Capillary HPLC-NMR Coupling: High-Resolution (1)H NMR Spectroscopy in the Nanoliter Scale

Coupling HPLC and NMR is one of the most powerful techniques for simultaneous separation and structural elucidation of unknown compounds in mixtures. To date, however, minimizing the detection volume, as is required when coupling NMR with miniaturized separation techniques, has been accompanied by a dramatic loss in resolution of the NMR spectra. Here, we report on the coupling of gradient capillary HPLC with on-column, high-resolution NMR detection. On-line stopped-flow and static (1)H NMR spectra were acquired with capillary columns of 75-315 μm i.d. With detection over a length of 1.2 cm, cell volumes cover a range of 50-900 nL. An on-line-detected NMR separation of dansylated amino acids was carried out in a 315 μm i.d. fused silica capillary packed to a length of 12 cm with C(18) stationary phase. The low solvent consumption makes the use of fully deuterated solvents economically feasible. NMR spectra with resolution on the order of 3 Hz were obtained using a 50 nL detection cell to measure 1.1 nmol of dansylated γ-aminobutyric acid under static conditions in a 75 μm i.d. capillary.     

Metabolite projection analysis for fast identification of metabolites in metabonomics. Application in an amiodarone study

The assignment of significantly changed NMR signals, which were identified with the help of multivariate models, to individual metabolites in biofluids is a manual and tedious task requiring knowledge in chemometrics and NMR spectroscopy. Metabolite projection analysis, introduced in this work, allows automatic linking of multivariate models with metabolites by skipping the level of manual NMR signal identification. The method depends on the projection of sets of metabolite NMR spectra from a database into PCA or PLS models of NMR spectra of biofluid samples. Metabolites that are significantly changed can be identified graphically in metabolite projection plots or numerically as projected virtual concentration. The method is demonstrated together with a newly introduced algorithm for refined nonequidistant binning using a metabonomics study with amiodarone as administered drug. Amiodarone can induce phospholipidosis in the lung and liver, which is accompanied by associated organ toxicity in these organs. It is shown how metabolite projection analysis allows easy and fast tentative assignment of all structures of metabolites whose concentrations in the urine samples significantly changed upon dosage. These metabolites had also been identified previously by manually interpreting the multivariate models and spectra. Among these metabolites, phenylacetylglycine was also identified as being significantly increased. This metabolite has recently been proposed as urinary biomarker for phospholipidosis.     

Quantitative 1H NMR spectroscopy of blood plasma metabolites

The absolute quantification of blood plasma metabolites by proton NMR spectroscopy is complicated by the presence of a baseline and broad resonances originating from serum macromolecules and lipoproteins. A method for spectral simplification of proton NMR spectra of blood plasma is presented. Serum macromolecules and metabolites are completely separated by utilizing the large difference in translational diffusion coefficients in combination with diffusion-sensitized proton NMR spectroscopy. The concentration of blood plasma metabolites can be quantified by using formate as an internal concentration reference. The results are compared with those obtained with ultrafiltration, a traditional method for separating macromolecules and metabolites, and demonstrate an excellent correlation between the two methods. The general nature of diffusion-sensitized NMR spectroscopy allows application on a wide range of biological fluids.     

Magic angle spinning NMR and 1H-31P heteronuclear statistical total correlation spectroscopy of intact human gut biopsies

Previously we have demonstrated the use of 1H magic angle spinning (MAS) NMR spectroscopy for the topographical variations in functional metabolic signatures of intact human intestinal biopsy samples. Here we have analyzed a series of MAS 1H NMR spectra (spin-echo, one-dimensional, and diffusion-edited) and 31P-{1H} spectra and focused on analyzing the enhancement of information recovery by use of the statistical total correlation spectroscopy (STOCSY) method. We have applied a heterospectroscopic cross-examination performed on the same samples and between 1H and 31P-{1H} spectra (heteronuclear STOCSY) to recover latent metabolic information. We show that heterospectroscopic correlation can give new information on the molecular compartmentation of metabolites in intact tissues, including the statistical "isolation" of a phospholipid/triglyceride vesicle pool in intact tissue. The application of 31P-1H HET-STOCSY allowed the cross-assignment of major 31P signals to their equivalent 1H NMR spectra, e.g., for phosphorylcholine and phosphorylethanolamine. We also show pathway correlations, e.g., the ascorbate-glutathione pathway, in the STOCSY analysis of intact tissue spectra. These 31P-1H HET-STOCSY spectra also showed different topographical regions, particular for minor signals in different tissue microenvironments. This approach could be extended to allow the detection of altered distributions within metabolic subcompartments as well as conventional metabonomics concentration-based diagnostics.     

Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra

One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) spectroscopy is used extensively for high-throughput analysis of metabolites in biological fluids and tissue extracts. Typically, such spectra are treated as multivariate statistical objects rather than as collections of quantifiable metabolites. We report here a two-dimensional (2D) (1)H-(13)C NMR strategy (fast metabolite quantification, FMQ, by NMR) for identifying and quantifying the approximately 40 most abundant metabolites in biological samples. To validate this technique, we prepared mixtures of synthetic compounds and extracts from Arabidopsis thaliana, Saccharomyces cerevisiae, and Medicago sativa. We show that accurate (technical error 2.7%) molar concentrations can be determined in 12 min using our quantitative 2D (1)H-(13)C NMR strategy. In contrast, traditional 1D (1)H NMR analysis resulted in 16.2% technical error under nearly ideal conditions. We propose FMQ by NMR as a practical alternative to 1D (1)H NMR for metabolomics studies in which 50-mg (extract dry weight) samples can be obtained.     

Partial least squares based decomposition of five spectrally overlapping factors

The development of a multi-sensory fiber-optic based fluence rate probe (MSP) for light monitoring and dosimetry during photodynamic therapy (PDT) created the need for a robust multivariate signal analysis algorithm capable of quantifying the intensity of five component spectra, representing the sensors, which display a large degree of spectral overlap. Partial least squares (PLS) analysis, as an option for such an analysis algorithm, was evaluated through simulations in the presence of three types of noise, which experimentally may limit the accuracy of PLS quantification of component spectra contributions. Random, or white noise, background was varied over a range of 0-15% relative intensity. A non-random (Gaussian) background vector was simulated, as an experimentally relevant spectral interferent, over a range of 0-7% relative intensity. Spectral variation was modeled by a spectral shift of +/-1-5 nm. Procedures for selecting preferred combinations of fluorophores, with minimum possible spectral overlap, were developed. To illustrate the importance of this selection process, PLS performance results were compared for two possible combinations of five fluorophores, as well as for the combination of three fluorophores currently in experimental use with MSPs. Experimentally anticipated worst-case quantifications were identified for all three types of simulated noise (5% random background, 0.5% Gaussian background, and +/-2 nm spectral shift). The effects of single and combined types of noise were evaluated by independently varying each type of simulated noise over the experimentally relevant range. The mean percentage error in determining the fluorophore contributions and hence quantification of the fluence rate were compared with the required performance standard of better than 10% error for optical power meters in medical use. The PLS algorithm provided an accuracy of 7 +/- 2% for five fluorophores and 3.3 +/- 0.8% for three fluorophores, indicating that PLS is an appropriate choice for this application.     

Structural elucidation and quantification of phenolic conjugates present in human urine after tea intake

In dietary polyphenol exposure studies, annotation and identification of urinary metabolites present at low (micromolar) concentrations are major obstacles. To determine the biological activity of specific components, it is necessary to have the correct structures and the quantification of the polyphenol-derived conjugates present in the human body. We present a procedure for identification and quantification of metabolites and conjugates excreted in human urine after single bolus intake of black or green tea. A combination of a solid-phase extraction (SPE) preparation step and two high pressure liquid chromatography (HPLC)-based analytical platforms was used, namely, accurate mass fragmentation (HPLC-FTMS(n)) and mass-guided SPE-trapping of selected compounds for nuclear magnetic resonance spectroscopy (NMR) measurements (HPLC-TOFMS-SPE-NMR). HPLC-FTMS(n) analysis led to the annotation of 138 urinary metabolites, including 48 valerolactone and valeric acid conjugates. By combining the results from MS(n) fragmentation with the one-dimensional (1D)-(1)H NMR spectra of HPLC-TOFMS-SPE-trapped compounds, we elucidated the structures of 36 phenolic conjugates, including the glucuronides of 3',4'-di- and 3',4',5'-trihydroxyphenyl-γ-valerolactone, three urolithin glucuronides, and indole-3-acetic acid glucuronide. We also obtained 26 h-quantitative excretion profiles for specific valerolactone conjugates. The combination of the HPLC-FTMS(n) and HPLC-TOFMS-SPE-NMR platforms results in the efficient identification and quantification of less abundant phenolic conjugates down to nanomoles of trapped amounts of metabolite corresponding to micromolar metabolite concentrations in urine.     

Quantitative analysis of low-field NMR signals in the time domain

Two novel methods are described for direct quantitative analysis of NMR free induction decay (FID) signals. The methods use adaptations of the generalized rank annihilation method (GRAM) and the direct exponential curve resolution algorithm (DECRA). With FID-GRAM, the Hankel matrix of the sample signal is compared with that of a reference mixture to obtain quantitative data about the components. With FID-DECRA, a single-sample FID matrix is split into two matrices, allowing quantitative recovery of decay constants and the individual signals in the FID. Inaccurate results were obtained with FID-GRAM when there were differences between the frequency or transverse relaxation time of signals for the reference and test samples. This problem does not arise with FID-DECRA, because comparison with a reference signal is unnecessary. Application of FID-DECRA to 19F NMR data, which contained overlapping signals from three components, gave concentrations comparable to those derived from partial least squares (PLS) analysis of the Fourier transformed spectra. However, the main advantage of FID-DECRA was that accurate (<5% error) and precise (2.3% RSD) results were obtained using only one calibration sample, whereas with PLS, a training set of 10 standard mixtures was used to give comparable accuracy and precision.     

On-line coupling of capillary electrochromatography, capillary electrophoresis, and capillary HPLC with nuclear magnetic resonance spectroscopy

A novel capillary NMR coupling configuration, which offers the possibility of combining capillary zone electrophoresis (CZE), capillary HPLC (CHPLC), and for the first time capillary electrochromatography (CEC) with nuclear magnetic resonance (NMR), has been developed. The hyphenated technique has a great potential for the analysis of chemical, pharmaceutical, biological, and environmental samples. The versatile system allows facile changes between these three different separation methods. A special NMR capillary containing an enlarged detection cell suitable for on-line NMR detection and measurements under high voltage has been designed. The acquisition of 1D and 2D NMR spectra in stopped-flow experiments is also possible. CHPLC NMR has been performed with samples of hop bitter acids. The identification and structure elucidation of humulones and isohumulones by on-line and stopped-flow spectra has been demonstrated. The suitability of the configuration for electrophoretic methods has been investigated by the application of CZE and CEC NMR to model systems.     

Application of directly coupled HPLC NMR to separation and characterization of lipoproteins from human serum

Disorders in lipoprotein metabolism are critical in the etiology of several disease states such as coronary heart disease and atherosclerosis. Thus, there is considerable interest in the development of novel methods for the analysis of lipoprotein complexes. We report here a simple chromatographic method for the separation of high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein from intact serum or plasma. The separation was achieved using a hydroxyapatite column and elution with pH 7.4 phosphate buffer with 100-microL injections of whole plasma. Coelution of HDL with plasma proteins such as albumin occurred, and this clearly limits quantitation of that species by HPLC peak integration. We also show, for the first time, the application of directly coupled HPLC 1H NMR spectroscopy to confirm the identification of the three major lipoproteins. The full chromatographic run time was 90 min with stopped-flow 600-MHz NMR spectra of each lipoprotein being collected using 128 scans, in 7 min. The 1H NMR chemical shifts of lipid signals were identical to conventional NMR spectra of freshly prepared lipoprotein standards, confirming that the lipoproteins were not degraded by the HPLC separation and that their gross supramolecular organization was intact.     

Exploring the analytical potential of NMR spectroscopy in chiral anisotropic media for the study of the natural abundance deuterium distribution in organic molecules

The deuterium/hydrogen (D/H)(i) ratio measurement by quantitative (2)H NMR spectroscopy is a method of choice for the analysis of kinetic isotopic effects associated with enzyme-catalyzed reactions during a biosynthetic pathway. However, the efficiency of the current isotropic (2)H-[(1)H] NMR can be limited by the rather small chemical shift dispersion of deuterium nuclei. In addition, this method does not allow the enantiotopic deuterons in prochiral molecules to be spectrally discriminated, hence precluding the quantification of isotopic fractionation on methylene prostereogenic sites. In this work, we explore another analytical strategy able to circumvent these disadvantages. This approach is based on the use of natural abundance (2)H 2D NMR experiments on solutes embedded in polypeptidic, chiral liquid crystalline solvent. Thus, we show that NMR in these oriented phases is a powerful way to separate deuterium signals on the basis of the quadrupolar interactions, providing a promising alternative to overcrowded (2)H NMR spectra obtained in liquid state. To illustrate our purpose, we have experimentally investigated the case of 1,1'-bis(phenylthio)hexane derived by cleavage from methyl linoleate of safflower. The (2)H NMR results in chiral liquid crystals are presented and discussed. We show, for the first time, that (D/H)(pro-R) and (D/H)(pro-S) can be measured at the same methylene position of a fatty acid chain.     

"Add to subtract": a simple method to remove complex background signals from the 1H nuclear magnetic resonance spectra of mixtures

Because of its highly reproducible and quantitative nature and minimal requirements for sample preparation or separation, (1)H nuclear magnetic resonance (NMR) spectroscopy is widely used for profiling small-molecule metabolites in biofluids. However (1)H NMR spectra contain many overlapped peaks. In particular, blood serum/plasma and diabetic urine samples contain high concentrations of glucose, which produce strong peaks between 3.2 ppm and 4.0 ppm. Signals from most metabolites in this region are overwhelmed by the glucose background signals and become invisible. We propose a simple "Add to Subtract" background subtraction method and show that it can reduce the glucose signals by 98% to allow retrieval of the hidden information. This procedure includes adding a small drop of concentrated glucose solution to the sample in the NMR tube, mixing, waiting for an equilibration time, and acquisition of a second spectrum. The glucose-free spectra are then generated by spectral subtraction using Bruker Topspin software. Subsequent multivariate statistical analysis can then be used to identify biomarker candidate signals for distinguishing different types of biological samples. The principle of this approach is generally applicable for all quantitative spectral data and should find utility in a variety of NMR-based mixture analyses as well as in metabolite profiling.     

Deconvolution of two-dimensional NMR spectra by fast maximum likelihood reconstruction: application to quantitative metabolomics

We have developed an algorithm called fast maximum likelihood reconstruction (FMLR) that performs spectral deconvolution of 1D-2D NMR spectra for the purpose of accurate signal quantification. FMLR constructs the simplest time-domain model (e.g., the model with the fewest number of signals and parameters) whose frequency spectrum matches the visible regions of the spectrum obtained from identical Fourier processing of the acquired data. We describe the application of FMLR to quantitative metabolomics and demonstrate the accuracy of the method by analysis of complex, synthetic mixtures of metabolites and liver extracts. The algorithm demonstrates greater accuracy (0.5-5.0% error) than peak height analysis and peak integral analysis with greatly reduced operator intervention. FMLR has been implemented in a Java-based framework that is available for download on multiple platforms and is interoperable with popular NMR display and processing software. Two-dimensional (1)H-(13)C spectra of mixtures can be acquired with acquisition times of 15 min and analyzed by FMLR in the range of 2-5 min per spectrum to identify and quantify constituents present at concentrations of 0.2 mM or greater.     

NMR detection with multiple solenoidal microcoils for continuous-flow capillary electrophoresis

Nuclear magnetic resonance (NMR) spectroscopy represents a promising on-line detector for capillary electrophoresis (CE). The inherent poor sensitivity of NMR mandates the use of NMR probes with the highest mass sensitivity, such as those containing solenoidal microcoils, for CE/NMR hyphenation. However, electrophoretic current degrades the resolution of NMR spectra obtained from solenoidal coils. A new method to avoid microcoil NMR spectral degradation during continuous-flow CE is demonstrated using a unique multiple solenoidal coil NMR probe. The electrophoretic flow from a single separation capillary is split into multiple outlets, each possessing its own NMR detection coil. While the CE electrophoretic flow is directed through one outlet, stopped-flow, high-resolution NMR spectra are obtained from the coil at the other outlet. The electrophoretic flow and NMR measurements are cycled between the outlets to allow a continuous CE separation with "stopped-flow" detection. As a new approach for improving multiple coil probe performance, the magnetic field homogeneity is automatically adjusted (via the shim coils of the magnet) for the active coil. The multiple microcoil CE/NMR coupling has been used to analyze a <3 nmole mixture of amines while obtaining between 1 and 2 Hz line width, demonstrating the ability to avoid electrophoretic current-induced line broadening.     

Heteronuclear 1H-31P statistical total correlation NMR spectroscopy of intact liver for metabolic biomarker assignment: application to galactosamine-induced hepatotoxicity

As part of our ongoing development of methods for enhanced biomarker information recovery from spectroscopic data we present the first example of a new hetero-nuclear statistical total correlation spectroscopy (HET-STOCSY) approach applied to intact tissue samples collected as part of a toxicological study. One-dimensional 1H and 31P-{1H} magic angle spinning (MAS) NMR spectra of intact liver samples after galactosamine (galN) treatment to rats and after cotreatment of galN plus uridine were collected at 275 K. Individual samples were also followed by 1H and 31P-{1H} MAS NMR through time generating time dependent modulations in metabolite signatures relating to toxicity. High-resolution 1H NMR spectra of urine and plasma and clinical chemical data were also collected to establish a biological framework in which to place these novel statistical heterospectroscopic data. In HET-STOCSY, calculation of the covariance between the 31P-{1H} and 1H NMR signals of phosphorus containing metabolites allows their molecular connectivities to be established and the construction of virtual two-dimensional heteronuclear correlation spectra that connect all protons on the molecule to the heteroatom. We show how HET-STOCSY applied to MAS NMR spectra of liver samples can be used to augment biomarker detection. This approach is generic and can be applied to correlate the covarying signals for any spin-active nuclei where there is parallel or serial collection of data.     

Targeted profiling: quantitative analysis of 1H NMR metabolomics data

Extracting meaningful information from complex spectroscopic data of metabolite mixtures is an area of active research in the emerging field of "metabolomics", which combines metabolism, spectroscopy, and multivariate statistical analysis (pattern recognition) methods. Chemometric analysis and comparison of 1H NMR1 spectra is commonly hampered by intersample peak position and line width variation due to matrix effects (pH, ionic strength, etc.). Here a novel method for mixture analysis is presented, defined as "targeted profiling". Individual NMR resonances of interest are mathematically modeled from pure compound spectra. This database is then interrogated to identify and quantify metabolites in complex spectra of mixtures, such as biofluids. The technique is validated against a traditional "spectral binning" analysis on the basis of sensitivity to water suppression (presaturation, NOESY-presaturation, WET, and CPMG), relaxation effects, and NMR spectral acquisition times (3, 4, 5, and 6 s/scan) using PCA pattern recognition analysis. In addition, a quantitative validation is performed against various metabolites at physiological concentrations (9 microM-8 mM). "Targeted profiling" is highly stable in PCA-based pattern recognition, insensitive to water suppression, relaxation times (within the ranges examined), and scaling factors; hence, direct comparison of data acquired under varying conditions is made possible. In particular, analysis of metabolites at low concentration and overlapping regions are well suited to this analysis. We discuss how targeted profiling can be applied for mixture analysis and examine the effect of various acquisition parameters on the accuracy of quantification.     

近红外光谱法测定茶多酚中总儿茶素含量

以高效液相色谱(HPLC)分析结果为参考值,建立了快速测量茶多酚中总儿茶素含量的近红外光谱定标模型.将48份茶多酚样品组成定标样品集,在1000～2500nm(4000～10000cm-1)的近红外漫反射光谱为定标波长范围内,光谱经一阶导数(Firstderivative)、二阶导数(Secondderivative)、标准归一化(Stan-dardnormalvariate,SNV)和多元散射校正(multiplicativesignalcorrection,MSC)处理后结合偏最小二乘回归(PLS)定标.经内部交叉验证表明,光谱经SNV处理后建模结果最佳.模型的相关系数Corr.Coeff=0.997,校正均方根RMSEC=1.71%.比较了经典最小二乘法(CLS)、偏最小二乘法(PLS)和主成分回归(PCR)等方法建模结果,以偏最小二乘回归建模效果最好.     

Direct On-Line Coupling of Capillary HPLC with (1)H NMR Spectroscopy for the Structural Determination of Retinyl Acetate Dimers: 2D NMR Spectroscopy in the Nanoliter Scale

This paper presents the application of directly coupled capillary high-performance liquid chromatography (capillary HPLC) and proton high-field nuclear magnetic resonance spectroscopy (NMR) for structural elucidation of a so-far unknown kitol isomer. One- and two-dimensional continuous- and stopped-flow NMR spectra were recorded in a 180 μm i.d. capillary, corresponding to a detection volume of only 200 nL. Unequivocal structural assignment on the basis of 1D and 2D stopped-flow capillary HPLC-NMR experiments was performed. The kitol isomer mixture was present in a sample of thermally isomerized retinyl acetate and separated on a capillary column.     

Combined analysis of C-18 unsaturated fatty acids using natural abundance deuterium 2D NMR spectroscopy in chiral oriented solvents

The quantitative determination of isotopic (2H/1H)i ratios at natural abundance using the SNIF-NMR protocol is a well-known method for understanding the enzymatic biosynthesis of metabolites. However, this approach is not always successful for analyzing large solutes and, specifically, is inadequate for prochiral molecules such as complete essential unsaturated fatty acids. To overcome these analytical limitations, we use the natural abundance deuterium 2D NMR (NAD 2D NMR) spectroscopy on solutes embedded in polypeptide chiral liquid crystals. This approach, recently explored for measuring (2H/1H)i ratios of small analytes (Lesot, P.; Aroulanda, C.; Billault, I. Anal. Chem. 2004, 76, 2827-2835), is a powerful way to separate the 2H signals of all nonequivalent enantioisotopomers on the basis both of the 2H quadrupolar interactions and of the 2H chemical shift. Two significant advances over our previous work are presented here and allow the complete isotopic analysis of four mono- and polyunsaturated fatty acid methyl esters: methyl oleate (1), methyl linoleate (2), methyl linolenate (3), and methyl vernoleate (4). The first consists of using NMR spectrometers operating at higher magnetic field strength (14.1 T) and equipped with a selective cryoprobe optimized for deuterium nuclei. The second is the development of Q-COSY Fz 2D NMR experiments able to produce phased 2H 2D maps after a double Fourier transformation. This combination of modern hardware and efficient NMR sequences provides a unique tool to analyze the (2H/1H)i ratios of large prochiral molecules (C-18) dissolved in organic solutions of poly(gamma-benzyl-L-glutamate) and requires smaller amounts of solute than previous study on fatty acids. For each compound (1-4), all 2H quadrupolar doublets visible in the 2D spectra have been assigned on the basis of 2H chemical shifts, isotopic data obtained from isotropic quantitative NAD NMR, and by an interspectral comparison of the anisotropic NAD spectra of four fatty acids. The NMR results are discussed in terms of (2H/1H)i isotopic distribution and molecular orientation in the mesophase. For the first time, we show that the investigation of natural isotopic fractionation of complete fatty acids is possible without the need of chemical modifications, hence providing an alternative method to probe the mechanisms of enzymes implied in the biosynthetic pathway of unsaturated fatty acids.