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Sensitivity and resolution are key considerations for NMR applications in general and for metabolomics in particular, where complex mixtures containing hundreds of metabolites over a large range of concentrations are commonly encountered. There is a strong demand for advanced methods that can provide maximal information in the shortest possible time frame. Here, we present the optimization and application of the recently introduced 2D real-time BIRD 1 H- 13 C HSQC experiment for NMR-based metabolomics of aqueous samples at 13 C natural abundance. For mouse urine samples, it is demonstrated how this real-time pure shift sensitivity-improved heteronuclear single quantum correlation method provides broadband homonuclear decoupling along the proton detection dimension and thereby significantly improves spectral resolution in regions that are affected by spectral overlap. Moreover, the collapse of the scalar multiplet structure of cross-peaks leads to a sensitivity gain of about 40-50% over a traditional 2D HSQC-SI experiment. The experiment works well over a range of magnetic field strengths and is particularly useful when resonance overlap in crowded regions of the HSQC spectra hampers accurate metabolite identification and quantitation.

Real-Time Pure Shift HSQC NMR for Untargeted Metabolomics