## 3 Discussion  
HS-SPME-GC-MS analysis of urine offers the potential for fast, non-invasive metabolite detection but standardisation of methods between laboratories remains a challenge. We have shown that altering urine sample pH via the addition of H\({}_{2}\)SO\({}_{4}\) is more effective at optimising detection of VOCs than  
Figure 4: The urinary VOC profiles using 2 mL (red) and 10 mL (blue) vials (\(n\) = 15). 0.5 mL urine was treated with 0.1 mL 5 M H\({}_{2}\)SO\({}_{4}\), to yield 0.83 M final solution, pH 0.075. (**a**) Paired boxplot to show the cumulative unique number of VOCs produced per sample for either vial volume. (**b**) Venn diagram showing the unique VOCs detected in each vial volume. Figure 5: The urinary VOC profiles using 0.5 mL (red) and 1 mL of urine (blue) (\(n\) = 15) treated with 5 M H\({}_{2}\)SO\({}_{4}\), to yield 0.83 M final solution, pH 0.075. (**a**) Paired boxplot to show the cumulative number of unique VOCs produced per sample for either volume of urine. (**b**) Venn diagram showing the unique VOCs detected in each volume of urine. NaOH or HCl. Ionic strength due to the addition of saturated NaCl solution was also examined in pooled QC samples and sample urine. We found less compounds were detected when using saturated NaCl compared to any other method of preparation (Figure S1). H2 SO4 resulted in a significantly larger mean number of VOCs identified per sample and more unique VOCs, produced a more diverse range of classes of VOCs, and led to less HS-SPME-GC-MS degradation. In addition, we found that resuspension of samples with 2.5 M and 5 M H2 SO4 yielded significantly better detection of VOCs compared to 1 M acid. Furthermore, using a 10 mL vial and 2.5 M or 5 M H2 SO4 further optimised VOC detection. Different urine sample treatment techniques alter the sample matrix. Firstly, change in pH may lead to an increase in decomposition or degradation of certain compounds present in the sample. In particular, using strong oxidizing acid will almost certainly change the sample composition. Secondly, changing either the ionic strength or pH may increase activity coefficients (how much a solution differs from an ideal solution), in turn, decreasing the partition coefficients (the ratio of the concentration of a sample in the liquid phase with the concentration of VOCs in the headspace), and, ultimately, resulting in more compounds transitioning into the gas phase. It known that an acid environment leads to a larger number of VOCs produced compared with a neutral or alkaline environment [6, 38]. Despite this, there is a lack of consistency in the approaches described in the literature with either acid, alkali, and/or salts used as a sample treatment [2, 8, 16, 31, 41, 42, 43, 44, 45, 46]. When acidification is chosen for HS-SPME-GC-MS, HCl appears to be the most common choice; although, the reason for this is typically not explained. We found here that HCl produced more degradation products than H2 SO4. Neither HCl nor H2 SO4 is very volatile and therefore they can accumulate at the front of the column. If they remain there, they will damage the stationary phase of the HS-SPME-GC-MS resulting in premature and excessive column bleed, reflected by an increased detection of degradation products. Both acids may lead to some deterioration of the SPME fibre; this requires further study. HCl is a stronger acid than H2 SO4, which may explain the greater number of compounds of degradation. Furthermore, samples resuspended with HCl yielded a lower number of VOCs than H2 SO4 from our urine samples. Notably, the only other paper that has described the use of H2 SO4 as a urine sample treatment technique for HS-SPME-GC-MS analysis, did not compare different acidification techniques for optimising urinary VOCs in HS-SPME-GC-MS [8]. After establishing H2 SO4 as the most effective treatment technique, we conducted further experiments altering other aspects of sample preparation, such as H2 SO4 concentration and phase ratio.