## 2 Materials and Methods
### Exhaled Breath Sampling and Analyses  
After obtaining informed written consent, the breath samples of twenty healthy subjects and ten CKD-affected patients (Table 2), enlisted to undergo kidney transplants from a living donor, were sampled to test our proposed TD-GC-MS protocol.  
Exhaled breath was collected with a ReCIVA(r) Breath Sampler (_Owlstone Medical_, Cambridge, UK), schematized in Figure 1.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline _Common_ & _Molecular Class_ & _Healthy_ & _CKD_ & _CAS_ & _M.W. (g\(\cdot\)mol\({}^{-1}\))_ & _Bibliographic Ref._ \\ \hline Hexanal & Aldehyde & * & * & 66-25-1 & 100.16 & [44; 45; 46; 47; 48; 49; 50] \\ \hline Hexanoic acid & Acid & * & & 142-62-1 & 116.16 & [47; 48; 49; 50] \\ \hline Octanal & Aldehyde & * & * & 124-13-0 & 128.21 & [44; 45; 46; 50] \\ \hline Octanol & Alcohol & * & & 111-87-5 & 130.23 & [47; 48; 49; 50] \\ \hline Propanal & Aldehyde & * & * & 123-38-6 & 58.08 & [44; 45; 50] \\ \hline Propylamine & Amine & * & 107-10-8 & 59.11 & [40; 41; 42; 47] \\ \hline Toluene & Benzene compound & * & * & 108-88-3 & 92.14 & [48; 49; 50] \\ \hline \end{tabular}  
* Find in the group.  
\end{table}
Table 1: _Cont._  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & _CKD Patients (n = 10) Healthy People (n = 20)_ \\ \hline Age (years) & 38 (IQR:32–41) & 58.7 (IQR: 49–72) \\ \hline Sex ratio (M:F) & 1:1 & 1:1 \\ \hline Hypertension & 1 & 0 \\ \hline Diabetes & 0 & 0 \\ \hline Hypothyroidism & 0 & 0 \\ \hline Smoker & 0 & 0 \\ \hline \end{tabular}
\end{table}
Table 2: Demographics and comorbidities in CKD and control groups.  
The device was connected to a breath-sampling kit (mask and TD tubes), ensuring reproducible collection of the VOCs during real-time monitoring of the patient's breathing. The exhaled breath of each patient was captured into four TD tubes (_Markes International_, Lantrisant, UK; biomonitoring sorbent tubes) capable of retaining a range of carbon compounds (from C4 to C30). The apparatus comprised infrared carbon dioxide detection with pressure sensors, permitting the selection of different volumes and fractions of the exhaled breath. A mask manufactured from medical grade silicone, which included a high-efficiency, low-resistance bacterial filter, was fixed onto the device before each sampling. This was connected to a medical air canister via a plastic pressure reducer and set to 15 L/min. A USB cable connected the ReCIVA(r) breath sampler to a laptop installed with breath-sampling software (_Owlstone Medical_), designed to ensure accurate monitoring of breathing air pressures (partial pressure of carbon dioxide). All subjects fasted for at least 4 h prior to breath sampling. Sampling was always performed in the same room, which was aerated for 30 min before each procedure. Patients were instructed to keep the mask securely adhered to their faces and to breathe normally with the air released by the medical air canister. After a 60 s ReCIVA(r) device washout with pure air (purity 99.99 percent; _SOL Group_, Monza, Italy), the patient's breath was collected for 10 min under a PC-dedicated program control. At the completion of the sampling, the sorbent tubes were removed, covered with a plastic cap, and delivered to the chemistry department within 24 h for GC-MS analysis.  
To exclude extraneous contamination, on each sampling day, three ReCIVA(r) steel tubes containing room air were sample-tested before the commencement of the breath sampling.