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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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Analytical evaluation of drugs using HPLC II.
Hanková, Eva ; Mokrý, Milan (advisor) ; Nobilis, Milan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Analysis Candidate: Eva Hanková Supervisor: RNDr. Milan Mokrý, CSc. Title of Diploma Thesis: Analytical evaluation of drugs using HPLC II. Abstract This diploma thesis dealt with analytical evaluation of tiaprofenic acid using high performance liquid chromatography. To determine tiaprofenic acid, a LiChroCART® 250-4 LiChrospher® 100 RP-18 (5 μm) column and a mobile phase consisting of acetonitrile and aqueous KH2PO4 buffer 0.01 mol/l in a ratio of 40 : 60 (v/v) were used. The pH of the buffer was adjusted to 3.0 with phosphoric acid; the flow rate was set to 1 ml/min and the temperature at 22 řC. Naproxen was chosen as the internal standard, and a 263 nm wavelength was used for detection. Since the tiaprofenic acid is a chiral substance present in the form of a racemate, the selection of chromatographic conditions for the separation of its enantiomers followed. Two chiral columns in both normal and reverse mode were tested. The cellulose-based Chiralcel OD-R 250 x 4.6 mm (10 μm) column was chosen as the most suitable, using a mobile phase of n-hexane : propan-2-ol : acetic acid in a ratio of 94 : 6 : 0,1 (v/v /v) at a flow rate of 0.5 ml/min and a temperature of 22 ř C. Enantiomers were eluted at...
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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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Analysis of Biologically Active Compounds by Capillary Electrophoresis
Petrů, Klára ; Polášek, Miroslav (advisor) ; Nobilis, Milan (referee) ; Urbánek, Marek (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate Mgr. Klára Petrů Supervisor Doc. RNDr. Miroslav Polášek, CSc. Title of Doctoral Thesis Analysis of biologically active compounds by capillary electrophoresis The thesis presented is dealing with the analysis of quaternary ammonium compounds of pharmaceutical importance by capillary electrophoresis (CE). Basic principles of CE and respective means of detection are discussed in the theoretical part. Special attention is paid to capacitively coupled contactless conductivity detection C4D and to the CE coupling with mass spectrometry (CE-MS). The chemical and pharmacological properties of the compounds of interest: carbethopendecinium bromide, pancuronium bromide (PM), vecuronium bromide (VM) and rocuronium bromide (RM) are characterized. The experimental work is focused on the development and validation of new analytical CE methods for the assay of such compounds. in pharmaceuticals. The C4D detection was applied because of poor UV adsorption capability of all compounds studied. I. The first part deals with the CE analysis of septonex, an antiseptic drug with the character of surfactant. The background electrolyte was 30 mM MES of pH 7 containing 12.5 mg/ml of 2-hydroxypropyl-β-...
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HPMC-based liposomal mucoadhesive films with model peptide as target API
Belcáková, Hedviga ; Nobilis, Milan (advisor) ; Doležal, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Kralove Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Hedviga Belcáková Supervisor: Prof. Milan Nobilis, CSc., Veronika Fidelj (Heidelberg University, Insitute of Pharmacy and Molecular Biotechnology) Diploma thesis title: HPMC-based liposomal mucoadhesive films with model peptide as target API This thesis describes the preformulation stage of mucoadhesive films intended for liposomal peptide delivery via buccal membrane. The evaluation consisted of thickness, maximum tensile strength, strain, moisture content, in vitro swelling and liposome integrity measurements. The chosen polymer (hypromellose, HPMC) was found to perform optimally in concentrations of 10 % with PEG 400 (5 %) acting as plasticizer and liposome concentration of 2 %. The developed preparation method showed good reproducibilty with room for improvement in the homogenization area. The choice of medium (H2O vs. PBS) showed strong influence on formulation's mechanical properties resulting in significant loss of elasticity and mucoadhesive strength. The addition of liposomes in the third stage had been carried out successfully with only occassional effect on their integrity after dissolution.
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Enantiomeric separation of drugs and their metabolites II
Verešpejová, Natália ; Nobilis, Milan (advisor) ; Mokrý, Milan (referee)
Tramadol is centrally acting opioid analgesic drug. It is converted in liver by the enzyme CYP2D6 into active substance O-desmethyltramadol, responsible for analgesia. In clinical practise it is used as racemate. Each enantiomer displays different properties: (-)-tramadol inhibits noradrenaline reuptake and (+)-tramadol, which is therapeutically more effective, inhibits serotonin reuptake. It is indicated for the treatment of mild to moderate pain. This Bachelor's thesis deals with possibilities of determination of drug tramadol and its main metabolite in plasma by chromatographic separation methods, which are currently modern and progressive. Using chiral HPLC analysis with different types of detection, the drug was determined with high sensitivity and selectivity. Individual publications provide pharmacokinetic data of tramadol and its metabolite after oral administration of a single dose of drug to healthy volunteers. The determinations differ by chromatographic conditions, sample preparation, chemicals used and mainly total time of analysis. The results are stated and compared in the summary table.
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Analytical evaluation of drugs using HPLC IV.
Mojšová, Kristína ; Mokrý, Milan (advisor) ; Nobilis, Milan (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Analysis Candidate: Kristína Mojšová Supervisor: RNDr. Milan Mokrý, CSc. Title of master's thesis: Analytical evaluation of drugs using HPLC IV. This thesis describes development of the suitable method for ketoprofen analysis by high performance liquid chromatography. For this aim a LiChro CART® 125-4 Lichrospher® 100 RP-18 (5 μm) column was used as a stationary phase and mobile phase contained mixture of methanol and aqueous KH2PO4 buffer (pH 3,4) in a ratio of 60:40 (v/v). The flow rate was set to 1 ml/min, temperature on the column was 25 řC and substances were detected at a wave length of 254 nm. Under these conditions final retention time of ketoprofen was 7,9 minutes. Benetazon was chosen as an appropriate internal standard for the quantitative evaluation. In the next part we were looking for suitable conditions for the chiral separation of the analyzed drug by using normal and reverse mode. Separation of ketoprofen's enantiomers was carried out on a Chiralcel OD-R, 250 x 4,6 mm, from Daicel Chemical Industries using a mobile phase of heptane and 2-propanol in a ratio of 97:3, with the addition of 0,6 ml formic acid. At a flow rate of 0,7 ml/min, retention times took about 20 minutes....
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Optimization of chromatographic parameters for chiral separation of biologically active compounds
Novák, Martin ; Kučera, Radim (advisor) ; Nobilis, Milan (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Chemistry and Pharmaceutical Analysis Student: Bc. Martin Novák Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Consultant: Mgr. et Mgr. Rafael Doležal, Ph.D. Title of diploma thesis: Optimization of chromatographic parameters for chiral separation of biologically active compounds. The diploma thesis was focused on the development of a HPLC-UV method for the determination of K 1277 enantiomers of systematic name N-(2-((6-chloro-1,2,3,4-tetra- hydroacridin-9-yl)amino)hexyl-2-amino-3-(1H-indole-3-yl) propylamide dihydrochloride, which is one of the compounds from the tacrine-tryptophan hybrids group. These tacrine-tryptophan hybrids could be considered as promising candidates of potential drugs against Alzheimer's disease. The thesis brings an explanation of basic characteristic of chiral molecules, principles of chiral separation, pathophysiology, clinical manifestation and treatment of Alzheimer's disease and short characteristic of tacrine-tryptophan hybrids in the theoretical section. The aim of my diploma thesis was to find the optimal chromatographic conditions for separation of K 1277 enantiomers synthesized from tacrine and tryptophan fragments. The experimental part deals with the development of the chiral...
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Modern separation techniques for the analysis of biological material in clinical research
Kasalová, Eva ; Solich, Petr (advisor) ; Nobilis, Milan (referee) ; Friedecký, David (referee)
Charles University, Faculty of Pharmacy in Hradec Kralove Department of Analytical Chemistry Candidate: Mgr. Eva Kasalová Supervisor: Prof. RNDr. Petr Solich, CSc. Supervisor specialist: Assoc. Prof. RNDr. Dagmar Solichová, Ph.D. Title of Doctoral Thesis: Modern separation techniques for the analysis of biological material in clinical research This doctoral thesis deals with development and validation of a chromatographic determination of liposoluble vitamins and with optimization of biological materials preparation. Clinical monitoring of these vitamins, especially vitamin D in different biological materials is one of the most discuss theme in the last decade. In the theoretical part of this dissertation the target analytes - vitamins A, E and D with their biological activity are characterized - as well as the biological material used in the Research Laboratory, especially a detailed description of the composition of breast milk, its irreplaceable role in neonatal nutrition and description of breast milk processing in Milk Bank. This part is followed by a comprehensive chapter dealing with the treatment of the biological matrix before analysis. The most commonly used sample pretreatment techniques such as extraction methods are described here, with a great description of new trends in these...
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Achiral and chiral high performance liquid chromatography of drugs and their metabolites III.
Horáček, Ondřej ; Nobilis, Milan (advisor) ; Mokrý, Milan (referee)
Nabumetone is non-steroidal anti-inflammatory prodrug that is converted into (6-methoxynaphthalen-2-yl)acetic acid in the liver. The differences of in vitro nabumetone biotransformation (rat vs human liver cytosolic and microsomal fractions) were studied in previous communication. Study of stereospecificity of enzymes and the calculation of enantiomeric ratios of the biotransformed chiral metabolites is an equally important characteristic of enzymes such as the Michaelis constant. Calculation of enantiomeric ratios of chiral metabolites biotransformed from prochiral drugs in vitro is used to identify new enzymes responsible for the biotransformation of a given prochiral drug. This work deals with the optimization of separation and detection conditions for achiral and chiral LLE-HPLC-PDA analysis of nabumetone and its six metabolites in extracts from various biomatrixes (human liver microsomal and cytosolic fractions and minipig blood plasma). Differences in biotransformation of nabumetone and racemic mixture of 3-HO-nabumetone between man and woman were studied in vitro in extracts from liver cytosolic and microsomal fractions, using chiral LLE-HPLC-PDA analysis. Enantiomeric ratios of all chiral metabolites of nabumetone were calculated. Pharmacokinetics of 6-MNA in minipig blood plasma after...
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