Intra- and inter-day variations in oral metabolites from mouth-rinsed water determined using capillary electrophoresis–mass spectrometry metabolomics

Background and aims: Collecting clinical samples without inconveniencing participants is desirable. The profile of metabolites in mouth-rinsed water is similar to that in saliva. However, the intra- and inter-day variations in unstimulated or stimulated saliva metabolites from mouth-rinsed water have yet to be clarified. Thus, we aimed to fill this research gap using capillary electrophoresis–mass spectrometry metabolomics. Materials and methods: We collected mouth-rinsed water from 15 healthy participants at 9:00, 11:30, 14:00, and 16:30 daily for 3 days. In total, 509 metabolite concentrations from 180 samples were obtained using capillary electrophoresis time-of-flight mass spectrometry. Variations in each metabolite were evaluated using the Wilcoxon signed-rank test to determine at which time/day significant differences occurred after removing metabolites without significant changes using the Friedman test. Results: Of 167 frequently detected metabolites, 100 exhibited intra-day variations, and none exhibited inter-day variations. Intra-day variations were classified into four patterns, and the intra-day variation in each metabolite was assessed. The variations may reflect elapsed time after meals, oral cleaning, or circadian rhythms. Conclusion: This study could serve as a reference for improving the design of future clinical trials and the accuracy of metabolome analysis of mouth-rinsed water samples collected at different dates and times.