Figure 3.

A. Glucuronidated anthocyanin conjugates are detectable in post BRB rinse saliva samples. The selected ion chromatograms (SIC) of the tandem mass spectrum of the specific ion of m/z 757 in the Rinse III solution (i) and the 5 min saliva sample post rinsed with Rinse III solution (ii). In contrast to the Rinse III solution (i.), there are three peaks with a retention time of 19.22, 21.88 and 25.65 labeled as Peak I, II and III in the SIC of the saliva samples. The tandem mass spectra of Peak I and II provide two expected product ions of m/z 287 and 449 (iii. and iv.), which are consistent with the presence of sambubioside glucuronides. The tandem mass spectrum of peak III showed a single peak of m/z 757.37 (v.), which shows a longer retention time relative to sambubioside, represents a novel metabolite.

Figure 3. B. The SIC of m/z 757 from the ex-vivo incubation (15 min) of 10% FBR and human liver microsomes in saliva collected from one of six healthy volunteers. Similar to data depicted in Figure 3.A., three peaks are also observed in the saliva-BRB-liver microsomal incubations. There are, however, slight variations in peak retention times and relative intensities which likely reflect different time frames when the samples were analyzed on the column as well as tissue specific variations i.e. liver relative to oral mucosal UGT polymorphisms.

Figure 3. C. Protocatechuic acid glucuronide (PCAOG) is also detectable in post-rinse saliva. The full scan (i) and collision induced dissociation (ii) mass spectra of protocatechuic acid O-glucuronide (PCAOG) under a negative mode demonstrated two predominant peaks with m/z 329.1 and 658.8, which correspond to the deprotonated ion of PCAOG and its dimer, respectively. The data dependent collision-induced dissociation spectrum of the ion of m/z 329.1 (ii) showed the most abundant peak with m/z 153, corresponding to the deprotonated protocatechuic acid formed via the labile cleavage of glycosidic bond, and two minor peaks with m/z 175.0 and 113.2. The total ion chromatograms (iii) of the ionic transition 329>153 of PCAOG (100 ng/mL) in 1% formic acid aqueous solution, the 5-min saliva sample (iv), and the 30 min saliva sample (v) from a healthy volunteer after rinse with rinse formulation III. A peak with retention time of 28.12 min labeled as Peak IV and a relative small peak with the retention time of 31.5 min labeled at Peak V was shown in PCAOG (iii), an identical peak IV was shown in the XIC 5 min saliva samples, but no peaks were shown in the 30 min saliva sample (v).