## 2 Results and Discussion  
### Optimization of the Extraction Parameters  
In the present study, the type of solvent, acid molarity and ionic strength were selected and evaluated to achieve the optimal condition of LLE, based on the maximum number of GC-MS peaks detected (Table 1). The solvents, acidic pH and amount of salt were preselected based on data from the literature [28; 29; 30]. Dichloromethane (DCM) was found to be the most efficient solvent for VC extraction from urine samples, followed by chloroform (Table 1 and Supplementary Figure S1). DCM is immiscible with water and can dissolve a wide range of organic compounds, hence its extensive use for LLE [31; 32]. Diethyl ether is a commonly used solvent for extracting organic compounds from aqueous solutions, however in this study on urine, it was found to be the least effective solvent for LLE, resulting in the detection of only circa 20 peaks. In contrast, [PERSON] et al. [28] noted the presence of 300 compounds in ether extracts (comparison to the efficiency of other solvents was not analyzed), out of which 40 have been identified. However, in their study, a very large amount of urine, 450 mL, was used for extraction with 80 mL of diethyl ether, as compared to 2 mL of urine used in our study. Use of 450 mL urine creates storage issues, and many patients could not produce such an amount. The use of 80 mL \begin{table}
\begin{tabular}{c c c} \hline \hline
**Factor Analyzed** & **Number of Peaks Detected** & **Constant Conditions** \\ \hline \multicolumn{3}{c}{Type of solvent (4 mL added)} \\
**DCM** * & **205.0 \(\pm\) 46.1 *** & \\ Chloroform & 121.0 \(\pm\) 47.6 \({}^{\text{b}}\) & 1 M acid, salt addition \\ Diethyl ether & 20.7 \(\pm\) 4.0 \({}^{\text{c}}\) & \\ \hline \multicolumn{3}{c}{Acid molarity (2 mL added)} \\
0.01 M & 137.0 \(\pm\) 2.7 \({}^{\text{b}}\) & \\
0.1 M & 120.0 \(\pm\) 21.1 \({}^{\text{b}}\) & solvent: DCM, salt addition \\
**1 M** & **205.0 \(\pm\) 46.1 \({}^{\text{a}}\)** & \\ \hline \multicolumn{3}{c}{Ionic strength} \\
**Salt addition (0.2 g)** & **205.0 \(\pm\) 46.1 \({}^{\text{a}}\)** & \\ No salt & 156.0 \(\pm\) 44.7 \({}^{\text{a}}\) & solvent: DCM, 1 M acid \\ \hline \hline \end{tabular} (*) DCM—dichloromethane; (*) Values are a mean number of peaks of three replicates \(\pm\) SD. Different letters (a, b, c) in a column for each parameter represents significantly different (\(p<0.05\)) values (Fisher’s Least Significant Difference (LSD), ANOVA) for solvent type and acid molarity and Student \(t\)-test for ionic strength.  
\end{table} 
Table 1: Comparison of the factors affecting the extraction efficiency of urine VCs using GC-MS analyses. Values in bold were selected as the most efficient and were used for the optimization of the next parameters. of ether for extraction requires more processing time, i.e., more drying agent, evaporation times, and it is undesirable from the health and safety perspective and impractical for certain processing steps such as centrifugation. 2 mL is a realistic volume of urine from a patient sample, which can be collected clinically, therefore the present study was optimized using that volume. In comparison to our methodology, the literature describes VC extraction using absorbents, and smaller numbers (75 and 147 peaks identified, respectively) of VCs were reported, even with larger amounts of urine (using single quadrupole GC-MS) [22; 30]. In the present study, the highest molarity of acid (1 M) with pH value near 0 was found to be significantly more efficient for LLE (Table 1). There was no significant difference between 0.1 M (pH value: \(\sim\)1.5) and 0.01 M (pH value: \(\sim\)2.5) acid addition. Acidic conditions were previously reported to be more suitable for VC analysis as compared to basic and neutral pH when using SPME [4; 14; 30]. This is related to the chemical properties of the compounds present in urine. Acidic pH increases the number of compounds in the non-conjugated form [4].