## 5 Discussion  
The principal findings of this study were the following: (i) saliva collected by the passive drool method presents the least contaminated collection method, with negligible variability between subjects, (ii) a volume of 500 \(\upmu\)L of saliva is acceptable for VOC headspace analysis, and (iii) salivary VOCs have an acceptable level of day-to-day and week-to-week variation. This study presents the simplest form of saliva collection with no preprocessing steps, which can introduce contamination. Salivary VOCs may therefore offer a simple, inexpensive, and acceptable method for noninvasive assessment of health and disease.  
The composition of saliva is influenced by factors such as flow rate and time of sampling, which are interlinked. Unstimulated saliva flows at 0.3-0.4 mL/min and is reduced by a further 0.1 mL/minute during sleep [9]. Therefore, morning saliva samples following an overnight fast are likely to be more concentrated with reduced flow. Stimulated saliva associated with higher flow rates has demonstrated a different electrolyte profile compared to unstimulated morning saliva [13]. [PERSON] et al. reported the expression of genes associated with circadian patterns and supported by varying concentrations of salivary electrolytes [14]. This was supported in this study with higher osmolality and reduced flow rates identified in unstimulated samples (i.e., passive drool; 99 mOsm, 0.7 mL/min) compared with stimulated samples with higher flow rates and reduced osmolality (64 mOsm, 1.9 mL/min). The passive drool and unstimulated method yielded comparable results; however, the Salivette(r) cotton roll introduced significant contamination of target compounds. Sample collection by passive drool is simple and inexpensive and minimises potential sources of bias. To establish a standardised method, we suggest a collection of morning saliva samples to reduce potential exogenous sources of bias/contamination introduced by daily activities.  
With a standardised methodology, the reproducibility of headspace salivary VOCs showed that longitudinal VOC variation was specific to target VOCs. Acetaldehyde and propanal showed wide variation over a three-week period with a high coefficient of variation \(>\) 50%. This suggests that certain VOCs may be labile either due to their volatility or systemic factors. However, the majority of compounds were stable, with a comparable median and low coefficient of variation. Comparisons between VOCs in healthy controls and disease-specific states should consider the range and variation. A narrow range of concentrations may not be as important as the differences in levels between both control and disease groups. Higher levels of certain VOCs in disease states relative to healthy states can mean that a wider variation is acceptable.  
Although this study presents a standardised model for saliva collection, the lack of normalisation of the results has been considered. Saliva normalisation has largely been established in the field of proteomics research, where osmolality or protein content, a measure of solute concentration within the given volume, can be used effectively. Consideration has been given to a variety of normalisation techniques; however, they did not yield valuable information. In this case, salivary flow rates and osmolality were within acceptable ranges supporting the reported study outcomes. The current study relied on two direct injection mass spectrometry methods for VOC analysis. Future biomarker discovery studies may benefit from using alternative analytical platforms, such as gas chromatography-mass spectrometry, that may provide broader untargeted analysis of the salivary volatilome.