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

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.