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 2 H, 13 C, 15 N, 18 O or 34 S. However, in the 1980s, the direct access to Site-specific Natural Isotope Fractionation by Nuclear Magnetic Resonance (SNIF-NMRTM ) 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-2 H NMR. The approach has been extended to a wide range of methodologies 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-13 C NMR approach delivers direct access to position-specific 13 C isotope content at natural abundance. After highlighting the application scope of irm-NMR (2 H and 13 C), this article describes the major improvements which made possible to reach the required accuracy of 1‰ (0.1%) in irm-13 C 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 © 2016 John Wiley & Sons, Ltd.

Robust 1D NMR lineshape fitting using real and imaginary data in the frequency domain.

Quantitative NMR is intrinsically dependent on precise, accurate, and robust peak area calculation. In this work, we demonstrate how the use of complex-valued peak descriptions can improve peak fitting in the frequency domain - incorporating phase and baseline correction as well as apodization while working with commonly used Fourier-transformed data. The method has been implemented in an open source R package called rnmrfit that is available for download on GitHub (https://github.com/ssokolen/rnmrfit). Application to real data suggests that this approach can also result in dramatically higher precision than can be achieved with existing software. Simulation data indicates that coefficients of variation below 0.1% can be readily achieved at signal to noise (SNR) ratios of approximately 100. The use of complex-valued data in the frequency domain is demonstrated as a relatively simple and effective means of improving peak fitting for quantitative NMR analysis.

Optimized slice-selective 1H NMR experiments combined with highly accurate quantitative 13C NMR using an internal reference method.

Isotope ratio monitoring by 13C NMR spectrometry (irm-13C NMR) provides the complete 13C intramolecular position-specific composition at natural abundance. It represents a powerful tool to track the (bio)chemical pathway which has led to the synthesis of targeted molecules, since it allows Position-specific Isotope Analysis (PSIA). Due to the very small composition range (which represents the range of variation of the isotopic composition of a given nuclei) of 13C natural abundance values (50‰), irm-13C NMR requires a 1‰ accuracy and thus highly quantitative analysis by 13C NMR. Until now, the conventional strategy to determine the position-specific abundance xi relies on the combination of irm-MS (isotopic ratio monitoring Mass Spectrometry) and 13C quantitative NMR. However this approach presents a serious drawback since it relies on two different techniques and requires to measure separately the signal of all the carbons of the analyzed compound, which is not always possible. To circumvent this constraint, we recently proposed a new methodology to perform 13C isotopic analysis using an internal reference method and relying on NMR only. The method combines a highly quantitative 1H NMR pulse sequence (named DWET) with a 13C isotopic NMR measurement. However, the recently published DWET sequence is unsuited for samples with short T1, which forms a serious limitation for irm-13C NMR experiments where a relaxing agent is added. In this context, we suggest two variants of the DWET called Multi-WET and Profiled-WET, developed and optimized to reach the same accuracy of 1‰ with a better immunity towards T1 variations. Their performance is evaluated on the determination of the 13C isotopic profile of vanillin. Both pulse sequences show a 1‰ accuracy with an increased robustness to pulse miscalibrations compared to the initial DWET method. This constitutes a major advance in the context of irm-13C NMR since it is now possible to perform isotopic analysis with high relaxing agent concentrations, leading to a strong reduction of the overall experiment time.