## 2 Material and Methods
### Optimal Saliva Sampling Parameters  
For methodological optimisation of salivary VOC analysis, a standardised approach was taken across all participants, who maintained a clear fluid diet for a minimum of six hours and subsequently provided whole saliva between 07:00 and 09:00 h. Each participant maintained their normal oral hygiene routines at least two hours prior to saliva sampling. Five sampling conditions were evaluated to establish the optimal sampling method for VOC headspace analysis of whole saliva: (i) method of collection; (ii) sample volume; (iii) sample dilution; (iv) sample acidification; (v) negative controls (background contamination)  
_Saliva collection method:_ Saliva (\(n\) = 11) was collected using three techniques, (i) passive drool into a sterile container over 5 min, (ii) unstimulated saliva by holding the Salivette(r) ([PERSON], Numbrecht, Germany) between the lateral teeth and buccal mucosa, or beneath the tongue, for two minutes, and (iii) stimulated saliva by chewing the Salivette(r) for one minute. The Salivette(r) is a commercially available saliva collection device composed of a cotton roll within a plastic collection tube suitable for centrifugation. As per the manufacturer's instructions, the tube was centrifuged for two minutes at 1000 \(g\). Salivette(r) was selected due to the ease of saliva sampling and simple instructions for potential patients. The osmolality of saliva was determined for each method using an osmometer (Advanced Instruments OsmoTech Promulti-Sample Micro Osmometer; Advanced Instruments, Wimborne, UK). Salivary flow rates (mL/min) were also calculated by saliva volume (mL) divided by time (minutes). _Saliva volume:_ The headspace of 500 \(\mu\)L and 1000 \(\mu\)L saliva (\(n\) = 11) was selected according to literature findings for headspace VOC assessment. _Saliva dilution:_ The headspace of 500 \(\mu\)L of whole saliva (\(n\) = 11) was compared to 500 \(\mu\)L of whole saliva diluted to 1 mL with deionised water. _Saliva acidification:_ A standardised protocol within the institution was used to acidify each sample (\(n\) = 11) with pure hydrochloric acid to pH 2.0 (FiveGoTM pH meter F2, Mettler Toledo, Columbus, OH, USA). An average of 3.7 \(\mu\)L hydrochloric acid was used (range 3-5 \(\mu\)L). Calibration checks were performed with standard buffer solutions of pH 4.0 and pH 7.0. Negative controlsA baseline of headspace VOCs produced by the experimental materials was assessed to define their potential contribution to background VOCs. Triplicate negative controls included: (i) a dry Salivette(r) (cotton roll); (ii) a Salivette(r) saturated with deionised water for two minutes; (iii) a Salivette(r) saturated with deionised water followed by centrifugation; (iv) 20 mL headspace vials containing 500 \(\mu\)L, 1000 \(\mu\)L and 1500 \(\mu\)L of deionised water. Saliva samples were stored at 4 \({}^{\circ}\)C and analysed within 2 h of collection. Samples were aliquoted into 20 mL glass headspace vials sealed with 18 mm screw-tops and a polytetrafluoroethylene (PTFE) septum. Vials were incubated for 30 min at 37 \({}^{\circ}\)C in a hot water bath to reflect the physiological body temperature. Septa were pierced with a sterile blunt needle (18 G, 1 \(\%\) (1.2 mm \(\times\) 40 mm)) for real-time direct headspace VOC analysis. AnalysisDirect injection mass spectrometry platforms provide soft chemical ionisation reducing fragmentation rates and allowing tentative compound annotation using specific molecular weights. The proton transfer reaction time of flight-mass spectrometer (PTR-ToF-MS; Ionicon Analytik GmbH, Innsbruck, Austria) uses an untargeted analytical approach with a hydronium ion (H\({}_{3}\)O\({}^{+}\)) as the primary reagent ion. The proton (H\({}^{+}\)) is transferred from the hydronium ion (H\({}_{3}\)O\({}^{+}\)) to the volatile compounds (VOC) due to their proton affinity being greater than water (VOC.H + H\({}_{2}\)O).