Stefano Caldarelli

Enhanced diffusion-edited NMR spectroscopy of mixtures using chromatographic stationary phases

We introduce an analytical method that combines in one pot the advantages of column chromatography separation and NMR structural analysis. The separation of the NMR spectra of the components of a mixture can be achieved according to their apparent diffusion rates [James, T. L. and McDonald, G. G. (1973) J. Magn. Reson. 58, 58–61]. We show that the separation of the spectral components, corresponding to single molecular species, can be enhanced by order of magnitudes upon addition of a typical stationary phase used in HPLC. The solid phase imbibed by the mixture for analysis is an heterogeneous ensemble, so that solid-state NMR methods (high-resolution magic angle spinning) are necessary to recover high-resolution spectra. We demonstrate applications of this combination of high-resolution magic angle spinning and NMR diffusometry on test mixtures for direct (silica gel) and inverse (C18) columns. However, many common chromatographic supports available for HPLC should be readily adaptable for use with this technique.

Evaluation of Fast 2D NMR for Metabolomics

Two-dimensional nuclear magnetic resonance (2D NMR) is increasingly explored as a tool for metabolomics because of its superior resolution compared to one-dimensional NMR (1D NMR). However, 2D NMR is characterized by longer acquisition times, which makes it less suitable for high-throughput studies. In this Article, we evaluated two methods for the acceleration of nD NMR, ultrafast (UF) and nonuniform sampling (NUS), in the context of metabolomics. To this end, model samples mimicking the metabolic profile variations in serum from subjects affected by colorectal cancer and controls were analyzed by 1D (1)H NMR along with conventional and accelerated DQF-COSY and HSQC. A statistical analysis (OPLS-DA) yielded similar results for the group separation with all techniques, but biomarker identification from 2D spectra was substantially enhanced, both in terms of number of molecules and easiness of assignment. Most interestingly, fast 2D NMR techniques lead to similar results as conventional 2D NMR, opening the way for high-throughput metabolomics studies using 2D NMR.

Increasing the robustness of heteronuclear decoupling in magic-angle sample spinning solid-state NMR

Proton decoupling is a crucial step to obtain high-resolution NMR spectra of macromolecules. Current methods employ phase (or amplitude)-modulated radio-frequency pulses to this end. To be effective, the modulation has to match some resonant condition. We demonstrate in this Communication a simple approach to expand the stability of the resonant condition by convolution of the basic modulation with a Gaussian envelope.

Chromatographic NMR: a tool for the analysis of mixtures of small molecules.

The addition of a solid to a mixture of small molecules introduces variations on the average translational mobility of these latter, proportional to the affinity of a given moiety for the less‐mobile phase. This effect is at the basis of chromatographic separation, but can be used to simplify the NMR analysis of mixtures as well. In fact, as the induced differences in molecular mobility can span orders of magnitude, it becomes a much easier task to split the overall NMR spectrum of the mixture into one of the pure components using pulsed field gradient (PFG) methods. We have demonstrated recently this approach, in the context of HRMAS (high‐resolution magic angle spinning) NMR, as required to recover high‐resolution spectra in solid/liquid mixtures. In this review, we shall cover some of the principles and the practicality of this HRMAS‐PFG approach for the study of mixtures. A comparison of the actual (in LC) and virtual (in NMR) separation capabilities of a few combinations of solid phase material/solvent composition shows similarities but also some striking differences: bare (not functionalized) silica expresses a superior potential for resolving spectral components than expected on the basis of the LC outcome. Copyright

A Solid-State NMR Study of Lead and Vanadium Substitution into Hydroxyapatite

A systematic study on cationic and anionic substitution in hydroxyapatite structures was carried out, with the aim of understanding the impact of ion exchange on the crystalline structure and properties of these materials. Lead and vanadium were chosen for the exchange, due to their known effects on the redox and catalytic properties of hydroxypatites. Hydroxyapatites with variable Pb and V contents, Pb(x)Ca(10-x)(VO(4))(y)(PO(4))(6-y)(OH)(2) (x = 0, 2, 4, 6, 8 and 10 for y = 1; y = 0, 0.5, 1, 2, 3 and 6 for x = 10) were synthesized and characterized by NMR spectroscopy. Solid-state NMR allowed an analysis of the chemical environment of every ion after substitution into the hydroxyapatite network. (43)Ca and (207)Pb NMR spectra at different lead concentrations provided clear evidence of the preferential substitution of lead into the Ca(II) site, the replacement of the Ca(I) site starting at x = 4 for y = 1. Two NMR distinguishable Pb(I) sites were observed in Pb(10)(PO(4))(6)(OH)(2), which is compatible with the absence of a local mirror plane perpendicular to the c direction. In contrast with (31)P NMR, for which only small variations related to the incorporation of Pb are observed, the strong change in the (51)V NMR spectrum indicates that lead perturbs the vanadium environment more than the phosphorus one. The existence of a wide variety of environments for OH in substituted apatites is revealed by (1)H NMR, and the mobility of the water molecules appears to vary upon introduction of lead into the structure.

General implementation of the ERETIC method for pulsed field gradient probe heads.

A capacitive coupling between a secondary radiofrequency (rf) channel and the gradient coil of a standard commercially available high resolution NMR spectrometer and probe head is described and used to introduce a low level exponentially damped rf signal near the frequency of the primary rf channel to serve as an external concentration standard, in analogy to the so-called ERETIC method. The stability of this inexpensive and simple to implement method, here referred to as the Pulse Into the Gradient (PIG) approach, is superb over a 14-h period and both gradient tailored water suppression and one-dimensional imaging applications are provided. Since the low level signal is introduced via the pulsed field gradient coil, the coupling is identical to that for a free induction signal and thus the method proves to be immune (within 5%) to sample ionic strength effects up to the 2M NaCl solutions explored here.

Demixing of Severely Overlapping NMR Spectra through Multiple-Quantum NMR

We introduce an NMR method to help in the analysis of complex mixtures. The spectra of molecular fragments are obtained as the traces of a correlation spectrum of the regular (1)H NMR spectrum on one dimension with the one of the highest possible (1)H multiple-quantum (MaxQ) order. As this latter is a function of the number of distinguishable protons in a given molecular fragment, the analysis of a series of multiple-quantum spectra is required to achieve a complete assignment. This MaxQ NMR approach is likely to perform best in the case of signals concentrated in a very narrow frequency range, which is a challenging situation commonly encountered in many relevant analytical problems such as the characterization of extraction fractions (oil, plants, tissues), biological fluids, or environmentally relevant samples. As a demonstration, we apply the MaxQ NMR analysis to a mixture of 11 poly- and monocyclic aromatic hydrocarbons.

Effective processing of pulse field gradient NMR of mixtures by blind source separation.

NMR diffusometry and its flagship layout, diffusion-ordered spectroscopy (DOSY), are versatile for studying mixtures of bioorganic and synthetic molecules, but a limiting factor of its applicability is the requirement of a mathematical treatment capable of distinguishing molecules with similar spectra or diffusion constants. We present here a processing strategy for DOSY, a synergy of two high-performance blind source separation (BSS) techniques: non-negative matrix factorization (NMF) using additional sparse conditioning (SC), and the JADE (joint approximate diagonalization of eigenmatrices) declination of independent component analysis (ICA). While the first approach has an intrinsic affinity for NMR data, the latter one can be orders of magnitude computationally faster and can be used to simplify the parametrization of the former.

Metabolomics approach to thyroid nodules: A high-resolution magic-angle spinning nuclear magnetic resonance–based study

BACKGROUND Proton magnetic resonance spectroscopy of operative specimens has been reported to successfully differentiate normal tissue from malignant thyroid tissue. We used a new high-resolution magnetic resonance spectroscopy technique for the differentiation of benign and malignant thyroid neoplasms. METHODS Histological specimens from 72 patients undergoing a total thyroidectomy were processed into a 4-mm ZrO(2) high-resolution magic angle spinning (HRMAS) rotor with 5 μL of D(2)O. A Bruker Avance spectrometer operating at 400 MHz for the (1)H frequency and equipped with a (1)H/(13)C/(31)P HRMAS probe was used. RESULTS Normal and neoplastic thyroid tissues could be discriminated from each other by different relative concentrations of several amino acids and lipids, as well as benign and malignant neoplasms, that differed in terms of a greater lactate and taurine and a lesser lipid choline, phosphocholine, myo-inositol, and scyllo-inositol levels in malignant samples. A statistical analysis with a receiver operating characteristic curve revealed that 77% of the samples were accurately predicted. Similar results were obtained with specimens obtained from ex vivo aspirates. CONCLUSION A further development of this project will be to use the metabolomics approach on specimens obtained from aspirates in vivo after the resolution of technical problems attributable to possible contamination.

Metabolomic and Lipidomic Analysis of Serum Samples following Curcuma longa Extract Supplementation in High-Fructose and Saturated Fat Fed Rats.

We explored, using nuclear magnetic resonance (NMR) metabolomics and fatty acids profiling, the effects of a common nutritional complement, Curcuma longa, at a nutritionally relevant dose with human use, administered in conjunction with an unbalanced diet. Indeed, traditional food supplements have been long used to counter metabolic impairments induced by unbalanced diets. Here, rats were fed either a standard diet, a high level of fructose and saturated fatty acid (HFS) diet, a diet common to western countries and that certainly contributes to the epidemic of insulin resistance (IR) syndrome, or a HFS diet with a Curcuma longa extract (1% of curcuminoids in the extract) for ten weeks. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) on the serum NMR profiles and fatty acid composition (determined by GC/MS) showed a clear discrimination between HFS groups and controls. This discrimination involved metabolites such as glucose, amino acids, pyruvate, creatine, phosphocholine/glycerophosphocholine, ketone bodies and glycoproteins as well as an increase of monounsaturated fatty acids (MUFAs) and a decrease of n-6 and n-3 polyunsaturated fatty acids (PUFAs). Although the administration of Curcuma longa did not prevent the observed increase of glucose, triglycerides, cholesterol and insulin levels, discriminating metabolites were observed between groups fed HFS alone or with addition of a Curcuma longa extract, namely some MUFA and n-3 PUFA, glycoproteins, glutamine, and methanol, suggesting that curcuminoids may act respectively on the fatty acid metabolism, the hexosamine biosynthesis pathway and alcohol oxidation. Curcuma longa extract supplementation appears to be beneficial in these metabolic pathways in rats. This metabolomic approach highlights important serum metabolites that could help in understanding further the metabolic mechanisms leading to IR.