Toward the General Mechanistic Model of Liquid Chromatographic Retention.

Large datasets of chromatographic retention times are relatively easy to collect. This statement is particularly true when mixtures of compounds are analyzed under a series of gradient conditions using chromatographic techniques coupled with mass spectrometry detection. Such datasets carry much information about chromatographic retention that, if extracted, can provide useful predictive information. In this work, we proposed a mechanistic model that jointly explains the relationship between pH, organic modifier type, temperature, gradient duration, and analyte retention based on liquid chromatography retention data collected for 187 small molecules. The model was built utilizing a Bayesian multilevel framework. The model assumes (i) a deterministic Neue equation that describes the relationship between retention time and analyte-specific and instrument-specific parameters, (ii) the relationship between analyte-specific descriptors (log P, pKa, and functional groups) and analyte-specific chromatographic parameters, and (iii) stochastic components of between-analyte and residual variability. The model utilizes prior knowledge about model parameters to regularize predictions which is important as there is ample information about the retention behavior of analytes in various stationary phases in the literature. The usefulness of the proposed model in providing interpretable summaries of complex data and in decision making is discussed.

Metabolic Evaluation of Urine from Patients Diagnosed with High Grade (HG) Bladder Cancer by SPME-LC-MS Method.

Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. 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. Moreover, this study's results may support a better understanding of bladder cancer development and progression mechanisms.

Quantitative determination of trigonelline in mouse serum by means of hydrophilic interaction liquid chromatography-MS/MS analysis: Application to a pharmacokinetic study.

Trigonelline is a pyridine alkaloid found in fenugreek seeds and coffee beans. Most of the previous studies are concerned with the quantification of trigonelline along with other constituents in coffee herbs or beverages. Only a few have focused on its determination in animal or human tissues by applying different modes of HPLC with UV or MS detection. The aim of the study was to develop and validate a fast and simple method for trigonelline determination in serum by the use of hydrophilic interaction liquid chromatography (HILIC) with ESI-MS/MS detection. Separation of trigonelline was achieved on a Kinetex HILIC column operated at 35°C with acetonitrile-ammonium formate (10 mm, pH = 3) buffer mixture (55:45, v/v) as the mobile phase. The developed method was successfully applied to determine trigonelline concentration in mouse serum after intravenous administration of 10 mg/kg. The developed assay is sensitive (limit of detection = 1.5 ng/mL, limit of quantification = 5.0 ng/mL) and linear in a concentration range from 5.0 to 250.0 ng/mL. Sample preparation is limited to deproteinization, centrifugation and filtration. The application of the HILIC mode of chromatography with MS detection and selection of deuterated trigonelline as internal standard allowed a rapid and precise method of trigonelline quantification to be to developed.

Determination of tocopherols and tocotrienols in human breast adipose tissue with the use of high performance liquid chromatography-fluorescence detection.

Tocopherols and tocotrienols have been extensively studied owing to their anticancer potential, especially against breast cancer. Therefore, the aim of this study was to quantitatively determine tocochromanols in human breast adipose tissue with the use of HPLC-FLD. The sample preparation procedure included homogenization and solvent extraction with isopropanol-ethanol-0.1% formic acid mixture prior to solid-phase extraction. After implementation of central composite design, satisfactory separation of all eight target compounds was achieved within 10.5 min. Chromatographic runs were carried out with the use of a naphthylethyl chromatographic column with methanol-water mixture (89:11, v/v) as the mobile phase. Fluorescence detection of tocochromanols was performed with excitation and emission wavelengths 298 and 330 nm, respectively. The method was validated in terms of linearity, carryover, recovery, precision, accuracy and stability. Extraction yield was also determined for accurate evaluation of vitamin E content in human breast adipose tissue samples. Finally, concentrations of particular tocochromanols compounds were assessed in human breast adipose tissue samples obtained from 99 patients, including women with breast cancer, healthy volunteers and deceased women who had died as a result of accidents. The raw data was transformed according to the newly developed equation for accurate estimation of the concentrations of tocochromanols in breast adipose tissue samples. Results obtained in the study indicated that the proposed analytical assay could be useful in breast cancer research.

[Prolonged-release drug formulations for parenteral administration. Part II. Microspheres and implants for injection]. / Pozajelitowe formy leków o przedluzonym dzialaniu Czesc II. Mikrosfery i implanty do wstrzykiwan.

Microspheres and implants are injectable drug forms, which by special design and selection of appropriate excipients, provide for a long time constant release rate of an active substance in the body. Development of both would not be possible without advances in polymer technology and invention of safe and biocompatible polymers such as: polyesters, vinyl acetate derivatives or silicones. Polymeric matrices provide retardation of drug release--for some implants up to a few years. In addition, this paper presents examples of all commercially available medicinal products containing microspheres and implants, currently registered in Poland, together with their characteristics: composition, time course and frequency of administration. Comments are also enclosed on frequently occurring inconsistent terminology in pharmaceutical forms.

[Prolonged-release drug formulations for parenteral administration. Part l. Suspensions and oily solutions for injection]. / Pozajelitowe formy leków o przedluzonym dzialaniu. Czesc I. Zawiesiny i roztwory olejowe do wstrzykiwan.

The article presents description of the most important parenteral forms, which release an active substance in sustained manner. Technological and biopharmaceutical information is supplemented with examples of commercial products, currently registered in Poland. Mechanism of drug release from the dosage form and duration of the process, role of other excipients and characteristics of certain medicinal products is presented. The information about suspensions and oily solutions for injection is summarized. Owing to specific chemical modifications and selection of suitable excipients, it was possible to develop these types of medicinal products for some antibiotics, hormones, antipsychotics and cytostatics. These formulations, after subcutaneous or intramuscular injection, release the active substances even for several weeks allowing to reduce the frequency of drug administration and finally help to improve patient's compliance. Here also the modified time course of insulin products achieved by selection of appropriate suspension form or insulin analogue is discussed.

Pre- and Post-Resection Urine Metabolic Profiles of Bladder Cancer Patients: Results of Preliminary Studies on Time Series Metabolomics Analysis.

The incidence of bladder cancer (BCa) has remained high for many years. Nevertheless, its pathomechanism has not yet been fully understood and is still being studied. Therefore, multiplatform untargeted urinary metabolomics analysis has been performed in order to study differences in the metabolic profiles of urine samples collected at three time points: before transurethral resection of bladder tumor (TURBT), the day after the procedure and two weeks after TURBT. Collected samples were analyzed with the use of high-performance liquid chromatography hyphenated with time-of-flight mass spectrometry detection (HPLC-TOF/MS) and gas chromatography coupled with triple quadrupole mass spectrometry detection (GC-QqQ/MS, in a scan mode). Levels of metabolites selected in our previous study were assessed in order to confirm their potential to differentiate the healthy and diseased samples, regardless of the risk factors and individual characteristics. Hippuric acid, pentanedioic acid and uridine confirmed their potential for sample differentiation. Based on the results of statistical analysis for the paired samples (comparison of metabolic profiles of samples collected before TURBT and two weeks after), a set of metabolites belonging to nucleotide metabolism and methylation processes was also selected. Longitudinal studies proved to be useful for the evaluation of metabolic changes in bladder cancer.

Molecular signature of renal cell carcinoma by means of a multiplatform metabolomics analysis.

Renal cell carcinoma (RCC) is a disease with no specific diagnostic method or treatment. Thus, the evaluation of novel diagnostic tools or treatment possibilities is essential. In this study, a multiplatform untargeted metabolomics analysis of urine was applied to search for a metabolic pattern specific for RCC, which could enable comprehensive assessment of its biochemical background. Thirty patients with diagnosed RCC and 29 healthy volunteers were involved in the first stage of the study. Initially, the utility of the application of the selected approach was checked for RCC with no differentiation for cancer subtypes. In the second stage, this approach was used to study clear cell renal cell carcinoma (ccRCC) in 38 ccRCC patients and 38 healthy volunteers. Three complementary analytical platforms were used: reversed-phase liquid chromatography coupled with time-of-flight mass spectrometry (RP-HPLC-TOF/MS), capillary electrophoresis coupled with time-of-flight mass spectrometry (CE-TOF/MS), and gas chromatography triple quadrupole mass spectrometry (GC-QqQ/MS). As a result of urine sample analyses, two panels of metabolites specific for RCC and ccRCC were selected. Disruptions in amino acid, lipid, purine, and pyrimidine metabolism, the TCA cycle and energetic processes were observed. The most interesting differences were observed for modified nucleosides. This is the first time that the levels of these compounds were found to be changed in RCC and ccRCC patients, providing a framework for further studies. Moreover, the application of the CE-MS technique enabled the determination of statistically significant changes in symmetric dimethylarginine (SDMA) in RCC.

A Robust Method for Sample Preparation of Gastrointestinal Stromal Tumour for LC/MS Untargeted Metabolomics.

Gastrointestinal stromal tumour has already been well explored at the genome level; however, little is known about metabolic processes occurring in the sarcoma. Sample preparation is a crucial step in untargeted metabolomics workflow, highly affecting the metabolome coverage and the quality of the results. In this study, four liquid-liquid extraction methods for the isolation of endogenous compounds from gastrointestinal stromal tumours were compared and evaluated. The protocols covered two-step or stepwise extraction with methyl-tert-butyl ether (MTBE) or dichloromethane. The extracts were subjected to LC-MS analysis by the application of reversed-phase and hydrophilic interaction liquid chromatography to enable the separation and detection of both polar and nonpolar analytes. The extraction methods were compared in terms of efficiency (total number of detected metabolites) and reproducibility. The method was based on the stepwise extraction with MTBE, methanol, and water proved to be the most reproducible, and thus, its robustness to fluctuations in experimental conditions was assessed employing Plackett-Burman design and hierarchical modelling. While most studied factors had no effect on the metabolite abundance, the highest coefficient value was observed for the volume of MTBE added during extraction. Herein, we demonstrate the application and the feasibility of the selected protocol for the analysis of gastrointestinal stromal tumour samples. The method selected could be considered as a reference for the best characterization of underlying molecular changes associated with complex tissue extracts of GIST.

Nanostructured Lipid Carriers Engineered as Topical Delivery of Etodolac: Optimization and Cytotoxicity Studies.

Etodolac (ETD), a nonsteroidal anti-inflammatory drug, exhibits antinflammatory, analgesic, and antipyretic activity. The main type of ETD administration is oral route, which is associated with significant systemic side effects. Nanostructured lipid carriers (NLC), a modern lipid formulation, are non-toxic, biocompatible, can improve the solubility and stability of drugs. Nanostructured lipid carriers (NLC) containing etodolac were prepared by a melt-emulsification and ultrasonication technique. Full factorial design (FFD) was applied to optimize the composition of NLC and their properties such as zeta potential, polidyspersity index, and entrapment efficiency. Formulations consisting of Capryol 90, glicerol monostearate, and Tween 20 displayed particle size below 300 nm, encapsulated drug with efficiency of approximately 87% and prolonged drug release up to 24 h. Stable formulations displayed moderately negative surface charge suggesting their limited ability to interact with skin surface but simultaneously presenting their lower risk to cause cell-membrane disruption. In fact, cytotoxicity assessment using human dermal fibroblasts and human epidermal keratinocytes revealed that etodolac-loaded NLC had no important impact on skin cells viability evaluated in vitro, which might evidence that NLC formulations are safe for dermal delivery. The studies developed were relatively fast and simple, requiring no specialized equipment method to prepare NLC as ETD carriers ensuring better solubility and prolonged drug release.

The Correlation between Physical Crosslinking and Water-Soluble Drug Release from Chitosan-Based Microparticles.

Microparticles containing water-soluble zidovudine were prepared by spray-drying using chitosan glutamate and beta-glycerophosphate as an ion crosslinker (CF). The Box-Behnken design was applied to optimize the microparticles in terms of their drug loading and release behavior. Physicochemical studies were undertaken to support the results from dissolution tests and to evaluate the impact of the crosslinking ratio on the microparticles' characteristics. The zidovudine dissolution behavior had a complex nature which comprised two phases: an initial burst effect followed with a prolonged release stage. The initial drug release, which can be modulated by the crosslinking degree, was primarily governed by the dissolution of the drug crystals located on the microparticles' surfaces. In turn, the further dissolution stage was related to the drug diffusion from the swollen polymer matrix and was found to correlate with the drug loading. Differential Scanning Calorimetry (DSC) studies revealed the partial incorporation of a non-crystallized drug within the polymer matrix, which correlated with the amount of CF. Although CF influenced the swelling capacity of chitosan glutamate microparticles, surprisingly a higher amount of CF did not impact the time required for 80% of the drug to be released markedly. The formulation with the lowest polymer:CF ratio, 3:1, was selected as optimal, providing satisfactory drug loading and displaying a moderate burst effect within the first 30 min of the study, followed with a prolonged drug release of up to 210 min.

Design of Experiments in metabolomics-related studies: An overview.

Nowadays, Design of Experiments (DoE) approach is a very popular methodology of planning and conducting experiments, where the effect of each tested factor on the studied responses is systematically examined and documented. The results obtained in such manner represent the design space more precisely than in the case of One-Variable-At-Time (OVAT) approach, leading to reliable and comprehensive results, while saving time and resources. Despite such a large increase of interest in this approach recently, its implementation in metabolomics research seems to be limited. Therefore, in this short overview, apart from summarizing some basic concepts of DoE, we wanted to provide a guideline for those who are about to plan metabolomics-related experiments. This overview is divided into four sections. In addition to the first section, which will introduce the history and basics of DoE, second part will provide concise description of the most popular experimental designs. Furthermore, third section will describe examples of DoE application in metabolomics and related studies. We will conclude with fourth section, providing you briefly with opportunities and trends in metabolomics research utilizing experimental design.

Ethylcellulose in Organic Solution or Aqueous Dispersion Form in Designing Taste-Masked Microparticles by the Spray Drying Technique with a Model Bitter Drug: Rupatadine Fumarate.

The taste of drugs is an important factor affecting pharmacotherapy effectiveness, and obtaining formulations with acceptable organoleptic properties is still an ongoing issue in pharmaceutical technology. One of the innovative methods of taste masking is preparation of microparticles by the spray drying technique, utilizing polymers with different physicochemical properties. Rupatadine fumarate (RUP) is one of the newest antihistamines, with an innovative and multidirectional mechanism of action, and an extremely bitter taste. The aim of this work was to investigate the feasibility of utilizing organic or aqueous forms of ethylcellulose (EC) for the preparation of microparticles with RUP by the spray drying technique. Spray dried samples at different drug:polymer ratios were prepared using organic solution (Ethocel®) or aqueous dispersions of EC (Surelease®, Aquacoat® ECD). Evaluation of the taste masking efficacy was performed in vivo in human taste panel, in vitro based on dissolution test, and by self-constructed electronic tongue. It was shown that microparticles obtained from aqueous dispersions of EC have superior pharmaceutical properties in terms of both morphology and taste masking efficacy in comparison to those obtained from organic solution.

Could spray-dried microbeads with chitosan glutamate be considered as promising vaginal microbicide carriers? The effect of process variables on the in vitro functional and physicochemical characteristics.

In order to improve efficacy and accessibility of vaginal microbicides, development of smart polymer-based delivery carriers appears essential. In scope of this study, the potential of chitosan glutamate in technology of microbicide multiunit formulations containing zidovudine-loaded microbeads was investigated. Spray-drying optimization was supported by statistical design of experiments. As polymer properties may alter upon processing, particularly important was to examine the influence of product composition and process variables on final microbeads characteristic. Data from ATR-FTIR, Raman, and DSC analysis confirmed drug compatibility with chitosan glutamate after spray-drying. Formulations with polymer:drug ratio 5:1 (w/w) prepared from azeotropic ethanol-water mixture were found to spread easily upon dilution with simulant vaginal fluid, forming viscous, shear-thinning barrier, which could impede direct contact of virus with mucus cells. Furthermore, the presence of ethanol was found crucial to overcome stickiness phenomenon by interrupting hydrogen bonding between drug and polymer. In vitro dissolution studies displayed an initial burst effect followed with prolonged (up to 4 h) drug release stage. By modifying spray-drying temperature, alterations in microbeads' swelling capacity and drug release were observed. Cytotoxicity studies using human vaginal cell line VK2/E6E7 revealed that drug-free formulations exerted no significant impact on mucosal cells, suggesting they are safe for vaginal delivery.

Urinary metabolomic signature of muscle-invasive bladder cancer: A multiplatform approach.

Bladder cancer (BCa) is ninth amongst the most common types of cancer in the human population worldwide. The statistics of incidence and mortality of BCa are alarming and the currently applied diagnostic methods are still not sensitive enough. This leads to a large number of undiagnosed BCa cases, usually among patients in the early stages of the disease. Despite the fact that many risk factors of BCa have been recognized, the pathomechanism of development of bladder cancer has not been fully explained yet. Therefore, in the present study, multiplatform urinary metabolomics has been implemented in order to scrutinize potential diagnostic indicators of BCa that might help to explain its pathomechanism and be potentially useful in diagnosis and determination of stage of the disease. Urine samples collected from muscle-invasive high grade BCa patients (n = 24) and healthy volunteers (n = 24) were matched in terms of most common BCa risk factors i.e. gender, age, BMI and smoking status. They were analyzed by high performance liquid chromatography coupled with time of flight mass spectrometry detection (HPLC-TOF/MS) using RP and HILIC chromatography, gas chromatography hyphenated with triple quadruple mass spectrometry detection (GC-QqQ/MS) in scan mode, and proton nuclear magnetic resonance (1H NMR). The six datasets obtained were submitted to univariate and multivariate statistical analyses. 17 metabolites significantly discriminated urinary profiles of BCa patients from urinary profiles of healthy volunteers. These metabolites are mainly involved in amino acid metabolism, pyrimidine and purine metabolism, as well as energy metabolism and might play a crucial role in the pathogenesis of BCa.

HPLC-APCI-MS/MS method development and validation for determination of tocotrienols in human breast adipose tissue.

For the last decade, significant attention has been paid to the potential role of tocotrienols in prevention and therapy of breast cancer. Therefore, the aim of this study was to develop and validate analytical method for quantitative determination of tocotrienols (α-, ß-, γ- and δ-tocotrienol) in human breast adipose tissue with the use of high performance liquid chromatography coupled with APCI-MS/MS detection. Separation of target compounds was achieved within 10min with the use of naphthylethyl Cosmosil 2.5π-NAP column with methanol/water mixture (90:10, v/v) under isocratic elution. Adipose tissue samples were obtained from breast cancer patients and women deceased as a result of accidents. Sample preparation procedure was optimized with the application of the Plackett-Burman design and included tissue homogenization with the use of isopropanol/ethanol/aqueous 0.1% FA mixture (13:3:8, v/v), centrifugation and solid phase extraction (SPE). The method was validated in terms of linearity, precision, accuracy, stability (bench top, autosampler, postpreparative, freeze and thaw stability), matrix effect (ME), recovery (RE) and process efficiency (PE). As for all four tocotrienols ME was negligible (< 15%), precision and accuracy tests were performed with the use of tocotrienols' standard solutions within the ranges of 10.0-400.0ng/g for all four tocotrienols. As the validation requirements were met, the validated method was applied for quantitative analysis of tocotrienols in breast cancer patients.

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.

Sample preparation procedures utilized in microbial metabolomics: An overview.

Bacteria are remarkably diverse in terms of their size, structure and biochemical properties. Due to this fact, it is hard to develop a universal method for handling bacteria cultures during metabolomic analysis. The choice of suitable processing methods constitutes a key element in any analysis, because only appropriate selection of procedures may provide accurate results, leading to reliable conclusions. Because of that, every analytical experiment concerning bacteria requires individually and very carefully planned research methodology. Although every study varies in terms of sample preparation, there are few general steps to follow while planning experiment, like sampling, separation of cells from growth medium, stopping their metabolism and extraction. As a result of extraction, all intracellular metabolites should be washed out from cell environment. What is more, extraction method utilized cannot cause any chemical decomposition or degradation of the metabolome. Furthermore, chosen extraction method should correlate with analytical technique, so it will not disturb or prolong following sample preparation steps. For those reasons, we observe a need to summarize sample preparation procedures currently utilized in microbial metabolomic studies. In the presented overview, papers concerning analysis of extra- and intracellular metabolites, published over the last decade, have been discussed. Presented work gives some basic guidelines that might be useful while planning experiments in microbial metabolomics.

Overactive bladder treatment: application of methylene blue to improve the injection technique of onabotulinum toxin A.

OBJECTIVE: The aim of this study was to test the addition of methylene blue (MB) to onabotulinum toxin A (BTX-A) solution in overactive bladder (OAB) treatment, as a means of facilitating observation of the injection site and assessing the distribution of the drug under the bladder mucosa during injection. Pharmacological interactions between BTX-A and MB were also evaluated. MATERIALS AND METHODS: The study was conducted between December 2014 and April 2016 on 30 patients: six males and 24 females (median age 57.7, range 23-80 years) diagnosed with OAB, who qualified for intravesical BTX-A injection. Each received 100 IU of BTX-A (Botox®; Allergan), dissolved in 9.5 ml of 0.9% NaCl with the addition of 0.5 ml of MB. Cystoscopy with submucosal injection of the solution was performed systematically, including the bladder triangle. For pharmacological evaluation, quantitative determination of MB was performed on a capillary electrophoresis system with diode array detection. RESULTS: In the course of 600 injections, the addition of MB facilitated the observation of the procedure; the exact distribution of the solution could not be observed in only 43 injections in seven patients. The range of distribution of the drug varied from 1 to 2.5 cm. Pharmacological evaluation based on visual observations and experiments showed that pharmaceutical interactions do not occur between MB and this commercially available formulation of BTX-A. CONCLUSIONS: Applying a coloured solution of BTX-A significantly facilitates observation of the procedure and assessment of drug distribution. There are no pharmaceutical interactions between MB and BTX-A.

Urine metabolic fingerprinting using LC-MS and GC-MS reveals metabolite changes in prostate cancer: A pilot study.

Prostate cancer (CaP) is a leading cause of cancer deaths in men worldwide. The alarming statistics, the currently applied biomarkers are still not enough specific and selective. In addition, pathogenesis of CaP development is not totally understood. Therefore, in the present work, metabolomics study related to urinary metabolic fingerprinting analyses has been performed in order to scrutinize potential biomarkers that could help in explaining the pathomechanism of the disease and be potentially useful in its diagnosis and prognosis. Urine samples from CaP patients and healthy volunteers were analyzed with the use of high performance liquid chromatography coupled with time of flight mass spectrometry detection (HPLC-TOF/MS) in positive and negative polarity as well as gas chromatography hyphenated with triple quadruple mass spectrometry detection (GC-QqQ/MS) in a scan mode. The obtained data sets were statistically analyzed using univariate and multivariate statistical analyses. The Principal Component Analysis (PCA) was used to check systems' stability and possible outliers, whereas Partial Least Squares Discriminant Analysis (PLS-DA) was performed for evaluation of quality of the model as well as its predictive ability using statistically significant metabolites. The subsequent identification of selected metabolites using NIST library and commonly available databases allows for creation of a list of putative biomarkers and related biochemical pathways they are involved in. The selected pathways, like urea and tricarboxylic acid cycle, amino acid and purine metabolism, can play crucial role in pathogenesis of prostate cancer disease.