## 2 Results and Discussion
### Method Application--\({}^{1}\)H-NMR  
\({}^{1}\)H-NMR analysis was performed in both extract parts, because the information obtained is not only complementary but also supportive to some of the metabolites from GC-MS. The \({}^{1}\)H-NMR spectra of the polar phase revealed approximately 60 proton signals originating from organic compounds within \begin{table}
\begin{tabular}{c c} \hline \hline
**Urine Sample** & **Number of Compounds Detected Using GC-MS** \\ \hline
1 & 336 \\
2 & 326 \\
3 & 330 \\
4 & 337 \\
5 & 338 \\
6 & 272 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: The number of deconvoluted peaks detected in urine samples from six apparently healthy individuals by GC-MS method. the urine. The signals are mostly located in the three areas of chemical shifts (5): (a) 0.77-1.94 ppm characteristic for CH\({}_{3}\) and CH\({}_{2}\) groups; (b) 1.95-4.33 ppm for the aliphatic proton signal of different CH and CH\({}_{2}\) groups, and (c) 6.8-8.07 ppm, distinctly visible peaks which in principle can exhibit protons originating from aromatic compounds. However, as is seen in the spectra (Figure 2), the signals from higher mass molecules are not filtered off completely by the CPMG pulse sequence. The signals in the polar fractions were assigned to the Chenomx references, where over 100 signals were detected, and among them 42 metabolites were found to be present in the urine aqueous phase (Supplementary Data Table S1). Interestingly, there were significant differences between the compounds tentatively identified by both GCMS and \({}^{1}\)H-NMR, which proves that simultaneous analysis of the same extracts with these two methods is complementary. The DCM (LLE) can extract metabolites with a range of polarities (particularly relatively non polar compounds), however it would not be expected to be that efficient in extracting polar compounds, such as ionic compounds [38]. The literature data related to non-polar compound analysis from biological fluids and tissues is very limited, usually including on the \({}^{1}\)H NMR spectra a general description of the groups of compounds [39]. In another report, the urine extracts were dissolved in different deuterated solvents (MeOD, DMSO, DMF, MeCN, Acetone, CDCl\({}_{3}\) and DCM), with subsequent monitoring of the levels of five metabolites: hippurate, creatinine, lactate, histidine and alanine [38]. Metabolites showed signal variation in the ppm scale depending on the solvent used. However, among the selected metabolites, only hippurate was found to be resolvable in CDCl\({}_{3}\). The study did not avoid some limitations. First, the method was optimized only for GC-MS, not both GC-MS and \({}^{1}\)H-NMR. However, the main aim of the study was the optimization of LLE extraction for VC analysis, and the \({}^{1}\)H-NMR part was the additional attempt, performed to check if the same extract can be used on multiple platforms. A second limitation is the small number of samples used for applicability testing. It is related to the character of the study, which is method development. The real urine sample analyses were included only to prove that the method is suitable for real sample analysis. Finally, the study does not contain the identification of all the compounds (however the main chemical groups were tentatively identified and mentioned). However, the authors decided not to undertake this exhaustive analysis because the main goal of this study was to detect the highest number of peaks corresponding to individual compounds that could be potential biomarkers.