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

Untargeted Metabolomic Approach to Study the Impact of Aging on Salivary Metabolome in Women

“…Our group previously reported on the importance of standardizing sample collection and treatment to allow for proper comparison of metabolomic profiles [ 16 ]. In view of the different partners, the samples were collected either by aspiration (24 samples) or by spitting (26 samples), two methods that were previously shown to give similar results for metabolomic analysis [ 16 ].…”

“…Our group previously reported on the importance of standardizing sample collection and treatment to allow for proper comparison of metabolomic profiles [ 16 ]. In view of the different partners, the samples were collected either by aspiration (24 samples) or by spitting (26 samples), two methods that were previously shown to give similar results for metabolomic analysis [ 16 ]. However, the following rules were defined to maximize standardization of sample collection: (i) All the volunteers were asked to refrain from having any food or drink or brushing their teeth for at least 2 h before saliva collection; (ii) The samples were collected without prior rinsing of the mouth; (iii) The collection time was set at 5 min; and (iv) Subjects were asked not to swallow, so that all oral fluid produced during the collection time would be collected.…”

“…Chromatographic separation and detection by mass spectrometry were performed according to Bosman et al [ 16 ]. The analyses were performed using a liquid chromatograph (1260 Infinity, Agilent Technologies, Waldbronn Germany), coupled to a tandem mass spectrometer (microOTOF2, Bruker Daltonics, Bremen, Germany), and equipped with an electrospray ionization source.…”

“…A stepping in the basic mode that alternates every 0.25 s between 70 and 500 Vpp for RF collision and between 50 and 120 µs for the transfer time was used to facilitate the transfer of ions on the range of m / z . As previously demonstrated, the widest possible coverage of salivary metabolites, and thus, the most comprehensive comparison possible for metabolomic profiles was obtained when each saliva sample was analyzed using four analytical methods [ 16 ]. Two chromatographic separation modes (RP-LC and HILIC) coupled with detection by HRMS used in positive or negative ionization modes, were respectively named RP-LC+, RP-LC−, HILIC+, and HILIC−, having been thus implemented.…”

“…Otherwise, a blank was inserted between each injection of a saliva sample to verify the absence of carry-over between the different samples. Finally, for quality control, we used a solution containing 90 compounds that had already been described in the literature and was confirmed by our group as being present in saliva [ 16 ], i.e., amino acids, fatty acids, and other small molecules, with m / z between 60.021 and 430.381 and belonging to a wide range of polarity (lop P values in the range −4.70–10.51). Indeed, in a previous study [ 16 ], we demonstrated that these compounds, which are representative of salivary metabolites in terms of molecular mass, structural family, and polarity, induced a response from the analytical system that was proportional to the analyte concentration and allowed us to obtain limits of quantification to the order of a hundred ng/mL.…”