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Determination of Selected Active Substance in the Preparation VIII
Shkodra, Blerina ; Kastner, Petr (advisor) ; Kovaříková, Petra (referee)
Objective: The purpose of this work was to convert conventional HPLC method to the conditions of UFLC for determination of related substances of Quetiapine Fumarate tablet; new method should need less organic solvents, shorten the analysis time and be validated to guarantee its suitability. Methods: The new chromatographic conditions were assigned using an online method transfer calculator to convert HPLC parameters to UFLC parameters. The suitability of the new method was validated by system suitability test, linearity, quantification limit, detection limit, selectivity, precision and accuracy. Results: The method was shown to be suitable as the tailing factor of the peaks was kept within the limit range of 0.8 - 1.5 and the resolution of each of the peak areas was in all cases greater than 2. In linearity testing, the correlation coefficient obtained from the procedure was 0.999, which showed that the two variables, the peak areas and the range of five different concentrations of the sample highly correlate with each other. The quantification limit obtained from the method was 0.03%, while the detection limit was 0.01%. The selectivity of the method was confirmed as the peak areas of two placebo solutions were shown not to interfere with the peak areas of quetiapine and the impurities dibenzo and...
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The alternative stationary phases in HPLC analysis of drugs
Holásková, Petra ; Kučera, Radim (advisor) ; Kovaříková, Petra (referee)
Rigorous thesis The Alternative HPLC Phases for Analysis of Pharmaceuticals Mgr. Petra Holásková Charles Univerzity in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Chemistry and Drug Control The solubility of silica gel at high pH is the cause for developing alternative HPLC columns based on metal oxides, such as titanium oxide, aluminium oxide, and zirconium oxide. Advantages of these columns are particularly high chemical stability and separation efficiency. In our project, zirconia-based stationary phases were tested in qualitative and quantitative analysis of doxazosine and its five impurities. Polystyrene-coated zirconia was chosen because of its low retention and appropriate selectivity. The separation was done within 16 min. Subsequently, chromatographic conditions were validated.
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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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Analysis of farnesylated peptides and proteins using LC-MS
Hessler, Filip ; Waisser, Karel (advisor) ; Kovaříková, Petra (referee)
Farnesylated proteins are important in transduction of signals in cell and therefore can figure in development of many diseases, mainly cancer. Electrospray mass spectrometry is a widely used method in analysis of peptides and proteins, thus it was chosen as a method to develop a simple way to detect farnesylated proteins in cell. A cleavage pattern of these proteins, when subjected to MS/MS, was found on examples of synthetically farnesylated simple peptides and bovine albumin. Distinctive features of MS/MS spectra of these peptides are two peaks, which both represent the virgin peptide fragment after the farnesyl moiety was cleaved off. These fragments have a different charge, because they originate from different type of cleavage. Homological cleavage of the bond between the farnesyl and sulfur leads to formation of a new charge on the peptide fragment, while the leaving farnesyl is also charged and has amu of 205. Second type of fragment rises from neutral loss of farnesyl moiety, there is no new charge generated on the peptide fragment and the amu of the leaving farnesyl moiety is 204, because it looses one of its hydrogen in favor of the peptide. This knowledge can be applied also to more complicated protein samples prepared from cells. When searching for farnesylated proteins in such a...
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HPLC analysis of daunorubicin and its metabolite
Adamíková, Klára ; Kovaříková, Petra (advisor) ; Kučera, Radim (referee)
At present, the High Performance Liquid Chromatography (HPLC) is still the dominant separation method, it is widely used in all areas of drug analysis. HPLC provides qualitative and quantitative evaluation of the separated components. This thesis describes the development of the method for HPLC analysis of daunorubicin (DAU) and its metabolite daunorubicinol (DAU-OL) in a rabbit plasma. The analyzed compounds were first separated from interfering substances in plasma due the deproteinization. For a precipitation was chosen methanol (600 μl) with the highest extraction efficiency. The separation of DAU and its metabolite was reached by Zorbax SB-Aq column (3,5 µm, 150 x 4,6 mm; Agilent Technologies, USA), the mobile phase consisted of a mixture of water with phosphate butter and phosphoric acid (pH 2,4) and acetonitril (74:26, v/v). The analysis ran at isocratic mode during 15 minutes. The mobile phase was delivered at a rate of 1,1 ml/min, an aliquot of 40 μl was injected into the chromatographic system. The fluorescence detector was operated at an excitation wavelength of 480 nm and an emission wavelength of 560 nm. Doxorubicin was used as an internal standard. The methodology was validated with respect to linearity (4 - 83 ng/ml for DAU, 25 - 500 ng/ml for DAU-OL), precision and accuracy. The...
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Employment of gas chromatography in the field of drug analysis IV.
Kratochvílová, Eva ; Kučera, Radim (advisor) ; Kovaříková, Petra (referee)
Employment of gas chromatography in the field of drug analysis IV. Diploma thesis Eva Kratochvílová Charles Univerzity in Pratur, Fakulty of Pharmacy in Hradec Králové, Department of Pharmaceutical Chemistry and Drug Control Heyrovského 1203, Hradec Králové This diploma thesis deals with the validation of analytical method for impurities determination in butane-1,3-diol by gas chromatography. Determination of ideal parameters for separation- temperature on column, temperature gradient, velocity of carrier gas and split ratio, allows ideal separation of the peak butane-1,3-diol and its impurities. For sample quantification was tested method of internal standard and method of normalization. The method of normalization was then validated. Validation of method includes linearity, reproducibility, robustness, LOD and LOQ. The found method meets all parametrs. The sample stability was confirmed by series of measurements during 24 hours.
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HPLC analysis of the clinically used drug - dexrazoxane
Pecuchová, Vladimíra ; Kovaříková, Petra (advisor) ; Vrbatová, Ivana (referee)
1. ABSTRACT High performance liquid chromatography (HPLC) is a progressive analytic method which belongs among the most frequently used separation techniques. Iron is an irreplaceable biogenous element, which is involved in many important biochemical processes in human body. However, under specific pathophysiological conditions its severe effects may occur. Anthracycline cytostatics belong to the frequently used drugs for the treatment of various hematologic malignity and solid tumors. Their most important toxic effect is cardiotoxicity. Anthracyclines are able to form complexes with iron that are very redox active. They produce by the cyclic mechanism the high amount of cytotoxic hydroxyl radicals. This mechanism is believed together with own redox ability of anthracyclines molecule to be responsible for the drugs cardiotoxicity. Dexrazoxane, bisdioxopiperazine derivative, is the only clinically used drug for the treatment of anthracycline induced cardiotoxicity. It is supposed, that dexrazoxane is bioactivated inside the cardiomyocytes to active metabolite - ADR-925. Iron chelating ability of ADR-925 is believed to be responsible for the cardioprotective effect of dexrazoxane. This work is aimed at development of the optimal mass spectrometry compatible chromatographic conditions for the separation of...
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HPLC analysis of drugs II
Juříčková, Markéta ; Sochor, Jaroslav (advisor) ; Kovaříková, Petra (referee)
HPLC ANALYSIS OF DRUGS II. Diploma thesis Markéta Juříčková Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Chemistry and Drug Control, Heyrovského 1203, Hradec Králové In diploma thesis the optimal condition for analytical determination of tiaprofenic acid in plasma using solid-phase microextraction (SPME) and high performance liquid chromatography (HPLC) was developed. The final analysis was performed on RP18 analytical column. The mobile phase was composed of a mixture of methanol and phosphate buffer. The flow rate of 0.7 ml/min was used. 20 µl of sample was injected on the column. Detection was performed in UV range at 306 nm. PDMS/DVB microextraction fiber was used for the isolation of tiaprofenic acid from the sample. At first tiaprofenic acid was isolated from the water solution. Recovery of the microextraction was 19.46 %. Thereafter, tiaprofenic acid was extracted from model plasma sample. Prior extraction, pH of plasma was adjusted on 2.8. Methanol was used for analyte desorption. Optimal time of sorption and desorption was also determined. Efficiency of the developed extraction was 9.74 %. Quantitative determination of tiaprofenic acid from the plasma sample was performed by calibration curve and using external standard. Key words: HPLC,...
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HPLC analysis of drug candidates from the group of aroylhydrazones II.
Stariat, Ján ; Kovaříková, Petra (advisor) ; Kučera, Radim (referee)
1. ABSTRACT High performance liquid chromatography (HPLC) is one of the most frequently used analytical techniques for the analysis of drugs. Although iron is a vital element, excessive amounts in the body are highly toxic. The search for highly selective and effective iron chelating agents has been mainly inspired by the need to mobilize iron from tissues that are chronically overloaded with iron. However, recent investigations focused on the possibility to use iron chelators for the treatment of many other pathologies. Salicylaldehyde isonicotinoyl hydrazone (SIH), a biocompatible iron chelator derived from aroylhydrazone, is under extensive investigation as a promising drug candidate. Besides ability to bind iron, it shows interesting pharmacological effects: antioxidative, antiproliferative, cardioprotective, antimalarical and antimicrobic. The aim of this study was to develop optimal HPLC conditions for the separation of SIH and its potential metabolites (isoniazide, acetylisoniazide, salicylaldehyde) and to apply the method to the study focused on the isolation of analytes from rabbit urine using SPE. The best chromatographic analysis was achieved on a HPLC column (Phenomenex 250 4.6 mm I. D.) packed with Prodigy 5u ODS3 100A (5 μm) as a stationary phase. The mobile phase was composed of methanol :...
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The Role of Antiangiogenic Therapy in Mantle Cell Lymphoma
Kovaříková, Petra ; Klener, Pavel (advisor) ; Kalinová, Markéta (referee) ; Boudová, Ludmila (referee)
Mantle cell lymphoma (MCL) is a subtype of B-non-Hodgkin's lymphoma, characterized by often relapses. Despite an Ibrutinib (a Bruton's kinase inhibitor) implementation into salvage therapy, these patients often relapse with biologically highly aggressive disease and very poor prognosis. An increased activation of alternative metabolic pathways was described as one of ibrutinib-resistance mechanisms. Some of these pathways have also significant proangiogenic activity (e.g. PI3K-AKT-mTOR). In presented study, we established and standardized a real-time ultrasound and photoacoustic imaging of neovascularization and tissue oxygenation of subcutaneous MCL tumors in mice. Ultrasound and photoacoustic imaging is a fast, non-invasive method for angiogenesis evaluation in subcutaneous tumors with huge preclinical potential. Using MCL mice models, we also demonstrated the importance of CD31/PECAM-1 expression for engraftment, growth and spread of MCL cells in vivo. The level of CD31 expression in primary MCL cell (obtained directly from MCL patients) positively correlates with extent of extranodal involvement. CD31 facilitates survival and regulates extranodal spread of mantle cell lymphoma. We found that increased VEGFA expression causes not only increased microvessel density due to higher sprouting...
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