Molecular signal suppression by in situ microextraction in nuclear magnetic resonance spectroscopy

The detailed characterization of complex mixtures by NMR is often hampered by the presence of signals from uninformative compounds, the resonances of which overlap with those of the molecules of interest. We provide here a proof of principle for an approach to NMR signal suppression in complex samples using Molecularly Imprinted Polymers (MIPS). Addition of a few milligrams of polymer to a solution traps the target molecule in typical micromolar to millimolar concentration, thus achieving in situ signal suppression, without altering any other spectral features. This method minimized any manipulation or perturbation of the spectrum and was applied to a complex mixture of known compounds and to a plant extract, in both cases spiked with a compound (bisphenol A), which was subsequently removed by selective binding to a complementary MIP. What is described in this report is comparable with microextraction and may in due course be applied to a large number of analytical challenges. Copyright © 2014 John Wiley & Sons, Ltd.     

Processing of high resolution magic angle spinning spectra of breast cancer cells by the filter diagonalization method

Proton nuclear magnetic resonance ((1)H NMR) spectroscopy for detection of biochemical changes in biological samples is a successful technique. However, the achieved NMR resolution is not sufficiently high when the analysis is performed with intact cells. To improve spectral resolution, high resolution magic angle spinning (HR-MAS) is used and the broad signals are separated by a T(2) filter based on the CPMG pulse sequence. Additionally, HR-MAS experiments with a T(2) filter are preceded by a water suppression procedure. The goal of this work is to demonstrate that the experimental procedures of water suppression and T(2) or diffusing filters are unnecessary steps when the filter diagonalization method (FDM) is used to process the time domain HR-MAS signals. Manipulation of the FDM results, represented as a tabular list of peak positions, widths, amplitudes and phases, allows the removal of water signals without the disturbing overlapping or nearby signals. Additionally, the FDM can also be used for phase correction and noise suppression, and to discriminate between sharp and broad lines. Results demonstrate the applicability of the FDM post-acquisition processing to obtain high quality HR-MAS spectra of heterogeneous biological materials.     

Residual water suppression by indirect covariance NMR

Residual water solvent signals in 2D NMR experiments adversely affect appearance and subsequent analysis of spectra. A method for water suppression that is based on indirect covariance processing is described. It produces a symmetric spectrum with a water signal that is substantially decreased or completely absent. The method, which can be combined with other water suppression schemes, is demonstrated for 2D TOCSY, NOESY, and ROESY spectra of the protein, ubiquitin in aqueous solution.     

Background‐free solution boron NMR spectroscopy

The appearance of background signals arising from the NMR probe and tube is a well‐known problem of boron NMR spectroscopy. Background suppression may be achieved by using DEPTH, which increases the signal‐to‐background (S/B) ratio. Although, the quality of such spectra is often adequate, but in the case of rapid relaxation broadened resonances (T₁ < 1 ms), the residual background signals may still hamper the interpretation of the spectra. It was observed that the background signals are practically invisible in solution ¹⁰B NMR. The unusual isotopic effect on the (S/B) ratio was interpreted as an inherent consequence of the integer versus half‐integer spin of ¹⁰B and ¹¹B, respectively. The practicability of ^10/11B NMR was compared for a selected set of boron compounds covering the typical range of (S/B) ratio. The application of ¹¹B is more favourable than ¹⁰B as long as it is possible to achieve the desired spectral quality by using DEPTH. Otherwise, the ‘background‐free’ appearance of ¹⁰B NMR spectra makes ¹⁰B a reasonable alternative of ¹¹B DEPTH. This was found typical for compounds having relaxation broadened resonances. The variable temperature (VT) NMR study of an adduct formation process was also presented here as an example of the advantage of ¹⁰B over ¹¹B. Copyright © 2012 John Wiley & Sons, Ltd.     

Chiral Phosphines: The Effect of Asymmetric Carbon Centers α or β to Phosphorus on the 31P NMR Spectra

Abstract

Chiral alkylphosphines containing asymmetric carbon centers either alpha or beta to the phosphorus are formed from the free radical addition of phosphine to prochiral olefins. The phosphorus NMR spectra of the primary phosphines have only one signal for the enantiomeric pair while the spectra of the secondary phosphines have three signals in a 1:2:1 ratio. This is the statistical ratio expected for the enantiomeric pair and two meso forms. Four enantiomeric pairs of tertiary phosphines can be formed from one prochiral olefin. One pair has alkyl groups of the same configuration while the remaining pairs have mixed configurations. The phosphorus NMR spectra show two signals in a 1:3 ratio. Dilution of the secondary phosphines with a polar solvent results in only one signal in the phosphorus NMR spectra. The chemical shifts and diluent effect can be explained in terms of the gamma shielding effect (1).     

Selective removal of interference signals for near-infrared spectra of biomedical samples by using region orthogonal signal correction

New approach for chemometrics algorithm named region orthogonal signal correction (ROSC) has been introduced to improve the predictive ability of PLS models for biomedical components in blood serum developed from their NIR spectra in the 1280-1849 nm region. Firstly, a moving window partial least squares regression (MWPLSR) method was employed to locate the region due to water as a region of interference signals and to find the informative regions of glucose, albumin, cholesterol and triglyceride from NIR spectra of bovine serum samples. Next, a novel chemometrics method named searching combination moving window partial least squares (SCMWPLS) was used to optimize those informative regions. Then, the specific regions that contained the information of water, glucose, albumin, cholesterol and triglyceride were obtained. When an interested component in the bovine serum solution, such as glucose, albumin, cholesterol or triglyceride is being an analyte, the other three interests and water are considered as the interference factors. Thus, new approach for ROSC has employed for each specific region of interference signal to calculate the orthogonal components to the concentrations of analyte that were removed specifically from the NIR spectra of bovine serum in the region of 1280-1849 nm and the highest interference signal for model of analyte will be revealed. The comparison of PLS results for glucose, albumin, cholesterol and triglyceride built by using the whole region of original spectra and those developed by using the optimized regions suggested by SCMWPLS of original spectra, spectra treated OSC for orthogonal components of 1-3 and spectra treated ROSC using selected removing the highest interference signals from the spectra for orthogonal components of 1-3 are reported. It has been found that new approach of ROSC to remove the highest interference signal located by SCMWPLS improves of the performance of PLS modeling, yielding the lower RMSECV and smaller number of PLS factors.     

Optimization of diffusion-filtered NMR experiments for selective suppression of residual nondeuterated solvent and water signals from 1H NMR spectra of organic compounds

Interpretation of 1H NMR spectra of organic compounds is sometimes hampered by the presence of strong peaks arising from residual nondeuterated solvent and water that obscure compound signals. Classical solvent suppression techniques such as presaturation or those based on pulsed field gradients are not effective in this regard because they also remove the compound resonances that overlap with the solvent signal being suppressed. Here, we propose an alternative scheme by using an optimized NMR diffusion filter that eliminates the nondesired peaks while retaining the signals of interest. This strategy has proved to be useful in three common deuterated solvents, namely, CDCl3, DMSO-d6, and CD3OD, resulting in clean spectra with no interference from solvent or water peaks.     

Use of the filter diagonalization method in the study of space charge related frequency modulation in Fourier transform ion cyclotron resonance mass spectrometry

The filter diagonalization method (FDM) is a recently developed computational technique capable of extracting resonance frequencies and amplitudes from very short transient signals. Although it requires stable resonance frequencies and is slower than the fast Fourier transform (FFT), FDM has a resolution and accuracy that is unmatched by the FFT or any other comparable techniques. This unique feature of FDM makes it an ideal tool for tracing space charge induced frequency modulations in Fourier transform ion cyclotron resonance (FT-ICR) cells, which are shown to reach +/-400 ppm even for such simple spectra as Substance P.     

Parahydrogen induced polarization of barbituric acid derivatives: 1H hyperpolarization studies

Homogeneous hydrogenation of barbituric acid derivatives with parahydrogen yields a substantial increase of the (1)H NMR signals of the reaction products. These physiologically relevant compounds were hydrogenated at both ambient and elevated temperatures and pressures using a standard cationic rhodium catalyst. The resulting nonthermal nuclear spin polarization (hyperpolarization) is limited by the spin-lattice relaxation time T(1) of the corresponding nuclei in the products, being shorter than the time constant of the hydrogenation. The signal-to-noise ratio of the NMR spectra could be further increased upon signal averaging the antiphase PHIP signals of 25 successive scans following 30 degrees pulse experiments and a delay of 10 s.     

Saturation transfer double-difference NMR spectroscopy using a dual solenoid microcoil difference probe

An experiment designed to collect a saturation transfer double difference (STDD) NMR spectrum using a solenoid microcoil NMR difference probe is reported. STDD-NMR allows the investigation of ligand-biomolecule binding, with moderate concentration requirements for unlabeled molecular targets and the ability to discern binding events in the presence of non-binding ligands. The NMR difference probe acquires the signals from two different samples at once, and cancels common signals automatically through a mechanism of switching between parallel excitation and serial acquisition of the sample signals. STDD spectra were acquired on a system consisting of human serum albumin and two ligands, octanoic acid and glucose. The non-binding ligand, glucose, was cancelled internally through phase cycling, while the protein signal was subtracted automatically by the difference probe. The proton NMR resonance signal from octanoic acid remained in the double difference spectrum. This work demonstrates that the double difference can be performed both internally and automatically through the utilization of the solenoid microcoil NMR difference probe and STDD-NMR pulse sequence, resulting in a clean signal from the binding ligand with good protein background subtraction and an overall favorable result when compared to the conventional approach.     

CRAFT (complete reduction to amplitude frequency table) – robust and time‐efficient Bayesian approach for quantitative mixture analysis by NMR

The intrinsic quantitative nature of NMR is increasingly exploited in areas ranging from complex mixture analysis (as in metabolomics and reaction monitoring) to quality assurance/control. Complex NMR spectra are more common than not, and therefore, extraction of quantitative information generally involves significant prior knowledge and/or operator interaction to characterize resonances of interest. Moreover, in most NMR‐based metabolomic experiments, the signals from metabolites are normally present as a mixture of overlapping resonances, making quantification difficult. Time‐domain Bayesian approaches have been reported to be better than conventional frequency‐domain analysis at identifying subtle changes in signal amplitude. We discuss an approach that exploits Bayesian analysis to achieve a complete reduction to amplitude frequency table (CRAFT) in an automated and time‐efficient fashion – thus converting the time‐domain FID to a frequency‐amplitude table. CRAFT uses a two‐step approach to FID analysis. First, the FID is digitally filtered and downsampled to several sub FIDs, and secondly, these sub FIDs are then modeled as sums of decaying sinusoids using the Bayesian approach. CRAFT tables can be used for further data mining of quantitative information using fingerprint chemical shifts of compounds of interest and/or statistical analysis of modulation of chemical quantity in a biological study (metabolomics) or process study (reaction monitoring) or quality assurance/control. The basic principles behind this approach as well as results to evaluate the effectiveness of this approach in mixture analysis are presented. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of the attached proton test technique in 31P NMR spectroscopy

Intensity modulated ³¹P NMR spectra were obtained using the pulse sequence published by Patt and Shoolery. This attached proton test (APT) technique for signal assignment could be applied to systems with long-range heteronuclear couplings in P? O? C? H fragments. In a model system derived from the alcoholysis of P₄S₁₀ the six reaction products were assigned to the six signals in the ³¹P NMR spectrum.     

Towards a Precise NMR Quantification of Acute Phase Inflammation Proteins from Human Serum

Nuclear Magnetic Resonance (NMR) spectra of human serum and plasma show, besides metabolites and lipoproteins, two characteristic signals termed GlycA and B arising from the acetyl groups of glycoprotein glycans from acute phase proteins, which constitute good markers for inflammatory processes. Here, we report a comprehensive assignment of glycoprotein glycan NMR signals observed in human serum, showing that GlycA and GlycB signals originate from Neu5Ac and GlcNAc moieties from N-glycans, respectively. Diffusion-edited NMR experiments demonstrate that signal components can be associated with specific acute phase proteins. Conventionally determined concentrations of acute phase glycoproteins correlate well with distinct features in NMR spectra (R² up to 0.9422, p-value <0.001), allowing the simultaneous quantification of several acute phase inflammation proteins. Overall, a proteo-metabolomics NMR signature of significant diagnostic potential is obtained within 10–20 min acquisition time. This is exemplified in serum samples from COVID-19 and cardiogenic shock patients showing significant changes in several acute phase proteins compared to healthy controls.     

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.     

免疫-遗传算法用于混合物重叠核磁共振信号解析

通过对免疫系统中抗体对外来抗原的识别、消除等过程的模拟,建立了一种新型的免疫算法模型.将标样信号作为抗体,混合物重叠信号作为抗原输入免疫算法模型,通过迭代运算,从抗原中消除抗体所表示的信息,当抗原被抗体完全消除时,即实现了混合物重叠信号的解析.对多组分混合氨基酸NMR谱图的解析结果证明,该算法可方便地用于多组分重叠信号的解析,为利用数据库解析混合物或生物大分子等物质的复杂NMR谱图开辟了一条全新的途径.     

Improved DECRA processing of DOSY data: correcting for non-uniform field gradients

Mixture analysis using PFG-NMR (DOSY) data is, for many chemists, a valuable and increasingly popular technique where the NMR signals of different species are separated according to their diffusion coefficients. Where NMR signals overlap, however, it is often difficult to extract the spectra of pure components from experimental data. In such situations, it can often be helpful to use multivariate methods, which exploit all the available signal covariance, to resolve the spectra of the components of a mixture. The best-established and by some way the quickest such method, DECRA (Direct Exponential Curve Resolution Algorithm), unfortunately requires that data conform to a pure exponential decay as a function of gradient strength squared, while experimental data typically deviate significantly from this. If this deviation is known, the performance of DECRA can be greatly improved for components with similar diffusion coefficients by adjusting the choice of gradient strengths used.     

Design considerations for a multiplexed spectrometer capable of simultaneous acquisition of multiple sample spectra

In order to make optimum use of expensive and vulnerable spectrometers in the industrial environment, it is desirable to obtain spectra from as many samples and/or spatially-separated sampling positions as possible using one spectrometer. This may be achieved by consecutive sampling; however, simultaneous sampling is possible if a suitable multiplexing scheme is devised. An outline design is presented for a multiplexed spectrometer capable of simultaneous acquisition of multiple sample spectra. Optical or electrical signals from an arbitrary number of individual samples are encoded by presenting them in various combinations to a single spectrometer or other signal detector. The switching of the signals may be dictated by the coefficients of the Hadamard S-matrix of the appropriate order; spectra of the individual samples may then be recovered by an inverse Hadamard transform. The method described, although general, is particularly suited to IR and Fourier transform Raman spectroscopy.     

Application of a new method for simultaneous phase and baseline correction of NMR signals (SINC)

Recently, we presented a new approach for simultaneous phase and baseline correction of nuclear magnetic resonance (NMR) signals (SINC) that is based on multiobjective optimization. The algorithm can automatically correct large sets of NMR spectra, which are commonly acquired when reactions and processes are monitored with NMR spectroscopy. The aim of the algorithm is to provide spectra that can be evaluated quantitatively, for example, to calculate the composition of a mixture or the extent of reaction. In this work, the SINC algorithm is tested in three different studies. In an in-house comparison study, spectra of different mixtures were corrected both with the SINC method and manually by different experienced users. The study shows that the results of the different users vary significantly and that their average uncertainty of the composition measurement is larger than the uncertainty obtained when the spectra are corrected with the SINC method. By means of a dilution study, we demonstrate that the SINC method is also applicable for the correction of spectra with low signal-to-noise ratio. Furthermore, a large set of NMR spectra that was acquired to follow a reaction was corrected with the SINC method. Even in this system, where the areas of the peaks and their chemical shifts changed during the course of reaction, the SINC method corrected the spectra robustly. The results show that this method is especially suited to correct large sets of NMR spectra and it is thus an important contribution for the automation of the evaluation of NMR spectra.     

Using indirect covariance processing for structure elucidation of small molecules in cases of spectral crowding

Indirect and unsymmetrical indirect covariance NMR provide powerful tools to compute and visualize correlation information by transforming component spectra into combined spectral data matrices. Sensitive component spectra such as TOCSY, HSQC and NOESY can be quickly converted into experimentally insensitive or time-consuming correlation spectra such as HSQC-NOESY. The comparison of illustrative series of spectra from four steroids, dexamethasone, testosterone, allylestrenol and tibolone, renders the effects of resonance overlap on the ease of interpretation visible. The compounds are selected such that signal overlap increases systematically in the proton and carbon domain. Spectra are defined as light, moderate and heavy signal overlap, based on signal density. The investigation suggests that moderate spectral congestion in either proton or carbon domain leads to a number of artifacts that does not hamper signal assignment but lowers the level of confidence on de novo structure elucidation. Since the number of correlations usually increases through covariance processing, component spectra with severe spectral congestion in both dimensions are not suitable for covariance processing and the resulting spectra do not support structure confirmation or structure elucidation. The calculated spectra are compared with the corresponding experimental spectra with respect to their application in structure elucidation laboratory environments.     

Acetate-methyl signals in the nuclear magnetic resonance spectra of peracetylated derivatives of some oligo- and polysaccharides

Peracetyl derivatives of oligo- and polysaccharides showed their own characteristic acetate-methyl signals in the NMR spectra measured in chloroform-d, which are useful for the identification and conformational analysis of oligo- and polysaccharides in solutions. An acetate-methyl signal in the lowest field among three acetate-methyl signals appeared in the NMR spectra of amylose acetate and was assigned to the acetate-methyl signal at C₆ on the basis of acetate-methyl signals of peracetylated derivatives of 6-O-tosylamylose and xylan. Monosaccharide moieties in oligo- and polysaccharides examined were found in the C1 (D ) conformation.