Targeted metabolomics in bladder cancer: From analytical methods development and validation towards application to clinical samples.

Bladder cancer constitutes the ninth most common cancer worldwide and, despite continuous development of new diagnostic approaches, the thirteenth leading cause of global cancer mortality. In our previous untargeted urine metabolomic investigation, seventeen metabolites were found to be statistically differentiating bladder cancer patients and healthy volunteers. Therefore, the main goal of this study was to develop and validate an analytical method for simultaneous quantitative determination of those metabolites using reversed phase high-performance liquid chromatography coupled with triple quadrupole mass spectrometry technique (RP-HPLC-QQQ/MS). Different chromatographic conditions, as well as various sample treatment procedures were tested in order to provide the best separation and the lowest limit of quantification (LOQ) values for studied compounds. The validation was performed according to the Food and Drug Administration guidelines (FDA). The limit of determination (LOD) and the LOQ values were in the range of 0.21–10.51 ng/ml and 0.69–35.02 ng/ml, respectively. The concentration range of compounds was developed between 2.5 and 12500 ng/ml. Only one compound (trimethyllysine) showed a significant matrix effect (61%) and consequently low process efficiency (64%). Overall, developed method presented recovery and precision values within the ranges proposed by FDA guidelines. The optimized and validated method was applied to urine samples obtained from 40 patients with bladder cancer and 40 healthy volunteers matched according to ones of the most important risk factors for developing urinary bladder tumors, e.i. age, gender and BMI. Afterwards, statistical analysis was provided by the use of Student's t-test or U-Mann Whitney test. The developed method was sensitive, selective and reproducible to be applied for the quantification of metabolites in the investigation of urine samples. As a consequence, ten out of previously chosen seventeen compounds, participating in different metabolites' pathways (gut floral metabolism, RNA degradation, purine metabolism, etc.), were found to be statistically significantly different in the urine concentration (p < 0.05) between cancer and control groups.