## 2 Materials and Methods
### Sample Preparation and Gas Chromatography-Mass Spectrometry (GC-MS) Analysis  
Human urine samples (first pass, morning, with 10-14 h of fasting and at least 4 to 6 h since the last urination) were collected from volunteers in 120 mL sterile urine specimen cups. Upon receipt (typically within 1 h of collection), all samples were stored at \(-20\)\({}^{\circ}\)C. Urine samples were stored for two weeks while all testing was performed. Prior to analysis, the samples were thawed at room temperature for 30 min and centrifuged at 12,000 rpm for 5 min. Afterward, samples were subjected to three different sample treatments, i.e., no treatment, lyophilization, and H\({}_{2}\)SO\({}_{4}\) addition. Samples without treatment were analyzed directly [25]. The required amount of NaCl (0.270 g) was added to 1 mL of urine in a 10 mL glass vial with a micro-stirring bar (PTFE bar, 5 mm diameter, 15 mm length). For the lyophilized samples, 1 mL of urine was lyophilized before being placed in glass vials [26].  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **Healthy Controls (\(n=32\))** & **COVID-19 Patients (\(n=35\))** \\ \hline
**Age [Median (IQR)]** & 52.5 (19.1) & 59 (20.0) \\
**Sex, distribution** & & \\  Male [\(n\) (\%)] & 9 (28.1) & 11 (31.4) \\  Female [\(n\) (\%)] & 23 (71.9) & 24 (68.6) \\ \hline \hline \end{tabular}
\end{table}
Table 1: Population and clinical data of the people whose urine have been analyzed.  
For the samples containing H\({}_{2}\)SO\({}_{4}\), 0.2 mL of 2.5 M H\({}_{2}\)SO\({}_{4}\) was added to 1 mL of urine in a 10 mL glass vial [25].  
The glass vials were sealed with aluminum crimp caps equipped with a needle-piercable polytetrafluoroethylene/silicone septa. The solid phase microextraction (SPME) fiber used was divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 \(\upmu\)m, StableFlex, 1 cm long, mounted on an SPME automatic holder assembly (Supelco, Bellefonte, PA, USA). The sample vial was then placed in a thermostatic water bath at 45 \({}^{\circ}\)C and stirred at 500 rpm for 30 min. After this equilibration, the SPME needle was exposed to the VOCs in the headspace for 30 min. Once the extraction was complete, the SPME needle was removed, and the fiber was immediately desorbed into the GC-MS injection port at 250 \({}^{\circ}\)C for 10 min. To avoid carryover, an additional step was added to our GC method. After analyzing each sample, the fiber was introduced into the oven with a helium flow (20 mL/min) for 2 min at 250 \({}^{\circ}\)C. Additionally, at the beginning and end of each sample processing batch, a blank analysis (empty, capped vials) was performed.  
Volatile analysis was carried out using an Agilent 6890N GC coupled to a 5973N MS (Agilent Technologies, Palo Alto, CA, USA) operating in electron impact ionization mode (EI 70 eV). The ion source and GC-MS transfer line temperatures were set to 230 \({}^{\circ}\)C and 280 \({}^{\circ}\)C, respectively. A DB-624 column (30 m length \(\times\) 0.25 mm internal diameter \(\times\) 1.4 \(\upmu\)m film thickness, Agilent Technologies, Palo Alto, CA, USA) was used. The oven temperature was programmed as follows: 50 \({}^{\circ}\)C initial temperature with a hold for 3 min, then from 50 to 250 \({}^{\circ}\)C at 5 \({}^{\circ}\)C min\({}^{-1}\), and finally hold for 10 min at 250 \({}^{\circ}\)C; the total run time was 53 min. Helium was used as the carrier gas (1 mL/min). Mass spectrum data were acquired in scan mode over a mass range of 30-350 mass-to-charge ratio (\(m/z\)), using MassHunter GCMS Acquisition B.07.06 (Agilent Technologies, Inc., 5301 Stevens Creek Boulevard. Santa Clara, CA, USA). Peak identification was based on a comparison of the mass spectrum data with spectra in the NIST/Wiley mass spectral library [27] using Enhanced ChemStation--MSD ChemStation F.01.03., Level 2 identification, according to the Metabolomics Standards Initiative (MSI) [28].