## 3 Discussion  
In recent years, the urology field has seen rapid development in terms of urine-based metabolomic analysis. Given that urine comes in direct contact with bladder epithelial cells and tumor tissue, it may therefore contain metabolites shed from cancerous cells in the bladder [6]. In metabolomics, sample preparation has appreciable influence on the quality of the obtained results, particularly for biofluid samples such as urine [18]. In the current study, a technology integrating sample preparation and extraction of metabolites, namely solid phase microextraction, was presented for metabolomics analysis of urine samples of bladder cancer patients. It has been already demonstrated that SPME is an effective tool for metabolomics studies in plasma and tissue [8; 10], but no direct immersion urinary metabolomics analyses have been reported to date to the best of our knowledge. However, extensive anti-doping multi-residue analyses in urine have been successfully carried out via the thin film SPME automated high throughput system [19], showing the potential of the technology for simultaneous determination of over 100 compounds with no matrix effect and good sensitivity in urine, all while affording a simple analytical protocol. Herein, thin film SPME followed by LC-HRMS analysis was used for the first time for urinary untargeted metabolomics in cancer patients. \begin{table}
\begin{tabular}{c c} \hline
**Pathway Name** & **Metabolites** \\ \hline \multicolumn{3}{c}{**Positive Ionization Mode**} \\ \hline \hline \multicolumn{3}{c}{Benzoic acid; Hippuric acid; 4-Hydroxycinnamic acid; \(N\)-Acetyl-phenylalanine} \\ Histidine metabolism & Histidine; Carrosine; \\ Caffe metabolism & Theophylline; 3-Methylxanthine; \\ beta-Alanine metabolism & Carrosine; Histidine \\ Glycerophospholipid metabolism & LysoPE(18:1); Phosphatidyl-N-dimethylylethanolamine; \\ Retinol; & Retinol; \\ Ether lipid metabolism & 2-Acetyl-l-alkyl-sn-glycero-3-phosphocholine; \\ Ubiquinone and other terpenoid-quinone biosynthesis & 4-Hydroxcinnamic acid; \\ Drug metabolism—other enzymes & Isoniazid; \\ Nitrogen metabolism & Histidine; \\ Folate biosynthesis & \(p\)-Aminobenzic acid \\ Aminoacyl-tRNA biosynthesis & Histidine \\ Tyrosine metabolism & Epinephrine; \\ Trypophan metabolism & 5-Hydroxyloideae acid \\ Purine metabolism & Adenine; \\ \hline \multicolumn{3}{c}{**Negative Ionization Mode**} \\ \hline \hline \multicolumn{3}{c}{Phenylalanine metabolism} & Hippuric acid; \\ Pentose phosphate pathway & Gluconic acid; \\ Nitrogen metabolism & Adenosine monophosphate; \\ Glycerophospholipid metabolism & Phosphatidyl-\(N\)-dimethylethanolamine; \\ Trypothpan metabolism & Indolelactic acid; \\ Purine metabolism & Adenosine monophosphate; \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Metabolic pathways and associated metabolites in positive and negative ionization mode. The metabolites selected as discriminant compounds and, subsequently, the results of the pathways analysis, are consistent with previous reports [13]. Compounds found present at decreased levels in BC patients in comparison to healthy controls included benzoic acid (BA) and hippuric acid (HA), which are known to be involved in the phenylalanine metabolism pathway. Hippuric acid is a normal component of urine as a metabolite of aromatic compounds from food [20]. While the serum concentration of HA is increased in uremic patients as a result of reduced renal clearance [21], lower levels of hippuric acid in urine samples from BC patients are in agreement with previous studies, making it characteristic for this group of patients [14; 15]. Benzoic acid, a compound commonly used as a food preservative, conjugates to glycine in the liver and is excreted as hippuric acid [20]. Benzoic acid and hippuric acid are also believed to play essential roles in gut microbial pathways [14; 22].