Development of analytical methods to study the salivary metabolome: impact of the sampling

Advances in metabolomics have allowed the identification and characterization of saliva metabolites that can be used as biomarkers. However, discrepancies can be noted with the content of the same biomarker being increased or decreased for a given disease. Differences in the way saliva is collected, stored, and/or treated could cause these discrepancies. Indeed, there is no standardized method for saliva sampling and analysis. In this work, two chromatographic modes were used, i.e., RP-LC and HILIC both coupled to MS used in positive and negative ionization modes. The analytical conditions were optimized with a mixture of 90 compounds naturally present in saliva, representative of the wide range of molecular mass and polarity of salivary metabolites and being described as having a differential expression in various pathologies. These four methods were applied to the analysis of saliva samples collected by spitting, aspiration, or Salivette® with or without prior rinsing of the mouth. Rinsing had an effect on some metabolite concentrations. As it can induce an additional parameter of variability to the sampling, it seems therefore preferable to use methods without rinsing while effects of these parameters on the metabolites are investigated. Saliva obtained by spitting and aspiration gave statistically equivalent results for 84% of the metabolites studied. Conversely, Salivette® gave different results since the majority of the metabolites chosen for the study were not quantified in the samples. The Salivette® does not seem therefore to be a suitable sampling method for an untargeted analysis of the salivary metabolome, unlike aspiration and spitting.