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Phase II biotransformation of NSAID flobufen
Babú, Yogeeta ; Wsól, Vladimír (advisor) ; Nobilis, Milan (referee) ; Kuchař, Miroslav (referee)
Xenobiotic chemicals are chemicals foreign to life that are usually derived synthetically or from an abiotic process. The synthetic xenobiotic chemicals are often of enormous value to human society and are usually the majority of the chemicals in such important groups of substances as petrochemicals, pesticides, plastics and pharmaceuticals, where the term drug is usually applied when referring to xenobiotics. Biotransformation is a major mechanism for drug elimination, as they undergo biotransformation after they enter the body. Biotransformation, which almost always produces metabolites that are more polar than the parent compound, usually terminates the pharmacologic action of the parent drug and, via excretion, increases removal of the drug from the body. However, other consequences are possible, including similar or different pharmacologic activity, or toxicological activity. The routes by which drugs may be biotransformed are many and varied and include oxidation, reduction, hydrolysis and conjugation reactions, among others. It is important that these pathways are understood, as the route of metabolism of a drug can determine its ultimate pharmacological or toxicological activity. Drug biotransformation is divided into two phases: Phase I, or functionalisation reactions and Phase II, or conjugative...
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Analytical evaluation of biologically active compounds using liquid chromatography.
Zerzaňová, Anna ; Klimeš, Jiří (advisor) ; Blešová, Marie (referee) ; Nobilis, Milan (referee)
Disertační práce Summary 133 This dissertation thesis is focused on the analytical evaluation of biologically active compounds with the aid of High Performance Liquid Chromatography, especially with selective and sensitive detection performed by mass and coulometric detector. In the theoretical part of this dissertation thesis, there is attention paid to the importance and status of mass spectrometry as a tool of the modern analytical method of the detection. In this dissertation thesis, there is also a view of electrochemical methods and applications, focusing on coulometric measurements in flow systems. The results of the experimental work have been compiled and presented in three articles published in scientific impact journals. The list of reached results is following: The first article deals with a comparison of four chromatographic columns for their use in bioanalytical studies. Numerous HPLC columns with different bonded stationary phases are nowadays available, and producers continue to expand the range of columns they offer. Four real mixtures of biologically active compounds were used for the testing. Every mixture contained a parent active compound, its real metabolites and proper internal standard. Measurements were performed on two monolithic and two particle-packed columns with quite new...
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Bioanalytical methodical approaches for the disposition monitoring of a new potential drug in organism using HPLC-PDA-MS-MS
Císař, Přemysl ; Nobilis, Milan (advisor) ; Chládek, Jaroslav (referee) ; Wsól, Vladimír (referee)
1. Summary The aim of this study consisted in the development and validation of a bioanalytical method for the identification and determination of dimefluron and its metabolites in biomatrices. For the purposes of this study, six expected potential dimefluron metabolites were prepared and identified by NMR and MS. Higher homologue of dimefluron (homodimefluron) was selected and synthesized as an internal standard. The chromatographic columns with various types of the stationary phases were tested. The best results were achieved using a pentafluorophenylpropylsilyl silica gel column rinsed with the mobile phase of acetonitrile- phosphate buffer pH = 3 in a gradient mode. After the validation of the bioanalytical method, this chromatographic system was applied to the disposition study of dimefluron in rats. After an intragastric administration of dimefluron to rats, samples of urine and faeces were collected each 24 hours. The elimination of dimefluron and its phase I and phase II metabolites in urine and faeces was studied. Maximum concentrations of dimefluron derivatives in the excrements were found in the time interval of 24 - 48 hours after the administration. 9-O-Desmethyldimefluron and 3-O-desmethyldimefluron were the principal metabolites found in the rat faeces, while the metabolic products of...
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Metabolic study of sibutramine
Link, Marek ; Wsól, Vladimír (advisor) ; Holčapek, Michal (referee) ; Nobilis, Milan (referee)
Obesity represents a serious problem especially in American and European populations. Pharmacotherapy in combination with a reduced calorie diet is recommended for obese patients as a multi-modal approach to weight loss. Sibutramine hydrochloride monohydrate represents one of the few established and well-proven agents available for treatment of obesity. It is sold as a racemic mixture under the trade-name Meridia, Reductil or Lindaxa. It acts as a monoamine reuptake inhibitor. The weight loss of patients induced by sibutramine is thought to be due to a combination of serotonin- and noradrenaline-mediated mechanisms that increase both satiety and energy expenditure. In organisms, sibutramine is rapidly demethylated to form metabolites M1 and M2. These metabolites contribute largely to the pharmacological effects of sibutramine and the pharmacokinetic characteristics of M1 and M2 were thoroughly studied in human plasma. Although sibutramine is widely used for the treatment of obesity almost ten years, the published information on the further metabolic fate of metabolites M1 and M2 as well as on the elimination of sibutramine from the body is almost exclusively limited to package inserts of the product. To address this issue we determined the routes of elimination of sibutramine in humans via urine. LC-API/MS...
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"The optimalization and validation of LC/MS method for the determination of selected diureticsin human blood serum"Optimization and validation of LC/MS method for quantitative determination of diuretics in human blood serum
Světlíková, Zuzana ; Nobilis, Milan (advisor) ; Kovaříková, Petra (referee)
A new method was developed and fully validated for the quantification of hydrochlorothoazide and chlorthalidone, both diuretic and anti-hypertensive agents, in human serum. This liquid chromatography-tandem mass spectrometry method (LC/MS/MS) is rapid, simple, accurate and selective. The analytes and the internal standard were extracted by liquid-liquid extraction (LLE) with ethylacetate-dichlormethane (80:20, v/v). The chromatographic separation was performed on a reversed-phase column C8 with a mobile phase acetonitrile-formic acid (0,2 %), the elution was gradient. The analytes were quantitated by linear ion trap mass spectrometry with an electrospray ionization interface (ESI). The diuretics were analysed in negative ion monitoring mode, the internal standard in positive ion monitoring mode. The assay exhibited a linear dynamic range of 0,5-200 ng/mL for hydrochlorothiazide and 0,5-500 ng/mL for chlorthalidone in human serum. Acceptable precision and accuracy were obtained for QC samples with concentration over the standard curve ranges. This developed and validated method has been used in the toxicological laboratory in the Institution of Clinical Biochemistry and Diagnosis in Hradec Králové.
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Analytical Evaluation of Biologically Active Substances by Chromatographic Methods
Pasáková, Ivana ; Sochor, Jaroslav (advisor) ; Nobilis, Milan (referee) ; Holčapek, Michal (referee)
Introduced dissertation thesis deals with the development and validation of the chromatographic methods for analytical evaluation of selected biologically active substances. HPLC coupled with UV and MS detection was chosen for determination of analytes, because of the dominant role of the HPLC in pharmaceutical analysis. The theoretical part is focused on the theory of chromatographic methods and topics of the experimental work. At first, summary of all the chromatographic methods is briefly introduced and subsequently, the most common analytical method HPLC is described in details. The next part deals with the mass spectrometry. Thank to its high sensitivity and ability to provide structural information about the analytes, MS became an indispensable tool not only in modern pharmaceutical analysis. Besides MS theory and instrumentation, its applications and new trends are also mentioned. Last chapters deal with the transdermal application of drugs, specifications of antiretroviral therapy and especially they provide basic information about the physical - chemical and biological properties of analysed substances. The experimental part is consisted of the original research papers with appropriate comments divided into two thematic sections. The first one is composed of three papers focused on analytical...
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Development and Validation of Novel HPLC Methods for the Determination of Specific Analytes in Biological Material.
Urbánek, Lubor ; Solich, Petr (advisor) ; Karlíček, Rolf (referee) ; Nobilis, Milan (referee)
1. SUMMARY The presented dissertation thesis shows an interesting connection of two research fields that seem totally different from the first sight: analytical chemistry and medicine. However, the second sight could show that both branches might exist very closely to each other and their cooperation can be even useful for both sides. The initial part of the thesis deals with theoretical basics of HPLC and chromatographic instrumentation bringing a short overview of important principles of liquid chromatography including a brief introduction of crucial parts of an HPLC instrument. Due to the fact that the heart of the chromatographic system is an analytical column, another part of the text deals with different types of stationary phases and analytical sorbents. A separate chapter is focused on monolithic columns and other materials representing a new and original trend of chromatographic techniques. The main advantages and disadvantages of monolithic columns are then summarized in the published papers listed in the Experimental part (Enclosures 1, 2 and 9). Finally, the technical section of my thesis is concluded by a review of new trends in the chromatographic instrumentation presented e.g. by Ultra Performance Liquid Chromatography, columns filled with sub-2-micron particles or automatization or...
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