Comparisons of different extraction methods and solvents for saliva samples.

Introduction: Changes in the categories and concentrations of salivary metabolites may be closely related to oral, intestinal or systemic diseases. To study salivary metabolites, the first analytical step is to extract them from saliva samples as much as possible, while reducing interferences to a minimum. Frequently used extraction methods are protein precipitation (PPT), liquid-liquid extraction (LLE) and solid-phase extraction (SPE), with various organic solvents. The types and quantities of metabolites extracted with different methods may vary greatly, but few studies have systematically evaluated them.

Objectives: This study aimed to select the most suitable methods and solvents for the extraction of saliva according to different analytical targets.

Methods: An untargeted metabolomics approach based on liquid chromatography-mass spectrometry was applied to obtain the raw data. The numbers of metabolites, repeatability of the data and intensities of mass spectrometry signals were used as evaluation criteria.

Results: PPT resulted in the highest coverage. Among the PPT solvents, acetonitrile displayed the best repeatability and the highest coverage, while acetone resulted in the best signal intensities for the extracted compounds. LLE with the mixture of chloroform and methanol was the most suitable for the extraction of small hydrophobic compounds.

Conclusion: PPT with acetonitrile or acetone was recommended for untargeted analysis, while LLE with the mixture of chloroform and methanol was recommended for small hydrophobic compounds.