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Sequential injection chromatography-testing and comparison of modern chromatographic columns
Molnárová, Petra ; Chocholouš, Petr (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Deparment of Analytical chemismy Candidate: Bc. Petra Molnárová Supervisor: PharmDr. Petr Chocholouš, PhD. Title of diploma thesis: Sequential injection chromatogramy - testing and comparison of modern chromatographic columns This diploma thesis deals with testing and comparison of two modern chromatoghraphic columns in Sequential injection chromatography system. They were used for separation of selected vitamins B (thiamine, nikotinamide, pyridoxine, riboflavin and folic acid) and vitamin C. Developed method was based on UV detection at 220, 272 and 290nm. The Ascentis Express HILIC column (3 cm x 4.6 mm, 2.7 µm) was tested by using acetonitrile with ammonium acetate as a mobile phase. The method was not siccessfully developed because of low retention times of vitamins. The separation of six vitamins was not able, because of low selectivity. There was developed a method with use of Ascentis Express RP- Amide column (3 cm x 4.6 mm, 2.7 µm) using gradient elution, which was able to separate all 6 vitamins. Acetonitrile and phosphoric acid (pH 1.8) were components of the mobile phases. There was made calibration, reproducibility and recovery tests, but there was not good results enough to validate the method. For recovery was used...
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Development and validation of method for determination of furosemide and its degradation product in pharmaceutical formulations
Břežná, Tereza ; Matysová, Ludmila (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Tereza Břežná Advisor: PharmDr. Ludmila Matysová, Ph.D. Thesis name: Development and validation of method for determination of furosemide and its degradation product in pharmaceutical formulations This thesis is focused on development and validation of a method for determination of furosemide and its degradation product in pharmaceutical formulations. The development was based on the HPLC method, which uses the mixture of 0.1 % formic acid and methanol in the 30:70 ratio as the mobile phase. The method development has been namely based on the mobile phase composition and analysis temperature conditions optimizing. In the final version was used the HPLC method with UV-VIS detection at 270 nm. The analysis was performed at the laboratory temperature using isocratic elution and C18 15 x 4.6 mm column with 5 μm particle size. The mixture of pH 5.75 formic acid and acetonitrile in the 65:35 ratio and at 1.5 ml/min flow rate has been selected as the mobile phase. The validation has proven that this method provides precise and acurate results and therefore it is suitable for furosemide determination in the solutions of medicinal products.
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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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Development of extraction and HPLC determination of vitamins A and D in pharmaceutical formulation Infadolan
Košvancová, Tereza ; Sklenářová, Hana (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Title of graduation theses: Development of extraction and HPLC determination of vitamins A and D in pharmaceutical formulation Infadolan Tereza Košvancová Supervisor : PharmDr. Hana Sklenářová, Ph.D. The High Performance Liquid Chromatography conditions for the determination of vitamins A and D in ointment Infadolan® were found. The analytical column Onyx Monolithic C18 (50 x 4,6 mm) with guard column (5 x 4,6 mm) was used for achievement of optimal results. A mobile phase composed of acetonitrile : methanol : water in ratio 49 : 49 : 2 (v/v/v), the flow-rate 2 ml/min, the sample volume 20 µl, the isocratic mode and laboratory temperature were chosen. The UV detection was set at 265 and 290 nm. Vitamin E acetate was used as an internal standard. Substance were eluted in the following order: vitamin A acetate, vitamin D and vitamin E acetate. During the search for suitable extraction agents for the development of an extraction method the ability of many basic analytical solvents was tested. Agents with the most suitable properties were used in five multi-stage extractions. Achieved results were compared and next procedure was proposed.
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HPLC method development for carotenoids determination in fruits
Růžička, Štěpán ; Šatínský, Dalibor (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Štěpán Růžička Supervisor: Doc. RNDr. Dalibor Šatínský, Ph.D. Title of the diploma thesis: HPLC method development for carotenoids determination in fruits In this thesis was optimized and developed a new HPLC method for the determination of specific carotenoids (betacarotene, lutein and zeaxanthin) content in selected species of sweet cherries. The method used Supelco Analytical RP-Amide (100 x 4.6 mm; 5 µm) column for separation and UV detection at a wavelength of 450 nm. There was used isocratic elution of the mobile phase acetonitrile:hexan:methylene chloride (96.6:1.7:1.7) at a flow rate of 1.409 ml/min during the measurement. The cherry samples were extracted to chloroform in ultrasound bath, centrifuged, and after filtration injected into HPLC system. The temperature was set at 30 řC and injected volume was 5 µl. The amount of betacarotene was quantified in a few cherry samples because of too low concentration of carotenoids.
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SEQUENTIAL INJECTION ANALYSIS CAPABILITY IN AUTOMATION OF ANALYTICAL PROCESSES
Novosvětská, Lucie ; Sklenářová, Hana (advisor) ; Polášek, Miroslav (referee) ; Hraníček, Jakub (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate (married name/maiden name): Mgr. Lucie Novosvětská / Mgr. Lucie Zelená Supervisor: assoc. prof. PharmDr. Hana Sklenářová, Ph.D. Consultant: PharmDr. Petr Chocholouš, Ph.D. Title of dissertation thesis: Sequential injection analysis capability in automation of analytical processes This dissertation, composed as a commented collection of four scientific articles and one undergoing project, introduces four novel applications in the field of automated flow methods and one application in the field of chromatographic methods used in analytical part of a complex pharmacological study. It the field of pharmaceutical analysis, three works were successfully optimized, validated, and published in scientific journals. Those works include: a) Automation of permeation studies within a sequential injection analysis system connected to a liberation unit and carrying out experiments with living cells. b) Pharmacological study of an antiretroviral drug efavirenz including its determination by fast chromatographic method in three types of sample matrices - in a medium from permeation studies with cellular models, in a Krebs solution from rat placenta perfusions, and in placenta tissue lysates. c) Determination of...
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Assessment of chelation of cupric ions with flavones by use of hematoxylin method
Tamborová, Barbora ; Karlíčková, Jana (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical botanic and ecology Candidate: Mgr. Barbora Tamborová Consultant: PharmDr. Jana Karlíčková, Ph.D. Title of Thesis: Assessment of chelation of cupric ions with flavones by use of hematoxylin method Copper is an essential trace element involved in many physiological processes. It is a part of several enzymes. Disruption of copper homeostasis results in pathological changes and diseases, excess of copper is highly toxic for human body. Flavonoids, the ubiquitous plant secondary metabolites, are highly abundant in the human diet. They represent a large group of polyphenolic compounds. They are characterized by a broad spectrum of biological activities. The anti-inflammatory, antiallergic, antimicrobial, hepatoprotective, immunomodulatory, cytotoxic and anticancer effects of flavonoids are well documented. Flavones belong to the group of flavonoids, they have chelating and antioxidative properties. They are known for their anti-inflammatory, antibacterial, antiviral and antiallergic activity. This thesis deals with chelating activity of six selected flavones, substituted on the A ring, namely 5-hydroxyflavone, baicalein, baicalin, chrysin, mosloflavone and negletein. Individual analyses were preformed using...
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