Isotopic NMR spectrometry as an efficient tool to fight against counterfeiting: Recent developments

242nd National Meeting of the American-Chemical-Society (ACS), Denver, CO, AUG 28-SEP 01, 201

The new face of isotopic NMR at natural abundance

The most widely used method for isotope analysis at natural abundance is isotope ratio monitoring by Mass Spectrometry (irm-MS) which provides bulk isotopic composition in H-2, C-13,N-15, O-18 or S-34. However, in the 1980s, the direct access to Site-specific Natural Isotope Fractionation by Nuclear Magnetic Resonance (SNIF-NMR (TM)) was immediately recognized as a powerful technique to authenticate the origin of natural or synthetic products. The initial - and still most popular - application consisted in detecting the chaptalization of wines by irm-H-2 NMR. The approach has been extended to a wide range ofmethodologies over the last decade, paving the way to a wide range of applications, not only in the field of authentication but also to study metabolism. In particular, the emerging irm-C-13 NMR approach delivers direct access to position-specific C-13 isotope content at natural abundance. After highlighting the application scope of irm-NMR (H-2 and C-13), this article describes the major improvements which made possible to reach the required accuracy of 1 parts per thousand (0.1%) in irm-C-13 NMR. The last part of the manuscript summarizes the different steps to perform isotope analysis as a function of the sample properties (concentration, peak overlap) and the kind of targeted isotopic information (authentication, affiliation). Copyright (C) 2016 John Wiley & Sons, Ltd

Position-Specific 13C Fractionation during Liquid−Vapor Transition Correlated to the Strength of Intermolecular Interaction in the Liquid Phase

International audienceThe relationship between the strength of the intermolecular interaction in liquid and the position-specific 13C fractionation observed during distillation was investigated. A range of molecules showing different intermolecular interactions in terms of mode and intensity were incorporated in the study. Although it had previously been suggested that during evaporation the diffusive 13C isotope effect in the thin liquid layer interfaced with vapor is not position-specific, herein we show that this is not the case. In particular, the position-specific effect was demonstrated for a series of alcohols. Our hypothesis is that intermolecular interactions in the liquid phase are the source of position-specific 13C fractionation observed on the molecule. A clear trend is observed between the 13C isotope effect of the carbon bearing the heteroatom of chemical function and the relative permittivity, the solvent hydrogen-bond acidity and the solvent hydrogen-bond basicity, while only a weak trend was observed when using the 13C content of the whole molecule. Furthermore two families of products appeared when using the hydrogen-bond acidity parameter for the correlation by distinguishing H-acceptors and H-donors molecules to those H-acceptors only. This strongly reinforces the hypothesis of an important role of the 13C positioned close to the interaction center

Biochemical and physiological determinants of intramolecular isotope patterns in sucrose from C-3, C-4 and CAM plants accessed by isotopic C-13 NMR spectrometry: a viewpoint

This paper discusses the biochemical and physiological factors underlying the site-specific, non-random distribution of C-13/C-12 isotope ratios within plant metabolites, which can be determined by isotopic C-13 NMR spectrometry. It focuses on the key metabolite glucose and on enzyme activities and physiological processes that are responsible for the carbon isotope patterns in glucose from different biological origins. It further considers how intramolecular C-13/C-12 isotope ratios in glucose can be exploited to understand fundamental aspects of plant biological chemistry, how these are related to environmental parameters and how these influence metabolites beyond central sugar metabolism. It does not purport to be an extensive overview of intramolecular isotopic patterns. Rather, the aim is to show how a full understanding of C-12/C-13 fractionations occurring during plant metabolism can only be possible once the factors that define intramolecular isotope values are better delineated

Insights into Mechanistic Models for Evaporation of Organic Liquids in the Environment Obtained by Position-Specific Carbon Isotope Analysis

International audiencePosition-specific isotope effects (PSIEs) have been measured by isotope ratio monitoring C-13 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

Position-Specific Isotope Analysis by Isotopic NMR Spectrometry: New Insights on Environmental Pollution Studies

11th International Symposium on Applied Isotope Geochemistry (AIG), French Geol Survey, Orleans, FRANCE, SEP 21-25, 2015International audienceThe common practice of measuring the change in isotope ratio for the whole molecule using isotope ratio measurement by mass spectrometry leads to the loss of significant information of potential interest, since it is the position-specific fractionation which most closely reflects the effect of physicochemical processes. We have shown for MTBE, a common ground water contaminant, that isotopic quantitative C-13 NMR spectrometry can be effectively applied to obtain this position-specific data. It is found that different characteristic position-specific isotope fractionation patterns are introduced by different types of evaporative process or by oxidative degradation. (C) 2015 Published by Elsevier B.V

Enhanced forensic discrimination of pollutants by position-specific isotope analysis using isotope ratio monitoring by C-13 nuclear magnetic resonance spectrometry

International audienceIn forensic environmental investigations the main issue concerns the inference of the original source of the pollutant for determining the liable party. Isotope measurements in geochemistry, combined with complimentary techniques for contaminant identification, have contributed significantly to source determination at polluted sites. In this work we have determined the intramolecular C-13 profiles of several molecules well-known as pollutants. By giving additional analytical parameters, position-specific isotope analysis performed by isotope ratio monitoring by C-13 nuclear magnetic resonance (irm-C-13 NMR) spectrometry gives new information to help in answering the major question: what is the origin of the detected contaminant? We have shown that isotope profiling of the core of a molecule reveals both the raw materials and the process used in its manufacture. It also can reveal processes occurring between the contamination site `source' and the sampling site. Thus, irm-C-13 NMR is shown to be a very good complement to compound-specific isotope analysis currently performed by mass spectrometry for assessing polluted sites involving substantial spills of pollutant. (C) 2015 Elsevier B.V. All rights reserved

Insights into the role of methionine synthase in the universal C-13 depletion in O- and N-methyl groups of natural products

International audienceMany O-methyl and N-methyl groups in natural products are depleted in C-13 relative to the rest of the molecule. These methyl groups are derived from the C-1 tetrahydrofolate pool via L-methionine, the principle donor of methyl units. Depletion could occur at a number of steps in the pathway. We have tested the hypothesis that methionine biosynthesis is implicated in this depletion by using a combined experimental and theoretical approach. By using isotope ratio monitoring C-13 NMR spectrometry to measure the position-specific distribution of C-13 within L-methionine of natural origin, it is shown that the S-methyl group is depleted in C-13 by similar to 20%o relative to the other positions in the molecule. In parallel, we have conducted a basic theoretical analysis of the reaction pathway of methionine synthase to assess whether the enzyme cobalamin-independent L-methionine synthase (EC 2.1.1.14) that catalyzes the synthesis of L-methionine from 5-methyl-tetrahydrofolate and homocysteine plays a role in causing this depletion. Calculation predicts a strong normal C-13 kinetic isotope effect (1.087) associated with this enzyme. Hence, depletion in C-13 in the S-methyl of L-methionine during biosynthesis can be identified as an important factor contributing to the general depletion seen in many O methyl and N-methyl groups of natural products

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

Synthetic vanillin is the flavouring most used in agro-food industries, but more and more frequently consumers are now turning to natural origin. Vanilla planifolia (Andrews) is the most commercialized species of pods in the world and is mainly produced in Madagascar. However, periodically the production of V. planifolia suffers from poor climatic conditions, with the resulting degradation of both the quality and the quantity of the pods. Consequently, the price of vanilla pods rose to record in 2017 and the risk of fraud has increased in response. Analytical methods based on isotopic compositions have already proven their ability to ensure the authenticity of natural vanillin. In 2006, H-2 SNIF-NMR (site-specific natural isotopic fractionation by nuclear magnetic resonance) was approved as official method by the Association of Official Analytical Chemists (AOAC 2006.05). However, this method is time consuming and the extraction of 1 g is cumbersome for the finished products. The information brought by combined H-2 and C-13 SNIF-NMR profiles was compared using chemometric tools to determine the best routine tool to improve both the global analysis time and the potential of detection. As a result of this work the ability of the SNIF-NMR method to verify the authenticity of vanillin has been improved, in particular by providing the first means to discern the geographical origin of vanilla pods. Furthermore, C-13 NMR using pulse sequences such as INEPT (insensitive nuclei enhanced by polarization transfer) offers the possibility to improve the sensitivity of the analysis, with a reduced quantity of product (less than 50 mg) and a shorter analysis time, which will facilitate the study of the finished matrices as well as clearly discriminating the origins of the vanilla flavourings