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Automated monitoring of Metridia luciferase released from genetically modified cell line
Jozičová, Natali ; Sklenářová, Hana (advisor) ; Horstkotte, Burkhard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of: Analytical Chemistry Candidate: Natali Jozičová Supervisor: doc. PharmDr. Hana Sklenářová, Ph.D. Consultant: prof. Manuel Miró Title of the diploma thesis: Automated monitoring of Metridia luciferase released from genetically modified cell line. The diploma thesis reports automatic real-time monitoring of the release of Metridia luciferase from genetically modified cells. A previously optimized method for monitoring the natural release of luciferase from cells was used during the experiment. At the same time, the toxic effect of bile acids (chenodeoxycholic and deoxycholic acid) on changes in cell membrane permeability was tested. Liver epithelial cell line modified with the Metridia luciferase plasmid were used during the experiment. After this genetic modification, the cells were able to synthesize the active Metridia luciferase enzyme. This enzyme generates luminescence upon reaction with substrate coelenterazine. The emitted light was detected in the flow cell of a spectrofluorometer at a wavelength of 485 nm. The measurement was taken in a sequential injection system connected to a 3D printed module containing an insert with the tested cell monolayer. This automated system allowed real-time monitoring of the luciferase...
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Study of BTEX determination by microextraction in the Lab-in-syringe system coupled with gas chromatography
Kohániová, Andrea ; Horstkotte, Burkhard (advisor) ; Šatínský, Dalibor (referee)
Charles university Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Andrea Kohániová Supervisor: M.Sc. Burkhard Horstkotte, Ph.D. Co-supervisor: PharmDr. Ivana Horstkotte Šrámková, Ph.D. Title: Study of BTEX determination by microextraction in the Lab-in-syringe system coupled with gas chromatography Benzene, toluene, ethylbenzene and xylenes are one of the major contaminants in the environmental water samples. Because these compounds are consider to be toxic to humans by World Health Organisation, their determination is critically important. The determination of BTEX using gas chromatography with flame ionisation detection combined with direct single drop microextraction using Lab-in-syringe system was studied. The effective paramateres such as microdroplet volume, extraction time, type and use of salt solution and chromatographic parameters were evaluated. The purpose of this work was automation of the entire process using 3D elements in the system.
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Automated monitoring of Metridia luciferase released from genetically modified cell line
Jozičová, Natali ; Sklenářová, Hana (advisor) ; Horstkotte, Burkhard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of: Analytical Chemistry Candidate: Natali Jozičová Supervisor: doc. PharmDr. Hana Sklenářová, Ph.D. Consultant: prof. Manuel Miró Title of the diploma thesis: Automated monitoring of Metridia luciferase released from genetically modified cell line. The diploma thesis reports automatic real-time monitoring of the release of Metridia luciferase from genetically modified cells. A previously optimized method for monitoring the natural release of luciferase from cells was used during the experiment. At the same time, the toxic effect of bile acids (chenodeoxycholic and deoxycholic acid) on changes in cell membrane permeability was tested. Liver epithelial cell line modified with the Metridia luciferase plasmid were used during the experiment. After this genetic modification, the cells were able to synthesize the active Metridia luciferase enzyme. This enzyme generates luminescence upon reaction with substrate coelenterazine. The emitted light was detected in the flow cell of a spectrofluorometer at a wavelength of 485 nm. The measurement was taken in a sequential injection system connected to a 3D printed module containing an insert with the tested cell monolayer. This automated system allowed real-time monitoring of the luciferase...
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Automation of solid phase extraction method for determination of flavonoids using a flow system
Krovová, Tereza ; Horstkotte, Burkhard (advisor) ; Sklenářová, Hana (referee)
Department: Department of Analytical Chemistry Candidate: Bc. Tereza Krovová Supervisor: M.Sc. Burkhard Hosrtkotte, Ph.D. Supervisor - specialist: PharmDr. Ivana Horstkotte Šrámková, Ph.D. Title of the diploma thesis: Automation of solid phase extraction method for determination of flavonoids using a flow system Development and optimization of a method for automated solid phase extraction in a flow system using the Lab-On-Valve platform for determination of flavonoids and metabolites is presented in this diploma thesis. Solid phase extraction performed in a bead injection format was followed by spectrophotometric detection in UV. The method was optimized for the determination of quercetin and five of its metabolites: rutin, 3,4-dihydroxyphenylacetic acid, homovanilic acid, 3-(3- hydroxyphenyl)-propionic acid and 4-methylcatechol. Optimized parameters included type of sorbent for extraction, amount of sorbent to build a column, pH of loading buffer, composition of eluent and weak wash solution, flow rates for loading and elution of sample, sample volume, sample-buffer ratio and influence of acidification of sample. Optimization of the flavonoid assay method was performed on standard solutions and is now ready for application to biological samples.
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Comparison of manual and dynamic extractions of selected transition metals from solid samples
Kucharčíková, Vendula ; Horstkotte, Burkhard (advisor) ; Horstkotte Šrámková, Ivana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Vendula Kucharčíková Supervisor: Burkhard Horstkotte, Ph.D. Consultant: Doc. PharmDr. Hana Sklenářová, Ph.D. Title of thesis: Comparison of manual and dynamic extractions of selected transition metals from solid samples The analysis of trace metals in soil is a subject of study in many laboratories. Due to large industrial areas soil can be highly contaminated. This work focused on two easy methods - manual extraction and single flow extraction with the same extractant -acetic acid. Two methods, two concentrations of acetic acid (0.11 M and 0.43 M), and two samples were used to evaluate, which conditions were more effective. As a detection device inductively coupled plasma - optical emission spectrometry was selected and two different wavelengths were used to prevent interferences. Manual extraction was more time-consuming and less reproducible than the dynamic extraction. Higher concentration of acetic acid extracted a larger amount of metals and the extraction showed higher reproducibility of the results. In both soils the contaminations with Cu outreached the permitted level for this metal (according to the requirement of the government of the Balearic Islands) and are potentially...
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Spectrophotometric determination of chlorhexidine in mouthwash employing Lab-In-Syringe automated ion-pair extraction and back-extraction
Řeháková, Pavla ; Horstkotte, Burkhard (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Pavla Řeháková Supervisor: Dr. Burkhard Horstkotte, Ph.D., M.Sc. Consultant: doc. PharmDr. Hana Sklenářová, Ph.D. Title of the diploma thesis: Spectrophotometric determination of chlorhexidine in mouthwash employing Lab-In-Syringe automated ion-pair extraction and back-extraction In this work, a magnetic stirring-assisted dispersive liquid-liquid micro-extraction (MSA-DLLME) automated by the Lab-In-Syringe technique is presented. MSA-DLLME is based on mixing the sample and an immiscible solvent by the action of a magnetic stirrer for the dispersion of the solvent into fine droplet to enhance the extraction process. In Lab-In-Syringe, the magnetic stirrer is placed inside a syringe void, which is used as extraction chamber. To allow the use of an extraction solvent lighter than water, the syringe was turned upside down in this work. This method was designed for the determination of chlorhexidine in commercial mouthwash samples. To allow the extraction of chlorhexidine, an ion-pair complex with the reagent methyl orange needed to be formed. After the extraction into an organic solvent, the aqueous solution was exchanged and the analyte was back-extracted into an acidic aqueous acceptor to yield...
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Monitoring of toxicological tests
Rosecká, Michaela ; Sklenářová, Hana (advisor) ; Horstkotte, Burkhard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Michaela Rosecká Supervisor: Doc. PharmDr. Hana Sklenářová, Ph.D. Title of Diploma Thesis: Monitoring of toxicological tests The aim of this work was to monitor kinetic profiles of permeation of fluorescent marker through cell monolayer (MDCKII-MDR) by sequential injection analysis. For this purpose, a new type of permeation module was prepared that could measure from 2 compartments - donor and acceptor. The module was thermostated at 37řC during the experiment and liquid in the acceptor compartment was continuously agitated. Experiments were repeated to observe difference in interaction of fluorescent marker with cell membrane transporter. MDCKII-MDR monolayer cell inserts were replaced after the experiments. The cell line used in this experiment include a genetically modified P-glycoprotein. Rhodamine 123, the fluorescent agent, was applied for on-line monitoring of extracellular transport. The permeability kinetics of this marker was monitored using a fully automated sequential injection analysis system with five minutes sampling intervals for the testing period of 120 minutes. The sample was taken from the donor compartment in one cycle, and from the acceptor one in the next cycle. Then,...
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Study of deproteinization of milk samples via salting-out induced phase separation and automation by Lab-In-Syringe
Machián, Daniel ; Horstkotte, Burkhard (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Daniel Machián Supervisor: M.Sc. Burkhard Horstkotte, Ph.D. Co-supervisor: PharmDr. Kateřina Fikarová, Ph.D. Diploma thesis title: Study of deproteinization of milk samples via salting-out induced phase separation and automation by Lab-In-Syringe A novel approach to sample deproteinization based on homogeneous liquid-liquid extraction with salting-out induced phase separation was explored. A methodology was developed, experimentally tested, and optimized that allows automated centrifuge-less deproteinization of milk samples using the flow-batch technique Lab-In-Syringe. As in other sample preparation methods, the aim was to eliminate the sample matrix and obtain an organic phase that could be submitted to analysis. The thesis includes a range of preliminary experiments performed offline, i.e. without the intended instrumentation, as well as optimization experiments performed online using a Lab-In-Syringe system. Salting out agents and three solvents were tested as well as phase ratios and operational parameters on the Lab-In-Syringe system, including system configuration. In the method optimized by the work reported in this thesis, milk proteins were found effectively denatured and...
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Separace vybraných anorganických iontů pomocí sekvenční injekční chromatografie
Jarošová, Patrícia ; Horstkotte, Burkhard (advisor) ; Chocholouš, Petr (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Patrícia Jarošová Supervisors: Dr. Burkhard Horstkotte, Ph.D., M.Sc. Doc. PharmDr. Hana Sklenářová, Ph.D. Title of Diploma Thesis: Separation of selected inorganic ions using the sequential injection chromatography The ability of the guard column Dionex Ionpac® CG5A (2 x 50 mm, P/N 046104) to separate some transition metal cations from water samples, using Sequential injection chromatography (SIC) system was demonstrated. The type of separation was based on ion exchange interaction and the column was filled with particles functionalized by a bilayer of anion-exchange and cation-exchange latex. The chelating agent used in eluent was pyridine- 2,6-dicarboxylic acid (PDCA). A post-column reagent with 4-(2-pyridylazo) resorcinol (PAR) was used for spectrophotometric detection. For optimization, two methods were used: Simplex and univariant studies. Ideal conditions of the separation were found. The mobile phase consisted of PDCA, formic acid, sodium sulphate, and sodium hydroxide in the concentrations 4 mmol/l, 40 mmol/l, 4 mmol/l and 2 mmol/l, respectively. The concentrations of the post-column reagent components were 0.3 mmol/l of PAR, 150 mmol/l of the ammonium hydroxide, 60 mmol/l of...
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Development of dispersive liquid-liquid microextraction coupled with capillary liquid chromatography for determination of fipronil and its two metabolites
Jirková, Tereza ; Nováková, Lucie (advisor) ; Horstkotte, Burkhard (referee)
1 ABSTRACT Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Tereza Jirková Supervisors: Dr. Francisco J. Lara prof. PharmDr. Lucie Nováková, Ph.D Title: Development of dispersive liquid-liquid microextraction coupled with capillary liquid chromatography for determination of fipronil and its two metabolites Fipronil is a phenylpyrazolic insecticide that is prohibited for use in food-producing animals. This diploma thesis proposes a green analytical methodology for the study of residual fipronil and its two degradation products, fipronil sulfone and fipronil sulfide. For their determination, a method of capillary liquid chromatography with UV detection was developed and validated. Correlation coefficients above 0.998 confirm good linearity and the study of repeatability with values not exceeding 2.9% RSD shows high precision of this method. For the dispersive liquid-liquid microextraction method used for sample preparation, the dispersing and extracting solvents were optimized. The results show that the highest recoveries 82% - 92% were achieved with dibromomethane as an extractant and acetonitrile as a dispersant. All the experiments were carried out with standard solutions and in further studies this methodology should be transferred to egg or other...
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