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.

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.

Biomarkers for cheese authentication by detailed and fast gas chromatographic profiling of triacylglycerol fatty acids.

Unsaturated fatty acid isomers and odd- and branched-chain fatty acids (OBCFAs) in milk triacylglycerols (TAGs) can be quantitated using gas chromatography (GC), providing access to biomarkers of animal species, breeds, diet, geographic origin, and environmental conditions. Such analysis requires expensive cyanopropyl siloxane or ionic liquid columns of at least 50 m in length, which increases the elution time. Aiming to use GC for cheese authentication and characterization while keeping the experiment time short and maintaining a good separation between fatty acid (FA) isomers, we considered using a 30 m polyethylene glycol-2-nitroterephthalate column. The FAs thus quantitated allowed the discovery of specific biomarkers for the origins of cheese varieties highly consumed in several countries. In addition, the simple and multivariate correlations we found between FAs in the cheese TAG matrix were alternative means for characterization and authentication purposes.

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).

Radio-frequency pulse calibration using the MISSTEC sequence.

NMR sequences are composed of multiple radio-frequency pulses. Probe adjustment, sample concentration and solvent influence the loading factor, therefore these parameters also impact the validity of flip angles. The commonly used method to calibrate RF pulses is to measure a nutation curve by varying the pulse duration. However, this method is impacted by off-resonance effects, radiation damping and B1 and B0 inhomogeneities. Furthermore, it is important to avoid partial saturation. In this work, the MISSTEC sequence is proposed for pulse calibration. This sequence takes only 8 s or 2 min for 1H or 13C calibration, respectively. High accuracy (with an error below 1%) was obtained for both nuclei. Therefore, the calibrations can be done rapidly and accurately. Furthermore, the MISSTEC measurement could be performed on each sample - in an automated way- before acquisitions, after which the calibration found could be automatically used.

Cheese characterization and authentication through lipid biomarkers obtained by high-resolution 1H NMR profiling.

Food quality and safety are at the heart of consumers' concerns across the world. Dairy products, because of their large consumption, are fertile ground for fraudulent acts. This fact justifies the development of effective, accessible, and rapid analytical methods for their authentication. A high-resolution spectral treatment method previously developed by our team was applied to 1H NMR spectra of cheese triacylglycerols. 178 Peaks were thus quantitated and successfully used in the construction of multivariate models for the quantitation of individual fatty acids and for the classification of cheese samples according to the producing species, to their origin and variety. Besides, several peaks related to the amount and position of anteisopentadecanoic, butyric, α-linolenic, myristoleic, rumenic, and vaccenic acids were, among others, specific biomarkers of cheese groups. For the first time in 1H NMR, we were able to identify and to quantitate signals related to minor fatty acids within cheese triacylglycerols.

High-resolution 1H NMR profiling of triacylglycerols as a tool for authentication of food from animal origin: Application to hen egg matrix.

Metabolomics of complex biological matrices conducted by means of 1H NMR leads to spectra suffering from severe signal overlapping. Previously, we have developed a high-resolution spectral treatment method to help solving this issue in 1H NMR of triacylglycerols. In this work, we tested the potential of the developed method in the characterization and authentication of food products from animal origin using egg yolk as a model matrix. The approach consisted in a spectral deconvolution guided by the precision obtained on the deconvoluted peaks after reference lineshape adjustment of spectra. Thus, 135 peaks were quantitated and successfully used as biomarkers of origin, of hens breed, and of farming system. This required multivariate statistical analyses for classification. The same pool of variables allowed construction of multivariate quantitation models for individual fatty acids. Furthermore, minute amounts of conjugated fatty acids were quantitated and used as fingerprints of samples from backyard and free-range farming.

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.

NMR-based isotopic and isotopomic analysis.

Molecules exist in different isotopic compositions and most of the processes, physical or chemical, in living systems cause selection between heavy and light isotopes. Thus, knowing the isotopic fractionation of the common atoms, such as H, C, N, O or S, at each step during a metabolic pathway allows the construction of a unique isotope profile that reflects its past history. Having access to the isotope abundance gives valuable clues about the (bio)chemical origin of biological or synthetic molecules. Whereas the isotope ratio measured by mass spectrometry provides a global isotope composition, quantitative NMR measures isotope ratios at individual positions within a molecule. We present here the requirements and the corresponding experimental strategies to use quantitative NMR for measuring intramolecular isotope profiles. After an introduction showing the historical evolution of NMR for measuring isotope ratios, the vocabulary and symbols - for describing the isotope content and quantifying its change - are defined. Then, the theoretical framework of very accurate quantitative NMR is presented as the principle of Isotope Ratio Measurement by NMR spectroscopy, including the practical aspects with nuclei other than 2H, that have been developed and employed to date. Lastly, the most relevant applications covering three issues, tackling counterfeiting, authentication, and forensic investigation, are presented, before giving some perspectives combining technical improvements and methodological approaches.

Forensic application of position-specific isotopic analysis of trinitrotoluene (TNT) by NMR to determine 13C and 15N intramolecular isotopic profiles.

2,4,6-trinitrotoluene (TNT) is a molecule which is easily identified with current instrumental techniques but it is generally impossible to distinguish between sources of the same substance (TNT). To overcome this difficulty, we present a multi stable isotope approach using isotope ratio monitoring by mass spectrometry (irm-MS) and Nuclear Magnetic Resonance spectrometry (irm-NMR). In the one hand, irm-MS provides bulk isotopic composition at natural abundance in 13C and 15N. The range of variation between samples is rather small particularly for 13C. In the other hand, irm-13C NMR and irm-15N NMR enable the determination of positional intramolecular 13C/12C ratios (δ13Ci) and 15N/14N ratios (δ15Ni) with high precision that lead to larger variation between samples. The present work reports an application of the recent methodology using irm-15N NMR to determine position-specific 15N isotope content of TNT. The interest of this methodology is compared to irm-13C NMR and irm-MS (13C and 15N) in terms of TNT samples discrimination. Thanks to the use of irm-NMR the results show a unique isotopic fingerprint for each TNT which enable origin discrimination between the samples without ambiguity.

Metabisotopomics of triacylglycerols from animal origin: A simultaneous metabolomic and isotopic profiling using 13C INEPT.

In previous works, we developed a 13C NMR method for analyzing triacylglycerols in olive oil using an adiabatic refocused INEPT sequence. This allowed spectral acquisition to be done in only 8 min with sufficient precision for isotopic measurements. In the present study, we made use of the same methodology to investigate the potential of triacylglycerols as source of biomarkers in animal origin matrices. To this end, egg yolk was taken as a model matrix. We called our profiling approach metabisotopomics since it was simultaneously metabolomic and isotopic profiling. Beside its ability to quantitate several fatty acids, metabisotopomics of triacylglycerols in egg yolk allowed the multivariate classification of samples according to the hen breed, to the farming system and origin. Achieved results confirmed our presumption that 13C metabisotopomics of triacylglycerols from animal sources is a powerful tool for metabolic studies as well as for food authentication processes.

Isotopomics by isotope ratio monitoring by 13 C nuclear magnetic resonance spectrometry on cutting agents in heroin: A new approach for illicit drugs trafficking route elucidation.

In the battle against the illicit drugs market, methodologies have been developed by forensic laboratories to address the determination of the origin and dismantlement of the trafficking route for various target molecules such as heroin and cocaine. These drug profiling methods are not straightforward, especially when the target molecules are synthetic and very pure, resulting in poorly informative impurity profiles, e.g. new psychoactive substances and cutting agents. A tool based on the determination of intramolecular isotopic profiles has been developed to provide origin discrimination with a new way to profile seized cutting agents and heroin samples. Whereas stable isotope analyses by mass spectrometry give the bulk isotopic composition, nuclear magnetic resonance gives direct access to the position-specific isotope content at natural abundance. This report shows how both 13 C NMR spectrometry and 13 C, 15 N MS might provide complementary and valuable information to link seized caffeine and paracetamol to their origin. Here, isotopic ratio monitoring by 13 C NMR (irm-13 C NMR) offers additional benefits over irm-MS in its capability to determine a detailed isotopic profile, leading to a better method to distinguish different caffeine and paracetamol batches.

Improved lipid mixtures profiling by 1H NMR using reference lineshape adjustment and deconvolution techniques.

ANALYSIS: of one-dimensional 1H NMR spectra of complex mixtures, such as lipids from natural extracts, is hampered by the small spectral width leading to a great number of overlapped signals. Additional complications including lineshape broadening and distortion may occur due to magnetic field inhomogeneity. Quantitation of such spectra is therefore challenging. We present in this work a quantitation approach based on deconvolution after correction of spectra by means of reference lineshape adjustment (RLA), also known as reference deconvolution. Spectral fit and precision obtained on deconvoluted peaks were used as indicators to iteratively improve the deconvolution process. This approach was tested on 1H NMR spectra of olive oil samples and allowed extraction of 77 peaks (available as peak intensities or areas), whereas spectral integration afforded 5 variables when only well-resolved signals were considered and 29 variables when a bucket around each discernible peak was integrated. Deconvoluted peak intensities and areas were obtained with improved precision after RLA of raw spectra. The use of these spectral variables as predictors in multivariate statistical analysis enhanced the classification of olive oil samples according to the altitude of the olive field or to the color of the olive drupes. The same variables allowed quantitation of oleic, palmitoleic, and vaccenic acids within triacylglycerols, which was not possible by 1H NMR, and improved quantitation of linoleic and linolenic acids. These results proved the high potential of the presented approach in the characterization and authentication of complex mixtures by 1H NMR spectroscopy.

Cholesterol, a powerful 13C isotopic biomarker.

Cholesterol is related to many health diseases and is considered as a metabolic disorder biomarker. This compound, present in all food products of animal origin, can also be used as food authentication biomarker. In this work and for the first time, positional 13C isotope contents were determined for such a high molecular weight compound. This was possible by means of NMR using adiabatic refocused INEPT. In order to test the potential of this approach for discrimination, hen eggs from different origins were collected. Quantitative extraction of egg yolk cholesterol was optimized, and partial reduced molar fractions of its different 13C isotopomers were used as predictors in discriminant analysis. Compared with the global 13C isotopic composition determined using isotope ratio monitoring by Mass Spectrometry, the relative content of cholesterol 13C isotopomers added valuable power to sample classifications according to their origins. This study paves the way to isotopomics of other steroids and similar molecular weight compounds.

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.

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.

Consequences of blunting the mevalonate pathway in cancer identified by a pluri-omics approach.

We have previously shown that the combination of statins and taxanes was a powerful trigger of HGT-1 human gastric cancer cells' apoptosis1. Importantly, several genes involved in the "Central carbon metabolism pathway in cancer", as reported in the Kyoto Encyclopedia of Genes and Genomes, were either up- (ACLY, ERBB2, GCK, MYC, PGM, PKFB2, SLC1A5, SLC7A5, SLC16A3,) or down- (IDH, MDH1, OGDH, P53, PDK) regulated in response to the drug association. In the present study, we conducted non-targeted metabolomics and lipidomics analyses by complementary methods and cross-platform initiatives, namely mass spectrometry (GC-MS, LC-MS) and nuclear magnetic resonance (NMR), to analyze the changes resulting from these treatments. We identified several altered biochemical pathways involved in the anabolism and disposition of amino acids, sugars, and lipids. Using the Cytoscape environment with, as an input, the identified biochemical marker changes, we distinguished the functional links between pathways. Finally, looking at the overlap between metabolomics/lipidomics and transcriptome changes, we identified correlations between gene expression modifications and changes in metabolites/lipids. Among the metabolites commonly detected by all types of platforms, glutamine was the most induced (6-7-fold), pointing to an important metabolic adaptation of cancer cells. Taken together, our results demonstrated that combining robust biochemical and molecular approaches was efficient to identify both altered metabolic pathways and overlapping gene expression alterations in human gastric cancer cells engaging into apoptosis following blunting the cholesterol synthesis pathway.

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.

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.