## 3 Discussion
A higher concentration of acid will lead to a greater increase in the activity coefficients, resulting in a larger concentration of compounds in the headspace. Previously, 1 M H2 SO4 has been used; however, we found that both 2.5 M and 5 M H2 SO4 were significantly more effective at producing VOCs detected using our HS-SPME-GC-MS method [8]. There was no significant difference in the number of VOCs produced between 5 M and 2.5 M H2 SO4; the reason for this is likely to be that the SPME fibre is saturated with VOCs at 2.5 M, and, thus, there was no difference when using a higher concentration [5]. We anticipate using a more concentrated acid may lead to more extensive damage to the column or SPME fibre, leading to further contamination of the samples. Future studies evaluating the effects of changing the volume of acid used at the same concentration would be beneficial. A recent report recommended the use of 1 M H2 SO4 for the optimisation of liquid-liquid extraction (LLE) coupled with GC-MS. This is an alternative to SPME but it is more tedious, time-consuming, and requires larger volumes of solvents [47, 48, 49]. Over the course of our study the repeated use of pooled urine QC samples at regular intervals indicated that there was no significant problem with volatile compound detection, suggesting adsorption was not being decreased over this period of time (Figure 6). Further, looking at the three most abundant compounds over time in a larger experiment using 5 M H2 SO4, over a period of 15 days, where we ran over 200 samples, indicated that there was no change to detection over this time period (Figure S2). As sulphuric acid is a strong oxidizing acid, its use will lead to the formation of sulphates, and whether these enter the gas phase and lead to fibre damage requires further study. Future studies should also include a study of the long-term stability of the fibre under acidic conditions. This study would physically measure the fibre and continue to use pooled QC samples in order to show that the fibre was intact and continuing to adsorb efficiently. This study would indicate the optimal time to change the fibre. These further studies on SPME fibre degradation are vital, since SPME fibres have a high cost associated with them. It is therefore important to consider how many samples can be run per fibre to make this a viable avenue moving forward. The phase ratio depends upon the sample volume and the vial size. Theoretically, an increased sample volume and a decreased vial size should result in more compounds in the headspace due to an increased phase ratio. However, the degree of change is dependent on the compounds' partition coefficients [50]. If the partition coefficient of a compound is low (prefers the headspace phase) then altering the phase ratio will significantly increase the concentration of a compound in the headspace, and vice versa. Therefore, increasing urine volume results in a larger concentration of VOCs in the headspace, as exemplified by one of the compounds we identified: \(n\)-hexane, which is known to have a low partition coefficient, and is present in more of the 1 mL compared to the 0.5 mL urine samples. Previous research found altering vial volume had no significance [5; 40]. The two vial sizes we selected are the most commonly used for HS-SPME analysis. We found the 10 mL vial was more effective than the 2 mL vial. It produced a significantly larger mean number of VOCs per sample and more unique VOCs. We propose that a larger headspace (as with the 10 mL vial) creates a larger surface area, and, thus, a greater opportunity for diffusion of gases into the headspace [50]. We hypothesise that because the 10 mL vial has a greater diameter than the 2 mL vial, the greater surface area enables more compounds to transition into the headspace from the liquid phase [50]. This study benefits from the use of duplicate or triplicate urine samples, to compare three simple treatment techniques (H\({}_{2}\)SO\({}_{4}\), HCl, and NaOH). Our sample preparation is straightforward.