## 4 Results  
### Optimal Saliva Sampling Parameters  
Eleven healthy participants were recruited with a median age of 25 years (IQR: 20-29 years), and 7 (58%) were female. Details of the concentrations (ppbv) of headspace saliva VOC across all four methodological parameters and saliva collection methods are presented in Table 1 and Figure 1. \begin{table}
\begin{tabular}{c c c c c c} \hline  & **Acetone** & **Acctic Acid** & **Butyric Acid** & **Pentanoic Acid** & **Hexanoic Acid** \\  & **(_m/z_ 59_)** & **(_m/z_ 61_)** & **(_m/z_ 89_)** & **(_m/z_ 103_)** & **(_m/z_ 117_)** \\ \hline \multicolumn{6}{c}{**Saliva collection**} \\ \hline Passive drool & 60 (46–86) & 14 (13–14) & 2.5 (2.4–4.0) & 1.0 (0.8–1.1) & 0.8 (0.7–0.9) \\ \hline Unstimulated saliva & 104 (61–1793) & 14 (14–37) & 3.2 (1.8–11.1) & 0.7 (0.6–3.2) & 0.7 (0.6–2.3) \\ \hline Stimulated saliva & 866 (278–1365) & 24 (16–27) & 6.1 (2.6–9.8) & 2.0 (0.8–2.3) & 1.1 (0.8–1.5) \\ \hline \multicolumn{6}{c}{**Volume**} \\ \hline Passive drool & & & & \\ \hline
500 \(\mu\)L & 57 (46–90) & 14 (13–14) & 2.5 (2.3–3.9) & 0.9 (0.8–1.1) & 0.8 (0.7–0.9) \\ \hline
1000 \(\mu\)L & 51 (37–58) & 13 (13–14) & 2.3 (1.6–3.2) & 0.8 (0.7–1.0) & 0.8 (0.5–0.8) \\ \hline Unstimulated saliva & & & & \\ \hline
500 \(\mu\)L & 1430 (390–1883) & 28 (16–44) & 8.2 (4.0–12) & 2.0 (0.9–3.9) & 1.8 (0.8–2.3) \\ \hline
1000 \(\mu\)L & 581 (213–1538) & 18 (15–44) & 4.2 (3.3–12) & 1.2 (0.8–4.3) & 1.0 (0.7–2.4) \\ \hline Stimulated saliva & & & & \\ \hline
500 \(\mu\)L & 866 (453–1365) & 24 (19–27) & 6.1 (3.2–9.7) & 2.0 (1.3–2.6) & 1.3 (0.9–1.6) \\ \hline
1000 \(\mu\)L & 173 (71–946) & 14 (13–26) & 4.5 (1.9–6.5) & 0.8 (0.6–2.9) & 0.7 (0.6–1.6) \\ \hline
**Dilution** & & & & \\ \hline Passive drool & & & & \\ \hline
500 \(\mu\)L saliva & 60 (46–86) & 14 (13–14) & 2.5 (2.4–4.0) & 1.0 (0.8–1.1) & 0.8 (0.7–0.9) \\ \hline
500 \(\mu\)L saliva + 1 mL & 41 (29–59) & 13 (13–14) & 2.3 (1.8–4.0) & 0.9 (0.7–0.9) & 0.8 (0.7–0.9) \\ deionised water & & & & \\ \hline Unstimulated saliva * & & & & \\ \hline
500 \(\mu\)L saliva & 104 (55–104) & 14 (14–14) & 1.8 (1.6–3.2) & 0.7 (0.6–0.7) & 0.6 (0.6–0.7) \\ \hline
500 \(\mu\)L saliva + 1 mL & 43 (40–55) & 14 (14–15) & 1.7 (1.7–1.9) & 0.7 (0.7–0.7) & 0.6 (0.6–0.7) \\ deionised water & & & & \\ \hline Stimulated saliva \(\dagger\) & & & & \\ \hline \end{tabular}
\end{table} 
Table 1: Influence of sampling methodology on the selected salivary VOC concentrations (ppbv). Figure 1: Four methodological parameters of saliva headspace optimised by VOC concentration (ppbv) assessment. Circles and asterisks denote outlying values. _Negative controls:_ Headspace concentrations (ppbv) and coefficient of variation (%) using the Salivette(r) (various treatment conditions) and aliquots of deionised water (\(n=3\)) were used as negative controls. Across all compounds of interest, all VOCs remained consistent with a low coefficient of variation (<10%) (Supplementary File S1). The dry Salivette(r) cotton roll demonstrated a high concentration of all SCFA (e.g., butyric acid at 23 ppbv) and acetone (3649 ppbv). The cotton roll saturated with deionised water showed reduced levels of butyric acid at 11 ppbv and acetone at 444 ppbv. Following centrifugation, headspace VOC analysis of the supernatant showed further reduction in butyric acid to 3.1 ppb. This was consistent across the remaining compounds suggesting this method of collection posed a potentially significant source of contamination to saliva samples. _Saliva collection method:_ Saliva collection method by passive drool and unstimulated saliva yielded comparable concentrations of acetone and SCFA of interest; acetic acid 14 (IQR: 13-14) vs. 14 (IQR: 14-37) ppbv, butyric acid 2.5 (IQR: 2.4-4.0) vs. 3.2 (1.8-11.1) ppbv, respectively. Stimulated saliva generated up to 2-fold higher VOC concentrations.