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Optimization of metabolomic workflow for a comparison of impurity profiles of levothyroxin tablets using UHPLC-HRMS
Hrušková, Anna ; Nováková, Lucie (advisor) ; Váňová, Nela (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Anna Hrušková Supervisor: prof. PharmDr. Lucie Nováková, Ph.D. Title of Diploma Thesis: Optimization of metabolomic workflow for a comparison of impurity profiles of levothyroxin tablets using UHPLC-HRMS The aim of this diploma thesis was to compare 3 designs of measuring the impurity profiles of levothyroxine tablets and to evaluate the most suitable procedure. Analyses were performed by ultra-high performance liquid chromatography coupled with high resolution mass spectrometry. Levothyroxine drug products are used to supplement reduced thyroid function. In this work, 23 batches of tablets from 2 different manufacturers were analysed. The optimization of the tablet sample and internal standard preparation method and the compilation of 3 measurement designs using data from the preliminary screening was the first step. The designs were compiled as targeted metabolomics analyses. Then, a targeted analysis of 4 known impurities in designs 1 and 2 was performed, which was semi-quantitative (relative content of impurity to levothyroxine content). Differences in impurity content between designs were also evaluated. The next step was to compare individual designs in terms of variability, which was...
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LC-MS/MS study of phase one in vitro biotransformation of potential drugs against Alzheimer's disease
Kuřátková, Aneta ; Kučera, Radim (advisor) ; Váňová, Nela (referee)
No treatment that would completely stop the progression of Alzheimer's disease has not been found yet. Recently used tacrine showed good results in the treatment of Alzheimer's disease, however long-term use led to chronic hepatotoxicity due to its metabolites. This master thesis deals with the compound 7-phenoxytacrine, one of the promising tacrine derivatives, which is one of the candidates for potential use in the therapy of Alzheimer's disease. Due to the formation of hepatotoxic metabolites of tacrine after the biotransformation in human liver, it appears necessary to identify the emerging metabolites of 7-phenoxytacrine molecule. Within this master's thesis in vitro biotransformation study of 7-phenoxytacrine using human liver microsomes was performed. High performance liquid chromatography with tandem mass spectrometry was used to determine the parent substance and the seventeen 7-phenoxytacrine metabolites. The analytical method showed the formation of six monohydroxylated and eleven dehydroxylated metabolites of 7-phenoxytacrine. Thus, we concluded that hydroxylation is the major metabolic reaction after in vitro microsomal biotransformation. In addition to the identification of metabolites, a quantification and microsomal stability study, including the determination of the amount of...
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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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UHPLC-MS/MS analysis of selected drugs in a biological material
Motyčka, Filip ; Štěrbová, Petra (advisor) ; Váňová, Nela (referee)
Dexrazoxane (DEX), a bisdioxopiperazine derivative, is the only clinically used drug effective against anthracycline-induced cardiotoxicity. First studies indicated that DEX is a pro-drug bioactivated in cardiomyocytes by enzymatic hydrolysis of piperazine rings to its active metabolite - ADR-925. However, further research revealed that effective cardioprotection induced by bisdioxopiperazine compounds is more likely related to topoisomerase IIβ depletion induced by DEX itself. The only bioanalytical method for simultaneous determination of DEX and its metabolite was developed using HPLC-MS/MS system. Nevertheless, the analysis requires 30 min for each run, which does not accomplish requirements for modern bioanalysis. The aim of this project is to develop and validate a fast UHPLC-MS/MS method for determination of DEX and ADR-925 in plasma. The analyses were performed using an UHPLC system coupled to triple quadrupole mass spectrometer with ESI source in positive ion mode (both Shimadzu). Following stationary phases were tested: ZORBAX Bonus-RP (100 mm × 3.0 mm, 1.8 μm), Luna Omega Polar C18 (100 mm × 2.1 mm, 1.6 μm) and Kinetex F5 column (100 mm × 2.1 mm, 1.7 μm). Mixtures of acetonitrile or methanol with different concentrations of ammonium formate or formic acid were tested as mobile phase with...
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The pharmacokinetics of the oxime acetylcholinesterase reactivator K869
Hojná, Anna ; Váňová, Nela (advisor) ; Bavlovič Piskáčková, Hana (referee)
Co-administration of oxime reactivators of AChE together with atropine and diazepam is the basic strategy for the pharmacological treatment of organophosphate (OP) poisoning. The aim is to develop such oxime, that would be effective against a wide range of OPs and simultaneously, that would penetrate the central nervous system. Bispyridinium oximes are among the structures with the most efficient acetylcholinesterase reactivation. However, they are charged molecules that poorly cross the blood-brain barrier. Researchers try to modify these molecules to be more lipophilic thus, enhancing their central effect. One of the possible approaches is to substitute the basic structure of the reactivator with suitable functional groups. K869 is an asymmetric bispyridinium oxime whose pyridinium ring is substituted by two chlorine atoms. An HPLC-UV method was developed and optimized for the determination of oxime K869 in plasma and kidneys. Since oxime K869 is a permanently charged molecule, ion- pair chromatography was applied, in which the molecule becomes uncharged with the addition of an ion-pairing agent (1 mM octane sulfonic acid) to the aqueous components of the mobile phase (citrate-phosphate buffer). It was then separated in a reverse chromatographic mode in isocratic elution mode where acetonitrile...
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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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