Advancing NMR sensitivity for LC-NMR-MS using a cryoflow probe: application to the analysis of acetaminophen metabolites in urine

Cryogenic cooling of the NMR radio frequency coils and electronics to give greatly enhanced sensitivity is arguably the most significant recent advance in NMR spectroscopy. Here we report the first cryogenic probe built in flow configuration and demonstrate the application to LC-NMR-MS studies. This probe provides superior sensitivity over conventional noncryogenic flow NMR probes, allowing the use of 100 microL of untreated urine (40% less material than previous studies that required preconcentration) and yet revealing drug metabolites hitherto undetected by LC-NMR-MS at 500 MHz. Besides the known sulfate and glucuronide metabolites, previously undetected metabolites of acetaminophen were directly observable in a 15-min on-flow experiment. Simultaneous MS data also provided knowledge on the NMR-silent functional moieties. Further, stop-flow LC-NMR-MS experiments were conducted for greater signal-to-noise ratios on minor metabolites. The cryoflow probe enables the NMR analysis of lower concentrations of metabolites than was previously possible for untreated biofluids. This strategy is generally applicable for samples containing mass-limited analytes, such as those from drug metabolism studies, biomarker and toxicity profiling, impurity analysis, and natural product analysis.     

Automated microflow NMR: routine analysis of five-microliter samples

A microflow CapNMR probe double-tuned for 1H and 13C was installed on a 400-MHz NMR spectrometer and interfaced to an automated liquid handler. Individual samples dissolved in DMSO-d6 are submitted for NMR analysis in vials containing as little as 10 microL of sample. Sets of samples are submitted in a low-volume 384-well plate. Of the 10 microL of sample per well, as with vials, 5 microL is injected into the microflow NMR probe for analysis. For quality control of chemical libraries, 1D NMR spectra are acquired under full automation from 384-well plates on as many as 130 compounds within 24 h using 128 scans per spectrum and a sample-to-sample cycle time of approximately 11 min. Because of the low volume requirements and high mass sensitivity of the microflow NMR system, 30 nmol of a typical small molecule is sufficient to obtain high-quality, well-resolved, 1D proton or 2D COSY NMR spectra in approximately 6 or 20 min of data acquisition time per experiment, respectively. Implementation of pulse programs with automated solvent peak identification and suppression allow for reliable data collection, even for samples submitted in fully protonated DMSO. The automated microflow NMR system is controlled and monitored using web-based software.     

Virtual chromatographic resolution enhancement in cryoflow LC-NMR experiments via statistical total correlation spectroscopy

A new approach to enhancing information recovery from cryogenic probe "on-flow" LC-NMR spectroscopic analyses of complex biological mixtures is demonstrated using a variation on the statistical total correlation spectroscopy (STOCSY) method. Cryoflow probe technology enables sensitive and efficient NMR detection of metabolites on-flow, and the rapid spectral scanning allows multiple spectra to be collected over chromatographic peaks containing several species with similar, but nonidentical, retention times. This enables 1H NMR signal connectivities between close-eluting metabolites to be identified resulting in a "virtual" chromatographic resolution enhancement visualized directly in the NMR spectral projection. We demonstrate the applicability of the approach for structure assignment of drug and endogenous metabolites in urine. This approach is of wide general applicability to any complex mixture analysis problem involving chromatographic peak overlap and with particular application in metabolomics and metabonomics.     

Trace level detection of compounds related to the chemical weapons convention by 1H-detected 13C NMR spectroscopy executed with a sensitivity-enhanced, cryogenic probehead

Two-dimensional 1H-13C HSQC (heteronuclear single quantum correlation) and fast-HMQC (heteronuclear multiple quantum correlation) pulse sequences were implemented using a sensitivity-enhanced, cryogenic probehead for detecting compounds relevant to the Chemical Weapons Convention present in complex mixtures. The resulting methods demonstrated exceptional sensitivity for detecting the analytes at trace level concentrations. 1H-13C correlations of target analytes at < or = 25 microg/mL were easily detected in a sample where the 1H solvent signal was approximately 58,000-fold more intense than the analyte 1H signals. The problem of overlapping signals typically observed in conventional 1H spectroscopy was essentially eliminated, while 1H and 13C chemical shift information could be derived quickly and simultaneously from the resulting spectra. The fast-HMQC pulse sequences generated magnitude mode spectra suitable for detailed analysis in approximately 4.5 h and can be used in experiments to efficiently screen a large number of samples. The HSQC pulse sequences, on the other hand, required roughly twice the data acquisition time to produce suitable spectra. These spectra, however, were phase-sensitive, contained considerably more resolution in both dimensions, and proved to be superior for detecting analyte 1H-13C correlations. Furthermore, a HSQC spectrum collected with a multiplicity-edited pulse sequence provided additional structural information valuable for identifying target analytes. The HSQC pulse sequences are ideal for collecting high-quality data sets with overnight acquisitions and logically follow the use of fast-HMQC pulse sequences to rapidly screen samples for potential target analytes. Use of the pulse sequences considerably improves the performance of NMR spectroscopy as a complimentary technique for the screening, identification, and validation of chemical warfare agents and other small-molecule analytes present in complex mixtures and environmental samples.     

Application of a microcoil probe head in NMR analysis of chemicals related to the chemical weapons convention

A 1.7-mm microcoil probe head was tested in the analysis of organophosphorus compounds related to the Chemical Weapons Convention. The microcoil probe head demonstrated a high mass sensitivity in the detection of traces of organophosphorus compounds in samples. Methylphosphonic acid, the common secondary degradation product of sarin, soman, and VX, was detected at level 50 ng (0.52 nmol) from a 30-microL water sample using proton-observed experiments. Direct phosphorus observation of methylphosphonic acid with (31)P{(1)H} NMR experiment was feasible at the 400-ng (4.17 nmol) level. Application of the microcoil probe head in the spiked sample analysis was studied with a test water sample containing 2-10 microg/mL of three organophosphorus compounds. High-quality (1)H NMR, (31)P{(1)H} NMR, 2D (1)H-(31)P fast-HMQC, and 2D TOCSY spectra were obtained in 3 h from the concentrated 1.7-mm NMR sample prepared from 1 mL of the water solution. Furthermore, a 2D (1)H-(13)C fast-HMQC spectrum with sufficient quality was possible to measure in 5 h. The microcoil probe head demonstrated a considerable sensitivity improvement and reduction of measurement times for the NMR spectroscopy in identification of chemicals related to the Chemical Weapons Convention.     

Strategy for automated analysis of dynamic metabolic mixtures by NMR. Application to an insect venom

Elucidation of the composition of chemical-biological samples is a main focus of systems biology and metabolomics. Due to the inherent complexity of these mixtures, reliable, efficient, and potentially automatable methods are needed to identify the underlying metabolites and natural products. Because of its rich chemical information content, nuclear magnetic resonance (NMR) spectroscopy has a unique potential for this task. Here we present a generalization and application of a recently introduced NMR data collection, processing, and analysis strategy that circumvents the need for extensive purification and hyphenation prior to analysis. It uses covariance TOCSY NMR spectra measured on a 1-mm high-temperature cryogenic probe that are analyzed by a spectral trace clustering algorithm yielding 1D NMR spectra of the individual components for their unambiguous identification. The method is demonstrated on a metabolic model mixture and is then applied to the unpurified venom mixture of an individual walking stick insect that contains several slowly interconverting and closely related metabolites.     

High-resolution capillary tube NMR. A miniaturized 5-microL high-sensitivity TXI probe for mass-limited samples,off-line LC NMR,and HT NMR

A new triple-resonance (TXI) (1H, 13C, 15N) high-resolution nuclear magnetic resonance (NMR) capillary probe with 2.5-microL NMR-active sample volume (V(obs)) was built and tested for applications with mass- and volume-limited samples and for coupling of microbore liquid chromatography to NMR. This is the first microliter probe with optimized coil geometry for use with individual capillary tubes with an outer diameter of 1 mm. The 90 degree pulse lengths of the 1-mm microliter probe were below 2 micros for proton, below 8 micros for carbon, and below 20 micros for nitrogen, and a spectral line width at signal half-height below 1 Hz was obtained. Compared to a conventional 5-mm probe, the new 600-MHz 1-mm TXI microliter probe with z-gradient shows an increase in mass sensitivity by a factor of 5, corresponding to a 25-fold reduction in measuring time. The consumption of costly deuterated solvent is reduced by at least 2 orders of magnitude. The 1-mm TXI microliter probe with z-gradient allows the measurement of one-dimensional 1H NMR and two-dimensional heteronuclear NMR spectra with a few nanomoles (micrograms) of compound with high sensitivity, speed, and quality. This is a breakthrough for discrete sample NMR spectroscopy with paramount importance for structure elucidation in natural compound chemistry and metabolic research. It offers also advantages for linking chromatographic methods to NMR in a nindustrial environment. Capillary tube NMR may find new applications in areas where high sample throughput is essential, e.g., in the quality control of large sample arrays from parallel chemistry, screening, and compound depositories. It has the potential to increase the sample throughput by 1 order of magnitude or more if new hardware for fast sample handling and exchange becomes available.     

Analysis of extracellular matrix production in artificial cartilage constructs by histology, immunocytochemistry, mass spectrometry, and NMR spectroscopy

Artificial cartilage constructs based on primary porcine chondrocytes embedded in agarose gel were cultivated for six weeks under static, free swelling conditions. Standard biochemical assays, immunocytochemical staining methods, MALDI-TOF mass spectrometry, and non-invasive 13C solid-state NMR spectroscopy were used to assess cell proliferation, chondrocyte metabolism, extracellular matrix composition, matrix production, and the nanoarchitecture of the macromolecules in the constructs. In particular the production of sulphated glycosaminoglycans such as chondroitin sulphate was investigated quantitatively. Standard methods such as histological and immunocytochemical tools as well as spectrophotometric assays indicated the production of extracellular matrix in the artificial cartilage constructs. In addition, MALDI-TOF mass spectrometric data allowed to clearly identify the production of chondroitin sulphate in the tissue engineered cartilage. While all these methods require invasive sample treatment, 13C NMR spectroscopy allows to study the composition of the artificial cartilage constructs without previous manipulations. Though lower in sensitivity, 13C NMR spectra clearly showed the presence of chondroitin sulphate in the constructs. To increase the sensitivity of the NMR method, a culture medium that contained uniformly 13C labelled glucose but no sodium pyruvate or L-glutamine was used. Thus, further insights into the chondrocyte metabolism ex vivo are possible. Therefore, MALDI-TOF mass spectrometry and 13C solid-state NMR are useful experimental techniques that can assist the quantitative evaluation and quality control of artificially engineered tissues.     

Self-consistent metabolic mixture analysis by heteronuclear NMR. Application to a human cancer cell line

Elucidation of the chemical composition of biological samples is a main focus of systems biology and metabolomics. In order to comprehensively study these complex mixtures, reliable, efficient, and automatable methods are needed to identify and quantify the underlying metabolites and natural products. Because of its rich information content, nuclear magnetic resonance (NMR) spectroscopy has a unique potential for this task. Here we present a generalization of the recently introduced homonuclear TOCSY-based DemixC method to heteronuclear HSQC-TOCSY NMR spectroscopy. The approach takes advantage of the high resolution afforded along the (13)C dimension due to the narrow (13)C line widths for the identification of spin systems and compounds. The method combines information from both 1D (13)C and (1)H traces by querying them against an NMR spectral database using our COLMAR query web server. The complementarity of (13)C and (1)H spectral information improves the robustness of compound identification. The method is demonstrated for a metabolic model mixture and is then applied to an extract from DU145 human prostate cancer cells.     

Development of a broadband picosecond infrared spectrometer and its incorporation into an existing ultrafast time-resolved resonance Raman, UV/visible, and fluorescence spectroscopic apparatus

We have constructed a broadband ultrafast time-resolved infrared (TRIR) spectrometer and incorporated it into our existing time-resolved spectroscopy apparatus, thus creating a single instrument capable of performing the complementary techniques of femto-/picosecond time-resolved resonance Raman (TR3), fluorescence, and UV/visible/infrared transient absorption spectroscopy. The TRIR spectrometer employs broadband (150 fs, approximately 150 cm(-1) FWHM) mid-infrared probe and reference pulses (generated by difference frequency mixing of near-infrared pulses in type I AgGaS2), which are dispersed over two 64-element linear infrared array detectors (HgCdTe). These are coupled via custom-built data acquisition electronics to a personal computer for data processing. This data acquisition system performs signal handling on a shot-by-shot basis at the 1 kHz repetition rate of the pulsed laser system. The combination of real-time signal processing and the ability to normalize each probe and reference pulse has enabled us to achieve a high sensitivity on the order of deltaOD approximately 10(-4) - 10(-5) with 1 min of acquisition time. We present preliminary picosecond TRIR studies using this spectrometer and also demonstrate how a combination of TRIR and TR3 spectroscopy can provide key information for the full elucidation of a photochemical process.     

Effect of magnetic field strength on NMR-based metabonomic human urine data. Comparative study of 250, 400, 500, and 800 MHz

Metabonomic analysis of urine utilizing high-resolution NMR spectroscopy and chemometric techniques has proven valuable in characterizing the biochemical response to an intervention. To assess the effect of magnetic field strength on information contained in NMR-based metabonomic data sets, 1H NMR spectra were acquired on 250-, 400-, 500-, and 800-MHz instruments, respectively, on the same set of human urine samples collected before and after dietary interventions with milk and with meat proteins. Partial least-squares regression discriminant analyses (PLS-DA) were performed in order to elucidate the ability of the 1H spectra acquired at various field strengths to identify possible spectral differences and discriminate between pre- and postintervention samples. The loadings from PLS-DA contained the same spectral regions, implying that the same metabolites were involved in the discrimination independent of magnetic field strength. The investigation revealed a strong increase in prediction performance and thereby spectral information content when increasing the magnetic field strength from 250 to 500 MHz, while from 500 to 800 MHz the increase was less pronounced.     

Micromixer-based time-resolved NMR: applications to ubiquitin protein conformation

Time-resolved NMR spectroscopy is used to studychanges in protein conformation based on the elapsed time after a change in the solvent composition of a protein solution. The use of a micromixer and a continuous-flow method is described where the contents of two capillary flows are mixed rapidly, and then the NMR spectra of the combined flow are recorded at precise time points. The distance after mixing the two fluids and flow rates define the solvent-protein interaction time; this method allows the measurement of NMR spectra at precise mixing time points independent of spectral acquisition time. Integration of a micromixer and a microcoil NMR probe enables low-microliter volumes to be used without losing significant sensitivity in the NMR measurement. Ubiquitin, the model compound, changes its conformation from native to A-state at low pH and in 40% or higher methanol/water solvents. Proton NMR resonances of the His-68 and the Tyr-59 of ubiquitin are used to probe the conformational changes. Mixing ubiquitin and methanol solutions under low pH at microliter per minute flow rates yields both native and A-states. As the flow rate decreases, yielding longer reaction times, the population of the A-state increases. The micromixer-NMR system can probe reaction kinetics on a time scale of seconds.     

LC-UV-solid-phase extraction-NMR-MS combined with a cryogenic flow probe and its application to the identification of compounds present in Greek oregano

Structure elucidation of natural products usually relies on a combination of NMR spectroscopy with mass spectrometry whereby NMR trails MS in terms of the minimum sample amount required. In the present study, the usefulness of on-line solid-phase extraction (SPE) in LC-NMR for peak storage after the LC separation prior to NMR analysis is demonstrated. The SPE unit allows the use of normal protonated solvents for the LC separation and fully deuterated solvents for flushing the trapped compounds to the NMR probe. Thus, solvent suppression is no longer necessary. Multiple trapping of the same analyte from repeated LC injections was utilized to solve the problem of low concentration and to obtain 2D heteronuclear NMR spectra. In addition, a combination of the SPE unit with a recently developed cryoflow NMR probe and an MS was evaluated. This on-line LC-UV-SPE-NMR-MS system was used for the automated analysis of a Greek oregano extract. Combining the data provided by the UV, MS, and NMR spectra, the flavonoids taxifolin, aromadendrin, eriodictyol, naringenin, and apigenin, the phenolic acid rosmarinic acid, and the monoterpene carvacrol were identified. This automated technique is very useful for natural product analysis, and the large sensitivity improvement leads to significantly reduced NMR acquisition times.     

"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.     

Multiple solenoidal microcoil probes for high-sensitivity, high-throughput nuclear magnetic resonance spectroscopy

Two designs for incorporating multiple solenoidal microcoils into a single probe head are presented to increase the throughput of high-resolution NMR. Through a combination of radio frequency switches and low-noise amplifiers, multiple NMR spectra can be acquired in the same time as a single spectrum from a conventional probe consisting of one coil. Since this method does not compromise sensitivity with regard to the individual microcoils, throughput increases linearly with the number of coils. Only one receiver is needed, and data acquisition parameters can be optimized for each sample. Specifically, a four-coil system has been implemented for proton NMR at 250 MHz using a wide-bore magnet, with an observe volume of 28 nL for each microcoil. Signal cross-contamination was approximately 0.2% between individual coils, and simultaneous one- and two-dimensional spectra have been obtained from samples of fructose, galactose, adenosine triphosphate, and chloroquine (7 nmol of each compound). A more compact two-coil configuration has also been designed for operation at 500 MHz, with observe volumes of 5 and 31 nL for the two coils. One- and two-dimensional spectra were acquired from samples of 1-butanol (55 nmol) and ethylbenzene (250 nmol).     

Chemical shift correlations from hyperpolarized NMR by off-resonance decoupling

Nuclear magnetic resonance, through observation of chemical shift, allows the separate identification of each atom in a molecule. Thus, NMR spectra impart an often unrivaled wealth of information on molecular structure. A particular advantage of NMR spectroscopy is the ability to record multidimensional spectra, which provide correlations between atoms. When compared to other techniques, such as optical spectroscopy, the acquisition of NMR spectra is however an insensitive process, requiring samples of high concentration and long acquisition times. Recently, it has been demonstrated that dynamic nuclear polarization, a hyperpolarization technique, can increase the NMR signal by several orders of magnitude. Here, we present a robust method that allows recording two-dimensional chemical shift correlations from such hyperpolarized molecules. The method makes use of an apparent scaling of the scalar coupling observed on one type of atom, when an off-resonance decoupling field is applied to another type of atom. Thus, two-dimensional chemical shift correlations can be read directly from a small number of scans acquired using a hyperpolarized sample. Due to the ease of implementing this technique on commercial hyperpolarization and NMR equipment, it appears ideally suited for routine application, for example, to obtain carbon-proton chemical shift correlations in organic molecules.     

Optically Polarized 129Xe in NMR Spectroscopy*

¹²⁹ Xe has found widespread applications in NMR spectroscopy, but its use is often restricted by its low sensitivity and long relaxation times. Optical pumping of rubidium followed by spin exchange with xenon provides a well understood technique to enhance the ¹²⁹ XePolarization by several orders of magnitude. The exploitation of this highly spin-polarized xenon as a surface probe in NMR studies of various meterials is discussed. Applications of this technique make use not only of ¹²⁹Xe NMR detection, but also of the-high spin polarization as a magnetization reservoir for selective cross-polarization experiments to surface nuclei. A diverse variety of materials has been studied, ranging from mesoscopic structures such as semiconductor nanocrystals and porous silicon to high surface area polymers.     

NMR approach to the quantification of nonstatistical 13C distribution in natural products: vanillin

Quantitative (13)C NMR conditions have been established that permit the precise determination of site-specific (13)C/(12)C ratios at low or natural abundance. Spectral acquisition parameters have been optimized in order to obtain minimum intensity distortions over the spectral width and in relation to the major sources of inaccuracy: the relaxation times, the decoupling pulse and power, and nuclear Overhauser effects. A major reduction in experimental time resulting from a study of the relaxation times and variance analysis has been achieved. The influence of (1)H decoupling conditions on peak areas was shown to be critical in that different relative peak areas are obtained according to the decoupling power. The efficiency with which the quantitative (13)C NMR method can determine site-specific (13)C/(12)C ratios in natural products has been tested for 12 independent samples of vanillin from different sources. Discriminatory analysis performed in the space defined by the site-specific carbon isotope ratios allows natural vanillin and that from different synthetic origins to be unambiguously distinguished.     

Femtosecond broadband stimulated Raman: a new approach for high-performance vibrational spectroscopy

Femtosecond stimulated Raman spectroscopy (FSRS) is a new technique that produces high-quality vibrational spectra free from background fluorescence. FSRS combines a narrow-bandwidth picosecond Raman pump pulse with an approximately 80 fs continuum probe pulse to produce stimulated Raman spectra from the pump-induced gain in the probe spectrum. The high intensity of the Raman pump combined with the broad bandwidth of the probe produces high signal-to-noise vibrational spectra with very short data acquisition times. FSRS spectra of standard solutions and solvents such as aqueous Na2SO4, aqueous KNO3, methanol, isopropanol, and cyclohexane are collected in seconds. Furthermore, stimulated Raman spectra can be obtained using just a single pump-probe pulse pair that illuminates the sample for only approximately 1 ps. Fluorescence rejection is demonstrated by collecting FSRS spectra of dyes (rhodamine 6G, chlorophyll a, and DTTCI) with varying degrees of fluorescence background and resonance enhancement. The high signal-to-noise, short data acquisition time, fluorescence rejection, and high spectral and temporal resolution of femtosecond stimulated Raman spectroscopy make it a valuable new vibrational spectroscopic technique.     

High-throughput nuclear magnetic resonance analysis using a multiple coil flow probe

An automated method for high-throughput nuclear magnetic resonance (NMR) spectroscopy has been developed using a four-coil Multiplex NMR probe. The probe is constructed with solenoidal microcoils optimized for detection of small volume, mass-limited samples and a flow-through design. Four samples can be simultaneously injected into the Multiplex probe with a robotics liquid handler and then analyzed in rapid succession using a selective excitation experiment. Due to the simultaneous injection of four samples and the reduced analysis time with rapid selective excitation, the analysis rate achieved thus far is as low as 1 sample/34 s for 1D 1H NMR.