## 5 Conclusions  
The main goals of this study were to test applicability of TFME for untargeted screening of urinary metabolites and the identification of compounds involved in tumor development and progression in bladder cancer patients. Multivariate OPLS-DA analysis was performed to differentiate BC patients from healthy individuals. This profiling has not only identified a group of metabolites that may contribute to bladder cancer development, but also compounds that show potential in the prevention of cancer. Pathway analysis allowed for the integration of metabolomics data and biological information to enhance knowledge about the biological links between selected metabolites and BC pathogenesis. Future quantitative targeted studies on a larger number of patients are required to validate current findings and to evaluate the predictive value of the selected metabolites. Moreover, the results of the present study demonstrated that thin film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research.  
**Supplementary Materials:** The following are available online. Figure S1: Total ion chromatogram of QC sample in positive (A) and negative (B) ionization mode. Figure S2: Chromatograms for selected compounds differentiating the studied groups: 2-Acetyl-1-alkyl-sn-glycero-3-phosphocholine (A), 4-Hydroxycinnamic acid (B), 5-Hydroxyindoleacetic acid (C), Adenosine monophosphate (D), Benzoic acid (E), Hippuric acid (F), Gluconic acid (G), Carnosine (H), Histidine (I), \(N\)-Acetyl-phenylalanine (J), Table S1: Differential metabolites in positive and negative ionization mode with information on the annotation in the ChemSpider database.  
**Author Contributions:** Conceptualization, M.J.M. and B.B.; formal analysis, data curation, K.L., N.W. and J.B.; investigation, methodology, S.O. and I.S.; writing--original draft preparation, K.L. and N.W.; writing--review and editing, J.J., R.W., W.S.-L., M.J.M. and B.B.; visualization, K.L. and N.W.; supervision, B.B.; project administration, B.B. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This research received no external funding.  
**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by Independent Bioethics Commission for Research of Medical University of Gdansk (NKBBN/49/2013) and the Bioethics Committee of Collegium Medium in Bydgoszcz at Nicolaus Copernicus University in Torun (KB 628/2015).  
**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.  
**Data Availability Statement:** Not applicable.  
**Acknowledgments:** The authors would like to thank Thermo Fisher Scientific for access to the Q-Exactive Focus Orbitrap mass spectrometer. We thank the Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdansk for urine samples.  
**Conflicts of Interest:** The authors declare no conflict of interest.  
**Sample Availability:** Extracts of the urine samples are available from the authors.