HSQC/F1-PSYCHE TOCSY NOAH Supersequence for High-Resolution NMR Analysis of Urine Metabolites.

Abstract

Accurate assignment of metabolites is the backbone of metabolomics studies. Two-dimensional (2D) NMR plays a critical role in the accurate assignment of metabolites. Characterization of 2D spectra such as ¹H-¹³C HSQC and ¹H-¹H TOCSY combined with database queries enables reliable metabolite identification for metabolic profiling and biological interpretation. However, recording a high-quality ¹H-¹³C HSQC spectrum at ¹³C natural abundance in biofluids requires extensive NMR signal averaging, often taking up to 24 h. Reducing the number of t₁ increments or scans is not useful in metabolomics as it compromises the sensitivity needed to detect low-abundance metabolites. "NMR by Ordered Acquisition using ¹H detection," or NOAH, supersequences are ideally suited for accelerated data collection in biofluids. Instead of shortening individual experiments, NOAH enables the simultaneous acquisition of multiple 2D experiments without compromising sensitivity. The principle of NOAH lies in utilizing the undisturbed magnetization from one experiment (e.g., ¹H-¹³C HSQC) for subsequent experiments (e.g., ¹H-¹H TOCSY) within the same scan. Previous studies have demonstrated the utility of the HSQC + TOCSY NOAH-2 supersequence for metabolomics applications. Nevertheless, due to the complexity of biofluids, even regular 2D TOCSY spectra often suffer from signal overlap, arising from numerous metabolite peaks, multiplet structures, and limited ¹H chemical shift dispersion. The pure shift F₁-PSYCHE TOCSY experiment addresses this challenge by offering a single peak per resonance, thereby greatly reducing signal overlap. In this work, we present HSQC + F₁-PSYCHE TOCSY NOAH-2 supersequence for the analysis of human urine.