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Development and validation of method for determination of clotrimazole in cream using HPLC
Poláčková, Jitka ; Matysová, Ludmila (advisor) ; Chocholoušová Havlíková, Lucie (referee)
The topic of my work was to optimalize and to validate the method for determination of capacity of the Clotrimazol, degradeted product (2-chlorophenyl) diphenylmethanol and conservations (methylparaben and propylparaben) in the preparation Clotrimazol ointment. My effort for this work was to develop the method that could half for total separation the particular defined substance in this sample, in the acceptable time, to find inside standard, to optimalize the conditions of separation and the method verify. The optimum chromatographic conditions were found on column Zorbax SB - Phenyl, 3,5 μm, 4,6 x 75 mm in the flow of mobile phase 0,5 ml per minute and wave - length 210 nm. Composition of the mobile phase was acetonitrile - water (55:45), pH water component was modified by acid phosphoric 5 % on 3,2. If these conditions are used, this method is validating. The testing parameters were accuracy, correctness, linearity, selectivity, robustness, detection and quantitative limit and test if the chromatographic system is acceptable.
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The optimization and validation of HPLC method for determination of triamcinolone acetonide in topical pharmaceutical product
Krivda, Anton ; Matysová, Ludmila (advisor) ; Nováková, Lucie (referee)
Author: Krivda A. Title: The optimalisation and validation of HPLC method for determination of triamcinolone acetonide in topical pharmaceutical product Language: Czech Keywords: HPLC Triamcinolone acetonide Triamcinolone Methylparaben Propylparaben Validation A novel reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination of active component triamcinolone acetonide, its degradation product triamcinolone and two preservatives presented in the cream - methylparaben and propylparaben, using estradiole as an internal standard. The chromatographic separation was performed on a Supelco Discovery HSF5 column. The mobile phase for separation of all compounds consists of a mixture of acetonitrile and water (45:55 v/v). The analysis time was less than 9 minutes, at a flow rate of 0,6 ml/min and detection at 240 nm. The method was found to be appicable for routine analysis (stability tests, homogeneity) in the pharmaceutical product topical cream Triamcinolone cream 0,1%.
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HPLC Method for Simultaneous Determination of Liposolubile Vitamines A, D and E
Nová, Alena ; Matysová, Ludmila (referee) ; Solich, Petr (advisor)
In this work the new HLPC method for simultaneous determination of vitamins D2, D3 and metabolite 25(OH)D3, vitamin A (retinol) and vitamin E (_-tocopherol) using the internal standard was developed. During the suggested assessment the monolith column Chromolith Performance RP-18e, 100 x 4,6 mm and Chromolith Performance RP-18e, 50 x 4,6 mm were used. The detection was carried out with the help of a diode array detector at wavelenght 264 nm for vitamins D2, D3 and metabolite 25(OH)D3, 295 nm for _-tocopherol and the internal standard tocol and 325 nm for retinol. The mixture of methanol : water : 2- propanol (75 : 15 : 10, v/v) was used as the mobile phase A in time 0,0-3,0 minutes. As the mobil phase B the mixture of methanol : water (95 : 5, v/v) was used in flow rate 3,5 ml/min in time 3,0-6,5 minutes. The injection volume of the sample was 20 μl. The total time of the analysis was 6,5 minutes including the equilibration of the column. This method was developed and partially validate. With the method will be continue on biological material.
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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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The comparison of different types of stationary phases in HPLC analysis of medicament Diclofenac emulgel
Poláčková, Jitka ; Karlíček, Rolf (referee) ; Matysová, Ludmila (advisor)
The aim of this rigorous work is a comparison of different types of stationary phases for the analysis of the drug Diclofenac emulgel. It is necessary to take into account the many specialized materials from the pharmacopoeias, ICH guidelines and professional scientific journals in solution of the development of HPLC methodology, validation and choice of stationary phase analytical columns. Several types of stationary phases were tested, with the testing of conditions of the separation of active substances from the pharmaceutical formulation - diclofenac emulgel. The best results were placed on Discovery HS-F5 (15 cm x 4.6 mm, 5 m), time analysis was less than 4 minutes; Zorbax SB - CN (4.6 x 150 mm, 5 m) time of analysis was less than 5 min; Chromolith Performance RP - 18e (100 - 3 mm) time analysis was less than 3 minutes.
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HPLC application in pharmaceutical analysis
Bajcurová, Lucie ; Sklenářová, Hana (referee) ; Matysová, Ludmila (advisor)
The HPLC method for determination of ketoprofen in therapeutic preparations "PRONTOFLEX" - a 10% skin spray and "Ketonal" - a 5% cream has been developed. This method was based on already developed and validated method for analyzing the preparation "KETOPROFEN" - a 2.5% gel. Both of the methods have been developed in the same department. The chromatographic separation was performed on SUPELCO Discovery C18 column (125 mm x 4.6 mm, 5 µm). The mobile phase consisted of a mixture of acetonitrile, water and a phosphate buffer pH 3.5 (40:58:2, v/v/v). At a mobile phase flow rate of 1.0 ml/min, injection volume of 10 μl and UV detection at a wavelength of 233 nm, the total time of analysis was less than 10 minutes. Ethylparaben was used as an internal standard.
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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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Determination of active substances in lubricant gel using gas chromatography.
Ryšavá, Pavlína ; Matysová, Ludmila (advisor) ; Šatínský, Dalibor (referee)
This thesis deals with the topic The determination of content substances in lubrication gel by method of gas chromatography and with lubrication gel Tea Tree Fantasy, which includes natural substance of tea tree oil. Tea tree oil has strong antiseptic, antimycotic and antifungal effects, and the terpinene-4-ol is its important germicidal component. This thesis is divided into eight chapters; its main part consists of chapters 3 - 6. The third chapter deals with the theoretical part, where the gas chromatography is described. This method is suitable for determination of terpinene-4-ol in lubrication gel. This chapter also deals with the validation of analytical method and with individual parameters and contains the characteristics of lubrication gel, tea tree oil and terpinene-4- ol. In the fourth chapter, there are worked out the search of used methods. The fifth chapter deals with the experimental part; all chemicals, instruments and conditions of isolation are written down there. The sixth chapter describes results and discussion from the development of analytical method, from the system suitability test and from the validation. The main goal of this thesis is the validation of gas chromatography method for the determination terpinene-4-ol in lubrication gel.
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Treatment of biological samples before HPLC analysis - determination of alfa-tocoferol in erythrocyte membrane
Pospíchalová, Naďa ; Solich, Petr (advisor) ; Matysová, Ludmila (referee)
α-Tocopherol is the principal membrane antioxidant in mammalian cells, although antioxidant is not the only mechanism of this vitamin. Many studies consider erytrocyte α-tocopherol levels to be more suitable to assess the tocopherol status of organism than its plasma levels. This thesis presents erytrocyte membranes α-tocopherol HPLC-UV analysis with diferential ultracentrigation and solid phase extraction sample pretreatment. Red cell samples were ultracentrifuged (288 000 × g, 3 minutes, 4 řC) in the presence of butylated hydroxytoluene (BHT), D-mannitol, HEPES and CaCl2. Then α-tocopherol was extracted from erytrocyte membranes by solid phase extraction with n-hexane in the presence of ascorbic acid and tocopherol acetate internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP-18e (100 4.6 mm) using a 100% methanol as the mobile phase. The absorbance of α-tocopherol was measured at 295 nm wavelength.
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