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Solid-phase extraction and its miniaturization by method Lab-On-Valve for determination of pharmaceutical substances
Jelínková, Iva ; Chocholouš, Petr (advisor) ; Vlčková, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical chemistry Candidate: Iva Jelínková Supervisor: PharmDr. Petr Chocholouš, Ph.D. Title of diploma thesis: Solid-phase extraction and its miniaturization by method Lab-On- Valve for determination of pharmaceutical substances This thesis deals with the development of the solid-phase extraction (SPE) method with the use of microextraction by packed sorbent (MEPS) for the determination of vitamins A, D, E and the possibility to automate this method. The determination was based on absorption of vitamins on microcolumn MEPS, interfering components were removed by washing solution (dilute acetic acid of pH 3) and extraction was performed by eluent solution (100% ACN). Detection was made by UV spectrophotometer at wave lengths of absorbing maximum for vitamin A - 325 nm, for vitamin D - 265 nm, for vitamin E - 295 nm. The method was optimized. There was developed a program, where concentrations of individual vitamins, dosing volumes, flow speed and composition solvents were tested. After the optimization method there was performed the extraction and the subsequent determination of blood plasma fortified with vitamins A, D, E by the sequential injection chromatography method. Recovery of the method for vitamin A was...
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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 UHPLC-MS/MS method and sample preparation procedure for the determination of steroid compounds in rat plasma
Hromádko, Jiří ; Kočová Vlčková, Hana (advisor) ; Chocholouš, Petr (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Jiří Hromádko Supervisor: RNDr. Hana Kočová Vlčková Ph.D. Title of Diploma Thesis: Development UHPLC-MS/MS method and sample preparation procedure for the determination of steroid compounds in rat plasm The diploma thesis deals with the development of the UHPLC-MS/MS method for the analysis of 37 steroids in plasma and optimization of sample preparation using PP. The analytes include structurally very similar substances with different numbers of hydroxyl or ketonic functional groups, isomeric and mutually isobaric compounds. Since these substances cleave water molecules very easily, precursor ions with one m/z for different substances can be found in the MS spectrum. For these reasons, careful and detailed characterization of the precursors, subsequent scanning of the product ions, and selection of additional SRM transitions were essential. Separation of critical compounds with the same m/z for precursor ions was of importance. The optimization started with the screening of 7 columns using gradient elution of the mobile phase ACN/0.1 % FA in the range of 5 - 98% ACN. Based on the results, an optimization of the mobile phase gradient was performed. The Cortecs C18 column was optimal. Most of the...
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A study of separation conditions and stability of selected phenolic compounds
Jandorová, Zuzana ; Nováková, Lucie (advisor) ; Kočová Vlčková, Hana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Zuzana Jandorová Supervisor: Prof. PharmDr. Lucie Nováková, Ph.D. Title of the diploma thesis: A study of separation conditions and stability of selected phenolic compounds The goal of this diploma thesis was to determine the most suitable separation conditions for the analysis of ten phenolic compounds by the ultra-high performance liquid chromatography with PDA detection. The thesis is focused mainly on the separation of critical pair tamarixetin and isorhamnetin. Another goal was to determine the short-term stability of all tested compounds for 24 hours. Analyses were performed using the ACQUITY UPLC chromatographic system with PDA detector at a wavelength of 275 nm. The first step in the study of separation conditions was the screening of 17 columns from several different manufacturers. Tested columns included ACE Excel C18, BEH C18, CSH C18, Arion Plus C18, Ascentis Express C18, Atlantis Premier BEH C18 AX, BEH Shield RP18, Cortecs Shield RP18, Ascentis Express RP-Amide, BEH Phenyl, CSH Phenyl-Hexyl, Kinetex Biphenyl, ACE Excel C18-AR, ACE Excel C18-PFP, CSH Fluoro-Phenyl, Ascentis Express F5, and Kinetex PFP. During the screening, 3 organic modifiers (acetonitrile, methanol and acetonitrile...
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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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Development UHPLC-MS/MS method and sample preparation procedure for the determination of steroid compounds in rat plasma
Hromádko, Jiří ; Kočová Vlčková, Hana (advisor) ; Chocholouš, Petr (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Jiří Hromádko Supervisor: RNDr. Hana Kočová Vlčková Ph.D. Title of Diploma Thesis: Development UHPLC-MS/MS method and sample preparation procedure for the determination of steroid compounds in rat plasm The diploma thesis deals with the development of the UHPLC-MS/MS method for the analysis of 37 steroids in plasma and optimization of sample preparation using PP. The analytes include structurally very similar substances with different numbers of hydroxyl or ketonic functional groups, isomeric and mutually isobaric compounds. Since these substances cleave water molecules very easily, precursor ions with one m/z for different substances can be found in the MS spectrum. For these reasons, careful and detailed characterization of the precursors, subsequent scanning of the product ions, and selection of additional SRM transitions were essential. Separation of critical compounds with the same m/z for precursor ions was of importance. The optimization started with the screening of 7 columns using gradient elution of the mobile phase ACN/0.1 % FA in the range of 5 - 98% ACN. Based on the results, an optimization of the mobile phase gradient was performed. The Cortecs C18 column was optimal. Most of the...
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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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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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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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