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HPLC study of the role of pyridoxal isonikotinoyl hydrazone in isoniazid intoxication
Gladziszová, Marcela ; Kovaříková, Petra (advisor) ; Kučera, Radim (referee)
High-performance liquid chromatography belongs nowadays among the most frequently used analytical methods in all fields of the pharmaceutical analysis. It makes both qualitative and quantitative evaluation possible. Isoniazid is used for the treatment and prevention of tuberculosis. One of the most serious symptoms of toxicity is a neurotoxicity which is connected with the B6 deficiency. The neurotoxicity probably relates to the formation of condenzation products isoniazid and vitamins B6 - such as pyridoxal isonikotinoyl hydrazone (PIH). Concrete evidence of the ongoing mechanisms and actions concerning this problemsis remains still elusive. The aim of this thesis was the optimalisation of the chromatography conditions for the separation of 8 analytes (isoniazid, acetylisoniazid, pyridoxal, pyridoxol, pyridoxamine, pyridoxal-5-phosphate, PIH a the internal standard o- 108). Therafter, the isolation of these compounds from plasma samples using precipiation was optimized. The separation was performed on a LiChroCART 250 × 4 mm I.D., analytical column with Lichrospher 100 RP-18, 5 µm packing, protected with a Purospher RP-18, 5µm guard column. As the mobile phase A was used aqueous solution of NaH2PO4 (0,01 mol/l), with the addition of EDTA (0,001 mol/l) and heptansulphonic acid (0,005 mol/l) with...
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Study of chiral aspects of drug metabolism using instrumental analytic methods
Holmanová, Veronika ; Nobilis, Milan (advisor) ; Kovaříková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Candidate: Bc. Veronika Holmanová Supervisor: Doc. PharmDr. Milan Nobilis, CSc. Title of diploma thesis: Study of chiral aspects of drug metabolism using instrumental analytic methods For the evaluation of stereospecificity of rat and human cytosolic carbonyl reductases and activity of microsomal oxidases involved in nabumetone biotransformation, a new chiral high-performance liquid chromatographic method was developed. The prepared LLE-HPLC-PDA method enabled extraction, separation and ultraviolet detection of prochiral nabumetone and its five phase I metabolites including enantiomers of two chiral biotransformation products of carbonyl reduction. Methyl ester of naproxen served as an internal standard. Diethyl ether was used for liquid-liquid extraction (LLE) of biomatrices. Chiralcel OD-R 250 mm×4.6 mm column with a mobile phase methanol-1M NaClO4/HClO4 aqueous solution pH=3 (75:25, v/v) were employed in isocratic sufficient separation of nine analytes. The whole analysis lasted 60 minutes at the flow rate of 0.5 ml/min. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at λ=265 nm). The results of in vitro nabumetone...
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Analysis of farnesylated peptides and proteins using LC-MS
Hessler, Filip ; Kovaříková, Petra (referee) ; Waisser, Karel (advisor)
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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Investigation of the stability of novel drug candidates in biological materials
Bechná, Lenka ; Kovaříková, Petra (advisor) ; Pilařová, Pavla (referee)
High performance liquid chromatography (HPLC) is one of the most frequently used analytical techniques for the analysis of drugs. Pyridoxal isonicotinoyl hydrazone (PIH) is a parental compound of the new group of the selective iron chelators derived from aroylhydrazone, which are currently intensively investigated as promising drug candidates. Besides the ability to bind iron, PIH was shown to posses other pharmacological effects: cardioprotective, antioxidative and cytoprotective. This work deals with the investigation of the stability of water-soluble salt of PIH (PIH·2HCl) in three biological materials (rabbit plasma, BSA solution and ultrafiltrate) using HPLC. The half-lives of PIH in these media were calculated as well. The measurement was executed with the guard column Lichrocart - 4×4 mm I.D. (Purospher RP-18C, 5 µm) and the analytical column Lichrocart - 250×4 mm I.D. (Lichrospher 100, RP-18 C, 5 µm). The mobile phase was composed of methanol/acetonitrile (in the ratio 24:14) and phosphate buffer (NaH2PO4 0,01mol/l; pH 6,0 - adjusted by 10% solution NaOH) + EDTA (1,0 mmol/l), in the ratio 50:50. Flow rate was 1 ml/min. The detection was executed in the ultraviolet spectrum at the wavelength of 297 nm. The stability of the PIH was characterized by its half-lives in plasma - t 1/2 = 29 min., BSA...
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Analytical evaluation of novel drug candidates from the group of iron chelators using HPLC I.
Schöngut, Michal ; Kovaříková, Petra (advisor) ; Kučera, Radim (referee)
Nowadays, high performance liquid chromatography (HPLC) belongs among the most modern and most frequently used analytical separation techniques. Biocompatible iron chelators are mainly used for the treatment of iron overload. Nevertheless, in the light of new information about physiological and patophysiological role of iron in organism, their potential use was widened into other diseases. Iron chelators were proved to have antioxidative, cardioprotective and cytostatic effects. Salicylaldehyde isonicotinoyl hydrazone is a biocompatible iron chelator, which also shows these activities. However, its short half life in plasma makes its use difficult. Nowadays, novel derivates of SIH were prepared to improve its stability in plasma. 2'-hydroxypropiophenone isonicotinoyl hydrazone (HPP-INH) is one of newly prepared SIH derivates. The aim of this study was to develop optimal chromatographic conditions for HPLC analysis of HPP-INH and to develop sample pretreatment method using precipitation for its analysis in plasma. The analysis was carried out on a Merck 250x4 mm column I.D. LiChrospher® 100, RP-18e (5 μm) protected with Purospher® 100, RP-18e (5 μm) guard column. The following mobile phase gradient condition was developed for HPP-INH purity evaluation: phosphate buffer (0,01 mol/l NaH2PO4 · 2 H20 water...
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Employment of gas chromatography in the field of drug analysis II.
Špalová, Klára ; Kučera, Radim (advisor) ; Kovaříková, Petra (referee)
Gas chromatography in drug analysis II. Thesis Klára Špalová Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control In this study, effect of various conditions on stability of 1,3-butanediol was studied. During these studies, 1,3-butanediol was found as a rather stable compound, susceptible only to oxidation using oxidative agents- including air oxygen or UV radiation. Various degradation products were detected during stress studies. There were peaks at retention times 6.0 and 6.9 at every chromatogram, which grew in time under some conditions. A new degradation product at retention time 3.0 was detected when UV radiation actuated on aqueous solution of 1,3- butanediol. The same peak was observed under oxidative conditions., Its concentration grew significantly in time.
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Development of an LC-MS method for determination of new antimalarial drugs in biological matrices.
Klimeš, Jiří ; Kučera, Radim (referee) ; Kovaříková, Petra (advisor)
Artemisinin a representative of Endoperoxide class of drugs and its derivatives particularly artesunate are the most important class of antimalarial drugs used in clinical practice. They are recommended as the first-line treatment of malaria in combination with other longer-acting antimalarial drugs (lumefantrine, piperaquine). As the main skeleton of these compounds lacks UV visible or fluorescent chromophore, earlier methods of detection have used post-column on-line derivatisation or electrochemical detection in the reductive mode. However, these methods suffer from poor sensitivity and selectivity. Within this thesis the whole LC-MS method development for the analysis of artesunate and its major metabolite dihydroartemisin in biological samples from the very beginning was performed. It included tuning of ESI - triple quadrupole MS detector and optimization of chromatographic conditions, particularly mobile phase pH. Artesunate (ARST) and dihydroartemisin (DHA) were assayed in human plasma using artemisisnin as an internal standard. Different approaches of plasma sample treatment, (protein precipitation and liquid- liquid extraction) were optimized and compared. Pre-validation data for liquid-liquid extraction revealed LLOQ as 2.5 and 3.0 ng/ml for DHA and ARST, respectively using 400 μl of...
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HPLC analysis of the clinically used drug - dexrazoxane
Pecuchová, Vladimíra ; Vrbatová, Ivana (referee) ; Kovaříková, Petra (advisor)
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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