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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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An influence of individual modes of TOF analyzer on linearity and sensitivity of UHPLC-HRMS methods
Halad, Jakub ; Vlčková, Hana (advisor) ; Khalikova, Maria (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Jakub Halad Supervisor: RNDr. Hana Vlčková, Ph.D. Title of thesis: An influence of individual modes of TOF analyzer on linearity and sensitivity of UHPLC-HRMS methods This diploma thesis deals with the comparison of four modes (sensitive mode, resolution mode, high resolution mode, enhanced resolution mode) of the hybrid analyser Q- TOF connected with UHPLC system. Totally, ten analytes were selected for this study. It was a group of 5 statins and their interconversion products. UHPLC method was borrowed from previous developed multistatin method however, ESI conditions were optimized. Developed method was used to measure standard calibration curves and matrix calibration curves in four analyser modes (sensitive, resolution, high resolution and enhanced mode). Spiked lyophilized serum treated by the protein precipitation was used for matrix calibration curve. Finally, the individual analyser modes were compared due to the linearity and sensitivity The effect of individual analyser modes on the correlation coefficient was not observed. On the other hand, analyser modes influenced the method sensitivity. In positive mode, lower LOQ values were obtained for the standard calibration curves...
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Development of Stage tips-UHPLC-MS/MS method for determination of statins in human serum
Otoupal, Lukáš ; Vlčková, Hana (advisor) ; Šatínský, Dalibor (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Lukáš Otoupal Supervisior: RNDr. Hana Vlčková, Ph.D. Title of Thesis: Development of Stage tips-UHPLC-MS/MS method for determination of statins in human serum This diploma thesis deals with development of a method for determination of statins in human serum. Extraction by Stage tips pipette tips was chosen and optimized for their extraction. The measurement was performed by the ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometer. The extraction procedure and important mass spectrometer parameters were optimized. Previously developed LC method for determination of statins was used for the analysis. The optimization of the mass spectrometer parameters was started by the selection of precursor ions, consequently the ion source parameters were tuned and finally appropriate ionization mode, SRM transition and collision energy for each analyte were chosen. The suitability of the LC-MS method was verified by the repeatability method (RSD ≤ 1 for the retention time and RSD ≤ 10 for the peak area), linearity (correlation coefficient ≥ 0,997) and sensitivity (LOQ in the range of 5 x 10-10 - 1 x 10- 9 g/ml). Firstly, the optimization employing mixed standard...
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Optimalization and validation of SPE method for determination of quercetin and its 9 metabolites in biological material
Chrenková, Lucia ; Nováková, Lucie (advisor) ; Vlčková, Hana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Lucia Chrenková Supervisor: doc. PharmDr. Lucie Nováková, Ph.D. Title of Graduation Thesis: Optimalization and validation of SPE method for the determination of quercetin and its 9 metabolites in biological material. The aim of this study was to develop and validate a new extraction method for the preparation of biological samples for the determination of quercetin and its 9 metabolites: phloroglucinol, 3,4-dihydroxyphenylacetic acid, homovanilic acid, 3-hydroxyphenylacetic acid, 3-(3-hydroxyphenyl)propionic acid, rutin, quercetin-3-glucuronide, tamarixetin and isorhamnetin. Due to the best retention and subsequent elution of all tested analytes the mixed mode (polymeric ion exchanging) cartridge MAX was chosen. The mixture of methanol and 0.5 % trifluoracetic acid in water (95/5) was chosen as optimal solvent for elution. The combination of 0.01M ammonium formate buffer pH 5.0 and 1% methanol in buffer pH 5.0 were chosen as washing solvent. The determination of quercetin and its 9 metabolites was performed using UHPLC- MS/MS. The best selectivity between the critical pair of analytes with the same molecular weight (tamarixetin and isorhamnetin) was achieved using BEH Shield RP18 column and...
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Study of HILIC separation mechanism and the influence of mobile phase composition on selectivity and retention of the analytes
Lepáček, Michal ; Vlčková, Hana (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Michal Lepáček Supervisor: RNDr. Hana Vlčková Ph.D. Title of Graduation Thesis: Study of HILIC separation mechanism and the influence of mobile phase composition on selectivity and retention of the analytes. This graduation thesis deals with a study of retention, selectivity and HILIC mechanisms depending on the mobile phase composition. The column Zorbax HILIC plus (2,1 x 100 mm, 3,5 μm, Agilent Technologies), one of the basic types of HILIC stationary phases, was chosen for the purposes of this study. The influence of acetonitrile concentration, buffer pH (3,8, 4,8 and 6,8) and buffer concentration (0,5 - 200 mM) on retention of selected analytes was tested. Based on the physical-chemical properties (log P and pKa) a set of 35 analytes including bases, acids and neutral compounds was chosen. Isocratic flow of 0,7 ml/min and injection volume of 2 μl were used. Detection of analytes was performed by a PDA detector at the wavelength of 254 nm. The data are presented in charts and tables for an easier evaluation. The effect of concentration of acetonitrile in mobile phase was evaluated by the dependence k' = f (% ACN). An increasing retention of analytes with increasing concentration of...
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Application of alternative mobile phases in Hydrophilic interaction chromatography method
Münsterová, Barbora ; Nováková, Lucie (advisor) ; Vlčková, Hana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Barbora Münsterová Supervisor: doc. PharmDr. Lucie Nováková, Ph.D. Title of Diploma thesis: Application of alternative mobile phases in Hydrophilic interaction chromatography method. This thesis was focused on the possibilities of using 1,3-dioxolan as an alternative organic component of the mobile phase in HILIC. The changes in selectivity and efficiency of the separation were observed when using dioxolan and acetonitrile as the organic components of the mobile phase. 0.5 % acetic acid or 50 mM ammonium acetate pH 3.8, pH 6.8 and pH 9.8 were used as the aqueous components for the conventional HILIC. The mobile phase consisting of acetonitrile-dioxolan, acetonitrile-dioxolan with 0.5 % acetic acid and acetonitrile- dioxolan modified with 50 mM ammonium acetate aqueous solution pH 6.8 were tested for non-aqueous HILIC. For the measurements on column ACQUITY UPLC BEH Amide (1.7 µm, 2.1 mm x 100 mm, Waters) 5 different groups of compounds were used, including of phenolic acids, purine and pyrimidine bases, pyridine derivates, water-soluble vitamins and beta-blockers. Retention, selectivity and the speed of analysis were competed. Measurements were performed using ACQUITY Ultra Performance...
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Study of influence of mobile phase composition on selectivity and retention of the analytes on HILIC stationary phases
Štětková, Kateřina ; 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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