Evaluation of non-uniform sampling 2D¹H–¹³C HSQC spectra for semi-quantitative metabolomics

Metabolomics is the comprehensive study of metabolism, the biochemical processes that sustain life. By comparing metabolites between healthy and disease states, new insights into disease mechanisms can be uncovered. NMR is a powerful analytical method to detect and quantify metabolites. Standard one-dimensional (1D)¹H-NMR metabolite profiling is informative but challenged by significant chemical shift overlap. Multi-dimensional NMR can increase resolution, but the required long acquisition times lead to limited throughput. Non-uniform sampling (NUS) is a well-accepted mode of acquiring multi-dimensional NMR data, enabling either reduced acquisition times or increased sensitivity in equivalent time. Despite these advantages, the technique is not widely applied to metabolomics. In this study, we evaluated the utility of NUS¹H–¹³C heteronuclear single quantum coherence (HSQC) for semi-quantitative metabolomics. We demonstrated that NUS improved sensitivity compared to uniform sampling (US).We verified that the NUS measurement maintains linearity, making it possible to detect metabolite changes across samples and studies. Furthermore, we calculated the lower limit of detection and quantification (LOD/LOQ) of common metabolites. Finally, we demonstrate that the measurements are repeatable on the same system and across different systems. In conclusion, our results detail the analytical capability of NUS and, in doing so, empower the future use of NUS¹H–¹³C HSQC in metabolomic studies.