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Optimization of metabolomics workflow: critical evaluation of sample clean-up step
Syřínek, Jakub Eduard ; Nováková, Lucie (advisor) ; Kočová Vlčková, Hana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Jakub Eduard Syřínek Supervisor: Assoc. Prof. PharmDr. Lucie Nováková, Ph.D. Title of Diploma Thesis: Optimization of metabolomics workflow: critical evaluation of sample clean-up step Metabolomics workflow involves following steps: experiment planning, sampling, sample preparation, sample analysis and both data processing and data interpretation. Metabolomics is focused on small molecules with mass lower than 1500 Da. This is reason, why sample preparation of biological samples is critical. This work deals with the optimization of sample preparation method for polyethylene glycol (PEG) elimination from sample. PEG can interfere with metabolites during the ionization in the ion source of mass spectrometer. Solid phase microextraction in pipette tips (SPE-PT) was used in this work for PEG elimination. Porous titanium dioxide was employed as a sorbent. Vaginal swabs were collected from patients suffering from vulvovaginal discomfort caused by Candida albicans. Samples were first dissolved in phosphate buffer saline solution. In SPE-PT method 5 different composition of binding solution, number of aspiration/expelling repeats in sample loading step, 5 different composition of washing buffer and...
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Comparison of two types of matrix effect evaluation for LC-MS/MS analysis
Lejnarová, Michaela ; Kočová Vlčková, Hana (advisor) ; Nováková, Lucie (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Michaela Lejnarová Supervisor: RNDr. Hana Kočová Vlčková, Ph.D. Title of Diploma Thesis: Comparison of two types of matrix effect evaluation for LC-MS/MS analysis This diploma thesis deals with a comparison of two methods of matrix effect evaluation in LC-MS/MS analysis using electrospray ionization and triple quadrupole. Specifically, it was a comparison of the method using slopes of a line of the calibration curves and the post-extraction addition method, where the main goal was to assess whether the obtained values of matrix effects correlate with each other or not. Where appropriate, to clarify the reasons for the different outcomes and define the conditions of both methods to obtain correct and accurate results. On the basis of physicochemical properties such as molecular weight, acid-base properties, and partition coefficient, a group of 28 diverse substances was selected. The actual evaluation of matrix effects was preceded by the selection of an appropriate LC method, optimization of the individual parameters of the ion source as well as the selection of appropriate SRM transition and collision energy for each substance. To evaluate the matrix effects, lyophilized serum was chosen as a...
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Development and validation of UHPLC-MS/MS method for determination of maraviroc in placental perfusion
Čapková Maxová, Eliška ; Nováková, Lucie (advisor) ; Kočová Vlčková, Hana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Eliška Čapková Maxová Supervisor: Assoc. Prof. PharmDr. Lucie Nováková, Ph.D. Title of Thesis: Development and validation of UHPLC-MS/MS method for the determination of maraviroc in placental perfusions The goal of this work was to develop fast and selective method for the determination of maraviroc in human placental perfusions. The ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) was used. BEH C18 column and gradient elution with the mobile phase A (water with 0.1% formic acid) and B (acetonitrile) at 0.35 mL/min flow-rate and 40 řC temperature were used for the separation. The mass spectrometry conditions were optimist and set up as follows: electrospray ionization in positive mode, capillary voltage 1.0 kV, cone voltage 35 V, extractor 3.0 V, RF lens 0.1 V, desolvatation gas flow 1000 L/h, temperature 450 žC, cone gas flow 100 l/h, ion source temperature 130 žC. Selected reaction monitoring (SRM) mode was used for quantitation. Liquid-liquid extraction (LLE) was chosen for sample preparation and was optimised. The best results were obtained when dichloromethane was used as the extraction agent (recovery >90%). The optimized method was fully validated in...
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Testing of stability of antivirotic drugs using ultra-high performance liquid chromatography
Pavlasová, Pavlína ; Kočová Vlčková, Hana (advisor) ; Chocholouš, Petr (referee)
Charles univerzity Fakulty of Pharmacy in Hradec Králové Department od Analytical Chemistry Candidate: Pavlína Pavlasová Supervisor: RNDr. Hana Kočová Vlčková, Ph.D. Title of thesis: Testing of stability of antivirotic drugs using ultra-high performance liquid chromatography. This thesis is concerned with determining the stability of five antiviral drugs (ledipasvir, sofosbuvir, tenofovir and the monoester and diester of tenofovir) under various temperature conditions. The study employed ultra-high performance liquid chromatography with a photodiode array detector detection at 259 nm. The tested antiviral drugs are new medicinal products used in treating serious diseases such as AIDS and hepatitis C. Two types of stability were tested: short-term and long-term. The short-term stability of the substances was tested in acetonitrile and methanol at 4 řC and 22 řC for 24 hours. The long- term stabilities of sofosbuvir and the monoester and diester of tenofovir in 50 % acetonitrile, ledipasvir in 100 % acetonitrile and tenofovir in water were determined while maintaining the substances at three temperatures in the medium: - 20 řC, 4 řC and 22 řC for a period of 28 days. In addition the substances were tested in phosphate buffers for 90 days for storage at - 20 řC. The storage conditions and the time...
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Study of influence of mobile phase composition on selectivity and retention of the analytes on HILIC stationary phases
Štětková, Kateřina ; Kočová Vlčková, Hana (advisor) ; Chocholoušová Havlíková, Lucie (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Kateřina Štětková Supervisor: RNDr. Hana Vlčková, Ph.D. Title of Diploma thesis: Study of influence of mobile phase composition on selectivity and retention of the analytes on HILIC stationary phases This graduation thesis deals with an influence of concentration of ACN, pH and buffer concentration in mobile phase on retention of selected analytes and on selectivity of Atlantis HILIC Silica column (3 μm, 2,1 mm x 100 mm) and Luna NH2 column (3 μm, 2,0 mm x 100 mm). A set of 35 analytes including neutral, basic and acidic compounds were chosen for this study. Detection was performed by PDA detector at the wave-length of 245 nm. Measurements were carried out using isocratic flow 0.4 ml.min-1 of mobile phase ACN/water (buffer). The column temperature was set up to 30 řC, auto sampler temperature to 4 řC and injection volume to 2 µl. For easier evaluation of results, measured data are presented in charts and tables. These were used to evaluation of columns in terms of selectivity and retention of the individual groups of analytes. The typical HILIC behavior, an increasing retention of analytes with increasing ACN concentration in mobile phase, was observed for Atlantis HILIC Silica column....
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