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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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Development and validation of HPLC methods for analysis of pharmaceutical formulations
Matysová, Ludmila ; Solich, Petr (advisor) ; Karlíček, Rolf (referee) ; Nobilis, Milan (referee)
7. Summary Development and validation of HPLC methods The doctoral thesis concerns with development of HPLC methods for analysis of pharmaceutical formulations, which meet all requirements for suitability, precision and reliability. Documentation of these parameters is called validation. At first theoretical principles of chromatography are described in this work, with the emphasis to liquid chromatography, included HPLC. The instrumentation in HPLC is described, mentioning the new trends in stationary phase's development - for example monolithic columns, zirkonia stationary phase, HILIC and sub-2-microns phases. In the other part UPLC system, one of the new trends in liquid chromatography development is discussed. Next part deals with the development of HPLC methods for analysis of pharmaceutics and is followed by chapter, in which analysis of pharmaceutical formulations is described, included preparation of the samples before analysis, stability monitoring and further the analysis of impurities. The last, relatively large chapter is dealing with the validation of analytical methods. Several institutions are concerned with validation requirements. They issue recommendation and/or guidelines, which are cogent for this process. In the Czech Republic there exists State Institute for Drug Control (SUKL -...
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Structure modifications of transkarbams - synthesis and evaluation of physico-chemical properties and biological activity focused on mechanism of biological activity
Klimentová, Jana ; Hrabálek, Alexandr (advisor) ; Csöllei, Jozef (referee) ; Nobilis, Milan (referee)
One of the subjects studied in the Department of Inorganic and Organic Chemistry of the Faculty of Pharmacy focuses on the synthesis and biological evaluation of potential transdermal permeation enhancers. Over the past few years, series of esters of 6-aminohexanoic acid of high enhancing activity and their analogues have been prepared. Presently, the structure-activity relationships are being studied. This work contributes to this problem by searching for the mechanism of action of one of the most active compounds, transkarbam 12 (T12). Transdermal permeation enhancers facilitate drug absorption through the skin. Generally, their mechanisms of action include the disturbance of highly ordered structure of stratum corneum, promotion of drug solubility in the vehicle or enhancing the partitioning of drug from the vehicle into stratum corneum. Nevertheless, the exact mechanism is usually unclear. T12 is a carbamic acid salt derived from two molecules of 6-aminohexanoic acid dodecyl ester. In slightly acidic environment (as in stratum corneum, its target place) it decomposes easily releasing a molecule of CO2 and free amino ester. To find out whether this ability contributes to its high activity, a series of T12 analogues with CO2 covalently bound in the polar head was prepared (esters of carbonic,...
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Separation and determination of pharmaceutically important compounds using electrophoretic methods
Hamoudová, Rafífa ; Pospíšilová, Marie (advisor) ; Nobilis, Milan (referee) ; Jokl, Vladimír (referee)
This thesis deals with the use of electrophoretic methods in the analysis of pharmaceutically important compounds. In the theoretical part, the classification of capillary electrophoretic techniques is discussed. Capillary zone electrophoresis (CZE, capillary isotachophoresis (ITP), micellar elektrokinetic chromatography (MEKC), microemulsion elektrokinetic chromatography (MEEKC), isoelectric focusation (IEF), capillary electrochromatography (CEC) and capillary gel electrophoresis (CGE) are described. Further, on-line pre-concentration methods are described. Particular attention is given to the on-line combination of capillary isotachophoresis with capillary zone electrophoresis using column coupling arrangement. This combination enables the improvement of the selectivity of separation and the improvement of the limit of detection of analytes present in complex matrices of biological and environmental origin. Afterwards, the results of the experimental analyses are presented: 1. The first paper deals with the use of CZE in the analysis of ibuprofen and flurbiprofen in pharmaceuticals. Separation was carried out in a fused silica capillary (60 cm x 100 m I.D effective length 45 cm) at 30 kV with UV detection at 232 nm. The optimized background electrolyte was 20mM N-(2- acetamido)-2-aminoethanesulfonic acid...
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Vliv vybraných látek ovlivňujících centrální nervový systém na kostní metabolismus
Fekete, Soňa ; Živný, Pavel (advisor) ; Horák, Petr (referee) ; Nobilis, Milan (referee)
The increase in life expectancy of the world population is associated with challenges regarding health issues. For instance, osteoporosis is a medical condition mostly observed in elderly people, in which the quality and quantity of the bone are severely affected. Not only for women but also for men, osteoporosis is recognized as an important public health issue. Osteoporosis is a systemic skeletal disorder and is a result of loss of skeletal mass. Osteoporosis is characterized by low bone mass, microarchitectural deterioration of bone tissue and an increase in bone fragility and susceptibility to fracture. Osteoporotic fractures are a significant cause of morbidity and mortality. The longterm use of drugs such as antiepileptics and antidepressants could affect the onset of osteoporosis. The aim of the study was to evaluate the effect of orchidectomy, the effect of newer antiepileptic (levetiracetam, lacosamide, topiramat, lamotrigine) and antidepressive drugs (mirtazapine, venlafaxine and trazodone) on bone metabolism in healthy male Wistar rats. The first specific aim was to determine the effect of orchidectomy on bone metabolism in rats. We found that after 12 weeks post-orchidectomy there was a negative effect on bone metabolism in rats. These results established these animals as suitable models for...
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Analytical evaluation of drugs and novel drug candidates using HPLC
Kovaříková, Petra ; Klimeš, Jiří (advisor) ; Nobilis, Milan (referee) ; Blešová, Marie (referee)
High performance liquid chromatography (HPLC) is one of the most frequently used analytical techniques for the analysis of drugs. HPLC methods are widely employed in all fields of the modern pharmaceutical analysis - new drugs development, quality control and assurance, the analysis of drugs and metabolites in a biological material (e.g. therapeutic drug monitoring, bioequivalence). The theoretical part of this thesis deals with the main specifics and aspects associated with HPLC analysis of drugs. The experimental part is consisted of six original research papers with appropriate comments divided in to two sections (The analysis of the drug candidates from the group of aroylhydrazone iron chelators and The stability study on selected drugs). The first section concerns with development, validation and application of new HPLC methods in the analysis of drug candidates from the group of iron chelators - pyridoxal isonicotinoyl hydrazone (PIH), salicylaldehyde isonicotinoyl hydrazone (SIH) and pyridoxal 2-chlorobenzoyl hydrazone (o-108). Aroylhydrazone iron chelators are under the investigation as promising drug candidates. Despite these chelators have demonstrated number of interesting pharmacological effects, in fact there were no modern analytical techniques suitable for the analysis of these drug...
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