Impact of Selected Pre-Analytical and Analytical Factors on Untargeted Salivary Metabolomics

With the growing interest in personalized medicine, alternative biological matrices to blood are increasingly explored as sources of diagnostic information. Saliva has emerged as a promising diagnostic matrix due to its non-invasive collection, suitability for home sampling, and minimal requirements for personnel training. Numerous studies have demonstrated the presence of metabolites in saliva that enable disease diagnosis and monitoring. However, the influence of pre-analytical and analytical factors on salivary metabolomics outcomes remains insufficiently characterized. In this study, we investigated factors potentially affecting the number and abundance of detected metabolites in untargeted salivary metabolomics using liquid chromatography coupled with mass spectrometry (LC–MS). The impact of chromatographic column type, extraction protocol, and saliva type (stimulated versus resting) was evaluated. Additionally, the effect of swab type on analyte recovery was assessed. The use of a synthetic swab for saliva collection yielded results most comparable to those obtained without swabs, for both resting and stimulated saliva samples, indicating minimal pre-analytical interference. The greatest metabolite coverage was obtained using ACN:MeOH (1:1, v/v), with a ZIC-HILIC column for polar metabolites and a C18 column for non-polar metabolite separation. These findings demonstrate that swab type, chromatographic column, extraction solvent, and saliva type critically shape metabolite coverage in untargeted salivary metabolomics. Importantly, the distinct metabolic profiles of resting and stimulated saliva suggest that these matrices may provide complementary clinical insights, underscoring the need for saliva type selection tailored to specific diagnostic and biomarker discovery objectives.