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HPLC method devolopment for betacarotene determination in nutraceuticals
Hlaváčková, Markéta ; Šatínský, Dalibor (advisor) ; Chocholoušová Havlíková, Lucie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Markéta Hlaváčková Consultant: Doc. RNDr. Dalibor Šatínský, Ph.D. Diploma Thesis Title: Development of HPLC Method for the Determination of Betacarotene in Nutritional Supplements A new HPLC method was optimized and developed for the determination of betacarotene contents in nutritional supplements Betakaroten Farmax, GS Betakaroten FORTE (GreenSwan pharmaceuticals), Walmark Betakaroten, Bioaktivní karoten (Pharma Nord Denmark), Beta karoten Max, BETAVID + LUTEIN (Naturvita), Karovit (Vitabalans Oy), SELZINK PLUS (PRO.MED.CS), Pupalkový olej (Aromatica). The method is based on using Ascentis Express C18 column (30 x 4.6 mm; 2.7 µm) and UV detection at 450 nm. There was used isocratic elution of the mobile phase 100% methanol at a flow-rate of 1.5 ml/min. Column oven temperature was set at 60 řC during the measurement. The retention time of betacarotene under the optimized and validated conditions was 2.185 min. Keywords: betacarotene, HPLC, Betakaroten Farmax, GS Betakaroten FORTE (GreenSwan pharmaceuticals), Walmark Betakaroten, Bioaktivní karoten (Pharma Nord Denmark), Beta karoten Max, BETAVID + LUTEIN (Naturvita), Karovit (Vitabalans Oy), SELZINK PLUS (PRO.MED.CS), Pupalkový olej...
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Two dimensionalk separations in Sequential Injection Chromatography low pressure system
Válová, Olga ; Šatínský, Dalibor (advisor) ; Chocholouš, Petr (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Olga Válová Supervisor: Doc. RNDr. Dalibor Šatínský, Ph.D. Title of diploma thesis: Two Dimensional Separation in the Low Pressure System of Sequential Injection Chromatography The subject of this work was to construct a new method for separation of two analytes, which are different in their chemical properties and lipophilicity. Newly described method named Two Dimensional Sequential Injection Chromatography (2D-SIC) for isocratic system was applied in this work for the analysis of pharmaceutical preparation Otobacid N, which contains active substances Dexamethasone and Cinchocaine, i.e. substances with different chemical properties. The separation was carried out in two dimensions of two columns. In the first dimension for separation of Dexamethasone was used column 25 x 4.6 mm C-18 monolithic column with mobile phase acetonitrile/water in ratio of 35/65, flow rate 15 µl/sec. In the second dimension for separation of Cinchocaine was used column 10 x 4.6 mm C-18 monolithic column with precolumn 5 x 4.6 mm C-18 with mobile phase acetonitrile/water in ratio 60/40, flow rate 15 µl/sec. Optimal wavelength at 240 nm was chosen by spectrophotometric detection. Relative standard deviation...
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Evaluation of properties of new HILIC stationary phases for pteridins using UHPLC-FD method
Kaufmannová, Iva ; Nováková, Lucie (advisor) ; Šatínský, Dalibor (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Iva Kaufmannová Supervisor: PharmDr. Lucie Nováková, Ph.D. Title of Doctoral Thesis: Evaluation of properties of new HILIC stationary phases for pteridins using UHPLC-FD method The subject of this thesis was to evaluate the characteristics of three new types of HILIC stationary phases for the identification and quantification of biologically active substances biopterin, neopterin, dihydrobiopterin and dihydroneopterin by UHPLC with fluorescence detection. Neopterin is used as a marker of immune system activation and inflammatory diseases. Its early detection in urine or plasma may indicate a pathological immune activity. Elevated concentration of neopterin is described in viral or bacterial diseases, in autoimmune diseases in HIV infection or in malignant tumors. 7,8 - dihydroneopterin is able to prevent proteins and lipoproteins from oxidative damage. Hydrophilic Interaction Liquid Chromatography (HILIC) is a chromatographic method that can be used to improve retention of very polar compounds. It uses a polar stationary phase and mobile phase containing a certain amount of water and polar solvents. UHPLC is the most advanced separation technology to develop a pressure of about 100 MPa....
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Using of HPLC for illegal colorants determination in food
Šmidrkalová, Tereza ; Šatínský, Dalibor (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Tereza Šmidrkalová Supervisor: Doc. RNDr. Dalibor Šatínský, Ph.D. Title of Diploma Thesis: Using of HPLC for illegal colorants determination in foods. This thesis deal with looking for optimal conditions for determination of Sudan dyes. High performance liquid chromatography (HPLC) was used for determination. During searching several various columns and mobile phases were tested. For validation of separation conditions Ascentis Express F5 column (100 x 4,6 mm, 2,7 μm) was used. Detection was measured at a wavelength of 500 nm and 426 nm. Separation used gradient elution with mobile phase of acetonitrile - water at a flow rate 1 ml/min. Injection volume was set to 5 μl. During measuring validation parametres was used on-line SPE extraction in HPLC system on precolumn Ascentis Express C18 (5 x 4,6 mm, 2,7 μm) with washing mobile phase 5% solution of acetonitril - water before separation. Optimalization and validation used only standards and method was suggested for other testing and using real samples. Keywords: high performance liquid chromatography (HPLC), Sudan dyes
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HPLC determination of selected incestisides in cosmetic
Kameníčková, Daniela ; Šatínský, Dalibor (advisor) ; Chocholouš, Petr (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Daniela Kameníčková Supervisor: Doc. RNDr. Dalibor Šatínský, Ph.D. Title of Diploma Thesis: HPLC determination of selected incestisides in cosmetic Active ingredients fenoxycarb and permethrin were determined in cosmetic anti- parasitic product Arpalit® Neo shampoo against parasites with bamboo extract. Analysis was performed by HPLC using RP-Amide column 100 x 3 mm with a particle size of 2.7 micron and detection by UV at a wavelength of 225 nm. Separation proceeded by isocratic elution with mobile phase of acetonitrile - water (55:45) at a flow rate 1.0 ml/min. Temperature during measurement was 60 řC. For evaluation method was used an internal standard method, as the internal standard was used dye sudan II. For separation was needed time interval of 6 min. The retention time of fenoxycarb was 1.01, sudan II 2.87 and permethrin 4.42 min. Resolution of chromatographic peaks fenoxycarb and sudan II was 10.612 and sudan II and permethrin 6.524. Keywords: fenoxycarb, permethrin, HPLC.
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Development of method for the determination of entecavir in biological materials using UHPLC-MS/MS
Gottvald, Tomáš ; Nováková, Lucie (advisor) ; Šatínský, Dalibor (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Tomáš Gottvald Supervisor: Doc. PharmDr. Lucie Nováková, Ph.D. Title of thesis: Development of method for the determination of entecavir in biological materials using UHPLC-MS/MS Entecavir is a synthetic guanosine nucleoside that plays an important role in treatment of chronical hepatitis B virus infection. This work was focused on development of highly sensitive method to determine entecavir concentrations in rat urine by ultra-high performance liquid chromatography-tandem mass spectrometry. Entecavir C2 13 N15 , stable isotopically labeled internal standard, was used for quantitation. Hydrophilic interaction chromatography seems to be a suitable technique for retention and separation of polar compounds present in analyzed sample. One of the main goals was to confirm this fact by comparison with commonly used reverse phase chromatography mode (RP-UHPLC) on BEH C18 stationary phase. Conditions for RP-UHPLC were optimized as follows: mobile phase composed of acetonitrile/0.01 % formic acid (4:96). The HILIC conditions on BEH Amide stationary phase were optimized using isocratic elution with mobile phase composed of acetonitrile/5 mM ammonium acetate pH 4.0 (75:25). HILIC method provided much...
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Using of separation methods in clinical research
Plíšek, Jiří ; Solich, Petr (advisor) ; Šatínský, Dalibor (referee) ; Friedecký, David (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Mgr. Jiří Plíšek Supervisor: prof. RNDr. Petr Solich, CSc. Supervisor specialist: assoc. prof. RNDr. Dagmar Solichová, Ph.D. Title of dissertation thesis: Using of separation methods in clinical research This thesis is devoted to development and validation of new LC methods for the determination of biologically active substances, specifically a fat-soluble vitamins (vitamin A, E and D) and theirs metabolites. Vitamins are essential micronutrients which cannot be synthesized by human organism with the exception of steroid hormone precursor of vitamin D. Vitamins are involved in biochemical processes of organism directly or as coenzymes. Interest in the clinical monitoring of these substances is increasing constantly over the last years. It needs to use modern instrumental techniques such as the UHPLC coupled with MS detection for the determination of vitamin D metabolites (25-hydroxy vitamin D3 and 25-hydroxy vitamin D2) due to their low concentrations in serum. The use of classical liquid chromatography with diode array detection is suitable in case of monitoring of levels of vitamin A (retinol) and E (α-tocopherol) in breast milk and vitamin E (α- tocopherol) in red blood cell...
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HPLC method development of active substances in nutraceuticals
Jägerová, Kateřina ; Šatínský, Dalibor (advisor) ; Matysová, Ludmila (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Kateřina Jägerová Supervisor: Doc. RNDr. Dalibor Šatínský, Ph.D. Title of Diploma Thesis: HPLC method development of active substances determination in nutraceuticals A high performance liquid chromatography (HPLC) method with diode array detection (DAD) was used and validated for the simultaneous determination rutin, troxerutin, diosmin and hesperidin. The method was used for direct determination of this flavonoids in pharmaceutical preparations and nutraceuticals, Cilkanol (Zentiva), Detralex (Les Laboratoires), Hemodin Prebio Forte (Valosun a. s.), MobiVen Micro (Vulm cz a.s.). Using Ascentis Express RP - Amide (10 cm x 3.0 mm, 2.7 μm) Supelco Analytical column, the following conditions were chosen as optimal: mobile phase acetonitril/water 30/70 containing acetic acid for pH adjustment to value pH 3. Method development was performed within a temperature of 50řC and pressure of 18.6 MPa. The flavonoids were detected by diode array detector (DAD) at the wavelength of 283 nm and 255 nm. Keywords: high performance liquid chromatography (HPLC), flavonoids, rutin, troxerutin, diosmin, hesperidin, nutraceuticals
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Development and optimization of chromatographic methods suitable for clinical application
Jenčo, Jaroslav ; Solich, Petr (advisor) ; Hroboňová, Katarína (referee) ; Šatínský, Dalibor (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of Analytical Chemistry Candidate: Mgr. Jaroslav Jenčo Supervisor: prof. RNDr. Petr Solich, CSc. Consultant: doc. RNDr. Lenka Kujovská Krčmová, Ph.D. Title of dissertation thesis: Development and optimization of chromatographic methods suitable for clinical application The presented dissertation thesis is divided into several parts, copying the workplaces, which I gradually worked on. The unifying element is the development and optimization of methods for the preparation of samples of biologically active substances and their subsequent analysis using separation methods. The theoretical part characterizes the target substances, from a biochemical, physicochemical, and analytical point of view. It describes the basic characteristics of extraction procedures and presents an overview of the methods used to determine the substances described in the thesis. In the first, main experimental part, of the dissertation thesis the development, optimization, and validation of an analytical method for chromatographic analysis of vitamin B1, its biologically active forms and the active form of vitamin B6 in blood is described. It illustrates the selection of sample preparation techniques, the optimization of the target methodology...
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Development of CE-C4D method for the analysis of saccharides in honey
Šebestová, Gabriela ; Jáč, Pavel (advisor) ; Šatínský, Dalibor (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Gabriela Šebestová Supervisor: PharmDr. Pavel Jáč, Ph.D. Title of the diploma thesis: Development of CE-C4D method for the analysis of saccharides in honey Honey is a commonly used natural mixture which quality must be monitored due to possible mishandling or adulteration. This diploma thesis is focused on the development of a capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE-C4D) for the determination of carbohydrates in honey samples. Design of Experiments (DoE) was used to optimize the separation conditions. This approach allows to find suitable separation conditions by means of multivariate optimization. During the optimization, the impact of the following factors was investigated: the composition of the background electrolyte, the separation voltage and temperature during separation and the inner diameter of the separation capillary. Two methods were optimized: one with normal electroosmotic flow (EOF) and the other using reversed EOF after CTAB addition. The method in the normal EOF mode was used for validation and analysis of honey samples. A fused silica capillary with an inner diameter of 20 m, a total length of 48.7 cm and an effective...
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