Extraction and identification of AHLs by HPLC-MS.

The identification of AHLs was done in different acidified saliva samples from the same healthy donor who provided a written informed consent that was approved by the Comité Ético de Investigación Clínica de Galicia (protocol 2009/319 modified in July 2017). The extraction of the sample (approximately 0.5 ml) was performed twice in an appropriate volume of organic solvents, ethyl acetate, and dichloromethane, and then it was evaporated to dryness at 40°C. AHLs present in the oral samples were reconstituted in acetonitrile and quantified by HPLC-MS methodology (45). Pure AHLs covering the whole range of AHL lengths (from C₄-HSL to C₁₈-HSL), both substituted and unsubstituted, were obtained from Sigma-Aldrich and from the University of Nottingham and used as external standards for quantification.