Application of high‐field NMR spectroscopy for characterization and quantitation of submilligram quantities of isolated natural products

We have investigated and compared a number of sample conditions on different NMR platforms in the search of maximum SNR and optimal experiment time efficiency for structure elucidation and quantitation of natural products. Using restricted volume 3 mm Shigemi microcell assembly in conjunction with a 900 MHz NMR spectrometer equipped with a 5 mm carbon‐sensitive inverse cryoprobe, it was possible to achieve a substantial increase in SNR (46‐fold) as compared with a conventional room temperature 400 MHz instrument. Switching from standard 5 mm NMR tube to 3 mm Shigemi microcell assembly typically improved SNR by threefold on either 600 or 900 MHz cryoplatform. A quantitation method that relies on a calibrated residual protonated NMR solvent signal as internal standard was developed using the same hardware setup and restricted sample volume tubes. Linearity of the method spans over 3 orders of magnitude, from low microgram to milligram quantities. We successfully applied this method to quantify a low micrgram sample of paclitaxel, verified by a UV/VIS quantitation measurement. Copyright © 2015 John Wiley & Sons, Ltd.     

A New Method for High-Resolution NMR Spectra in Inhomogeneous Fields with Efficient Solvent Suppression

High-resolution nuclear magnetic resonance （NMR） is one of the most powerful tools for analyzing molecular structures and dynamics. Magnetic field homogeneity is required for conventional high-resolution spectra. However, there are many chemical and/or biological circumstances where the spatial homogeneities of the magnetic fields are degraded. Intense solvent signal is another obstacle for obtaining high-resolution spectra, especially in in vivo and in situ NMR spectroscopy. In this paper, a new pulse sequence based on intermolecular multiple quantum coherence （iMQC） was reported. This sequence can effectively remove the effect of magnetic field inhomogeneity and suppress the solvent signal. It can recover the spectral information such as chemical shifts, coupling constants, multiplet patterns, and relative peak areas in inhomogeneous fields. Theoretical analyses and experimental verifications are presented to demonstrate the feasibility of this method.     

Construction and 13C hyperpolarization efficiency of a 180 GHz dissolution dynamic nuclear polarization system

Dynamic nuclear polarization (DNP) via the dissolution method has become one of the rapidly emerging techniques to alleviate the low signal sensitivity in nuclear magnetic resonance (NMR) spectroscopy and imaging. In this paper, we report on the development and ¹³C hyperpolarization efficiency of a homebuilt DNP system operating at 6.423 T and 1.4 K. The DNP hyperpolarizer system was assembled on a wide‐bore superconducting magnet, equipped with a standard continuous‐flow cryostat, and a 180 GHz microwave source with 120 mW power output and wide 4 GHz frequency tuning range. At 6.423 T and 1.4 K, solid‐state ¹³C polarization P levels of 64% and 31% were achieved for 3 M [1‐¹³C] sodium acetate samples in 1 : 1 v/v glycerol:water glassing matrix doped with 15 mM trityl OX063 and 40 mM 4‐oxo‐TEMPO, respectively. Upon dissolution, which takes about 15 s to complete, liquid‐state ¹³C NMR signal enhancements as high as 240 000‐fold (P=21%) were recorded in a nearby high resolution ¹³C NMR spectrometer at 1 T and 297 K. Considering the relatively lower cost of our homebuilt DNP system and the relative simplicity of its design, the dissolution DNP setup reported here could be feasibly adapted for in vitro or in vivo hyperpolarized ¹³C NMR or magnetic resonance imaging at least in the pre‐clinical setting. Copyright © 2017 John Wiley & Sons, Ltd.     

On‐Line Monitoring of Chemical Reactions by using Bench‐Top Nuclear Magnetic Resonance Spectroscopy

Real‐time nuclear magnetic resonance (NMR) spectroscopy measurements carried out with a bench‐top system installed next to the reactor inside the fume hood of the chemistry laboratory are presented. To test the system for on‐line monitoring, a transfer hydrogenation reaction was studied by continuously pumping the reaction mixture from the reactor to the magnet and back in a closed loop. In addition to improving the time resolution provided by standard sampling methods, the use of such a flow setup eliminates the need for sample preparation. Owing to the progress in terms of field homogeneity and sensitivity now available with compact NMR spectrometers, small molecules dissolved at concentrations on the order of 1 mmol L^?1 can be characterized in single‐scan measurements with 1 Hz resolution. Owing to the reduced field strength of compact low‐field systems compared to that of conventional high‐field magnets, the overlap in the spectrum of different NMR signals is a typical situation. The data processing required to obtain concentrations in the presence of signal overlap are discussed in detail, methods such as plain integration and line‐fitting approaches are compared, and the accuracy of each method is determined. The kinetic rates measured for different catalytic concentrations show good agreement with those obtained with gas chromatography as a reference analytical method. Finally, as the measurements are performed under continuous flow conditions, the experimental setup and the flow parameters are optimized to maximize time resolution and signal‐to‐noise ratio.     

Ternary deep eutectic solvent magnetic molecularly imprinted polymers for the dispersive magnetic solid‐phase microextraction of green tea

Ternary deep eutectic solvent magnetic molecularly imprinted polymers grafted on silica were developed for the selective recognition and separation of theophylline, theobromine, (+)‐catechin hydrate, and caffeic acid from green tea through dispersive magnetic solid‐phase microextraction. A new ternary deep eutectic solvent was adopted as a functional monomer. The materials obtained were characterized by FTIR spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, NMR spectroscopy, and powder X‐ray diffraction. The practical recovery of the theophylline, theobromine, (+)‐catechin hydrate, and caffeic acid isolated with ternary deep eutectic solvent magnetic molecularly imprinted polymers in green tea were 91.82, 92.13, 89.96, and 90.73%, respectively, and the actual amounts extracted were 5.82, 4.32, 18.36, and 3.69 mg/g, respectively. The new method involving the novel material coupled with dispersive magnetic solid‐phase microextraction showed outstanding recognition, selectivity and excellent magnetism, providing a new perspective for the separation of bioactive compounds.     

Level Anti‐Crossings are a Key Factor for Understanding para‐Hydrogen‐Induced Hyperpolarization in SABRE Experiments

Various hyperpolarization methods are able to enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) by several orders of magnitude. Among these methods are para‐hydrogen‐induced polarization (PHIP) and signal amplification by reversible exchange (SABRE), which exploit the strong nuclear alignment of para‐hydrogen. Several SABRE experiments have been reported but, so far, it has not been possible to account for the experimentally observed sign and magnetic‐field dependence of substrate polarization. Herein, we present an analysis based on level anti‐crossings (LACs), which provides a complete understanding of the SABRE effect. The field‐dependence of both net and anti‐phase polarization is measured for several ligands, which can be reproduced by the theory. The similar SABRE field‐dependence for different ligands is also explained. In general, the LAC concept allows complex spin dynamics to be unraveled, and is crucial for optimizing the performance of novel hyperpolarization methods in NMR and MRI techniques.     

Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl‐Nitroxide Biradicals

Cross‐effect (CE) dynamic nuclear polarization (DNP) is a rapidly developing technique that enhances the signal intensities in magic‐angle spinning (MAS) NMR spectra. We report CE DNP experiments at 211, 600, and 800 MHz using a new series of biradical polarizing agents referred to as TEMTriPols, in which a nitroxide (TEMPO) and a trityl radical are chemically tethered. The TEMTriPol molecule with the optimal performance yields a record ¹H NMR signal enhancement of 65 at 800 MHz at a concentration of 10 mM in a glycerol/water solvent matrix. The CE DNP enhancement for the TEMTriPol biradicals does not decrease as the magnetic field is increased in the manner usually observed for bis‐nitroxides. Instead, the relatively strong exchange interaction between the trityl and nitroxide moieties determines the magnetic field at which the optimum enhancement is observed.     

Complete 1H NMR spectral analysis of ten chemical markers of Ginkgo biloba

The complete and unambiguous ¹H NMR assignments of ten marker constituents of Ginkgo biloba are described. The comprehensive ¹H NMR profiles (fingerprints) of ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, quercetin, kaempferol, isorhamnetin, isoquercetin, and rutin in DMSO‐d₆ were obtained through the examination of 1D ¹H NMR and 2D ¹H,¹H‐COSY data, in combination with ¹H iterative full spin analysis (HiFSA). The computational analysis of discrete spin systems allowed a detailed characterization of all the ¹H NMR signals in terms of chemical shifts (δ_H) and spin‐spin coupling constants (J_HH), regardless of signal overlap and higher order coupling effects. The capability of the HiFSA‐generated ¹H fingerprints to reproduce experimental ¹H NMR spectra at different field strengths was also evaluated. As a result of this analysis, a revised set of ¹H NMR parameters for all ten phytoconstituents was assembled. Furthermore, precise ¹H NMR assignments of the sugar moieties of isoquercetin and rutin are reported for the first time. Copyright © 2012 John Wiley & Sons, Ltd.     

Standardizing the use of fast-field cycling NMR relaxometry for measuring hydrological connectivity inside the soil

Hydrological connectivity inside the soil (HCS) is applied to study the effects of heterogeneities in complex environmental systems. It refers to both the spatial patterns inside the soil (i.e., structural connectivity [SC]) and the physical–chemical processes at a molecular level (i.e., functional connectivity [FC]). NMR relaxometry has been already applied to assess both SC and FC components of the HCS by defining SC and FC indexes. Here, fast-field cycling NMR relaxometry has been applied on a water suspended soil and a sediment to optimize the conditions to standardize the technique. Proton Larmor frequencies (ω_L) from 0.01 to 25 MHz were used on samples suspended in three different rates of Milli-Q grade water. The application of different magnetic fields revealed that the T₁ values of the sediment sample are always shorter than those measured for the soil sample. This difference was attributed to the soil erosion processes limiting FC by reducing the size of macropores. For the soil sample, analyses showed that both structural and functional connectivity indexes can be assumed ω_L independent. For the sediment sample, the connectivity indexes resulted ω_L independent only for ω_L ≥ 0.1 MHz. This could be due either to instrumental problems for ω_L < 0.1 MHz or to a real magnetic field effect when a finer textured sample is examined. Further research is required in this area. Finally, the experiments revealed that the optimal water amount to obtain reliable results was corresponding to the water holding capacity.     

Early stages of fat crystallisation evaluated by low-field NMR and small-angle X-ray scattering

Low-field time-domain nuclear magnetic resonance (NMR; 20 MHz) is commonly used in the studies of fats in the form of solid fat content (SFC) measurements. However, it has the disadvantage of low sensitivity to small amounts of crystalline material (0.5%), thus often incorrectly determining crystallisation induction times. From spin–lattice relaxation rate measurements (R₁) during the isothermal crystallisation measurements of cocoa butter between 0.01 and 10 MHz using fast field cycling NMR, we learnt previously that the most sensitive frequency region is below 1 MHz. Thus, we focused on analysing our 10-kHz data in detail, by observing the time dependence of R₁ and comparing it with standard SFC_NMR and SFC determinations from small-angle X-ray scattering (SFC_SAXS). Although not reflecting directly the SFC, the R₁ at this low frequency is very sensitive to changes in molecular aggregation and hence potentially serving as an alternative for determination of crystallisation induction times. Alongside R₁, we also show that SFC_SAXS is more sensitive to early stages of crystallisation, that is, standard SFC_NMR determinations become more relevant when crystal growth starts to dominate the crystallisation process but fail to pick up earlier crystallisation steps. This paper thus demonstrates the potential of studying triacylglycerols at frequencies below 1 MHz for obtaining further understanding of the early crystallisation stages of fats and presents an alternative and complementary method to estimate SFC by SAXS.     

Bridging the gap between metabolic profile determination and visualization in neurometabolic disorders: a multivariate analysis of proton magnetic resonance in vivo spectra

The purpose of this work was to check the degree of overlap between rare inborn errors of metabolism and other neurological disorders using principal component analysis of proton magnetic resonance spectroscopy (¹H MRS) in vivo data. We examined 60 patients (median age of 22 months). Fourteen of them were diagnosed with neurometabolic disorders (three cases of metachromatic leukodystrophy, two cases of Canavan disease, two cases of megalencephalic leukoencephalopathy with subcortical cysts, three cases of mitochondrial cytopathy, one case of nonketotic hyperglycinemia, one case of globoid leukodystrophy, one case of congenital disorders of glycosylation, and one case of ethylmalonic encephalopathy). The remaining 46 patients were diagnosed with epilepsy, cerebral palsy, and developmental delay. Results obtained from principal component analysis of complete unresolved ¹H MRS in vivo spectra were interpreted parallelly with LCModel‐derived metabolite levels. The main attention was paid to the following metabolites: N‐acetylaspartate, glutamate + glutamine, creatine, choline, myo‐inositol signal with an uncertain contribution of glycine, and glucose. ¹H MRS in vivo coupled with multivariate analysis is an efficient tool in visualization of metabolic abnormalities in several inborn errors of metabolism (metachromatic leukodystrophy, globoid leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts, and Canavan disease). Copyright © 2013 John Wiley & Sons, Ltd.     

13C CP MAS NMR of halogenated (Cl,Br, I) pharmaceuticals at ultrahigh magnetic fields

This work reports significantly improved spectral resolution of ¹³C CP MAS NMR spectra of chlorinated, brominated and iodinated solid organic compounds when such spectra are recorded at ultrahigh magnetic field strengths. The cause of this is the residual dipolar coupling between carbon atoms and quadrupolar halogen nuclides (chlorine‐35/37, bromine‐79/81 or iodine‐127), an effect inversely proportional to the magnetic field strength which declines in importance markedly at 21.1 T as compared to lower fields. In favorable cases, the fine structure observed can be used for spectral assignment, e.g. for Cl‐substituted aromatics where the substituted carbon as well as the ortho‐carbons show distinct doublets. The experimental results presented are supported by theoretical modeling and calculations. The improved spectral resolution in the studied systems and similar halogenated materials will be of particular interest and importance for polymorph identification, drug discovery and quality control in the pharmaceutical industry. Copyright © 2009 John Wiley & Sons, Ltd.     

Harnessing polarisation transfer to indazole and imidazole through signal amplification by reversible exchange to improve their NMR detectability

The signal amplification by reversible exchange (SABRE) approach has been used to hyperpolarise the substrates indazole and imidazole in the presence of the co‐ligand acetonitrile through the action of the precataysts [IrCl(COD)(IMes)] and [IrCl(COD)(SIMes)]. ²H‐labelled forms of these catalysts were also examined. Our comparison of the two precatalysts [IrCl(COD)(IMes)] and [IrCl(COD)(SIMes)], coupled with ²H labelling of the N‐heterocyclic carbene and associated relaxation and polarisation field variation studies, demonstrates the critical and collective role these parameters play in controlling the efficiency of signal amplification by reversible exchange. Ultimately, with imidazole, a 700‐fold¹H signal gain per proton is produced at 400 MHz, whilst for indazole, a 90‐fold increase per proton is achieved. The co‐ligand acetonitrile proved to optimally exhibit a 190‐fold signal gain per proton in these measurements, with the associated studies revealing the importance the substrate plays in controlling this value. Copyright © 2017 The Authors. Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd.     

Rapid preparation of methyltrimethoxy‐modified magnetic mesoporous silica as an effective solid‐phase extraction adsorbent

A time‐saving method was applied to synthesize methyltrimethoxy‐modified magnetic mesoporous silica with or without p‐toluenesulfonic acid as the catalyst for magnetic solid‐phase extraction. The synthesized materials were systematically characterized. Results demonstrated that methyltrimethoxy modified magnetic mesoporous silica with p‐toluenesulfonic acid as the catalyst has a relatively smaller aperture and extreme hydrophobicity (water contact angle of 135°). To evaluate the feasibility of these prepared materials as effective adsorbents, it was combined with gas chromatography and electron capture detection to determine 26 polychlorinated biphenyls in environmental water. The result revealed that methyltrimethoxy modified magnetic mesoporous silica with p‐toluenesulfonic acid as the catalyst had the best extraction efficiency and recovery. Under the optimized extracted conditions, the proposed method showed good linearity within the concentration range of 5 to 200 ng/L with correlation coefficients of 0.9969 to 0.9999. The limits of detection and quantification based on signal‐to‐noise ratios of 3 and 10 were in the range of 0.16 to 0.91 and 0.52 to 3.0 ng/L, respectively. The polychlorinated biphenyl concentrations in environmental water samples were successfully determined using the developed method. PCB008 and PCB110 were 4.05 and 8.52 ng/L in Red‐Star lake water (Hubei Province, China), respectively.     

NMR spectroscopy goes mobile: Using NMR as process analytical technology at the fume hood

Nuclear magnetic resonance (NMR) is potentially a very powerful process analytical technology (PAT) tool as it gives an atomic resolution picture of the reaction mixture without the need for chromatography. NMR is well suited for interrogating transient intermediates, providing kinetic information via NMR active nuclei, and most importantly provides universally quantitative information for all species in solution. This contrasts with commonly used PAT instruments, such as Raman or Flow-infrared (IR), which requires a separate calibration curve for every component of the reaction mixture. To date, the large footprint of high-field (≥400 MHz) NMR spectrometers and the immobility of superconducting magnets, coupled with strict requirements for the architecture for the room it is to be installed, have been a major obstacle to using this technology right next to fume hoods where chemists perform synthetic work. Here, we describe the use of a small, lightweight 60 MHz Benchtop NMR system (Nanalysis Pro-60) located on a mobile platform, that was used to monitor both small and intermediate scale Grignard formation and coupling reactions. We also show how low field NMR can provide a deceptively simple yes/no answer (for a system that would otherwise require laborious off-line testing) in the enrichment of one component versus another in a kilogram scale distillation. Benchtop NMR was also used to derive molecule specific information from Flow-IR, a technology found in most manufacturing sites, and compare the ease at which the concentrations of the reaction mixtures can be derived by NMR versus IR.     

Solid‐state 11B and 13C NMR,IR, and X‐ray crystallographic characterization of selected arylboronic acids and their catechol cyclic esters

Nine arylboronic acids, seven arylboronic catechol cyclic esters, and two trimeric arylboronic anhydrides (boroxines) are investigated using ¹¹B solid‐state NMR spectroscopy at three different magnetic field strengths (9.4, 11.7, and 21.1 T). Through the analysis of spectra of static and magic‐angle spinning samples, the ¹¹B electric field gradient and chemical shift tensors are determined. The effects of relaxation anisotropy and nutation field strength on the ¹¹B NMR line shapes are investigated. Infrared spectroscopy was also used to help identify peaks in the NMR spectra as being due to the anhydride form in some of the arylboronic acid samples. Seven new X‐ray crystallographic structures are reported. Calculations of the ¹¹B NMR parameters are performed using cluster model and periodic gauge‐including projector‐augmented wave (GIPAW) density functional theory (DFT) approaches, and the results are compared with the experimental values. Carbon‐13 solid‐state NMR experiments and spectral simulations are applied to determine the chemical shifts of the ipso carbons of the samples. One bond indirect ¹³C‐¹¹B spin‐spin (J) coupling constants are also measured experimentally and compared with calculated values. The ¹¹B/¹⁰B isotope effect on the ¹³C chemical shift of the ipso carbons of arylboronic acids and their catechol esters, as well as residual dipolar coupling, is discussed. Overall, this combined X‐ray, NMR, IR, and computational study provides valuable new insights into the relationship between NMR parameters and the structure of boronic acids and esters. Copyright © 2012 John Wiley & Sons, Ltd.     

Targeted projection NMR spectroscopy for unambiguous metabolic profiling of complex mixtures

Unambiguous identification of individual metabolites present in complex mixtures such as biofluids constitutes a crucial prerequisite for quantitative metabolomics, toward better understanding of biochemical processes in living systems. Increasing the dimensionality of a given NMR correlation experiment is the natural solution for resolving spectral overlap. However, in the context of metabolites, natural abundance acquisition of ¹H and ¹³C NMR data virtually excludes the use of higher dimensional NMR experiments (3D, 4D, etc.) that would require unrealistically long acquisition times. Here, we introduce projection NMR techniques for studies of complex mixtures, and we show how discrete sets of projection spectra from higher dimensional NMR experiments are obtained in a reasonable time frame, in order to capture essential information necessary to resolve assignment ambiguities caused by signal overlap in conventional 2D NMR spectra. We determine optimal projection angles where given metabolite resonances will have the least overlap, to obtain distinct metabolite assignment in complex mixtures. The method is demonstrated for a model mixture composition made of ornithine, putrescine and arginine for which acquisition of a single 2D projection of a 3D ¹H–¹³C TOCSY‐HSQC spectrum allows to disentangle the metabolite signals and to access to complete profiling of this model mixture in the targeted 2D projection plane. Copyright © 2010 John Wiley & Sons, Ltd.     

Nuclear magnetic resonance study of Li and H diffusion in the high-temperature solid phase of LiBH4

In order to study the atomic jump motions in the high-temperature solid phase of LiBH₄, we have measured the ¹H and ¹¹B nuclear magnetic resonance (NMR) spectra and the ¹H, ⁷Li and ¹¹B spin–lattice relaxation rates in this compound over the resonance frequency range of 14–34.4 MHz. In the temperature range 384–500 K, all the spin–lattice relaxation data are satisfactorily described in terms of a thermally activated jump motion of Li ions with the pre-exponential factor τ₀=1.1×10⁻¹⁵ s and the activation energy E_a=0.56 eV. The observed frequency dependences of the spin–lattice relaxation rates in this temperature range exclude a presence of any distributions of the Li jump rate or any other jump processes on the frequency scale of 10⁷–10¹⁰ s⁻¹. The strong narrowing of the ¹H and ¹¹B NMR lines above 440 K is consistent with the onset of diffusive motion of the BH₄ tetrahedra.     

Electroless‐coated magnetic three‐dimensional graphene with silver nanoparticles used for the determination of pesticides in fruit samples

A new type of adsorbent composed of magnetic three‐dimensional graphene coated with silver nanoparticles was synthesized by an electroless technique and used in the magnetic solid‐phase extraction of selected pesticides (fenitrothion, chlorpyrifos, and hexaconazole) before gas chromatography with a micro‐electron capture detector. The adsorbent was characterized using Fourier‐transform infrared spectroscopy, X‐ray diffraction, vibrating sample magnetometry, and field‐emission scanning electron microscopy. The important extraction parameters such as pH, adsorbent dose, extraction time, and desorption conditions were investigated. Under the optimal conditions, the analytical figures of merit were obtained as: linear dynamic range of 0.1–5 ng/g with determination coefficients of 0.991–0.996; limit of detection of 0.07–0.13 ng/g; limit of quantification of 0.242–0.448 ng/g; and the intraday and interday relative standard deviations (C = 5 ng/g, n = 3) were 3.8–8.7 and 6.6–8.9%, respectively. The developed method was successfully applied for analysis of the selected pesticides in tomato and grape with extraction recoveries in the range of 72.8–109.6%.     

Accurate grading of brain gliomas by soft independent modeling of class analogy based on non‐negative matrix factorization of proton magnetic resonance spectra

Hydrogen magnetic resonance spectroscopy (¹H‐MRS) is a non‐invasive technique which provides a ‘frequency‐signal intensity’ spectrum of biochemical compounds of tissues in the body. Although this method is currently used in human brain studies, accurate classification of in‐vivo ¹H‐MRS is a challenging task in the diagnosis of brain tumors. Problems such as overlapping metabolite peaks, incomplete information on background component and low signal‐to‐noise ratio disturb classification results of this spectroscopic method. This study presents an alternative approach to the soft independent modeling of class analogy (SIMCA) technique, using non‐negative matrix factorization (NMF) for dimensionality reduction. In the adopted strategy, the performance of SIMCA was improved by application of a robust algorithm for classification in the presence of noisy measurements. Total of 219 spectra from two databases were taken by water‐suppressed short echo‐time ¹H‐MRS, acquired from different subjects with different stages of glial brain tumors (Grade II (26 cases), grade III (24 cases), grade IV (41 cases), as well as 25 healthy cases). The SIMCA was performed using two approaches: (i) principal component analysis (PCA) and (ii) non‐negative matrix factorization (NMF), as a modified approach. Square prediction error was considered to assess the class membership of the external validation set. Finally, several figures of merit such as the correct classification rate (CCR), sensitivity and specificity were calculated. Results of SIMCA based on NMF showed significant improvement in percentage of correctly classified samples, 91.4% versus 83.5% for PCA‐based model in an independent test set. Copyright © 2015 John Wiley & Sons, Ltd.