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Development of method for the determination of entecavir in biological materials using UHPLC-MS/MS
Gottvald, Tomáš ; Nováková, Lucie (advisor) ; Šatínský, Dalibor (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Tomáš Gottvald Supervisor: Doc. PharmDr. Lucie Nováková, Ph.D. Title of thesis: Development of method for the determination of entecavir in biological materials using UHPLC-MS/MS Entecavir is a synthetic guanosine nucleoside that plays an important role in treatment of chronical hepatitis B virus infection. This work was focused on development of highly sensitive method to determine entecavir concentrations in rat urine by ultra-high performance liquid chromatography-tandem mass spectrometry. Entecavir C2 13 N15 , stable isotopically labeled internal standard, was used for quantitation. Hydrophilic interaction chromatography seems to be a suitable technique for retention and separation of polar compounds present in analyzed sample. One of the main goals was to confirm this fact by comparison with commonly used reverse phase chromatography mode (RP-UHPLC) on BEH C18 stationary phase. Conditions for RP-UHPLC were optimized as follows: mobile phase composed of acetonitrile/0.01 % formic acid (4:96). The HILIC conditions on BEH Amide stationary phase were optimized using isocratic elution with mobile phase composed of acetonitrile/5 mM ammonium acetate pH 4.0 (75:25). HILIC method provided much...
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Using of separation methods in clinical research
Plíšek, Jiří ; Solich, Petr (advisor) ; Šatínský, Dalibor (referee) ; Friedecký, David (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Mgr. Jiří Plíšek Supervisor: prof. RNDr. Petr Solich, CSc. Supervisor specialist: assoc. prof. RNDr. Dagmar Solichová, Ph.D. Title of dissertation thesis: Using of separation methods in clinical research This thesis is devoted to development and validation of new LC methods for the determination of biologically active substances, specifically a fat-soluble vitamins (vitamin A, E and D) and theirs metabolites. Vitamins are essential micronutrients which cannot be synthesized by human organism with the exception of steroid hormone precursor of vitamin D. Vitamins are involved in biochemical processes of organism directly or as coenzymes. Interest in the clinical monitoring of these substances is increasing constantly over the last years. It needs to use modern instrumental techniques such as the UHPLC coupled with MS detection for the determination of vitamin D metabolites (25-hydroxy vitamin D3 and 25-hydroxy vitamin D2) due to their low concentrations in serum. The use of classical liquid chromatography with diode array detection is suitable in case of monitoring of levels of vitamin A (retinol) and E (α-tocopherol) in breast milk and vitamin E (α- tocopherol) in red blood cell...
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HPLC method development of active substances in nutraceuticals
Jägerová, Kateřina ; Šatínský, Dalibor (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Kateřina Jägerová Supervisor: Doc. RNDr. Dalibor Šatínský, Ph.D. Title of Diploma Thesis: HPLC method development of active substances determination in nutraceuticals A high performance liquid chromatography (HPLC) method with diode array detection (DAD) was used and validated for the simultaneous determination rutin, troxerutin, diosmin and hesperidin. The method was used for direct determination of this flavonoids in pharmaceutical preparations and nutraceuticals, Cilkanol (Zentiva), Detralex (Les Laboratoires), Hemodin Prebio Forte (Valosun a. s.), MobiVen Micro (Vulm cz a.s.). Using Ascentis Express RP - Amide (10 cm x 3.0 mm, 2.7 μm) Supelco Analytical column, the following conditions were chosen as optimal: mobile phase acetonitril/water 30/70 containing acetic acid for pH adjustment to value pH 3. Method development was performed within a temperature of 50řC and pressure of 18.6 MPa. The flavonoids were detected by diode array detector (DAD) at the wavelength of 283 nm and 255 nm. Keywords: high performance liquid chromatography (HPLC), flavonoids, rutin, troxerutin, diosmin, hesperidin, nutraceuticals
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Development and optimization of chromatographic methods suitable for clinical application
Jenčo, Jaroslav ; Solich, Petr (advisor) ; Hroboňová, Katarína (referee) ; Šatínský, Dalibor (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of Analytical Chemistry Candidate: Mgr. Jaroslav Jenčo Supervisor: prof. RNDr. Petr Solich, CSc. Consultant: doc. RNDr. Lenka Kujovská Krčmová, Ph.D. Title of dissertation thesis: Development and optimization of chromatographic methods suitable for clinical application The presented dissertation thesis is divided into several parts, copying the workplaces, which I gradually worked on. The unifying element is the development and optimization of methods for the preparation of samples of biologically active substances and their subsequent analysis using separation methods. The theoretical part characterizes the target substances, from a biochemical, physicochemical, and analytical point of view. It describes the basic characteristics of extraction procedures and presents an overview of the methods used to determine the substances described in the thesis. In the first, main experimental part, of the dissertation thesis the development, optimization, and validation of an analytical method for chromatographic analysis of vitamin B1, its biologically active forms and the active form of vitamin B6 in blood is described. It illustrates the selection of sample preparation techniques, the optimization of the target methodology...
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Development of CE-C4D method for the analysis of saccharides in honey
Šebestová, Gabriela ; Jáč, Pavel (advisor) ; Šatínský, Dalibor (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Gabriela Šebestová Supervisor: PharmDr. Pavel Jáč, Ph.D. Title of the diploma thesis: Development of CE-C4D method for the analysis of saccharides in honey Honey is a commonly used natural mixture which quality must be monitored due to possible mishandling or adulteration. This diploma thesis is focused on the development of a capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE-C4D) for the determination of carbohydrates in honey samples. Design of Experiments (DoE) was used to optimize the separation conditions. This approach allows to find suitable separation conditions by means of multivariate optimization. During the optimization, the impact of the following factors was investigated: the composition of the background electrolyte, the separation voltage and temperature during separation and the inner diameter of the separation capillary. Two methods were optimized: one with normal electroosmotic flow (EOF) and the other using reversed EOF after CTAB addition. The method in the normal EOF mode was used for validation and analysis of honey samples. A fused silica capillary with an inner diameter of 20 m, a total length of 48.7 cm and an effective...
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Studying the distribution of substituents in derivatives based on hyaluronane
Sedláček, Jakub ; Šatínský, Dalibor (advisor) ; Zimčík, Petr (referee) ; Chmelař, Josef (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate Ing. Jakub Sedláček Supervisor doc. RNDr. Dalibor Šatínský, Ph.D. Title of Doctoral Thesis Studying the distribution of substituents in derivatives based on hyaluronan Derivatives based on hyaluronan have found applications in various areas of medicine. Prepared derivatives are commonly characterized by their molar mass and their degree of substitution, however, such parameters are unable to distinguish among derivatives with different distributions of substituents along the chain, which may exhibit different macroscopic properties. For this reason, methods to determine the distribution of substituents along the chain are needed. The presented thesis deals with the determination of distribution of amino groups in deacetylated hyaluronan. The theoretical part starts with a description of hyaluronan structure and of its derivatives. Next, the application, the preparation, and the characterization of deacetylated hyaluronan are discussed, focusing particularly on its depolymerisation into oligosaccharides using deaminative cleavage or enzymes that degrade hyaluronan. Finally, examples of liquid chromatographic methods used to analyse hyaluronan oligosaccharide are presented. The experimental part...
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Development and optimization of chromatographic methods suitable for clinical application
Jenčo, Jaroslav ; Solich, Petr (advisor) ; Hroboňová, Katarína (referee) ; Šatínský, Dalibor (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of Analytical Chemistry Candidate: Mgr. Jaroslav Jenčo Supervisor: prof. RNDr. Petr Solich, CSc. Consultant: doc. RNDr. Lenka Kujovská Krčmová, Ph.D. Title of dissertation thesis: Development and optimization of chromatographic methods suitable for clinical application The presented dissertation thesis is divided into several parts, copying the workplaces, which I gradually worked on. The unifying element is the development and optimization of methods for the preparation of samples of biologically active substances and their subsequent analysis using separation methods. The theoretical part characterizes the target substances, from a biochemical, physicochemical, and analytical point of view. It describes the basic characteristics of extraction procedures and presents an overview of the methods used to determine the substances described in the thesis. In the first, main experimental part, of the dissertation thesis the development, optimization, and validation of an analytical method for chromatographic analysis of vitamin B1, its biologically active forms and the active form of vitamin B6 in blood is described. It illustrates the selection of sample preparation techniques, the optimization of the target methodology...
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Development of on-line SPE HPLC method for determination of ochratoxin A in Tokaj wines
Uhrová, Adéla ; Šatínský, Dalibor (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Adéla Uhrová Supervisor, consultant: Doc. RNDr. Dalibor Šatinský, Ph.D. Title of Diploma Thesis: Development of on-line SPE HPLC method for determination of ochratoxin A in Tokaj wines This diploma thesis deals with the optimization and validation of chromatographic method for determination of ochratoxin A in Tokaj wine with subsequent application on real samples. A high- performance liquid chromatography method with fluorescence detection using on-line extraction by column switching has been developed. 50µl of sample was injected directly in the extraction column, where at 1st minute the analyte was extracted on the Ascentis® Express RP-Amide precolumn in the presence of a mobile phase of 40% MeOH in 0.5% aqueous acetic acid. After the extraction, the valve was switched and the extracted substances were separated on a YMC Triart C18 ExRS analytical column. The separation was carried out under gradient elution with an initial acetonitrile ratio of 45% in the mobile phase, which reached a concentration of 80% in the 5th minute. Fluorimeter detection was optimized for excitation and emission wavelengths ʎEx 335 nm and ʎEm 463 nm. In the last part of the work, ochratoxin A was analyzed in real...
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Development of HPLC method for determination of selected phenolic acids and flavonoids in Tokaj wines
Moravcová, Pavlína ; Šatínský, Dalibor (advisor) ; Lhotská, Ivona (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical chemistry Candidate: Bc. Pavlína Moravcová Supervisor: Doc. RNDr. Dalibor Šatínský, Ph.D. Title of the diploma thesis: Development of HPLC method for determination of selected phenolic acids and flavonoids in Tokaj wines The aim of this diploma thesis was to develop and optimize HPLC method for simultaneous determination of 15 phenolic compounds, protocatechic acid, fertaric, coffee, p-hydroxybenzoic, syringic, gallic, p-coumaric, ferulic, chlorogenic, caftaric, 2-hydroxy-4-methoxybenzoic , polydatin, resveratrol, catechin hydrate and epicatechin in Tokaj wines. The quantity and content of selected phenolic compounds was analyzed in 25 samples of Tokaj wines, which come from vineyards from the Slovak part of Tokaj wine region. The work was also focused on the description of the specific characteristics of this unique area, the unique method of Tokaj wine production, its chemical composition, and namely the spectrum of phenolic substances. Analysis of selected phenolic compounds was performed using an Ascentis® Express F5 column (150 x 4.6 mm; 5 µm) using a mobile phase consisting of 0.1% phosphoric acid and acetonitrile in gradient elution. The substances were analyzed at 30 řC and flow rate of 1 ml/min and detected...
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Optimization of neonicotinoids extraction using nanofibers in sequential injection analysis systém
Zouharová, Monika ; Horstkotte Šrámková, Ivana (advisor) ; Šatínský, Dalibor (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Monika Zouharová Supervisor: PharmDr. Ivana Horstkotte Šrámková, Ph.D. Title: Optimization of neonicotinoids extraction using nanofibers in sequential injection analysis system In this diploma thesis, we tested the use of various nanofibers as sorbents for solid phase extraction of neonicotinoids connected with sequential injection analysis system. Seven types of nanofibers were tested, specifically polyamide (PA6), polycaprolactone (PCL), polyimide (PID) and polydopamine coated polyamide (PA6)coated, polycaprolactone (PCL)coated, polyimide (PID)coated and a combination of polyimide and polycaprolactone (PID/PCL)coated. These nanofibers were produced by electrospinning. The studied nanofibers were placed in the 3D printed holder, which was connected into sequential injection analysis system. UV spectrophotometry was used as a detection method. The substances to analyse were neonicotinoid insectisides. The retention of these analytes was examinated with all seven types of nanofibers. Retention of the analytes on the tested nanofibers depending on their chemical structures and physical-chemical properties was studied in these measurements. The most experiments were performed with polydopamine coated...
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