Optimization of heteronuclear ultrafast 2D NMR for the study of complex mixtures hyperpolarized by dynamic nuclear polarization.

Hyperpolarized 13C NMR at natural abundance, based on dissolution dynamic nuclear polarization (d-DNP), provides rich, sensitive and repeatable 13C NMR fingerprints of complex mixtures. However, the sensitivity enhancement is associated with challenges such as peak overlap and the difficulty to assign hyperpolarized 13C signals. Ultrafast (UF) 2D NMR spectroscopy makes it possible to record heteronuclear 2D maps of d-DNP hyperpolarized samples. Heteronuclear UF 2D NMR can provide correlation peaks that link quaternary carbons and protons through long-range scalar couplings. Here, we report the analytical assessment of an optimized UF long-range HETCOR pulse sequence, applied to the detection of metabolic mixtures at natural abundance and hyperpolarized by d-DNP, based on repeatability and sensitivity considerations. We show that metabolite-dependent limits of quantification in the range of 1-50 mM (in the sample before dissolution) can be achieved, with a repeatability close to 10% and a very good linearity. We provide a detailed comparison of such analytical performance in two different dissolution solvents, D2O and MeOD. The reported pulse sequence appears as an useful analytical tool to facilitate the assignment and integration of metabolite signals in hyperpolarized complex mixtures.

Hyperpolarized 13 C†NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization.

Hyperpolarized nuclear magnetic resonance (NMR) offers an ensemble of methods that remarkably address the sensitivity issues of conventional NMR. Dissolution Dynamic Nuclear Polarization (d-DNP) provides a unique and general way to detect 13 C NMR signals with a sensitivity enhanced by several orders of magnitude. The expanding application scope of d-DNP now encompasses the analysis of complex mixtures at natural 13 C abundance. However, the application of d-DNP in this area has been limited to metabolite extracts. Here, we report the first d-DNP-enhanced 13 C NMR analysis of a biofluid -urine- at natural abundance, offering unprecedented resolution and sensitivity for this challenging type of sample. We also show that accurate quantitative information on multiple targeted metabolites can be retrieved through a standard addition procedure.

Hyperpolarized NMR metabolomics.

Hyperpolarized NMR is a promising approach to address the sensitivity limits of conventional NMR metabolomics approaches, which currently fails to detect minute metabolite concentrations in biological samples. This review describes how tremendous signal enhancement offered by dissolution-dynamic nuclear polarization and parahydrogen-based techniques can be fully exploited for molecular omics sciences. Recent developments, including the combination of hyperpolarization techniques with fast multi-dimensional NMR implementation and quantitative workflows are described, and a comprehensive comparison of existing hyperpolarization techniques is proposed. High-throughput, sensitivity, resolution and other relevant challenges that should be tackled for a general application of hyperpolarized NMR in metabolomics are discussed.

Ultrafast 2D NMR for the analysis of complex mixtures.

2D NMR is extensively used in many different fields, and its potential for the study of complex biochemical or chemical mixtures has been widely demonstrated. 2D NMR gives the ability to resolve peaks that overlap in 1D spectra, while providing both structural and quantitative information. However, complex mixtures are often analysed in situations where the data acquisition time is a crucial limitation, due to an ongoing chemical reaction or a moving sample from a hyphenated technique, or to the high-throughput requirement associated with large sample collections. Among the great diversity of available fast 2D methods, ultrafast (or single-scan) 2D NMR is probably the most general and versatile approach for complex mixture analysis. Indeed, ultrafast NMR has undergone an impressive number of methodological developments that have helped turn it into an efficient analytical tool, and numerous applications to the analysis of mixtures have been reported. This review first summarizes the main concepts, features and practical limitations of ultrafast 2D NMR, as well as the methodological developments that improved its analytical potential. Then, a detailed description of the main applications of ultrafast 2D NMR to mixture analysis is given. The two major application fields of ultrafast 2D NMR are first covered, i.e., reaction/process monitoring and metabolomics. Then, the potential of ultrafast 2D NMR for the analysis of hyperpolarized mixtures is described, as well as recent developments in oriented media. This review focuses on high-resolution liquid-state 2D experiments (including benchtop NMR) that include at least one spectroscopic dimension (i.e., 2D spectroscopy and DOSY) but does not cover in depth applications without spectral resolution and/or in inhomogeneous fields.