NMR-Based Method for Intramolecular 13C Distribution at Natural Abundance Adapted to Small Amounts of Glucose.

Quantitative nuclear magnetic resonance (NMR) for isotopic measurements, known as irm-NMR (isotope ratio measured by NMR), is well suited for the quantitation of 13C-isotopomers in position-specific isotope analysis and thus for measuring the carbon isotope composition (δ13C, mUr) in C-atom positions. Irm-NMR has already been used with glucose after derivatization to study sugar metabolism in plants. However, up to now, irm-NMR has exploited a "single-pulse" sequence and requires a relatively large amount of material and long experimental time, precluding many applications with biological tissues or extracts. To reduce the required amount of sample, we investigated the use of 2D-NMR analysis. We adapted and optimized the NMR sequence so as to be able to analyze a small amount (10 mg) of a glucose derivative (diacetonide glucofuranose, DAGF) with a precision better than 1 mUr at each C-atom position. We also set up a method to correct raw data and express 13C abundance on the usual δ13C scale (δ-scale). In fact, due to the distortion associated with polarization transfer and spin manipulation during 2D-NMR analyses, raw 13C abundance is found to be on an unusual scale. This was compensated for by a correction factor obtained via comparative analysis of a reference material (commercial DAGF) using both previous (single-pulse) and new (2D) sequences. Glucose from different biological origins (CO2 assimilation metabolisms of plants, namely, C3, C4, and CAM) was analyzed with the two sequences and compared. Validation criteria such as selectivity, limit of quantification, precision, trueness, and robustness are discussed, including in the framework of green analytical chemistry.

Site-specific carbon isotope measurements of vanillin reference materials by nuclear magnetic resonance spectrometry.

Vanillin, one of the world's most popular flavor used in food and pharmaceutical industries, is extracted from vanilla beans or obtained (bio)-synthetically. The price of natural vanillin is considerably higher than that of its synthetic alternative which leads increasingly to counterfeit vanillin. Here, we describe the workflow of combining carbon isotope ratio combustion mass spectrometry with quantitative carbon nuclear magnetic resonance spectrometry (13C-qNMR) to obtain carbon isotope measurements traceable to the Vienna Peedee Belemnite (VPDB) with 0.7‰ combined standard uncertainty (or expanded uncertainty of 1.4‰ at 95% confidence level). We perform these measurements on qualified Bruker 400 MHz instruments to certify site-specific carbon isotope delta values in two vanillin materials, VANA-1 and VANB-1, believed to be the first intramolecular isotopic certified reference material (CRMs).

A precise and rapid isotopomic analysis of small quantities of cholesterol at natural abundance by optimized 1H-13C 2D NMR.

Cholesterol, the principal zoosterol, is a key metabolite linked to several health complications. Studies have shown its potential as a metabolic biomarker for predicting various diseases and determining food origin. However, the existing INEPT (insensitive nuclei enhanced by polarization transfer) 13C position-specific isotope analysis method of cholesterol by NMR was not suitable for very precise analysis of small quantities due to its long acquisition time and therefore is restricted to products rich in cholesterol. In this work, a symmetric and adiabatic heteronuclear single quantum coherence (HSQC) 2D NMR sequence was developed for the high-precision (few permil) analysis of small quantities of cholesterol. Adiabatic pulses were incremented for improving precision and sensitivity. Moreover, several strategies such as the use of non-uniform sampling, linear prediction, and variable recycling time were optimized to reduce the acquisition time. The number of increments and spectral range were also adjusted. The method was developed on a system with a cryogenically cooled probe and was not tested on a room-temperature system. Our new approach allowed analyzing as low as 5 mg of cholesterol in 31 min with a long-term repeatability lower than 2‰ on the 24 non-quaternary carbon atoms of the molecule comparing to 16.2 h for the same quantity using the existing INEPT method. This result makes conceivable the isotope analysis of matrices low in cholesterol. Graphical abstract.

Position-specific 15 N isotope analysis in organic molecules: A high-precision 15 N NMR method to determine the intramolecular 15 N isotope composition and fractionation at natural abundance.

The position-specific 15 N isotope content in organic molecules, at natural abundance, is for the first time determined by using a quantitative methodology based on 15 N Nuclear Magnetic Resonance (NMR) spectrometry. 15 N NMR spectra are obtained by using an adiabatic "Full-Spectrum" INEPT sequence in order to make possible 15 N NMR experiments with a high signal-to-noise ratio (>500), to reach a precision with a standard deviation below 1‰ (0.1%). This level of precision is required for observing small changes in 15 N content associated to 15 N isotope effects. As an illustration, the measurement of an isotopic enrichment factor ε for each 15 N isotopomer is presented for 1-methylimidazole induced during a separation process on a silica column. The precision expressed as the long-term repeatability of the methodology is good enough to evaluate small changes in the 15 N isotope contents for a given isotopomer. As observed for 13 C, inverse and normal 15 N isotope effects occur concomitantly, giving access to new information on the origin of the 15 N isotope effects, not detectable by other techniques such as isotope ratio measured by Mass Spectrometry for which bulk (average) values are obtained.

The FAQUIRE Approach: FAst, QUantitative, hIghly Resolved and sEnsitivity Enhanced 1H, 13C Data.

The targeted analysis of metabolites in complex mixtures is a challenging issue. NMR is one of the major tools in this field, but there is a strong need for more sensitive, better-resolved, and faster quantitative methods. In this framework, we introduce the concept of FAst, QUantitative, hIghly Resolved and sEnsitivity enhanced (FAQUIRE) NMR to push forward the limits of metabolite NMR analysis. 2D 1H, 13C 2D quantitative maps are promising alternatives for enhancing the spectral resolution but are highly time-consuming because of (i) the intrinsic nature of 2D, (ii) the longer recycling times required for quantitative conditions, and (iii) the higher number of scans needed to reduce the level of detection/quantification to access low concentrated metabolites. To reach this aim, speeding up the recently developed QUantItative Perfected and pUre shifted HSQC (QUIPU HSQC) is an interesting attempt to develop the FAQUIRE concept. Thanks to the combination of spectral aliasing, nonuniform sampling, and variable repetition time, the acquisition time of 2D quantitative maps is reduced by a factor 6 to 9, while conserving a high spectral resolution thanks to a pure shift approach. The analytical potential of the new Quick QUIPU HSQC (Q QUIPU HSQC) is evaluated on a model metabolite sample, and its potential is shown on breast-cell extracts embedding metabolites at millimolar to submillimolar concentrations.

Full Spectrum Isotopic 13C NMR Using Polarization Transfer for Position-Specific Isotope Analysis.

For the last ten years, quantitative isotope ratio monitoring 13C NMR (irm-13C NMR) has been successfully tested and proven as an efficient tool for the determination of position-specific 13C/12C ratios. Several applications in different domains have shown the interest in this technique. In the context of origin assignment, the possibility to track the distribution network of illicit drugs or cutting agents is of prime importance. However irm-13C NMR still suffers from a relative lack of sensitivity limiting its dissemination among control laboratories. Improvements were proposed to reduce experiment time by using the INEPT sequence ("Insensitive Nuclei Enhanced by Polarization Transfer") based on polarization transfer from highly sensitive 1H to less sensitive 13C. Several applications based on the use of the one bond scalar coupling between 1H and 13C (1 JCH) have shown the potential of this methodology in terms of short experimental duration. However, the isotopic information given by quaternary carbons was lost. The aim of this study is to extend this approach by using short- and long-range coupling (1 JCH, 2 JCH, and 3 JCH) in order to have access to all 13C/12C position-specific ratios, i.e., acquisition of the full spectrum (FS-INEPT). It is shown that this innovative tool provides both sensitivity gain-thanks to the long-range polarization transfer-and appropriate repeatability. The relative isotopic profiles allowed the classification of two cutting agents, caffeine and paracetamol (acetaminophen), according to their origin, as it was previously observed with "classical" irm-13C NMR but consuming much less sample and/or reducing the experimental time.

A strategy for simultaneous determination of fatty acid composition, fatty acid position, and position-specific isotope contents in triacylglycerol matrices by 13C-NMR.

Triacylglycerols, which are quasi-universal components of food matrices, consist of complex mixtures of molecules. Their site-specific 13C content, their fatty acid profile, and their position on the glycerol moiety may significantly vary with the geographical, botanical, or animal origin of the sample. Such variables are valuable tracers for food authentication issues. The main objective of this work was to develop a new method based on a rapid and precise 13C-NMR spectroscopy (using a polarization transfer technique) coupled with multivariate linear regression analyses in order to quantify the whole set of individual fatty acids within triacylglycerols. In this respect, olive oil samples were analyzed by means of both adiabatic 13C-INEPT sequence and gas chromatography (GC). For each fatty acid within the studied matrix and for squalene as well, a multivariate prediction model was constructed using the deconvoluted peak areas of 13C-INEPT spectra as predictors, and the data obtained by GC as response variables. This 13C-NMR-based strategy, tested on olive oil, could serve as an alternative to the gas chromatographic quantification of individual fatty acids in other matrices, while providing additional compositional and isotopic information. Graphical abstract A strategy based on the multivariate linear regression of variables obtained by a rapid 13C-NMR technique was developed for the quantification of individual fatty acids within triacylglycerol matrices. The conceived strategy was tested on olive oil.

Gradient-based solvent suppression methods on a benchtop spectrometer.

Benchtop NMR emerges as an appealing alternative to widely extend the scope of NMR spectroscopy in harsh environments and for on-line monitoring. Obviously, the use of low-field magnets induces a dramatic reduction of the spectral resolution leading to frequent peak overlaps. This issue is even more serious because applications such as chemical process monitoring involve the use of non-deuterated solvents, leading to intense and broad peaks overlapping with the signals of interest. In this article, we highlight the need for efficient suppression methods compatible with flowing samples, which is not the case of the common pre-saturation approaches. Thanks to a gradient coil included in our benchtop spectrometer, we were able to implement modern and efficient solvent suppression blocks such as WET or excitation sculpting to deliver quantitative spectra in the conditions of the on-line monitoring. While these methods are commonly used at high field, this is the first time that they are investigated on a benchtop setting. Their analytical performance is evaluated and compared under static and on-flow conditions. The results demonstrate the superiority of gradient-based methods, thus highlighting the relevance of implementing this device on benchtop spectrometers. The comparison of major solvent suppression methods reveals an optimum performance for the WET-180-NOESY experiment, both under static and on-flow conditions. Copyright © 2016 John Wiley & Sons, Ltd.

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.

Nonstatistical 13C distribution during carbon transfer from glucose to ethanol during fermentation is determined by the catabolic pathway exploited.

During the anaerobic fermentation of glucose to ethanol, the three micro-organisms Saccharomyces cerevisiae, Zymomonas mobilis, and Leuconostoc mesenteroides exploit, respectively, the Embden-Meyerhof-Parnas, the Entner-Doudoroff, and the reductive pentose phosphate pathways. Thus, the atoms incorporated into ethanol do not have the same affiliation to the atomic positions in glucose. The isotopic fractionation occurring in each pathway at both the methylene and methyl positions of ethanol has been investigated by isotopic quantitative (13)C NMR spectrometry with the aim of observing whether an isotope redistribution characteristic of the enzymes active in each pathway can be measured. First, it is found that each pathway has a unique isotope redistribution signature. Second, for the methylene group, a significant apparent kinetic isotope effect is only found in the reductive pentose phosphate pathway. Third, the apparent kinetic isotope effects related to the methyl group are more pronounced than for the methylene group. These findings can (i) be related to known kinetic isotope effects of some of the enzymes concerned and (ii) give indicators as to which steps in the pathways are likely to be influencing the final isotopic composition in the ethanol.

Internal Referencing for ¹³C Position-Specific Isotope Analysis Measured by NMR Spectrometry.

The intramolecular (13)C composition of a molecule retains evidence relevant to its (bio)synthetic history and can provide valuable information in numerous fields ranging from biochemistry to environmental sciences. Isotope ratio monitoring by (13)C NMR spectrometry (irm-(13)C NMR) is a generic method that offers the potential to conduct (13)C position-specific isotope analysis with a precision better than 1‰. Until now, determining absolute values also required measurement of the global (or bulk) (13)C composition (δ(13)Cg) by mass spectrometry. In a radical new approach, it is shown that an internal isotopic chemical reference for irm-(13)C NMR can be used instead. The strategy uses (1)H NMR to quantify both the number of moles of the reference and of the studied compound present in the NMR tube. Thus, the sample preparation protocol is greatly simplified, bypassing the previous requirement for precise purity and mass determination. The key to accurate results is suppressing the effect of radiation damping in (1)H NMR which produces signal distortion and alters quantification. The methodology, applied to vanillin with dimethylsulfone as an internal standard, has an equivalent accuracy (<1‰) to that of the conventional approach. Hence, it was possible to clearly identify vanillin from different origins based on the (13)C isotopic profiles.

Understanding J-Modulation during Spatial Encoding for Sensitivity-Optimized Ultrafast NMR Spectroscopy.

Ultrafast (UF) NMR spectroscopy is an approach that yields 2D spectra in a single scan. This methodology has become a powerful analytical tool that is used in a large array of applications. However, UF NMR spectroscopy still suffers from an intrinsic low sensitivity, and from the need to compromise between sensitivity, spectral width, and resolution. In particular, the modulation of signal intensities by the spin-spin J-coupling interaction (J-modulation) impacts significantly on the intensities of the spectral peaks. This effect can lead to large sensitivity losses and even to missing spectral peaks, depending on the nature of the spin system. Herein, a general simulation package (Spinach) is used to describe J-modulation effects in UF experiments. The results from simulations match with experimental data and the results of product operator calculations. Several methods are proposed to optimize the sensitivity in UF COSY spectra. The potential and drawbacks of the different strategies are also discussed. These approaches provide a way to adjust the sensitivity of UF experiments for a large range of applications.

Real-time reaction monitoring by ultrafast 2D NMR on a benchtop spectrometer.

Reaction monitoring is widely used to follow chemical processes in a broad range of application fields. Recently, the development of robust benchtop NMR spectrometers has brought NMR under the fume hood, making it possible to monitor chemical reactions in a safe and accessible environment. However, these low-field NMR approaches suffer from limited resolution leading to strong peak overlaps, which can limit their application range. Here, we propose an approach capable of recording ultrafast 2D NMR spectra on a compact spectrometer and of following in real time reactions in the synthetic chemistry laboratory. This approach--whose potential is shown here on a Heck-Matsuda reaction--is highly versatile; the duration of the measurement can be optimized to follow reactions whose time scale ranges from between a few tens of seconds to a few hours. It makes it possible to monitor complex reactions in non-deuterated solvents, and to confirm in real time the molecular structure of the compounds involved in the reaction while giving access to relevant kinetic parameters.

Comparative study of ¹³C composition in ethanol and bulk dry wine using isotope ratio monitoring by mass spectrometry and by nuclear magnetic resonance as an indicator of vine water status.

The potential of wine (13)C isotope composition (δ(13)C) is presented to assess vine water status during grape ripening. Measurements of δ(13)C have been performed on a set of 32 authentic wines and their ethanol recovered after distillation. The data, obtained by isotope ratio monitoring by mass spectrometry coupled to an elemental analyser (irm-EA/MS), show a high correlation between δ(13)C of the bulk wine and its ethanol, indicating that the distillation step is not necessary when the wine has not been submitted to any oenological treatment. Therefore, the ethanol/wine δ(13)C correlation can be used as an indicator of possible enrichment of the grape must or the wine with exogenous organic compounds. Wine ethanol δ(13)C is correlated to predawn leaf water potential (R(2) = 0.69), indicating that this parameter can be used as an indicator of vine water status. Position-specific (13)C analysis (PSIA) of ethanol extracted from wine, performed by isotope ratio monitoring by nuclear magnetic resonance (irm-(13)C NMR), confirmed the non-homogenous repartition of (13)C on ethanol skeleton. It is the δ(13)C of the methylene group of ethanol, compared to the methyl moiety, which is the most correlated to predawn leaf water potential, indicating that a phase of photorespiration of the vine during water stress period is most probably occurring due to stomata closure. However, position-specific (13)C analysis by irm-(13)C NMR does not offer a greater precision in the assessment of vine water status compared to direct measurement of δ(13)C on bulk wine by irm-EA/MS.

Fast hybrid multi-dimensional NMR methods based on ultrafast 2D NMR.

Conventional multi-dimensional (nD) NMR experiments are characterized by inherent long acquisition durations, while ultrafast (UF) NMR makes it possible to reduce to a few hundreds of milliseconds the overall acquisition duration of a complete nD NMR dataset. Although extremely promising for a number of specific applications, the UF strategy suffers from significant limitations compared with its conventional counterpart. The main limitations concern the sensitivity, the resolution, and the accessible spectral width. However, when the targeted applications are compatible with an acquisition duration between a few seconds and a few minutes, hybrid UF techniques can be used to improve the performance of UF nD NMR while remaining faster than conventional acquisitions. Much better results in terms of signal-to-noise ratio can be achieved with the multi-scan single-shot approach or with interleaved acquisitions. Even more, for the same experimental duration, and in the case of homonuclear 2D NMR, the multi-scan single-shot approach has a much higher precision than conventional 2D NMR. Interleaved 2D NMR overcomes the drawbacks of single-scan UF NMR in terms of spectral width and provides spectra for which the quality is not significantly different from that obtained with conventional 2D NMR. Finally, high spectral qualities have been demonstrated from hybrid conventional/UF 3D approaches capable of recording a whole 3D spectrum in the time needed to record a conventional 2D spectrum. This mini-review aims at describing the principles, the recent advances and the latest applications of these hybrid techniques. Copyright © 2015 John Wiley & Sons, Ltd.

MISSTEC-S: A fast 1H pulse calibration from spectra simultaneously produced by a spin echo and a stimulated echo.

Radio-Frequency (RF) pulse calibration is an essential step in guaranteeing both optimum acquisition quality in multi-pulse NMR and accurate results in quantitative experiments. Most existing methods are based on a series of spectra for which the flip angle of one or more pulses is progressively incremented, implying a significant experiment time. In order to circumvent this drawback, we have previously proposed an approach based on the acquisition of a spin echo and a stimulated echo - the MISSTEC sequence - which requires only 8 s to determine the PW90-1H, while it is several minutes in the case of the use of a nutation curve. In this work, a new sequence for RF calibration is presented: MISSTEC-S. It is derived from the previously proposed MISSTEC sequence, but the observation of echoes in presence of magnetic field gradient is replaced by the observation of FIDs. This modification allows both spectra to be phased, while imposing a strong constraint on the Mixing Time (TM). However, the relationship used to calculate the flip angle is only correct when TM is small enough to neglect longitudinal relaxation during this delay. In order to reduce TM, the first FID is truncated during acquisition and subsequently lengthened using points from the second FID. Results obtained with MISSTEC-S were compared to those obtained from a complete nutation curve and an excellent correlation was observed, although the experimental time to obtain the PW90 is dramatically reduced.

Recovery time reduction to decrease experimental duration (R2D2): A simple and universal method to accelerate NMR experiments.

Acceleration techniques for one dimensional Nuclear Magnetic Resonance (1D NMR) are very useful, both for NMR enthusiasts and for chemists that use NMR for structural elucidation. To the latter, such techniques need to be straightforward. Recovery time Reduction to Decrease the experimental Duration (R2D2) relies on the incremental reduction of a pulse sequence's Recycle Time (TR). A pseudo-2D spectrum is acquired and after two Fourier transform, extraction and addition of the central rows, a 1D spectrum is obtained. Not only can it be applied to any pulse sequence that contains a TR, but it also requires only a list of recovery times and 2D processes to operate. With this method, we were able to easily reduce the experimental time by a factor of 2 and up to 4 using single-pulse, APT and DEPT 13C sequences. Moreover, R2D2 has the potential to be used on other low abundance nuclei (such as 15N or 2H) and numerous other pulse sequences.

Exploring the complementarity of fast multipulse and multidimensional NMR methods for metabolomics: a chemical ecology case study.

This study investigates the potential and complementarity of high-throughput multipulse and multidimensional NMR methods for metabolomics. Through a chemical ecology case study, three methods are investigated, offering a continuum of methods with complementary features in terms of resolution, sensitivity and experiment time. Ultrafast 2D COSY, adiabatic INEPT and SYMAPS HSQC are shown to provide a very good classification ability, comparable to the reference 1D 1H NMR method. Moreover, a detailed analysis of discriminant buckets upon supervised statistical analysis shows that all methods are highly complementary, since they are able to highlight discriminant signals that could not be detected by 1D 1H NMR. In particular, fast 2D methods appear very efficient to discriminate signals located in highly crowded regions of the 1H spectrum. Overall, the combination of these recent methods within a single NMR metabolomics workflow allows to maximize the accessible metabolic information, and also raises exciting challenges in terms of NMR data analysis for chemical ecology.

Fast quantitative 1H-13C two-dimensional NMR with very high precision.

Quantitative analysis by nuclear magnetic resonance (NMR) requires highly precise measurements to achieve reliable quantification. It is particularly true in (13)C site-specific natural isotope fractionation studied by nuclear magnetic resonance, where the range of values of (13)C isotopic deviations at natural abundance is highly restricted. Consequently, an NMR method capable of measuring δ(13)C ‰ values with a very high precision (a few per mil) is indispensable. This high degree of precision has already been achieved by one-dimensional (13)C acquisitions; however, this approach is limited by peak overlaps which reduce the precision of the isotope content determination, even for certain small molecules. It is therefore necessary to extend this promising methodology to a higher dimensionality. In this context, this paper aims at determining conditions that allow the achievement of two-dimensional (2D) (1)H-(13)C heteronuclear experiments with a precision of a few per mil in a reasonable time. Our results demonstrate that a high precision (repeatability of 2 per mil) can be reached with the (1)H-(13)C HSQC (Heteronuclear Single Quantum Correlation) experiment, thus satisfying the conditions needed to perform (13)C isotope analysis by 2D NMR. We also consider the impact of several approaches which have been proposed to reduce the duration of heteronuclear 2D experiments. Two of these common time-saving strategies, spectral aliasing and linear prediction, are fully compatible with the high-precision requirements of isotopic NMR, while a third one, nonuniform sampling, leads to dramatic precision losses. In conclusion, this study demonstrates the feasibility of very precise 2D NMR measurements and opens a number of application perspectives.

Fast spatially encoded 3D NMR strategies for (13)C-based metabolic flux analysis.

The measurement of site-specific (13)C enrichments in complex mixtures of (13)C-labeled metabolites is a powerful tool for metabolic flux analysis. One of the main methods to measure such enrichments is homonuclear (1)H 2D NMR. However, the major limitation of this technique is the acquisition time, which can amount to a few hours. This drawback was recently overcome by the design of fast COSY experiments for measuring specific (13)C-enrichments, based on single-scan 2D NMR. However, these experiments are still limited by overlaps because of(1)H-(13)C splittings, thus limiting the metabolic information accessible for complex biological mixtures. To circumvent this limitation, we propose to tilt the (1)H-(13)C coupling into a third dimension via fast-hybrid 3D NMR methods combining the speed of ultrafast 2D NMR with the high resolution of conventional methods. Two strategies are described that allow the acquisition of a complete 3D J-resolved-COSY spectrum in 12 min (for concentrations as low as 10 mM). The analytical potentialities of both methods are evaluated on a series of (13)C-enriched glucose samples and on a biomass hydrolyzate obtained from Escherichia coli cells. Once optimized, the two complementary experiments lead to a trueness and a precision of a few percent and an excellent linearity. The advantages and drawbacks of these approaches are discussed and their potentialities are highlighted.