Metabolites provide energy and biomass for life to exist, and alterations in metabolites have been linked to nearly all diseases. Thus, metabolite-based biomarkers have the potential to transform how diseases are diagnosed and treated. However, the exquisite sensitivity of metabolites to non-disease-related factors (diet, ethnicity, sample handling, bias from analytical measurement and more), poses significant challenges to discovering and developing robust clinical diagnostics. Indeed, recent meta-studies have demonstrated low concordance from metabolomic studies in diseases such as pancreatic cancer and kidney transplant. We have pioneered a multi-dimensional NUS NMR-based metabolomics discovery platform that enables the detection and relative quantification of a broad range of metabolites (µM to mM), in a highly repeatable and reproducible manner (%CV< 10%) and with turnaround time amenable to clinical testing (~1 hour/sample). This platform is enabling diagnostic development and aiding in biopharma drug development. High-quality clinical samples are a prerequisite for this discovery. However, the quality of metabolite data is largely set during biospecimen collection, storage, and sample processing. While sample collection/storage is often beyond our control, we have been developing improved quality control and quality assessment techniques to monitor sample preparation. Here, we report the development of “Navigators,” which include a proprietary combination of molecule(s) that can be added at the onset of sample processing to ensure quality metabolite extraction. These molecule(s) do not influence sample pH nor interfere with metabolite resonances. We have shown that high-quality sample preparation leads to consistent levels of the Navigators even with different operators, while low-quality sample preparation leads to dramatic changes, and thus the Navigators can be used as a flag for potential processing errors. Further, the influence of initial pH on urine metabolites is an area of active debate. With the demands of clinical testing, it is not often feasible to adjust the pH of individual samples prior to analysis. Here, we show that Navigators can be used to monitor pH even after metabolite extraction. Finally, by the addition of paramagnetic relaxation enhancement (PRE) agents to our NMR samples, we can further increase the apparent signal-to-noise ratio and reproducibility of sample measurements. These tools (Navigators, NUS, and signal enhancement agents), coupled with standard operating procedures, provide a workflow for reliable and robust NMR-based metabolite data, shaping a new path forward for the accessible use of NMR for metabolite-based diagnostics.
