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Method development for determination of 8-hydroxy-2-deoxy guanosine in urine for clinical research
Šestáková, Veronika ; Kujovská Krčmová, Lenka (advisor) ; Matysová, Ludmila (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Veronika Šestáková Supervisor: assoc. prof. RNDr. Lenka Kujovská Krčmová, Ph.D. Title of the Diploma Thesis: Method development for determination of 8-hydroxy-2-deoxy guanosine in urine for clinical research This diploma thesis is based on the method development for determination of 8- hydroxy-2-deoxy guanosine, 8-hydroxyguanosine and creatinine using UHPLC. The aim was to develop optimal conditions for clinical research. Experiments were carried out using UHPLC Nexera with mass spectrometer LCMS- 8030, (Shimadzu, Japan). Two stationary phases were tested. The chromatographic separation was achieved using a Meteoric core C18 BIO 4.6 × 50 mm stationary phase with core-shell particles, particle size 2.7 μm (YMC, Germany) secured with a KrudKatcher Ultra 0.5 μm in-line filtr (Phenomenex, Germany). The used mobile phase consisted of water (pH 3 using acetic acid) and methanol (using formic acid 0.2 mM) in the ratio 90:10 (v/v). The temperature was maintained at 25 řC, a flow rate was set at 0.5 mL/min and 4 µl of sample was injected. After optimization of separation conditions, the method was applied to biological material (urine). The samples were prepared using solid-phase extraction. The method...
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The comparison of particle stationary phases with monolith in analysis of selected medicaments
Šestakova, Veronika ; Chocholoušová Havlíková, Lucie (referee) ; Matysová, Ludmila (advisor)
The performance of monolithic HPLC columns Chromolith® (made by Merck, Germany) and Onyx™ (made by Phenomenex, USA) and conventional C18 columns Discovery® (made by Supelco, Sigma-Aldrich, Prague, Czech Republic) and LiChroCart® 125-4 Purospher 5 µm (made by Merck, Germany) was tested and the comparison for two topical preparation Estrogel gel and Aphlox-K paste was made. The composition of mobile phase - for Estrogel gel analysis a mixture of acetonitrile, methanol and water (23:24:53, v/v/v) and for Aphlox-K paste analysis a mixture of methanol, water and acetic acid (50:50:1, v/v/v) - was usually not optimal for analysis at all types of columns. Thus an adjustment of components ratio was necessary for sufficient resolution of the analyzed compounds. Various flow rates (0.3 - 5.0 ml/min) and mobile phase composition (usually increasing ratio of water content) were applied. Determination of active substances, preservatives and impurities and comparison of retention times and system suitability test parameters was accomplished. For Estrogel gel analysis, following chromatographic conditions were found: using Chromolith® Flash RP18-e (25 mm x 4.6 mm), monolith column mobile phase was acetonitrile, methanol, water (16:24:60 v/v/v) and flow-rate 2.0 ml/min. Using monolith column Onyx™ Monolithic C18-e (50 mm x...
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Application of HPLC in pharmaceutical analysis.
Šestakova, Veronika ; Chocholoušová Havlíková, Lucie (referee) ; Matysová, Ludmila (advisor)
Determination of hexachlorophene in the product SEPTONEX ointment to the Department of Analytical Chemistry performed by HPLC. This method has many advantages over previously used titrate determination. The conditions of analysis are as follows: mobile phase is composed of 85% methanol with the addition of 1% acetic acid, column Purospher Merck RP C18, 5 mm (12.5 cm x 4 mm), flow rate of the mobile phase is 1.5 ml / min, sample injection volume is 20 µl. The aim was to optimize the use of methods, i.e. find the column using the modern trends in HPLC, which would allow obtaining precise, rapid and well reproducible results under similar chromatographic conditions. Based on the literature research were selected six different analytical columns with more or less similar characteristics as the column still used. In each of these columns have been measured solutions of standards hexachlorophene and solutions of ointment SEPTONEX samples at different flow rate of mobile phase. Other conditions of analysis have been retained. From the experiment showed that the tested columns is the most appropriate column Chromolith Performance RP18-e (10 cm x 4,6 mm) at a flow rate of mobile phase 2.0 ml / min. Another challenge was to find a suitable internal standard for faster and more accurate determination of...
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