Article pubs.acs.org/ac
Ultrahigh-Resolution NMR Spectroscopy for Rapid Chemical and Biological Applications in Inhomogeneous Magnetic Fields Yuqing Huang, Shuohui Cao, Yu Yang, Shuhui Cai, Haolin Zhan, Chunhua Tan, Liangjie Lin, Zhiyong Zhang, and Zhong Chen* Department of Electronic Science, State Key Laboratory of Physical Chemistry of Solid Surfaces, Xiamen University, Xiamen, Fujian 361005, China S Supporting Information *
ABSTRACT: NMR spectroscopy is a commonly used analytical technique in practical applications, and its applicability is further promoted by pure chemical shift techniques based on spectral simplification for analyses. Unfortunately, magnetic field inhomogeneity caused by adverse experimental conditions remains an obstacle restricting NMR applications. In this study, we introduce a new NMR method for high-resolution pure shift proton (1H) NMR measurements in inhomogeneous magnetic fields. We demonstrate that the method allows one to perform chemical analyses on complex solutions in deshimmed magnetic fields, to obtain metabolite information on intact biological tissues with intrinsic field inhomogeneities and to achieve in situ electrochemical detection under externally adverse field conditions. This approach is readily implemented on common commercial NMR instruments without field shimming and locking procedures, specialized hardware requirements as well as complicated sample pretreatments. It provides an effective tool for NMR applications to high-resolution chemical and biological measurements under inhomogeneous magnetic field conditions.
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pure shift NMR strategies have been proposed.8−16 From classical techniques (e.g., 2D J-resolved experiment8 and constant-time decoupling evolution9) to current mainstream approaches (e.g., bilinear rotation scheme,10 Zangger-Sterk strategy,11 and PSYCHE strategy12), researchers have spent massive effort to continuously refine 1H pure shift NMR, aiming for a perfect approach that fulfills easy experiment setting, simple data processing, high sensitivity, reliable quantitative analysis, and flexible sequence combination. According to individual features, existing 1H pure shift NMR approaches have found applications on enhancing spectral resolution and simplifying spectral analyses.17,18 Except for basic 1D versions, pure shift 1H NMR is also extended to other spectroscopic types, such as 2D J-resolved spectroscopy,19 TOCSY,20 DOSY,21 and HSQC.22 It is worth noting that favorable performances of these 1H pure shift NMR approaches generally rely on homogeneous magnetic fields. In addition to JHH couplings, magnetic field inhomogeneity constitutes another factor lowering spectral resolution in 1H NMR applications. Field inhomogeneity gives rise to spectral line broadening and directly distorts spectral information,23,24 which is even more serious than spectral congestion caused by JHH couplings. The development in advanced magnets and the implementation of novel field shimming25 and deuteriumsolvent locking26 techniques can guarantee high-degree field
uclear magnetic resonance (NMR) spectroscopy presents a powerful tool for revealing structural and dynamic properties of molecules, and it has been widely applied in fields of chemistry, structural biology and life science.1−3 In general NMR measurements, spectral resolution and sensitivity constitute two key indexes determining the usability of acquired spectra. Along with the continuous development of NMR hardware (e.g., stronger superconducting magnet4) and experimental techniques (e.g., spin hyperpolarization5), sensitivity has been greatly improved in modern NMR. In comparison, enhancement on spectral resolution by virtue of advanced NMR hardware techniques remains inapparent, particularly in proton (1H) NMR spectroscopy. Under commercial NMR spectrometers available today, spectral congestion is generally observed in conventional 1D 1H NMR applications due to limited chemical shift ranges and appended scalar coupling (JHH) splittings. This phenomenon becomes worse in measurements on complex chemical and biological samples containing abundant compositions and extensive JHH couplings.6 The JHH couplings, mediated through chemical bonds among atoms, provide information for structural elucidation and conformation determination. However, it also induces peak splittings that lead to low spectral resolution in 1H NMR spectra. A 1H NMR technique, called “pure shift NMR spectroscopy”, emerges for eliminating JHH couplings and for achieving remarkable spectral resolution enhancement with only chemical shift information present.7 On the basis of the pulse sequence design and related data processing, numerous © 2017 American Chemical Society
Received: March 21, 2017 Accepted: June 5, 2017 Published: June 5, 2017 7115
DOI: 10.1021/acs.analchem.7b01036 Anal. Chem. 2017, 89, 7115−7122
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Analytical Chemistry homogeneity for 1H pure shift NMR experiments on homogeneous liquid samples. However, there exist adverse experimental conditions in which magnetic fields suffer from spatial or temporal inhomogeneity,27,28 for example, measurements on biological tissues with intrinsic magnetic susceptibility variations and on in situ electrochemistry samples containing heterogeneous electrodes within the detected volume. It is generally difficult to circumvent these field inhomogeneities by routine field shimming and locking approaches. Under these situations, existing 1H pure shift NMR generally faces the challenge on extracting the desired spectral information for analyses. Therefore, a great demand for an NMR method available for high-resolution 1H pure shift measurements in inhomogeneous magnetic fields caused by adverse experimental conditions has arisen. In this study, we present an NMR method, dubbed as UPSIF (Ultrahigh-resolution Pure Shifts in Inhomogeneous Fields), to achieve the aforementioned demand. The UPSIF method is designed based on the combination of intermolecular zeroquantum coherence (iZQC)29 and constant-time decoupling9 schemes to overcome influences of magnetic field inhomogeneity and JHH couplings. It has been proven that iZQC, evolving at the frequency difference of a dipole−dipole coupling spin pair, are intrinsically immune to field inhomogeneity.30,31 The constant-time decoupling scheme presents a simple way to collapse JHH couplings and preserve high decoupling signal intensity. Moreover, iZQC and constant-time schemes are implemented based on 2D acquisition, thereby achieving a perfect fusion and sharing the same indirect evolution period. For data processing, we apply the DMUSIC (Damped MUltiple SIgnal Classification) algorithm to resolve the resolution limitation caused by the constant-time evolution.32 In addition, we introduce the fold-over correction (FOC) scheme14 into the UPSIF method to accelerate 2D acquisition for rapid measurements by reducing the spectral width of the indirect dimension. In addition, we extend the 1D UPSIF to its 2D J-resolved version, UPSIF-2DJ, by simply substituting the echo-train acquisition module33 for the direct acquisition period of the UPSIF sequence. The UPSIF-2DJ can retrieve JHH couplings orthogonally added to each chemical shift site and yield high-resolution 2D J-resolved spectra free of 45° spectral shearing in inhomogeneous fields. The UPSIF and its extended version UPSIF-2DJ are readily implemented for highresolution measurements in common commercial NMR instruments without tedious field shimming and locking procedures, specialized hardware requirements (e.g., magicangle spinning device) as well as complicated sample pretreatments (e.g., sample extraction and deuterium solvent injection).
Figure 1. Pulse sequence diagram for UPSIF and UPSIF-2DJ. The signal excitation part and acquisition part are shown in the blue box and the purple box, respectively. The iZQC and constant-time schemes are combined into the evolution period t1. UPSIF and UPSIF2DJ share the same signal excitation part, and they break up in the signal acquisition part. Standard direct acquisition is used for UPSIF and echo-train acquisition is utilized for UPSIF-2DJ. Full vertical bars indicate nonselective π/2 and π pulses, Gaussian shaped pulses represent solvent-selective (π/2)I pulse, dashed rectangles represent coherence selection gradient Gz, and “SS” indicates optional solvent suppression module. Related pulse sequence codes are given in Supporting Information.
signal evolution in the indirect evolution period t1. The selected iZQC term evolves at the frequency difference between solvent and solute spins, resulting in the elimination of field inhomogeneity in the t1 period. To collapse JHH couplings, we introduce the constant-time decoupling scheme into the t1 period, which is made up of a hard π pulse and two intervals t1/ 2 and τ − t1/2 (τ ≥ t1max/2, where τ is a constant time). It is clear that a 1D spectrum free of inhomogeneous line broadening and JHH coupling splitting can be extracted from the indirect dimension. After the signal evolution in the t1 period, the solvent-selective (π/2)I pulse transfers the solvent magnetization into the z direction to generate the desired distant dipolar field (DDF) which convents the iZQC term I+S− into the observable term S− for signal acquisition. A spin echo module (ΔπΔ, where Δ is a constant interval) is applied to adjust the action time of the DDF and prevent signals from dephasing by field inhomogeneity before signal acquisition. The optional SS module, consisting of two W5 binomial pulses acting as solvent-exclusive π pulses and their suited crusher gradients added bilaterally, is used to suppress the solvent signal.34 Once the desired signal is generated from the signal excitation part, it is ready for acquisition by the signal acquisition part. For the UPSIF sequence, the generated signal is directly acquired in the acquisition period t2, yielding a standard 2D spectrum after direct Fourier transform. A 1D spectrum free of effects of field inhomogeneities and JHH couplings is accessible along the indirect dimension (F1). In principle, full chemical shift range should be covered along the F1 dimension and numerous t1 increments are required for satisfactory resolution, which would lead to long acquisition time. The FOC scheme is applied to convert the F1 dimension to be only related to the field inhomogeneity range, thus greatly accelerating 2D acquisition of UPSIF experiments. To circumvent the resolution limitation of residual line broadening caused by the constant-time evolution, we further combine the DMUSIC algorithm into data processing to achieve desired 1D 1 H pure shift spectra. For the UPSIF-2DJ sequence extended from the 1D UPSIF, the signal acquisition part is switched to the echo-train acquisition module, consisting of a series of acquisition periods t2 and π pulses. In the echo-train acquisition, sequentially acquired signals form a complete spin echo evolution with an increment of t2max (the maximum time of a single acquisition t2 unit) and yield JHH couplings along an additional dimension, analogous to the second indirect evolution dimension (F3). Different from standard incremental
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EXPERIMENTAL METHODS Ultrahigh-Resolution NMR Methods in Inhomogeneous Magnetic Fields. Pulse sequences for UPSIF method and UPSIF-2DJ are shown in Figure 1. The UPSIF and UPSIF2DJ sequences share the same signal excitation part (marked by blue box in Figure 1) and break up in the signal acquisition part (marked by purple box in Figure 1). In the signal excitation part, four modules of iZQC, constant-time, spin echo, and solvent suppression (SS) are included. The sequences start with a nonselective π/2 pulse for exciting both solvent and solute spins. Under the effect of the coherence selection gradient Gz in the iZQC module, only the iZQC spin term, I+S− (I and S denote solvent and solute spins, respectively), is selected for 7116
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Figure 2. High-resolution 1H NMR measurements on a mimic brain phantom. Measurements in a well-shimmed homogeneous field, including (a) standard 1D single-pulse NMR spectrum expanded with metabolite peaks in the spectral region between 1.14 and 4.58 ppm; (b) 1D PSYCHE spectrum without JHH couplings; (c) standard water-presaturated 2D J-resolved spectrum and its 1D projection along the F2 dimension. Measurements in a deshimmed inhomogeneous field with 250 Hz line broadening, including (d) standard 1D water-presaturated spectrum; (e) 1D UPSIF spectrum presenting high-resolution pure shift NMR; (f) high-resolution 2D J-resolved spectrum obtained from the UPSIF-2DJ experiment. Peak assignments of metabolites are given in panels a, b, and e.
(Figure S2) are given. We further test UPSIF and UPSIF-2DJ on a mimic brain phantom that contains various complex chemical compositions (Figure 2). Under the well-shimmed field with the full width at half-maximum (fwhm) of water signal of 2.5 Hz, numerous peaks of brain phantom compositions are observed in the expanded region from 1.20 to 4.60 ppm in the standard 1D single-pulse spectrum (Figure 2a). Because of sample complexity and extensive JHH coupling splittings, spectral congestion and even peak overlap are observed, for example, Glu/Gln and NAS around 2.05 ppm. With the aid of collapsing JHH couplings by the PSYCHE experiment,12 the peak discriminability is greatly improved and all 28 peaks are assigned to 11 chemical compositions in the resulting 1D PSYCHE spectrum (Figure 2b). Measurements on JHH coupling constants and multiplet structures are available in the water-presaturated 2D J-resolved spectrum (Figure 2c). However, when the field homogeneity is degraded to 250 Hz inhomogeneous broadening, spectral information conveyed by the standard 1D single-pulse spectrum is concealed and only several broad peak envelopes are observed (Figure 2d). In contrast, a high-resolution 1D pure shift NMR spectrum displayed in absorption Lorentzian line shape is recovered by the UPSIF experiment from the same inhomogeneous field (Figure 2e). Compared to the standard 1D spectrum (Figure 2d), spectral line width in the 1D UPSIF spectrum (Figure 2e) is narrowed from 250 to 1.9 Hz. Similar to the 1D PSYCHE spectrum (Figure 2b), multiplets are collapsed into singlets in the 1D UPSIF spectrum, presenting an efficient manner for complex composition analyses. Most compositions assigned in the 1D UPSIF spectrum in the inhomogeneous field are the same as those observed in the 1D PSYCHE spectrum in the well-shimmed field. Because of the complexity of chemical compositions and the setting limitation on constant-time τ, some highly adjacent peaks such as m-I at 3.22 and 4.17 ppm, and the obvious differences in line width of the peak at 2.97 ppm cannot be recognized in the resulting 1D UPSIF spectrum. Furthermore, the UPSIF-2DJ provides a high-resolution 2D J-
evolution, the spin echo evolution in the echo-train acquisition period is achieved by a single magnetization excitation, thus UPSIF-2DJ experiments hold similar acquisition efficiency as UPSIF experiments. By combining pure chemical shifts and JHH couplings orthogonally added to each chemical shift site, the desired high-resolution 2D J-resolved spectrum free of field inhomogeneity is obtained. Experiments. Three types of samples, (a) solution mixtures in deshimmed inhomogeneous fields, (b) biological samples with intrinsic field inhomogeneities, and (c) an in situ electrochemical reaction solution under externally adverse field conditions, were adopted to illustrate the applicability of UPSIF and UPSIF-2DJ on extracting high-resolution NMR for chemical and biological analyses in inhomogeneous magnetic fields. Detailed experimental settings are given in Supporting Information. NMR Data Processing. The processing procedure for UPSIF data includes 2D Fourier transform, FOC shearing, 1D projection, and DMUSIC processing. The processing procedure for UPSIF-2DJ data includes 3D data reconstruction, 3D Fourier transform, FOC shearing, 2D projection, and DMUSIC processing. The full details of the data processing are given in Supporting Information. All data processing is carried out by using a custom-written program on MATLAB 7.8 (MathWorks, Natick, MA). Related data processing codes for UPSIF and UPSIF-2DJ are given in Supporting Information.
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RESULTS Solution Mixtures under Deshimmed Inhomogeneous Magnetic Fields. To demonstrate implementation details of UPSIF and UPSIF-2DJ methods and their feasibilities on extracting high-resolution pure shift NMR spectra from inhomogeneous magnetic fields, we first perform experiments on a simple solution of ethyl 3-bromopropionate and methanol in acetone under a deshimmed inhomogeneous field with 2500 Hz spectral broadening. Major data processing steps for UPSIF and UPSIF-2DJ experiments (Figure S1) and resulting spectra 7117
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Figure 3. High-resolution NMR results on grape samples. (a) Standard 1D water-presaturated spectrum of a piece of intact grape sarcocarp fitted into a 5 mm NMR tube. A photo of the grape sample is given. (b) High-resolution pure shift 1D spectrum acquired from the grape sarcocarp by the UPSIF experiment. (c) High-resolution 2D J-resolved spectrum of the grape sarcocarp by the UPSIF-2DJ experiment. (d) Standard 1D waterpresaturated spectrum of grape juice extracted from grape sarcocarp in a well-shimmed field. The crowded spectral region between 3.09 and 4.18 ppm is expanded in panels b, c, and d. Peak assignments of metabolites in grape are given in panels b and d.
Figure 4. In situ electrochemical NMR detection. (a) The schematic diagram of the electrochemical device on a sample of 200 mM aqueous solution of butyl alcohol. (b) Standard 1D water-presaturated spectra before and after the electrochemical reaction. (c) A series of high-resolution 1D UPSIF spectra acquired at different time points of electrochemical reaction from initial state to 6 h later, in situ recording the electro-oxidation process of butyl alcohol. The corresponding electrochemical reaction equation based on 1D UPSIF spectra is given on the top of (c).
mixture solution under random magnetic fields without tedious field shimming procedures. Biological Tissues with Intrinsic Field Inhomogeneity. In this section, UPSIF and UPSIF-2DJ methods are applied to a challenging case of grape sample, which suffers from intrinsic field inhomogeneity caused by magnetic susceptibility variations and complex metabolites with extremely congested spectral peaks. UPSIF and UPSIF-2DJ experiments directly on an intact grape sarcocarp fitted into a 5 mm NMR tube (as displayed in the photo of Figure 3a). Experiments are performed without any field shimming and locking because it is difficult to overcome the field inhomogeneity in biological samples by routine field shimming approaches. The resulting field inhomogeneity from intact grape sarcocarp is shown in the
resolved spectrum for JHH coupling analyses under the same inhomogeneous field (Figure 2f), similar to the standard waterpresaturated 2D J-resolved spectrum acquired from the wellshimmed field (Figure 2c). Both these two 2D J-resolved spectra are displayed in absolute-value mode. Compared to the 1D UPSIF spectrum (Figure 2e), the larger spectral width and the slight line shape distortion are observed on the 1D projection along the F2 dimension of the 2D J-resolved spectrum obtained by UPSIF-2DJ (Figure 2f). This is mainly due to parameter settings in the DMUSIC processing on UPSIF-2DJ data. Although field inhomogeneity in solution samples can be eliminated by routine shimming methods, UPSIF and UPSIF-2DJ methods enjoy an advantage of enhancing spectral resolution in applications to complex 7118
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distinguished for assignments. It can be seen that the original butyl alcohol (peaks marked by i to iv in Figure 4c) decreases gradually while new products appear and increase gradually during the electro-oxidation process. According to chemical assignments in resulting 1D UPSIF spectra, butyl alcohol is gradually oxidated to acetaldehyde (marked by v in Figure 4c), butyric acid (marked by vi, vii and viii in Figure 4c) and acetic acid (marked by ix in Figure 4c).41 Note that two adjacent peaks (marked by i and vi in Figure 4c) with only 12 Hz frequency difference can also be identified. These peak changes observed in resulting 1D UPSIF spectra can be explained by the reaction equation in the upper part of Figure 4c. Therefore, the UPSIF method may provide an opportunity to monitor in situ electrochemical reaction process under adverse experimental conditions, which is unexpected in standard NMR measurements.
standard 1D water-presaturated spectrum (Figure 3a), in which a broad peak envelope is observed in the spectral region between 3.09 and 4.18 ppm. The broad peak envelope contains numerous peaks from sugar components of the grape, whose detail information is ruined by the inhomogeneous broadening. By contrast, a high-resolution pure shift 1D spectrum is recovered by the UPSIF experiment (Figure 3b). Spectral resolution is significantly improved and multiplets are decoupled into singlets, facilitating metabolite assignments of grape tissues, even for the congested spectral region between 3.09 and 4.18 ppm. In addition, the UPSIF-2DJ experiment presents a 2D J-resolved spectrum for analyses of JHH coupled metabolites (Figure 3c). Although spectral resolution along the F2 dimension of the UPSIF-2DJ spectrum is lower than that in the 1D UPSIF spectrum, major metabolites are identified. From the expanded region marked in the dotted red box in Figure 3c, related multiplet structures and JHH coupling constants are available for analyses on the congested peak region of sugar components. Tissue extraction35 and magic-angle spinning technique36 are two classical solutions to circumvent intrinsic field inhomogeneity of biological samples for high-resolution NMR analyses. In this application, we adopt tissue extraction for comparison and perform a standard 1D water-presaturated experiment on a grape juice that is extracted from the same grape tissue under a well-shimmed field (Figure 3d). Most metabolites assigned in the 1D UPSIF spectrum from intact grape sarcocarp (Figure 3b) are the same as those obtained in standard 1D spectrum of grape tissue extraction (Figure 3d). To further show the superiority of UPSIF and UPSIF-2DJ in biological applications, we also perform the UPSIF experiment on intact pig brain tissue which has more intense water and lower metabolite concentrations (Figure S3). In Situ NMR Detection on Electrochemical Reaction Systems. Recently, NMR has been recognized as a useful tool for analyses on electrochemical reactions.37−39 However, homogeneity of the magnetic field would be greatly disturbed when an electrochemical device is placed inside the probe and the electric current is switched on, thus spectral resolution is reduced and NMR capability on in situ detecting reaction products and intermediates is undermined.40 Herein, we applied the UPSIF method to an in situ electrochemical reaction in the NMR spectrometer, showing its ability to overcome the unavoidable field inhomogeneity, caused by in situ electrochemical conditions, and recover desired highresolution 1D pure shift spectra for analyses (Figure 4). As shown in the schematic diagram of electrochemical device (Figure 4a), three electrodes, including (a) platinum black decorated carbon electrode as working electrode, (b) Pt wire as counter electrode, and (c) Ag/AgCl as reference electrode, were directly immersed into the butyl alcohol solution in a 5 mm NMR tube, locating inside the detection area of the probe. The magnetic field homogeneity is significantly degraded by the electrochemical device. It is generally difficult to compensate this field inhomogeneity by routine shimming techniques. Although careful field shimming has been performed, inhomogeneous line broadening up to 200 Hz remains in the standard 1D single-pulse spectra during the electro-oxidation process (Figure 4b). However, the UPSIF method can recover high-resolution NMR spectra under the same adverse experimental condition (Figure 4c). Compared to standard 1D single-pulse spectra (Figure 4b), the spectral resolution is greatly improved and the line width is reduced from 200 to 2.0 Hz in the resulting 1D UPSIF spectra. All peaks are well
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DISCUSSION In aforementioned experiments, we demonstrate the applicability of UPSIF and UPSIF-2DJ on high-resolution measurements under different inhomogeneous magnetic field conditions, such as those induced by the artificial detuning on spectrometer’s shimming coils in chemical solutions (Figure 2 and Figure S2), intrinsic magnetic susceptibility variations in biological samples (Figure 3 and Figure S3), and externally structural components in in situ electrochemical samples (Figure 4). In UPSIF and UPSIF-2DJ experiments, the mechanism of removing field inhomogeneity derives from the iZQC evolution, thus yielding the chemical-shift frequency difference between coupled solvent and solute spins.42 Under the inhomogeneous fields, the chemical-shift frequency difference from the iZQC evolution eliminate inhomogeneous broadening, namely, (ωI − ωS ± JHH), in which ωI and ωS denote chemical shifts of solvent and solute in samples, and JHH is coupling constant of solutes. In iZQC experiments, the solute corresponds to objective components (e.g., chemical compositions or metabolites), while the solvent refers to a single component (e.g., water) as a source of the DDF in transferring the iZQC signals into observable signals. When the spectrometer reference frequency is set to the resonant frequency of the solvent, that is, ωI = 0, a high-resolution 1D spectrum with chemical shifts and JHH couplings of solutes can be extracted along the indirect evolution dimension, namely (−ωS ± JHH). To eliminate JHH coupling splittings for pure shift NMR, we introduce the constant-time decoupling scheme into the iZQC evolution in the t1 period, thus simplifying the 1D iZQC spectrum of (−ωS ± JHH) into a 1D iZQC pure shift spectrum of (−ωS). In constant-time experiments, the constant time τ is set for signal evolution in the t1 period, and t1 increments should be limited to 2τ (t1max ≤ 2τ). Therefore, spectral resolution of the 1D iZQC pure shift spectrum directly depends on 1/2τ. Intuitively, large τ and sufficient t1 increments can achieve high spectral resolution. However, large τ value generally leads to serious signal intensity variations on JHH coupled resonances. Thus, the short τ is generally adopted in constant-time decoupling experiments, while the short τ inevitably limits spectral resolution due to signal truncations with limited t1 increments. For UPSIF and UPSIF-2DJ experiments aiming for applications in inhomogeneous fields, there are two basic principles of setting τ, (a) τ should be large enough to ensure the distinction of all singlet peaks, and (b) τ should be kept within a reasonable range to minimize signal 7119
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Analytical Chemistry intensity variations. In our experiments, τ is carefully optimized following these two principles. To further break the spectral resolution limitation with residual line broadening caused by constant-time evolution, we introduce the DMUSIC algorithm into the data processing of UPSIF and UPSIF-2DJ experiments and achieve the desired pure shift 1H NMR spectra with sharp lines. The DMUSIC algorithm presents an effective processing for reconstructing NMR spectra with ultrahigh spectral resolution.43 As a parametric method, the efficacy of the DMUSIC generally relies on the model order, p value, which is related to the number of frequency components in the original data or the number of peaks in the resulting spectra. In this algorithm, the original data is modeled as a sum of exponentially damping harmonics, that is, the frequency spectrum is approximated and reconstructed with a group of peaks with Lorentzian line shape. To ensure reliable parameter retrieval, we should set the model order p to be slightly larger than the number of frequency components of the original data. Because of the defect on extracting proper peak intensities in resulting spectra, the DMUSIC processing has not been applied widely in NMR fields, in spite of its superiority on spectral resolution. To solve this problem, we first apply DMUSIC to extract frequencies, phases, and relaxation rates of frequency components in the original data, and we estimate amplitudes (i.e., peak intensities) of each frequency components by least square fitting. On the basis of these parameters, resulting spectra with zero noise and flat baseline can be reconstructed, in which frequencies and intensities of peaks are approximately consistent with the original Fourier spectrum. In our data processing of UPSIF experiments (Figure S1), the first processing procedure with Fourier transform and FOC yields a 1D iZQC pure shift spectrum with the spectral resolution limitation. In the DMUSIC processing on UPSIF data, the reference information, including frequencies, amplitudes, phases, and relaxation rates of all frequency components, are first extracted from the data of the 1D iZQC pure shift spectrum. Then, in the spectral reconstruction, frequencies and amplitudes of frequency components are preserved for reconstructed peaks, while phases of frequency components are set to zero to obtain absorption Lorentzian line shape free of phase distortion. In addition, spectral line widths are proportional to relaxation rates, therefore relaxation rates of frequency components are adjusted to narrow residual line broadening and obtain desired peaks with sharp line shape. After the DMUSIC processing, the resulting high-resolution 1D pure shift spectrum free of spectral resolution limitation is obtained. The effectiveness of the data processing combined with the DMUSIC processing is verified in our 1D UPSIF experiments (see Figure S2d and S2e). The DMUSIC processing is also applied to the UPSIF-2DJ data to further enhance the spectral resolution along the chemical shift dimension. Considering the accuracy of JHH couplings along the second dimension, we preserve original frequencies, amplitudes and phases of frequency components in the DMUSIC processing on UPSIF-2DJ data. Relaxation rates of frequency components are carefully set to guarantee JHH coupling accuracy. Otherwise, JHH couplings may be overestimated. Different from the optimal setting on phases and relaxation rates for absorption Lorentzian line shape in the DMUSIC processing on UPSIF data, parameter settings in the DMUSIC processing on UPSIF-2DJ data is generally limited. Thus, the slight line shape distortion on the 1D projection along the chemical dimension of resulting 2D UPSIF-2DJ spectra is
observed (Figures 2f and 3c). In addition, spectral resolution along the chemical shift dimension in 2D UPSIF-2DJ spectra is also larger than that in 1D UPSIF spectra (see Figure S2e and S2f). Clearly, three major schemes of iZQC evolution on removing field inhomogeneity, constant-time evolution on hindering JHH coupling, and the DMUSIC processing on further eliminating residual line broadening are combined to establish the UPSIF method for high-resolution pure shift NMR applications in inhomogeneous magnetic fields. UPSIF and PSYCHE experiments are also performed for comparison under magnetic field conditions that vary from homogeneity to severe inhomogeneity (Figure S4). Compared to the PSYCHE, the UPSIF method is immune to magnetic field homogeneity variations and resulting spectra acquired from different field homogeneities show the same high spectral resolution. Our results convincingly demonstrate the superiority of UPSIF and UPSIF2DJ methods for pure shift NMR applications to inhomogeneous magnetic field conditions. Similar to existing pure shift 1 H NMR experiments, 2D acquisition is also required in UPSIF and UPSIF-2DJ experiments. However, the FOC processing scheme is utilized to accelerate the acquisition process of UPSIF and UPSIF-2DJ experiments by reducing the spectral width along the indirect dimension. In addition, the short τ value setting can further aid efficient acquisition in UPSIF and UPSIF-2DJ experiments since the maximum t1 incremental value is limited to 2τ (t1max ≤ 2τ) for constant-time evolution. Consequently, the acquisition efficiency of UPSIF experiments is related to the field inhomogeneity range and the limited t1 increments. Benefiting from the performance of echo-train acquisition module on extracting JHH couplings in a single scan, the acquisition efficiency of UPSIF-2DJ is similar to that of UPSIF. In our experiments, the acquisition time is reasonably kept within 6 min, and it is appropriate for rapid chemical and biological applications. There exist several limitations in UPSIF and UPSIF-2DJ applications. First, according to the iZQC mechanism, a concentrated solvent component with a single resonance should be contained in investigation samples and desired components are dissolved as solutes into the solvent.44 It is relatively easy to meet this requirement in practical applications since most chemical solution and biological samples usually contain such a solvent. However, this requirement may be limited in some measurements in which the solvent is not contained, such as oil samples. Moreover, since the solvent is used as a source of DDF in UPSIF and UPSIF-2DJ experiments and its peak intensity increases in the resulting spectrum, this increased solvent signal is generally ignored in analyses. An optional solvent-suppression (SS) module is also provided to suppress the solvent signal, which is prerequisite for measurements on biological samples. Second, because of the intrinsic disadvantage of constant-time scheme, that is, relative signal intensity variations with different JHH coupling modulations of cosn(πJHHτ), in which n is the JHH coupling number related to objective signals, UPSIF and UPSIF-2DJ methods has the limitation on signal quantitative evaluations for analyses. The short τ value setting aids to minimize the JHH coupling modulation effect. For example, in our UPSIF and UPSIF-2DJ experiments on the solution of ethyl 3-bromopropionate and methanol in acetone (Figure S2), τ is set to 23 ms which generates a modulation of cos2(0.16π) = 0.77 on methyl signals and a modulation of cos3(0.16π) = 0.68 on methylene signals for the ethyl 3-bromopropionate with JHH coupling constant of 7120
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around 7.0 Hz. We perform the signal quantification comparison by calculating integral intensity ratios of solute peaks among standard 1D spectrum, 1D PSYCHE spectrum, and 1D UPSIF spectrum. Similar quantitative evaluations on peak intensity ratios in these three spectra can be obtained. Although peak intensity variations exist in UPSIF and UPSIF2DJ experiments on samples with complex JHH couplings, relatively quantitative analyses remain practicable. There exists some NMR measurements in which variations of sample components during the given process of chemical or biological reactions are desired for analyses. When experimental and processing parameters of UPSIF and UPSIF-2DJ are set consistently during the whole measurements, a series of spectra with the same peak intensity variations are obtained by UPSIF and UPSIF-2DJ. Then relative variations of peak intensities among these spectra is identified, providing relatively quantitative analyses. For example, from in situ electrochemical NMR detection on the butanol aqueous solution (Figure 4), it can be seen that relative variations of observed peak intensities through the 1D UPSIF array spectra is obtained for corresponding electrochemical analyses during the electrooxidation process. Third, according to the sensitivity calculation on the sample of ethyl 3-bromopropionate and methanol in acetone (Figure S2), the sensitivities for UPSIF and UPSIF-2DJ is around 12−14% in comparison to standard 1D NMR method, which is mainly due to 2D acquisition manner adopted in UPSIF and UPSIF-2DJ experiments. The UPSIF and UPSIF2DJ remain available for measurements on samples with low concentration, such as intact pig brain tissue with the metabolite concentrations of 1.0−15.0 mM (Figure S4). Therefore, the detectable low concentration of UPSIF and UPSIF-2DJ is around 1.0 mM. Fourth, the performance of the DMUSIC processing on peak distinctions depends on the reference information provided by intermediate 1D iZQC pure shift spectra. If peaks are recognized in intermediate 1D iZQC pure shift spectra, then they can be well-resolved in resulting 1D pure shift spectra after the DMUSIC processing. Generally, the DMUSIC processing can work well for most practical samples by optimizing the τ value, but it may fail on some complex samples with extremely crowded peaks and extensive JHH couplings.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b01036. Additional information includes experimental settings for UPSIF and UPSIF-2DJ experiments, detail data processing procedures for UPSIF and UPSIF-2DJ experiments, experiments on the solution of ethyl 3-bromopropionate and methanol in acetone under the deshimmed inhomogeneous field with 2500 Hz spectral broadening, applications to intact pig brain tissues fitted into a 5 mm NMR tube, comparison among standard 1D single-pulse NMR, 1D PSYCHE, and 1D UPSIF experiments under different magnetic field conditions, pulse sequence codes for UPSIF and UPSIF-2DJ, and data processing codes for UPSIF and UPSIF-2DJ (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Zhong Chen: 0000-0002-1473-2224 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge supports from the National Natural Science Foundation of China (11675135, 21327001, 11375147, and 11474236) and Key Science and Technique Project of Fujian Province of China (2014Y0013 and 2017H0040).
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REFERENCES
(1) Chow, W. Y.; Rajan, R.; Muller, K. H.; Reid, D. G.; Skepper, J. N.; Wong, W. C.; Brooks, R. A.; Green, M.; Bihan, D.; Farndale, R. W.; Slatter, D. A.; Shanahan, C. M.; Duer, M. J. Science 2014, 344, 742− 746. (2) Zhang, Y.; Kitazawa, S.; Peran, I.; Stenzoski, N.; McCallum, S. A.; Raleigh, D. P.; Royer, C. A. J. Am. Chem. Soc. 2016, 138, 15260− 15266. (3) Simpson, A. J.; Simpson, M. J.; Soong, R. Environ. Sci. Technol. 2012, 46, 11488−11496. (4) Nakamura, T.; Tamada, D.; Yanagi, Y.; Itoh, Y.; Nemoto, T.; Utumi, H.; Kose, K. J. Magn. Reson. 2015, 259, 68−75. (5) Frydman, L.; Blazina, D. Nat. Phys. 2007, 3, 415−419. (6) Tredwell, G. D.; Bundy, J. G.; De Iorio, M.; Ebbels, T. M. D. Metabolomics 2016, 12, 152. (7) Aguilar, J. A.; Faulkner, S.; Nilsson, M.; Morris, G. A. Angew. Chem., Int. Ed. 2010, 49, 3901−3903. (8) Aue, W. P.; Karhan, J.; Ernst, R. R. J. Chem. Phys. 1976, 64, 4226−4227. (9) Bax, A.; Mehlkopf, A. F.; Smidt, J. J. Magn. Reson. 1979, 35, 167− 169. (10) Donovan, K. J.; Frydman, L. Angew. Chem., Int. Ed. Engl. 2015, 54 (2), 594−598. (11) Zangger, K.; Sterk, H. J. Magn. Reson. 1997, 124, 486−489. (12) Foroozandeh, M.; Adams, R. W.; Meharry, N. J.; Jeannerat, D.; Nilsson, M.; Morris, G. A. Angew. Chem., Int. Ed. 2014, 53, 6990− 6992. (13) Sinnaeve, D.; Foroozandeh, M.; Nilsson, M.; Morris, G. A. Angew. Chem., Int. Ed. 2016, 55, 1090−1093. (14) Pell, A. J.; Edden, R. A. E.; Keeler, J. Magn. Reson. Chem. 2007, 45, 296−316.
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CONCLUSIONS In this study, we present a previously unreported NMR method to obtain high-resolution pure shift NMR for rapid chemical and biological analyses in inhomogeneous magnetic fields. We deem that the methods will broaden the scope of pure shift NMR applications with an extension to the field inhomogeneity problem caused by adverse experimental conditions, which is generally encountered in chemical and biological applications. Basic blocks of pulse sequences and data processing can be easily introduced to other higher dimension NMR experiments, facilitating analyses of complex samples. Moreover, we envision combining other pure shift techniques with our experiments for relatively quantitative measurements in practical applications. As demonstrated in this work, our methods provide an effective way for high-resolution NMR measurements under adverse experimental conditions that are unexpected for conventional NMR approaches, thus offering extremely interesting prospects for studying metabolites in in vivo living organisms, characterizing intermediate products in in situ chemical reactions, etc. 7121
DOI: 10.1021/acs.analchem.7b01036 Anal. Chem. 2017, 89, 7115−7122
Article
Analytical Chemistry (15) Sorensen, O. W.; Griesinger, C.; Ernst, R. R. J. Am. Chem. Soc. 1985, 107, 7778−7779. (16) Glanzer, S.; Zangger, K. J. Am. Chem. Soc. 2015, 137, 5163− 5169. (17) Brennan, L. Prog. Nucl. Magn. Reson. Spectrosc. 2014, 83, 42−49. (18) Rachineni, K.; Kakita, V. M. R.; Dayaka, S.; Vemulapalli, S. P. B.; Bharatam, J. Anal. Chem. 2015, 87, 7258−7266. (19) Foroozandeh, M.; Adams, R. W.; Kiraly, P.; Nilsson, M.; Morris, G. A. Chem. Commun. 2015, 51, 15410−15413. (20) Foroozandeh, M.; Adams, R. W.; Nilsson, M.; Morris, G. A. J. Am. Chem. Soc. 2014, 136, 11867−11869. (21) Hamdoun, G.; Sebban, M.; Cossoul, E.; Harrison-Marchand, A.; Maddaluno, J.; Oulyadi, H. Chem. Commun. 2014, 50, 4073−4075. (22) Timari, I.; Szilagyi, L.; Koever, K. E. Chem. - Eur. J. 2015, 21, 13939−13942. (23) Fu, R. Q.; Wang, X. S.; Li, C. G.; Santiago-Miranda, A. N.; Pielak, G. J.; Tian, F. J. Am. Chem. Soc. 2011, 133, 12370−12373. (24) Yuk, J.; Simpson, M. J.; Simpson, A. J. Ecotoxicology 2012, 21, 1301−1313. (25) Koch, K. M.; Sacolick, L. I.; Nixon, T. W.; McIntyre, S.; Rothman, D. L.; de Graaf, R. A. Magn. Reson. Med. 2007, 57, 587−591. (26) Yanagisawa, Y.; Nakagome, H.; Hosono, M.; Hamada, M.; Kiyoshi, T.; Hobo, F.; Takahashi, M.; Yamazaki, T.; Maeda, H. J. Magn. Reson. 2008, 192, 329−337. (27) Pelupessy, P.; Rennella, E.; Bodenhausen, G. Science 2009, 324, 1693−1697. (28) de Graaf, R. A.; Behar, K. L. NMR Biomed. 2014, 27, 802−809. (29) Galiana, G.; Branca, R. T.; Warren, W. S. J. Am. Chem. Soc. 2005, 127, 17574−17575. (30) Lin, Y. Y.; Ahn, S.; Murali, N.; Brey, W.; Bowers, C. R.; Warren, W. S. Phys. Rev. Lett. 2000, 85, 3732−3735. (31) Vathyam, S.; Lee, S.; Warren, W. S. Science 1996, 272, 92−96. (32) Li, Y. G.; Razavilar, J.; Liu, K. J. R. IEEE Trans. Biomed. Eng. 1998, 45, 78−86. (33) Chinthalapalli, S.; Bornet, A.; Segawa, T. F.; Sarkar, R.; Jannin, S.; Bodenhausen, G. Phys. Rev. Lett. 2012, 109, 047602. (34) Zheng, G.; Price, W. S. Prog. Nucl. Magn. Reson. Spectrosc. 2010, 56, 267−288. (35) de Graaf, R. A.; Chowdhury, G. M. I.; Behar, K. L. Anal. Chem. 2011, 83, 216−224. (36) Wind, R. A.; Hu, J. Z. Prog. Nucl. Magn. Reson. Spectrosc. 2006, 49, 207−259. (37) Boisseau, R.; Bussy, U.; Giraudeau, P.; Boujtita, M. Anal. Chem. 2015, 87, 372−375. (38) Bayley, P. M.; Trease, N. M.; Grey, C. P. J. Am. Chem. Soc. 2016, 138, 1955−1961. (39) Bussy, U.; Giraudeau, P.; Tea, I.; Boujtita, M. Talanta 2013, 116, 554−558. (40) Bussy, U.; Boujtita, M. Talanta 2015, 136, 155−160. (41) Li, N. H.; Sun, S. G.; Chen, S. P. J. Electroanal. Chem. 1997, 430, 57−67. (42) Branca, R. T.; Warren, W. S. J. Lipid Res. 2011, 52, 833−839. (43) Liao, W. IEEE Trans. Signal Proces. 2015, 63, 6395−6406. (44) Chen, Z.; Cai, S.; Huang, Y.; Lin, Y. Prog. Nucl. Magn. Reson. Spectrosc. 2015, 90−91, 1−31.
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DOI: 10.1021/acs.analchem.7b01036 Anal. Chem. 2017, 89, 7115−7122
