Optimized NMR-Based Metabolomic Protocol for Salivary Analysis in Clinical Practice

Saliva remains underexplored in metabolomics compared with widely used biofluids such as blood and urine. However, its noninvasive, rapid, and cost-effective collection, together with its suitability for self-sampling, makes it attractive for clinical and personalized medicine. Moreover, saliva is expected to provide complementary metabolic information on other biofluids. In this context, this study aims to identify an optimal sample collection and preparation protocol for maximizing informative metabolomic data from salivary nuclear magnetic resonance (NMR) profiles. Four preparation protocols, selected and adapted from the literature, were systematically compared using the number of identified metabolites, their concentrations, and intraday repeatability as evaluation criteria. Among them, an in-house-adapted method combining centrifugation, freeze-drying, and ultrafiltration proved most effective. This approach provided the broadest metabolome coverage, with 42 metabolites quantified. This method was further assessed using analysis of variance to determine intra- and interday precision. Most metabolites demonstrated excellent repeatability (coefficients of variation below 10%), confirming the protocol reliability for quantitative metabolomics. Overall, the optimized approach yields high-quality spectra, wide metabolic coverage, and strong analytical precision, supporting reproducible salivary NMR metabolomics. Beyond its methodological contribution, this work highlights saliva as a promising biofluid for diagnostics, disease monitoring, and personalized medicine, provided that collection and preparation procedures are appropriately standardized.