Novel determination of the total phenolic content in crude plant extracts by the use of H NMR of the –OH spectral region

A novel method for the determination of the total phenolic content using ¹H NMR spectroscopy in the –OH spectral region is presented. The use of DMSO-d₆, which is an aprotic and strongly hydrogen bonding solvent, allows the “appearance” of the relative sharp resonances of phenolic hydroxyl protons in the region of 8–14 ppm. The determination of the total phenolic –OH content requires three steps: (i) a 1D ¹H NMR spectrum is obtained in DMSO-d₆; (ii) a subsequent 1D ¹H NMR spectrum is recorded with irradiation of the residual water signal which results in the elimination or reduction of the phenolic –OH groups, due to proton exchange; and (iii) 1D ¹H NMR spectra are recorded with the addition of a progressively increased amount of salt, NaHCO₃, which results in extensive linebroadening of the COOH resonances thus allowing the discrimination of the phenolic from the carboxylic acid signals. Integration, with respect to the internal standard TSP-d₄, of the signal resonances between 14 and 8 ppm in spectrum (i) which are either eliminated or reduced in intensity in steps (ii) and (iii) allows the quantitation of the total phenolic content. The method was applied to model compounds, a mixture of them and several extracts of natural products. The results of the proposed ¹H NMR method were compared to the Folin-Ciocalteu (FC) reagent method. Additionally, since ¹H NMR refers to the total phenolic hydroxyl protons, a reaction factor, A_e, is proposed that corresponds to the hydroxyl reactivity. The ¹H NMR method is rapid and accurate bearing the inherent advantages of the NMR spectroscopy and can be applied directly in complex extracts. Furthermore, it can be applied in a wide range of matrixes from crude plant extracts and food products to biological samples.     

Post‐interface signal suppression,a phenomenon observed in a single‐stage Orbitrap mass spectrometer coupled to an electrospray interfaced liquid chromatograph

Signal suppression is a common issue when analyzing compounds by liquid chromatography coupled to mass spectrometry (LC/MS/MS). Suppression of signals is caused by co‐eluting matrix compounds and is thought to take place in the interface. This paper reports strong signal suppression effects which were observed when using a single‐stage Orbitrap instrument which was coupled by an electrospray interface to a liquid chromatograph. This type of signal suppression (often the complete loss of certain analyte signal) is observed in addition to signal suppression originating in the electrospray interface. The location of where this phenomenon occurs was shown to be clearly beyond the interface region. It was suspected that not the Orbitrap cell itself, but the C‐trap, which is an integral part within the Orbitrap instrument, was the probable location. Such post‐interface signal suppression was observed – and could be experimentally induced – when multiply charged ions (e.g. electrospray protonated proteins) were co‐eluting with the analytes. High concentrations of proteins, yet not exceeding the maximum ion capacity of the trap, can cause a complete loss of all low m/z masses. This paper describes the practical implication when analyzing heavy matrix samples and discusses strategies to reduce such detrimental effects. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of metabolites in coriander seeds (Coriandrum Sativum L.) aided by ultrahigh resolution total correlation NMR spectroscopy

NMR is a fast method for obtaining a holistic snapshot of the metabolome and also offers quantitative information without separating the compounds present in a complex mixture. Identification of the metabolites present in a plant extract sample is a crucial step for all plant metabolomics studies. In the present work, we used various two dimensional (2D) NMR methods such as J-resolved NMR, total correlation spectroscopy (TOCSY), and heteronuclear single quantum coherence sensitivity enhanced NMR spectroscopy for the identification of 36 common metabolites present in Coriandrum sativum L. seed extract. The identified metabolites belong to the following classes: organic acids, amino acids, and carbohydrates. ¹H NMR spectra of such complex mixtures in general display tremendous signal overlap due to the presence of a large number of metabolites with closely resonating multiplet signals. This signal overlapping leads to ambiguity in an assignment, and hence, identification of metabolites becomes tedious or impossible in many cases. Therefore, the utility of pure-shift proton spectrum along the indirect (F₁) dimension of the F₁-PSYCHE-TOCSY spectrum is demonstrated for overcoming ambiguity in assignment of metabolites in crowded spectral regions from Coriandrum sativum L. seed extract sample. Because pure-shift NMR methods yield ultrahigh resolution spectrum (i.e., a singlet peak per chemical site) along one or more dimensions, such spectra provide better identification of metabolites compared with regular 2D TOCSY where signal overlap and peak distortions lead to ambiguity in the assignment. Nine metabolites were unambiguously assigned by pure-shift F₁-PSYCHE-TOCSY spectrum, which was unresolved in regular TOCSY spectrum.     

Reaction control in radical polymerization of di‐n‐butyl itaconate utilizing a hydrogen‐bonding interaction

We have reported that intramolecular chain‐transfer reaction takes place in radical polymerization of itaconates at high temperatures and/or at low monomer concentrations. In this article, radical polymerizations of di‐n‐butyl itaconate (DBI) were carried out in toluene at 60 °C in the presence of amide compounds. The ¹³C‐NMR spectra of the obtained poly(DBI)s indicated that the intramolecular chain‐transfer reaction was suppressed as compared with in the absence of amide compounds. The NMR analysis of DBI and N‐ethylacetamide demonstrated both 1:1 complex and 1:2 complex were formed at 60 °C through a hydrogen‐bonding interaction. The ESR analysis of radical polymerization of diisopropyl itaconate (DiPI) was conducted in addition to the NMR analysis of the obtained poly(DiPI). It was suggested that the suppression of the intramolecular chain‐transfer reaction with the hydrogen‐bonding interaction was achieved by controlling the conformation of the side chain at the penultimate monomeric unit of the propagating radical with an isotactic stereosequence. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4895–4905, 2004     

NMR‐based analysis of the chemical composition of Japanese persimmon aqueous extracts

Japanese persimmon (Diospyros kaki L.) is recognized as an outstanding source of biologically active compounds relating to many health benefits. In the present study, NMR spectroscopy provided a comprehensive metabolic overview of Japanese persimmon juice. Detailed signal assignments of Japanese persimmon juice were carried out using various 2D NMR techniques incorporated with broadband water suppression enhanced through T₁ effects (BB‐WET) or WET sequences, and 26 components, including minor components, were identified. In addition, most components were quantitatively evaluated by the integration of signals using conventional ¹H NMR and BB‐WET NMR. This is the first detailed analysis combined with quantitative characterization of chemical components using NMR for Japanese persimmon. Copyright © 2015 John Wiley & Sons, Ltd.     

Novel determination of the total phenolic content in crude plant extracts by the use of 1H NMR of the -OH spectral region

A novel method for the determination of the total phenolic content using (1)H NMR spectroscopy in the -OH spectral region is presented. The use of DMSO-d(6), which is an aprotic and strongly hydrogen bonding solvent, allows the "appearance" of the relative sharp resonances of phenolic hydroxyl protons in the region of 8-14 ppm. The determination of the total phenolic -OH content requires three steps: (i) a 1D (1)H NMR spectrum is obtained in DMSO-d(6); (ii) a subsequent 1D (1)H NMR spectrum is recorded with irradiation of the residual water signal which results in the elimination or reduction of the phenolic -OH groups, due to proton exchange; and (iii) 1D (1)H NMR spectra are recorded with the addition of a progressively increased amount of salt, NaHCO(3), which results in extensive linebroadening of the COOH resonances thus allowing the discrimination of the phenolic from the carboxylic acid signals. Integration, with respect to the internal standard TSP-d(4), of the signal resonances between 14 and 8 ppm in spectrum (i) which are either eliminated or reduced in intensity in steps (ii) and (iii) allows the quantitation of the total phenolic content. The method was applied to model compounds, a mixture of them and several extracts of natural products. The results of the proposed (1)H NMR method were compared to the Folin-Ciocalteu (FC) reagent method. Additionally, since (1)H NMR refers to the total phenolic hydroxyl protons, a reaction factor, A(e), is proposed that corresponds to the hydroxyl reactivity. The (1)H NMR method is rapid and accurate bearing the inherent advantages of the NMR spectroscopy and can be applied directly in complex extracts. Furthermore, it can be applied in a wide range of matrixes from crude plant extracts and food products to biological samples.     

Preparative three-dimensional GC and nuclear magnetic resonance for the isolation and identification of two sesquiterpene ethers from Dictyota Dichotoma

Separation science plays a crucial role in the isolation of novel compounds contained in complex matrices. Yet their rationale employment needs preliminary structure elucidation, which usually requires sufficient aliquots of grade substances to characterize the molecule by nuclear magnetic resonance experiments. In this study, two peculiar oxa-tricycloundecane ethers were isolated by means of preparative multidimensional gas chromatography from the brown alga species Dictyota dichotoma (Huds.) Lam., aiming to assign their 3D structures. Density functional theory simulations were carried out to select the correct configurational species matching the experimental NMR data (in terms of enantiomeric couples). In this case, the theoretical approach was crucial as the protonic signal overlap and spectral overcrowding were preventing any other unambiguous structural information. Just after the identification through the density functional theory data matching of the correct relative configuration it was possible to verify an enhanced self-consistency with the experimental data, confirming the stereochemistry. The results obtained further pave the way toward structure elucidation of highly asymmetric molecules, whose configuration cannot be inferred by other means or strategies.     

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.     

The Silicides M4Si4 with M = Na,K, Rb,Cs, and Ba2Si4 – NMR Spectroscopy and Quantum Mechanical Calculations

The Zintl phases M₄Si₄ with M = Na, K, Rb, Cs, and Ba₂Si₄ feature a common structural unit, the Si₄^4– anion. The coordination of the anions by the cations varies significantly. This allows a systematic investigation of the bonding situation of the anions by ²⁹Si NMR spectroscopy. The compounds were characterized by powder X‐ray diffraction, differential thermal analysis, magnetic susceptibility measurements, ²³Na, ²⁹Si, ⁸⁷Rb, ¹³³Cs NMR spectroscopy, and quantum mechanical calculation of the NMR coupling parameter. The chemical bonding was investigated by quantum mechanical calculations of the electron localizability indicator (ELI). Synthesis of the compounds results for all of them in single phase material. A systematic increase of the isotropic ²⁹Si NMR signal shift with increasing atomic number of the cations is observed by NMR experiments and quantum mechanical calculation of the NMR coupling parameter. The agreement of experimental and theoretical results is very good allowing an unambiguous assignment of the NMR signals to the atomic sites. Quantum mechanical modelling of the NMR shift parameter indicates a dominant influence of the cations on the isotropic ²⁹Si NMR signal shift. In contrast to this a negligible influence of the geometry of the anions on the NMR signal shift is obtained by these model calculations. The origin of the systematic variation of the isotropic NMR signal shift is not yet clear although an influence of the charge transfer estimated by calculation using the QTAIM approach is indicated.     

Application of automated eightfold suppression of water and ethanol signals in 1H NMR to provide sensitivity for analyzing alcoholic beverages

The 400 MHz (1)H NMR analysis of alcoholic beverages using standard pulse programs lacks the necessary sensitivity to detect minor constituents such as methanol, acetaldehyde or ethyl acetate. This study investigates the application of a shaped pulse sequence during the relaxation delay to suppress the eight (1)H NMR frequencies of water and ethanol (the OH singlet of both water and ethanol, as well as the CH(2) quartet and CH(3) triplet of ethanol). The sequence of reference measurement for frequency determination followed by the suppression experiment is controlled by a macro in the acquisition software so that a measurement under full automation is possible (12 min per sample total time). Additionally, sample preparation was optimized to avoid precipitation, which is facilitated by 1:1 dilution with ethanol and pH 7.4 buffer. Compared with the standard water presaturation pulse program, the eightfold suppression allowed a significantly higher setting of receiver gain without receiver overflow, which significantly increased the signal-to-noise ratio by an average factor of 10. The signal intensities increased by a factor of 20. The resulting limits of detection (below 1 g/hl of pure alcohol) now allow the control of legal requirements for minor compounds in alcoholic beverages.     

Enantiomeric NMR signal separation mechanism and prediction of separation behavior for a praseodymium (III) complex with (S,S)-ethylenediamine-N,N-disuccinate

Because choice of chiral nuclear magnetic resonance (NMR) shift reagents and concentration conditions have been made empirically by trials and errors for chiral NMR analyses, the prediction of NMR signal separation behavior is an urgent issue. In this study, the separation of enantiomeric and enantiotopic ¹H and ¹³C NMR signals for α-amino acids and tartaric acid was performed by using the praseodymium(III) complex with (S,S)-ethylenediamine-N,N′-disuccinate ((S,S)-EDDS). All the present D-amino acids exhibited larger downfield shift of their α-protons and α-carbons compared with those for the corresponding L-amino acids in common. This regularity is applicable to absolute configurational assignment and determination of optical purity of amino acids. The chemical shifts of β-protons of d - and l -alanine fully bound with the Pr(III) ((S,S)-EDDS) complex (δ_bs) and the adduct formation constants of both enantiomers (Ks) were obtained by dependences of the observed downfield shifts of the β-protons on the total concentrations of the respective enantiomers in the presence of a constant concentration of the Pr(III) complex. The difference in the K values was found to be predominant determining factor for the enantiomeric signal separation. The chemical shifts of both enantiomers (δs) and the enantiomeric signal separations (Δδs) under given conditions could be calculated from the δ_b and K values. Furthermore, prediction of the signal separation behavior was enabled by using the calculated δ values and the signal broadening obtained by dependences of the half-height widths of the observed signals on the bound/free substrate concentration ratios for the respective enantiomers.     

Identification of complex structures of paintings on canvas by NMR: Correlation between NMR profile and stratigraphy

Paintings on canvas are complex structures created by superimposing layers of different composition. Investigations on the structure of these artworks can provide essential information on their state of conservation, pictorial technique, possible overpaintings, and in planning a proper conservation plan. Standard methods of investigation consist in sampling a limited number of fragments for stratigraphic analyses. Despite the recognized validity of these methods, they are affected by evident limitations. Nuclear magnetic resonance (NMR) profiling, often named NMR stratigraphy, is an NMR relaxometry technique applied by single-sided portable devices developed to overcome the disadvantages of microinvasive stratigraphic analyses. The potential of this approach on artworks, including wall paintings and a few examples of painted canvas, is described in the literature. In this study, NMR profiles of painting on canvas were examined by analyzing transverse relaxation time data by T₂ quasi-continuous distributions and the results compared with standard stratigraphic cross-sections analysis. Combining signal intensity and T₂ quasi-continuous distributions, the identification of textile, preparatory, and paint layers was enhanced. The diction “NMR stratigraphy” for these inhomogeneous layered artworks is also discussed. Indeed, unlike the stratigraphic cross-sections, NMR profiles provide information on a volume (flat slice), rather than on a surface, and the collected signal can derive from nonuniform and partially overlapping layers. This study paves the way for extensive investigations on relaxation time quasi-continuous distributions in various binder/pigment mixtures in order to improve the reliability of NMR profile as an innovative, non–invasive, and nondestructive method for analyzing paintings on canvas.     

NMR Characterization of Complex p‐Oligophenyl Scaffolds by Means of Aliasing Techniques to Obtain Resolution‐Enhanced Two‐Dimensional Spectra

The usefulness of computer‐assisted aliasing to secure maximal resolution of signal clusters in ¹H‐ and ¹³C‐NMR spectra (which is essential for structure determination by HMBC 2D NMR spectroscopy) in minimal acquisition time is exemplified by the complete characterization of the two complementary p‐octiphenyls 1 and 2 with complex substitution patterns. The need for digital resolution near 1 Hz/pt to dissect the extensive signal clusters in the NMR spectra of these refined oligomers excluded structure determination under routine conditions. High resolution was secured by exploiting the low signal density in the ¹³C dimension of HMBC spectra by using computer‐assisted aliasing to maximize signal density. Based on the observed shifts in DEPT and ¹H‐decoupled ¹³C‐NMR spectra of 1 and 2 , computer‐assisted aliasing allowed to reduce the number of required time increments by a factor of 20 to 30 compared to full‐width spectra with identical resolution. Without signal‐to‐noise constraints, this computer‐assisted aliasing reduced the acquisition time for high‐resolution NMR spectra needed for complete characterization of refined oligomers 1 and 2 by the same factor (e.g., from over a day to about an hour). With resolved signal clusters in fully aliased HSQC and HMBC spectra, unproblematic structure determination of 1 and 2 is demonstrated by unambiguous assignment of all C‐ and H‐atoms. These findings demonstrate that computer‐assisted aliasing of the underexploited ¹³C dimension makes extensive molecular complexity accessible by conventional multidimensional heteronuclear NMR experiments without extraordinary efforts.     

Effect of liquid chromatography separation of complex matrices on liquid chromatography-tandem mass spectrometry signal suppression

The effect of liquid chromatography separation on liquid chromatography-tandem mass spectrometry (LC-MS-MS) signal response for the characterization of low-molecular-mass compounds in a complex matrix was investigated. Matrix induced signal suppression appears throughout the entire LC-MS-MS analysis of wheat forage extract, with greatest suppression occurring at early retention times. Experimental results show that co-elution of matrix components and analytes from the LC column may be most strongly attributed to column overloading rather than similar analyte and matrix retention behavior. As a result, two-dimensional (LC-LC) separation can be a highly effective approach to address signal suppression effects for the quantitative LC-MS-MS analysis of complex matrix samples.     

Gem-diol Generation in Copper and Zinc N-methyl-2-imidazolecarboxaldehyde Complexes: Solid-state NMR,EPR and Single-Crystal X-ray Studies

Novel coordination compounds, mono- ( 1 ) and binuclear copper complexes ( 2 ) and a zinc complex ( 3 ) were synthesized and studied through single-crystal X-ray crystallography, solid-state NMR and EPR techniques to determine the chemical functionalization of the carbonyl group in N-methyl-2-imidazolecarboxaldehyde ligand. Particularly, molecules containing carbonyl groups are versatile ligands that give rise to a wide range of new materials due to the high reactivity of the carbonyl group. However, the chemical identification of the functional group present in these coordination compounds is a challenge because the copper ion affects the NMR signals. In this sense, X-ray crystallography becomes an indispensable tool for the analysis. The imidazole ligands in copper complexes 1 and 2 were found to be the aldehyde and the gem-diol forms, respectively. Furthermore, the gem-diol and carboxylate moieties were detected in the crystal lattice for the zinc complex 3 and studied by solution- and solid-state NMR.     

Detection of Potential TNA and RNA Nucleoside Precursors in a Prebiotic Mixture by Pure Shift Diffusion‐Ordered NMR Spectroscopy

In the context of prebiotic chemistry, one of the characteristics of mixed nitrogenous‐oxygenous chemistry is its propensity to give rise to highly complex reaction mixtures. There is therefore an urgent need to develop improved spectroscopic techniques if onerous chromatographic separations are to be avoided. One potential avenue is the combination of pure shift methodology, in which NMR spectra are measured with greatly improved resolution by suppressing multiplet structure, with diffusion‐ordered spectroscopy, in which NMR signals from different species are distinguished through their different rates of diffusion. Such a combination has the added advantage of working with intact mixtures, allowing analyses to be carried out without perturbing mixtures in which chemical entities are part of a network of reactions in equilibrium. As part of a systems chemistry approach towards investigating the self‐assembly of potentially prebiotic small molecules, we have analysed the complex mixture arising from mixing glycolaldehyde and cyanamide, in a first application of pure shift DOSY NMR to the characterisation of a partially unknown reaction composition. The work presented illustrates the potential of pure shift DOSY to be applied to chemistries that give rise to mixtures of compounds in which the NMR signal resolution is poor. The direct formation of potential RNA and TNA nucleoside precursors, amongst other adducts, was observed. These preliminary observations may have implications for the potentially prebiotic assembly chemistry of pyrimidine threonucleotides, and therefore of TNA, by using recently reported chemistries that yield the activated pyridimidine ribonucleotides.     

Resolving NMR signals of short‐chain fatty acid mixtures using unsupervised component analysis

Nuclear magnetic resonance (NMR) is a very powerful instrumental technique suited to identify and characterize organic compounds. NMR has been successfully used in the analysis of complex biological and environmental samples; however, these applications are still rather limited. In this work, we describe unsupervised component analysis as a multivariate unsupervised method suited to identify the number of relevant NMR signal contributions and to deconvolve mixed signals into signal individual sources and respective contributions. Using this approach, we were able to advance further in the field of quantification of NMR spectra, and this methodology will help in the characterization of complex biological samples. Copyright © 2017 John Wiley & Sons, Ltd.     

31P NMR assessment of the phosvitin-iron complex in mayonnaise

Lipid oxidation is the main reason for the limited shelf life of mayonnaise. One of the main catalysts of this process is iron, which is introduced in its ferric (Fe(III)) form via phosvitin, an egg yolk phosphoprotein rich in phosphoserines. The binding of Fe(III) to phosvitin and its ability to establish a redox couple with Fe(II) is believed to determine the oxidation rate of unsaturated lipids. In this work, a ³¹P NMR based method was developed to quantify loading of phosvitin with Fe(III) and its reductive release. Both features could be quantified in model phosvitin solutions by exploiting the paramagnetic broadening of ³¹P NMR signal of phosphoserine residues by Fe(III). This method was then successfully applied to quantify the phosvitin-Fe(III) loading in mayonnaise water phase by liquid NMR, whereas ³¹P NMR MAS could only provide a qualitative measure. The ³¹P NMR method showed a direct relation between loading of the Fe(III)-phosvitin complex and lipid oxidation.     

Solution and solid state13C-NMR of barley straw lignins

Grass lignins are formed by the polymerization of phenoxy radicals and contain a variety of carbon-carbon and carbon-oxygen bonds. They are similar to the hardwood lignins, but differ by containing a substantial proportion of esterified cinnamic acids. Detailed nuclear magnetic resonance studies in conjunction with chemical analysis have given new information on the structure of grass lignins. Milled straw lignins (MSL) from barley were examined by both solution and solid-state (CP/MAS) NMR before and after acetylation. The assignment of the carbon-13 (100 MHz) solution spectra was achieved using model compound data, nuclear Overhauser enhancement (NOE) suppression, and insensitive nuclei enhanced by polarization transfer (INEPT) techniques. The NOE suppression permitted quantitative analysis of lignin, giving information on the ratio of specific carbon atoms. Use of the relaxation agent, chromium acetylacetonate, enabled accumulation of sufficient spectral data to give a spectrum suitable for integration after 90 h. The INEPT technique, which had not previously been used for lignin analysis, was successfully applied to acetylated MSL. This technique increased signal intensities 3–4-fold and simplified the spectrum by inverting methylene carbons and eliminating or inverting quaternary carbons. Comparison of this spectrum with the normal spectrum permitted accurate assignment of quaternary and methine carbons. The solid-state carbon-13 CP/MAS NMR was used to examinein situ lignin and the isolated MSL. The¹³C-CP/MAS spectrum ofin situ lignin shows that cellulose and hemicellulose resonances dominate with little evidence ot the aromatic structure of lignin. the¹³C-CP/MAS of MSL shows reduced carbohydrate resonances and increased aromatic resonances. The extent of modification to the barley straw was estimated and results indicate the presence oflignin-carbohydrate complexes. Detailed information on the nature of the linkages between lignin components and between lignin and carbohydrate components has been obtained from these spectra.