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UHPLC analysis of degradation products of tetracaine hydrochloride
Horáková, Michaela ; Matysová, Ludmila (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Michaela Horáková Supervisor: Ludmila Matysová Title of Diploma Thesis: Determination of Degradation Product of Tetracaine hydrochloride by Ultra-High-Performance Liquid Chromatography This diploma thesis deal with using UHPLC for determination of tetracaine hydrochloride and its degradation products, p-aminobenzoic acid and p-n-butylaminobenzoic acid. Separation was achieved by using Kinetex 5 µ XB- C18 100A column and the eluent, acetonitrile-buffer, when buffer containing water with phosphoric acid (pH=2.2) at a flow rate of 1.7 ml/min. Gradient elution was chosen, which began at 35 % of acetonitrile, in the second minute rised to 95 % of organic phase, and then descended to 35 % in the fourth minute. Ethylparaben was used as an internal standard. The wavelengths 300 nm for tetracaine hydrochloride and its degradation products, and 240 nm for internal standard were chosen for detection. Validation approved that this method provides accurate and precise results.
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Determination of tocopherols in giological samples (review)
Koutová, Jana ; Kujovská Krčmová, Lenka (advisor) ; Sklenářová, Hana (referee)
The aim of this diploma thesis is to create the list of laboratory methods used for the determination of vitamin E in biological materials. Vitamin E is important antioxidant and its deficit is connected with major cardiovascular, neural and immunity disorders. The frequency of its determination is increasing in the last years; the emphasis is put on the speed, universality and possibility of automation. Theoretical part of this work is dealing with chemical and physical characteristics and biological function of vitamin E. These findings are important to understand the role of vitamin E in the organism and also important for choice of determination method. The main methods of analysis are chromatographic (UHPLC, HPLC, GC), electrochromatographic (CEC) as well as spectrofotometric methods.The comparison of used methods is also included in the diploma thesis. Key words: tocopherols, vitamin E, biological material.
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Testing antioxidant activity using luminol chemiluminescence system
Chládková, Gabriela ; Sklenářová, Hana (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Gabriela Chládková Supervisor: Doc. PharmDr. Hana Sklenářová, Ph.D. Consultant: Doc. RNDr. Miroslav Polášek, Csc. Title of Diploma Thesis: Testing antioxidant activity using luminol chemiluminescence systém The aim of the thesis was to compare various conditions for the testing of the antioxidant activity by means of the luminol chemiluminescent system in the flow system, and evaluation of dependence of the antioxidant efficacy on concentration. The antioxidant efficacy was described as dependency of the decrease of a chemiluminescence signal on the used concentration of an antioxidant. Efficacy of three substances - resveratrol, p-coumaric acid and trolox was observed. Trolox was used as a standard substance in evaluation of the antioxidant activity. The chemiluminescence system was based on a chemiluminescent reagent. In this particular case we used luminol in a sodium hydroxide solution or in a sodium carbonate solution adjusted to pH 10 or without pH adjustment. Luminol demonstrated chemiluminescence emission after oxidation with hydrogen peroxide in the presence of catalysts. As the catalyst, potassium hexacyanoferrate(III) or potassium hexacyanoferrate(II) were used. The working...
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Development of HPLC method for determination of vancomycine in clinical research
Kučerová, Kateřina ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Kateřina Kučerová Supervisor: RNDr. Lenka Kujovská Krčmová, Ph.D. Title of diploma thesis: Development of HPLC method for determination of vancomycine in clinical research The aim of this diploma thesis was to develop optimal conditions for the chromatographic determination of vancomycin for clinical routine practice. The chromatographic separation was carried out by KinetexTM C18 column, 2.6 μm particle size, 50 × 4.6 mm (Phenomenex, USA) in combination with potassium phosphate buffer (pH 4.5) and acetonitrile (90 : 10, v/v) as the mobile phase. As internal standard was chosen cefuroxime. For analytes determination UV detection at 220 nm wavelength was applied. After optimization of separation conditions, the method was applied to biological material. Samples were easily modified using the protein precipitation. The method was partially validated. Keywords: vancomycin, HPLC-UV, plasma samples
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Stanovení acetylcholinu pomocí LC-MS ve vzorcích mozkových mikrodialyzátů LC-MS/MS
Vrobel, Ivo ; Solich, Petr (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Ivo Vrobel Supervisors: Prof. RNDr. Petr Solich, CSc; Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague Prof. Seppo Auriola, MSc.(Chem.) Marko Lehtonen; Department of Pharmaceutical Chemistry, School of Pharmacy, University of Eastern Finland in Kuopio Title of master's thesis: LC-MS/MS analysis of acetylcholine in brain microdialysis samples Novel fast and simple LC-MS/MS method of ACh quantification in brain microdialysis samples utilizing stable-isotope-labeled IS was developed. The chromatographic step is based on revered-phase mode of pentafluorophenylpropyl (PFPP) column. The satisfactory retention of ACh is achieved with highly aqueous mobile phase containing 0.05% of the ion-pairing agent TFA and 4% of ACN in 4 min analytical run. Ionization of ACh and IS with low background noise and tolerant towards use of TFA was performed with atmospheric pressure thermospray ionization (APTSI). The selectivity of ACh and IS detection was obtained by SRM modes of MS/MS in the linear ion trap mass analyzer. The performance of developed method was cross validated to the validated method used in the laboratory for ACh measurements. The set of microdialysis...
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Forced degradation studies of dimethindene maleate using HPLC
Pannyová, Anna ; Chocholoušová Havlíková, Lucie (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Student: Anna Pannyová Supervisor: PharmDr. Lucie Havlíková, Ph.D. Title of diploma thesis: Forced degradation studies of dimethindene maleate using HPLC The aims of this thesis were to perform forced degradation of dimethindene maleate and to optimize the chromatographic conditions for the determination of dimethindene maleate and its related compounds. The stress testing was conducted with drug product/drug in order to exam its stability. Decomposition of the product can lead to loss of the content of the active substance, loss of their pharmacological activity or forming of degradation products. Dimethindene maleate is an antihistamine drug. He affects H1 histamine receptors. Nowadays, high performance liquid chromatography (HPLC) is one of the most used separation techniques for the determination of the drug stability. The stability of dimethindene maleate was investigated using stress testing (forced degradation studies). Dimethindene was stable in aqueous and acid solution as well as under heating (70řC). Significant degradation was observed when the drug was subjected to basic and oxidation stress conditions; this led to the formation of two degradation products DP1 and DP2 (described using...
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Metabolomic analysis of plasma samples using ultra performance liquid chromatography and high resolution mass spectrometry
Machová, Karolína ; Solich, Petr (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Karolína Machová Consultant: Prof. RNDr. Petr Solich, CSc. Diploma Thesis Title: Metabolomic analysis of plasma samples using ultra performance liquid chromatography and high resolution mass spectrometry Metabolomics is a novel type of science which represents a complex methods of metabolite determination in biofluid e.g. plasma, urine, saliva, cerebrospinal fluid and sperm. The method describes cell mechanisms and finds wide assertion in disease diagnostics, biomarkers description, disease, pathophysiology and in clinical practice. It is vital to have a sensitive method because levels of metabolites are quite low. Metabolomic analysis uses mainly high-performance liquid chromatography combined with mass spectrometry for detection. However, other methods such as nuclear magnetic resonance spectroscopy and gas chromatography can also be used. Presented work is mainly focused on method standardization for identifying metabolites in clinical practice with the use of ultra-performance liquid chromatography and mass spectrometry. The primary outcome was to establish searching parameters for the operating software to automatically identify xenobiotics in provided sample and compare them with...
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Development and Validation of New HPLC Methods for Determination of Biologically Active Compounds in Clinical Practice
Krčmová, Lenka ; Solich, Petr (advisor) ; Sochor, Jaroslav (referee) ; Malíř, František (referee)
Abstroct Charles University inPrague, Foculty of Phormocy in Hrodec Králové Deportment of Ánolytícol Chamistry Condídote: M9r. Lenko Krčmovó Supervisor: prof. RNDr. Petr Solich, CSc. Title oÍDoctorol Thesis: Developmant ond volidotion of new HPLC methods for determinotion of bíolo9icolly octive compounds in clinicol proctísa The presented dissertotion thesis is daolin9 with developmenŤ ond volidotion of HPLC methods for clinical research ond clinicol proctice in Reseorch loborotory of Daportment of Metobolic Care and Gerontology (GMC) in University Hospitol in Hrodec Krolove (UHHK). For this purpose modern technologies deoling with lorge sequences of biologicol moteriol, speed seporotion, smoll volum es of organic solvalt ond somple were used. Naw HPLC method for deŤerminotion of 13-cis ond oll-trons retinoic acid, retinol, alpho- ond gommo-tocopherol using intarnol colibrotion for potients suffers from ocute promyelocytic leukemio treated with retinoic ocid wos daveloped ond volidoted. Solid phose extroction (SPE) ond monoliths were selected for somple praporotion ond seporotion of the target compounds. New method for determinotion of neopterin, creatinine, kynuranine ond tryptophon in humon serum using two monolithic column os stotionory phose wos developed ond volidoted. Thís method wos developed...
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Evaluation of new "fused-core" analytical columns in UHPLC system - a comparison with BEH UHPLC analytical columns
Nežádalová, Jitka ; Nováková, Lucie (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical chemistry Candidate: Jitka Nežádalová Supervisor: PharmDr. Lucie Nováková, Ph.D. Title of Diploma thesis: Evaluation of properties of new "fused - core" analytical columns in UHPLC system and a comparison with the BEH UPLC analytical columns This thesis was focused on testing properties of new analytical columns filled with porous shell particles (so-called fused-core) in UHPLC system and a comparison with the BEH UPLC analytical column. The following analytical columns were tested: Kinetex PFP particle size of 1,7 µm and 2,6 µm (Phenomenex) and Ascentis Express Phenyl-Hexyl particle size of 2,7 µm (Sigma- Aldrich), which are columns with porous shell particles and Acquity BEH Phenyl column with totally porous particles size of 1,7 µm. To compare the properties of the columns, 7 estrogen steroids were used using PDA detector at a wavelength of 280 nm for detection. Measurements were carried out using isocratic elution. The Mobile phase consisted of ultrapure water and acetonitrile. Columns were evaluated in terms of efficiency, peak symmetry, resolution and the back pressure, which was generated on the columns. The efficiency of the columns was expressed as a number of theoretical plates and by van Deemter...
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Development and validation of HPLC method for determination of retinyl esters in human serum using monolithic columns.
Krčmová, Lenka ; Solich, Petr (advisor) ; Šatínský, Dalibor (referee)
DEVELOPMENT AND VALIDATION OF HPLC METHOD FOR DETERMINATION OF RETINYL ESTERS IN HUMAN SERUM USING MONOLITHIC COLUMN Absorption test of vitamin A is used for monitoring of intestinal permeability and may represent a sensitive indicator of intestinal damage. In this study, a simple and rapid reversed- phase high-performance liquid chromatography (RP-HPLC) procedure for selective and sensitive determination of retinol, -tocopherol, retinyl-palmitate and retinyl-stearate in blood serum has been developed and used for the monitoring of intestinal mucosal damage in cancer patients treated with cytotoxic drugs after absorption test of vitamin A. The HPLC instrumentation Series 200 LC from Perkin Elmer (Norwalk, USA) with diode- array detector (DAD) was used for the analysis. Separation of retinol, -tocopherol, retinyl- palmitate and retinyl-stearate were performed using the monolithic column Chromolith Performance RP-18e, 100 x 4.6 mm (Merck, Darmstadt, Germany). The gradient elution was used at the flow rate 3 ml min-1 ; mobile phase methanol:water (95:5) in 0-2.1 min and methanol:2-propanol (60:40) in 2.1-4.9 min. The total time of analysis was 6 min. The injection volume of sample was 20 μl and analysis was done at ambient temperature. The detection of retinol, -tocopherol and retinyl esters was carried out...
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