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Analytical determination of active compounds by liquid chromatography II
Raška, Martin ; Sochor, Jaroslav (advisor) ; Kovaříková, Petra (referee)
ANALYTICAL DETERMINATION OF ACTIVE COMPOUNDS BY LIQUID CHROMATOGRAPHY II. The Preliminary Study of SPME Master's Thesis Martin Raška Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Chemistry and Drug Control, Heyrovského 1203, Hradec Králové This thesis deals with the issue of analytical determination of active compounds by liquid chromatography. It's specifically focused on determination of benzodiazepines in biological material by using solid-phase microextraction (SPME) and high performance liquid chromatography (HPLC). HPLC is one of the most important chromatographic methods today. Its main advantages are sensitivity, selectivity and speed of separation and creation of reproducible record. SPME can be described as a simple and effective sample preparation technique integrating sorption, desorption and concentration of an analyte. This technique is solvent-free and requires no use of a complicated apparatus. Its basic principle is the exposure of liquid or fluid sample to a small amount of extraction phase. The following chromatographic conditions were proved to be optimal. Methanol:water (80:20) was used as a mobile phase with pH value adjusted to 7.5, flow rate was 0.8 ml/min and detection wavelength was 254 nm. Polydimethylsiloxane fiber...
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Analytical and bioanalytical evaluation of drug candidates from the group of aroylhydrazones
Kresová, Jana ; Kovaříková, Petra (advisor) ; Stariat, Ján (referee)
High-performance liquid chromatography (HPLC) is a progressive analytical method used for qualitative and quantitative drug analysis. This work describes various modifications of the chromatographic method for determining of BSIH (isonicotinic acid [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)- benzylidene]-hydrazide) and SIH (salicylaldehyde isonicotinoyl hydrazone) in rabbit plasma. BSIH belongs to prochelators which convert to iron chelator SIH in the presence of hydrogen peroxide. Increased stability in plasma and lower toxicity even during repetitive administration is the advantage of BSIH. Separation of BSIH, SIH and internal standard (o-108) was tested on various stationary phases using different chromatographic conditions. However, none of them provided better results than the initial one. Tha analysis was performed on chromatographic column Zorbax Bonus-RP (150 mm x 3,0 mm, particle size 3,5 µm,) with guard column using identical sorbent at a flow rate of 0,3 ml/min. Phosphate buffer (10 mM monosodium phosphate, pH6, adjusted with sodium hydroxide) : acetonitrile with methanol (60:40) (v/v) with the ratio of 60:40 (v/v)) as the mobile phase. Detector response was registered at 297 nm. Linearity of the method was verified in the concentration range of 10 µM to 100 µM for both BSIH and...
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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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Studium bioaktivace vybraných proléčiv
Vernerová, Monika ; Kovaříková, Petra (advisor) ; Kastner, Petr (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Student: Monika Vernerová Supervisor: Assoc. Prof. PharmDr. Petra Kovaříková, Ph.D. Consultant: Kristiina Huttunen, Ph.D., School of Pharmacy, Faculty of Health Sciences, Kuopio, University of Eastern Finland Title of thesis: Bioactivation study of selected pro-drugs This thesis deals with an enzymatic bioactivation study of the valproic acid's pro- drug. The evaluation was based on the data obtained from the HPLC analysis with appropriate method. The incubation reactions were carried out in a presence of different biological tissues, but also with isolated enzymatic fractions. The half-life of each bioactivation reaction was calculated from the gained results. It was also observed, if the bioconversion of the pro-drug to the parent drug, valproic acid, had been occured. These in vitro results will help to analyse samples from following in vivo experiments and help in designing better pro-drugs in future.
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HPLC analysis of drugs and drug candidates in biological material
Luňáková, Veronika ; Kovaříková, Petra (advisor) ; Mokrý, Milan (referee)
High-performance liquid chromatography (HPLC) is currently one of the most progressive separation methods, permitting, simultaneously, both quantitative and qualitative analysis. An important area for the utilisation of HPLC is the identification of drugs and their metabolites in biological materials. This study was focused on the development of chromatographic conditions appropriate for HPLC analysis of two new potential drugs derived from aroyl-hydrazone, HNAF-INH and 4,6DAR-INH, in biological material and its subsequent application in the assessment of the stability of these substances in plasma. Analysed chelators were prepared by the modification of the structure of salicylaldehyde isonicotinoyl hydrazone (SIH) with the purpose of improving the stability of the substance in plasma. As a suitable stationary phase the HPLC column LiChroCART® HPLC - cartridge LiChrospher®100 RP - 18e (15μm) with the pre-column LiChroCART® 4-4 Purospher® RP - 18 (5μm) was chosen. The mobile phase in this case represented by a HNAF-INH compound: phosphate buffer (0,01 mol/l aqueous solution of NaH2PO4 · 2 H2O with 2 mmol/l EDTA, pH 3) : methanol, 42:58 (v/v). The temperature in the column was 25řC, the flow rate 1,0 ml/min and UV detection was performed at 288 nm; SIH was used as an internal standard. The mobile...
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Bioanalytical evaluation of novel drug candidates from the group of thiosemicarbazones I.
Šesták, Vít ; Kovaříková, Petra (advisor) ; Kučera, Radim (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of pharmaceutical chemistry and drug analysis Candidate: Vít Šesták Supervisor: PharmDr. Petra Kovaříková, PhD. Title of the Master's Thesis: Bioanalytical assessment of new prospective drugs derived from thiosemicarbazone I. Abstract Development of novel anti-cancer drugs which would boost current therapeutic regimes and/or overcome the resistance against common chemotherapy ranks among the most progressive fields of modern drug discovery. Selective targeting of intracellular iron (Fe) has been recognized as a new strategy for anti-cancer drug development. Biocompatible Fe chelators structurally derived from thiosemicarbazone belong to the most intensively studied anti-cancer agents. Apart from Triapine, currently in phase II of a clinical trial, the systematic investigation led to development of a new generation of iron chelators - di-2-pyridylketone thiosemicarbazones (DpTs). Based on the outcomes of numerous in vitro and in vivo experiments and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) was selected as the lead compound for further development. The aim of this study was to develop an LC-MS method for the analysis of Dp44mT and its main phase I metabolites in biological materials (plasma, urine and faeces)....
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Development of an LC-MS method for determination of new antimalarial drugs in biological matrices.
Klimeš, Jiří ; Kovaříková, Petra (advisor) ; Kučera, Radim (referee)
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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Study of retention behavior of polar analytes on novel types of the stationary phases
Buková, Jana ; Kovaříková, Petra (advisor) ; Pilařová, Pavla (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Candidate: Jana Buková Supervisor: PharmDr. Petra Kovaříková, Ph.D. Title of diploma thesis: Study of retention behavior of polar analytes on novel types of the stationary phases. The retention behavior of selected polar analytes (sulphanilic acid, p-hydroxybenzoic acid, p-aminobenzoic acid, 3,4-diaminobenzoic acid, 3-nitrophenol, 3-aminophenol) on two different types of stationary phases in HILIC mode was studied in this diploma thesis. The following stationary phases were used in these experiments: column packed with pure silica Atlantis HILIC Silica, which is widely used for separation in HILIC mode and zirconia column with filling ZirChrom ® - PHASE packing, which is a possible alternative to silica based columns for analysis in HILIC mode, especially due to their high thermal and chemical stability in the whole pH range. The influence of the mobile phase strength on the retention of the selected analytes was tested on both columns. The influence of buffer strength, mobile phase pH and temperature was tested only on zirconia column (due to its higher stability). The chromatographic conditions used in this work were selected according to results of initial experiments.
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Analytical evaluation of novel drug candidates from the group of iron chelators using HPLC II.
Tomalová, Kateřina ; Kovaříková, Petra (advisor) ; Kučera, Radim (referee)
High performance liquid chromatography (HPLC) has a dominant position among the modern techniques in both the qualitative and quantitative drugs analysis. After adequate sample preparation it is also commonly used for the determination of drugs and metabolites in biological material. This study is focused on the development and validation of a chromatographic method suitable for the analysis of 2',4'-dihydroxyacetophenon isonicotinoyl hydrazone (DHAP-INH) - a novel biocompatible iron chelator, and its application for stability evaluation of this drug candidate in rabbit plasma in vitro. DHAP-INH is among newly synthesized salicylaldehyde isonicotinoyl hydrazone (SIH) analogues, which were prepared to improve compound stability in plasma and thus make pharmacokinetic profile of aroylhydrazones more favorable. Sufficient separation of DHAP-INH, its synthetic precursors/degradation products (2,4-dihydroxyacetophenon and isoniazide) and internal standard (SIH) was achieved on LiChrospher 100, RP-18 column (250 mm × 4,6 mm ID, 5 mm) employing a mobile phase composed of a mixture of phosphate buffer (0.01 M NaH2PO4.2H2O; 2 mM EDTA; pH 6,0) and methanol in a ratio of 54:46 (v/v). UV detection was performed at 254 and 326 nm. Different organic solvents were tested as precipitation agents, however...
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