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The influence of solubility and adsorption on plastic materials on transport experiments
Šilhanová, Marie ; Kučera, Radim (advisor) ; Váňová, Nela (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department: Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Student: Marie Šilhanová Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Title of diploma thesis: The influence of solubility and adsorption on plastic materials on transport experiments From transport experiments on cell culture models we get valuable information about transport mechanism of drugs in organism. In vitro experiments are conducted for example on Transwell type inserts. During the experiment it was discovered that the results are not homogeneous, and the quantity of a substance in the solution decreases apparently, the reason behind this is inadequate solubility of lipophilic substances or their adsorption on the surface of plastic materials used in the experiment. Due to these problems we experience significant bias. This thesis is focused on antivirotics that did not perform well during transport experiments. First, HPLC/MS methods were developed, and they were used for concentration measurement of samples containing individual antivirotics. The drugs were tested under wide range of conditions so possible changes in effects of adsorption on plastic surfaces and solubility of drugs could be observed. The substances were divided into groups based on...
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LC-HRMS analysis of selected drugs in biological material I.
Tomanová, Jana ; Kučera, Radim (advisor) ; Váňová, Nela (referee)
Charles Univesity, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Jana Tomanová Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Consultant: PharmDr. Vít Šesták, Ph.D. Title of the Diploma Thesis: LC-HRMS analysis of selected drugs in biological material I. High performance liquid chromatography is one of the most widespread separation analytical techniques. It is used in various medical, pharmaceutical and industrial laboratories. It uses different affinities of the substances in the analysed mixture to the mobile and stationary phases. Mass spectrometry is one of the instrumental techniques by which charged particles are separated in the gas phase based on the mass- to-charge ratio. Nowadays, great emphasis is placed on the fact that the individual analytical methods which are being developed are subsequently validated according to a guideline issued by national or international authorities. Validation is a process that verifies that a method is suitable for its intended use. In this thesis, the conditions for the determination of warfarin in patient's serum by means of mass spectrometer with LTQ XL linear ion trap were optimized and validated. The optimization of the method was based on the already developed and validated...
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The monitoring of agent BZ after intramuscular administration
Čechová, Lenka ; Váňová, Nela (advisor) ; Bavlovič Piskáčková, Hana (referee)
The compound 3-quinuclidinyl benzylate (agent BZ) belongs to the group of incapacitating warfare agents with anticholinergic activity. Today, for its ability to induce functional cognitive impairment (i.e. coordination and memory disorders), the agent BZ is used mainly for scientific purposes in the study of Alzheimer's disease. Despite this fact, its pharmacokinetics has not been fully examined yet. In order to determine the agent BZ in biological material, LC-MS / MS method and sample preparation procedure for body fluids (plasma, bile) and tissues (brain, liver, kidneys) were developed, optimized and validated. The sample preparation procedure for body fluid employed solid phase extraction using a C18 column eluted with methanol and for body tissues it was precipitation with acetonitrile. The chromatographic separation was performed on Gemini NX-C18 reverse phase column (150 × 4.6 mm, 5 μm). The mobile phase consisted of a 10mM solution of ammonium acetate at pH 11 and methanol in a ratio of 30 : 70. Elution of the analytes was performed under isocratic elution. The total analysis time was 5 minutes. Mass spectrometric detection was performed by a linear ion trap using electrospray ionization. Sample preparation procedure and chromatographic analysis methods were successfully applied to real...
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Determination of oxidative stress biomarkers by HPLC
Váňová, Nela ; Jun, Daniel (advisor) ; Doležal, Rafael (referee) ; Hroch, Miloš (referee)
and keywords Although reactive oxygen and nitrogen species have a fundamental role in physiological processes occurring in living organisms, their overproduction induced by endogenous and/or exogenous sources may lead to serious imbalance in redox homeostasis, damage to intracellular components and thus, dramatically alter their function or even trigger cell death. Oxidative stress is believed to be very important mechanism of toxicity of xenobiotics, including drugs, and may be responsible for the development of their unwanted side effects. Considering a very low number of studies evaluating oxidative stress after the treatment with oxime reactivators of acetylcholinesterase (AChE) in vivo and in vitro, the relationship between their toxicity and generation of specific biomarkers of oxidative damage is not still fully understood. In order to monitor antioxidant/prooxidant properties of drugs, high performance liquid chromatography method coupled with tandem mass spectrometry (LC-MS/MS) for simultaneous determination of two biomarkers of oxidative stress, malondialdehyde (MDA) and 3-nitrotyrosine (3-NT), in biological matrices was developed. Validation of this LC-MS/MS method demonstrated the acceptable appreciable selectivity, accuracy, intra- and interday precission, and recovery of sample...
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Analysis of oxidative and free radical induced DNA damage
Váňová, Nela ; Kučera, Radim (advisor) ; Kovaříková, Petra (referee)
Analysis of oxidative and free radical induced DNA damage Diploma thesis Nela Váňová Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Free radicals and reactive oxygen species (ROS) are highly reactive molecules capable of modifications of biomolecules, including DNA. 5',8-cyclopurine-2'deoxynucleosides represent a group of DNA lesions characterized by concomitant damage to both sugar and base moieties of the same purine nucleoside that are together with 8-oxo-2'-deoxypurines among the major lesions formed by attack of free radicals (e.g. hydroxyl radical). Quantification of oxidative and free radical induced DNA lesions as biomarkers of oxidative stress has a high importance in study of their role in human health and disease. For quantification of these DNA lesions in gamma irradiated samples, high performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) will be utilized. Before injection into the LC/MS/MS, irradiated samples, treated by enzymatic digestion in order to gain free nucleosides, have to be desalted and DNA lesions have to be separated from undamaged nucleosides. A new HPLC/UV method was developed for separation of (5'R)-5',8-cyclo-2'- deoxyadenosine;...
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