the enzyme-dependent fractionations satisfactorily predict the observed intramolecular patterns. These results demonstrate that glucose and sucrose metabolism is the primary determinant of the 13 C abundance in source and sink tissue and is, therefore, of fundamental importance to the interpretation of plant isotopic signals.

In order to understand (13)C isotope distributions in glucose and its metabolites, it is necessary to measure the internal (13)C distribution at natural abundance. These data, however, are not directly accessible, even by quantitative isotopic (13)C NMR spectrometry, due to anomerization at the C-1 position. A strategy has been developed that overcomes this difficulty by converting glucose via a three-step synthesis into 3,5,6-triacetyl-1,2-O-isopropylidene-alpha-D-glucofuranose (TAMAGF). This compound provides a satisfactory molecular probe to measure the site-specific (13)C/(12)C ratios in glucose by (13)C NMR. It is shown that the isotopic (13)C NMR signal gives sufficient precision (repeatability standard deviation < or = 0.8 per thousand) for routine use for the determination of the (13)C abundance of each carbon atom position in glucose. Thus, it can be seen that the internal (13)C distribution of glucose biosynthesized by the C3 and C4 metabolic pathways differs markedly. Furthermore, the method is suitable for determining the isotope ratio in the glucose moiety of sucrose and, possibly, in free fructose and the fructose moiety of sucrose.

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