New approach in determination of urinary diagnostic markers for prostate cancer by MALDI-TOF/MS.

In our study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is proposed as a novel tool, which can be applied to analyze lipids in urine samples. For this reason, the main aim of the study was to develop and optimize the preparation protocol for urine samples in lipidomics, using urine samples obtained from patients with diagnosed cancer and non-cancer controls. Several conditions like extraction method and types of matrices were evaluated. For this purpose, two methods for the extraction of lipids, namely modified Folch and Bligh & Dyer were employed. Furthermore, two types of matrices (α-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxybenzoic acid (DHB)) for the separation of lipids into individual components was tested. The results of this study can serve as an essential source for the selection of appropriate extraction methods and the appropriate choice of a matrix for the purification and identification of a particular class of lipid in human biological fluids. Based on it, Bligh & Dyer method associated with the usage of HCCA matrix was found to be the most effective for lipidomics using MALDI-TOF/MS. The optimized method was applied to compare the lipid profile of 139 urine samples collected from both healthy individuals and patients with prostate cancer. The tandem spectroscopic analysis allowed to identify lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and triacylglycerols in urine samples. Finally, MALDI-TOF/MS analysis enabled to discriminate between the two tested groups (healthy individuals and patients with prostate cancer). A preliminary statistical model suggested that classification accuracy ranging from 83.3 to100.0% may be achieved by using pre-selected MS signals.

Robust HPLC-MS/MS method for levofloxacin and ciprofloxacin determination in human prostate tissue.

Fluoroquinolones are the drugs of choice in the prevention of bacterial infections after transrectal ultrasound guided prostate biopsy. In order to improve assessment of antibacterial efficacy in the target tissue a simple, selective, rapid and robust HPLC-ESI-MS/MS method for the determination of levofloxacin and ciprofloxacin concentrations in human prostate bioptates was developed and validated. Preparation procedure for prostate samples (10mg) was carried out using homogenization and filtration steps. Analyses were performed within 3.5min using RP C18 column in the isocratic elution mode with mobile phase composed of a mixture of 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution (v/v; 79:21). The method was linear between 0.3µg/g and 15µg/g for levofloxacin and ciprofloxacin with coefficient of correlation (r) ≥0.999. The limit of detection and the limit of quantification for levofloxacin were 0.06µg/g and 0.2µg/g and for ciprofloxacin were 0.04µg/g and 0.13µg/g, respectively. Average concentrations (±SD) of levofloxacin and ciprofloxacin obtained from patients tissue were 5.4±2.2µg/g and 3.9±1.5µg/g, respectively. Additionally, during validation procedure a novel, experimental design approach was applied for the robustness study. For evaluation of analytical method robustness, Plackett-Burman design was employed and for sample preparation method robustness Fractional Factorial design was used. The developed and validated method was successfully applied to examine prostate tissue samples obtained from patients enrolled into a clinical study. Up to now, there has been no other HPLC-ESI-MS/MS method reported for the simultaneous determination of levofloxacin and ciprofloxacin in human prostatic tissue.

Ultra-high performance liquid chromatographic determination of levofloxacin in human plasma and prostate tissue with use of experimental design optimization procedures.

Fluoroquinolones are considered as gold standard for the prevention of bacterial infections after transrectal ultrasound guided prostate biopsy. However, recent studies reported that fluoroquinolone- resistant bacterial strains are responsible for gradually increasing number of infections after transrectal prostate biopsy. In daily clinical practice, antibacterial efficacy is evaluated only in vitro, by measuring the reaction of bacteria with an antimicrobial agent in culture media (i.e. calculation of minimal inhibitory concentration). Such approach, however, has no relation to the treated tissue characteristics and might be highly misleading. Thus, the objective of this study was to develop, with the use of Design of Experiments approach, a reliable, specific and sensitive ultra-high performance liquid chromatography- diode array detection method for the quantitative analysis of levofloxacin in plasma and prostate tissue samples obtained from patients undergoing prostate biopsy. Moreover, correlation study between concentrations observed in plasma samples vs prostatic tissue samples was performed, resulting in better understanding, evaluation and optimization of the fluoroquinolone-based antimicrobial prophylaxis during transrectal ultrasound guided prostate biopsy. Box-Behnken design was employed to optimize chromatographic conditions of the isocratic elution program in order to obtain desirable retention time, peak symmetry and resolution of levofloxacine and ciprofloxacine (internal standard) peaks. Fractional Factorial design 2(4-1) with four center points was used for screening of significant factors affecting levofloxacin extraction from the prostatic tissue. Due to the limited number of tissue samples the prostatic sample preparation procedure was further optimized using Central Composite design. Design of Experiments approach was also utilized for evaluation of parameter robustness. The method was found linear over the range of 0.030-10µg/mL for human plasma and 0.300-30µg/g for human prostate tissue samples. The intra-day and inter-day variability for levofloxacine from both plasma and prostate samples were less than 10%, with accuracies between 93 and 108% of the nominal values. The limit of detection and the limit of quantification for human plasma were 0.01µg/mL and 0.03µg/mL, respectively. For the prostate tissue, the limit of detection and the limit of quantification were 0.1µg/g and 0.3µg/g, respectively. The average recoveries of levofloxacin were in the range from 99 to 106%. Also, the method fulfills requirements of robustness what was determined and proved by Design of Experiments. The developed method was successfully applied to examine prostate tissue and plasma samples from 140 hospitalized patients enrolled into the clinical study, 12h after oral administration of LVF at a dose of 500mg. The mean (±SD) LVF concentration in prostate was 6.22±3.52µg/g and in plasma 2.54±1.14µg/mL. Due to simplicity of the method and relative small amount of sample needed for the assay, the method can be applied in clinical practice for monitoring of LVF concentrations in plasma and prostate gland.

The simultaneous determination of hydrophobicity and dissociation constant by liquid chromatography-mass spectrometry.

Convenient methods for testing drug candidates' lipophilicity and acidity are highly requested in modern pharmaceutical research and drug development strategies. Reversed-phase high-performance liquid chromatography (RP HPLC) might be particularly useful for the determination of both dissociation constant and the (pH-dependent) partition coefficient related parameters, applicable in high-throughput analysis of multi-component mixtures. The general theory of combined pH/organic modifier gradient has recently provided equations relating gradient retention time and pH of the mobile phase. The purpose of this work was to facilitate the identification of analytes in this technique by its transfer to RP HPLC coupled with time-of-flight mass spectrometry with electrospray ionization source (ESI-TOF-MS). The accuracy of the proposed methodology was assessed by analyzing a set of known drugs. The ammonium formate, ammonium acetate or ammonium bicarbonate buffers were used to control pH during chromatographic analysis. In result, the pKa and hydrophobicity parameters were determined and the accuracy of the estimated values was assessed by comparing them with literature data. The gradient RP HPLC coupled with ESI-TOF-MS methods allowed for the rapid determination of dissociation constant and hydrophobicity and was shown to be especially applicable for complex mixtures. The use of ESI-TOF-MS detection allowed to achieve the medium-throughput screening rate (100 compounds/day) and provided a simple approach to assess pharmacokinetically important physicochemical properties of drugs.

Chromatographic behaviour of ionic liquid cations in view of quantitative structure-retention relationship.

The availability of ionic liquids (ILs) in wide areas of application often results in the requirement on their determination. The attention is also often focused on the knowledge of hydrophobicity as it plays a key role in the biological effects, in the assessment of environmental risk and in the prediction of the fate of chemicals in the environment and of its influence on retention in RP HPLC. One can get information regarding hydrophobicity and retention mechanism if quantitative structure-retention relationships (QSRRs) are identified. The QSRRs were derived for logarithms of retention factors extrapolated to a pure water (or aqueous buffer) eluent, log k(w), determined for the pyridinium and imidazolium ionic liquid (IL) cations on two C8 (Supelcosil LC-8-DB, Symmetry C8) and two C18 (ACE 5 C18, Symmetry C18) stationary phases with isocratic elution by a mobile phase consisting of acetonitrile/40 mM phosphate buffer. The analyses of ILs were performed at a flow rate of 1 mL min(-1) with UV detection at 218 nm. The QSRRs were derived based on the retention parameters determined experimentally and the structural descriptors of test analytes from molecular modeling. Separations of ILs were obtained with aqueous acetonitrile buffered at pH 3.55 mobile phases. The statistically most significant two-parameter QSRR regression equations related log k(w) to the solvent accessible surface (SAS) of the analytes and the differences in the energies of the highest occupied and the lowest unoccupied molecular orbitals (diffHL). These equations were especially good in case of columns with the highest carbon loads and larger specific surface areas, i.e. Symmetry C18 and Symmetry C8. On the other hand, the column ACE 5 C18 appeared to produce the best quality separations of the ILs studied. The QSRRs derived in the research shed light on the molecular mechanism of HPLC separation of ILs and helped to predict their relative separations.

Increasing conclusiveness of metabonomic studies by chem-informatic preprocessing of capillary electrophoretic data on urinary nucleoside profiles.

Nowadays, bioinformatics offers advanced tools and procedures of data mining aimed at finding consistent patterns or systematic relationships between variables. Numerous metabolites concentrations can readily be determined in a given biological system by high-throughput analytical methods. However, such row analytical data comprise noninformative components due to many disturbances normally occurring in analysis of biological samples. To eliminate those unwanted original analytical data components advanced chemometric data preprocessing methods might be of help. Here, such methods are applied to electrophoretic nucleoside profiles in urine samples of cancer patients and healthy volunteers. The electrophoretic nucleoside profiles were obtained under following conditions: 100 mM borate, 72.5 mM phosphate, 160 mM SDS, pH 6.7; 25 kV voltage, 30 degrees C temperature; untreated fused silica capillary 70 cm effective length, 50 microm I.D. Different most advanced preprocessing tools were applied for baseline correction, denoising and alignment of electrophoretic data. That approach was compared to standard procedure of electrophoretic peak integration. The best results of preprocessing were obtained after application of the so-called correlation optimized warping (COW) to align the data. The principal component analysis (PCA) of preprocessed data provides a clearly better consistency of the nucleoside electrophoretic profiles with health status of subjects than PCA of peak areas of original data (without preprocessing).