Exploring nuclear magnetic resonance spectroscopy for the analysis of dried blood spots

Dried blood spots (DBS) offer a practical and relatively non-invasive method for sample collection. Here, we evaluate the feasibility of applying ¹H NMR spectroscopy to metabolomic analysis of DBS. Various solvent suppression techniques and extraction protocols were tested using aqueous and methanolic solvents. Methanol was found to provide heightened metabolite recovery with fewer interfering macromolecular signals compared to aqueous buffers, supporting its use as a preferred extraction solvent. Additional experimental variables, such as extraction time and the number of DBS punches, were optimized to improve spectral quality. Potentially interfering contaminations from sampling materials and consumables were identified. As a demonstration of practical application, DBS cards were distributed and returned via international and local transport, and their metabolic profiles were assessed. Despite some variation among transported samples, individual-specific metabolic signatures remained discernible, suggesting a robustness of the method for comparative analyses. Overall, these initial results support the use of NMR as a complementary technique to mass spectrometry for DBS-based metabolomics, particularly when simplicity, reproducibility, and robustness is prioritized, and when overall sensitivity is less of a factor.