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HPLC method development for determination of active compounds in Arpalit preparation.
Švestková, Petra ; Šatínský, Dalibor (advisor) ; Pospíšilová, Marie (referee)
A HPLC method was developed for the separation and determination of the substances fenoxycarb and permethrin. The method is based on using HS F5 column (10 x 4 mm, 3 μm particle) and UV detection at 225 nm. The compounds were separated using isocratic elution of the mobile phase acetonitril - water (65:35) at a flow-rate of 1,0 ml/min. There was temperature 70 řC during the measurement. The system enabled successful separation of both compounds in time less than 5 min. The retention time of fenoxycarb was 1,53 min and the retention time of permethrin was 3,68 min. The chromatographic resolution between both compounds was 11,012. The method was applied to analysis of the active substances fenoxycarb and permethrin in veterinary preparations Arpalit® Neo mechanical spray, Arpalit® Neo spray a Arpalit® Neo foam. Developed method was compared with the method available on Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University in Prague (HPLC, Chromolith Performance RP-18, 100 x 4,6 mm, gradient elution of mobil phase acetonitril + water/acetonitril (60:40), flow rate 1,0 ml/min, temperature 30 řC, analysis time 11 min). Keywords: fenoxycarb, permethrin, HPLC
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Study of retention behaviour of neopterine in HILIC chromatographic system with different stationary phases
Matula, Aleš ; Nováková, Lucie (advisor) ; Šatínský, Dalibor (referee)
TITLE: The study of neopterin retention characteristics in HILIC chromatography system with different types of stacionary phases SUMMARY: This graduation thesis was dealing with the development of an analytical method for the identification and quantification of biologically active substances biopterin, neopterin and 7,8-dihydroneopterin by hydrophilic liquid interaction chromatography (HILIC) connected to fluorescence detection. Neopterin represents an important and useful parameter for monitoring of the immune system activation, that accompanies different infectious and autoimmune diseases. Increased level of neopterin concentration can be observed in several types of malignant tumors. Recently the coherency between neopterin, 7,8-dihydroneopterin and intracelular oxidative stress and apoptosis of the cells has been demonstrated. Totally reduced form of biopterin serves as a cofactor of some enzymes in human organism. HILIC is an option of high performance liquid chromatography (HPLC). Three chromatographic columns (LUNA HILIC, OBELISK R, OBELISK N) were tested at different chromatografic conditions. Mobile phase consisted of mixture of aqueous component (ammonium acetate buffer, acetic acid, water) and water miscible organic component (acetonitrile). The mixture of acetonitrile and 100mM ammonium acetate...
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Chromatography method optimization for HPLC determination of carotenoids.
Dvořáková, Petra ; Šatínský, Dalibor (advisor) ; Solich, Petr (referee)
This thesis deals with searching for optimal conditions for determination of carotenoids lutein, betacarotene and zeaxanthin. High performance liquid chromatography - HPLC was used for determination. During searching for optimal conditions many various columns and mobile phases were tested. Primary information about carotenoids separation was obtained from scientific literature. There were tested both types of chromatography - normal phases chromatography and reversed phases chromatography. The most frequently tested columns were C18 columns from different producers. The biggest problem for HPLC determination and separation of these three substance were position isomers of lutein and zeaxanthin. Separation of these substances is relatively difficult and it wasn't completely solved in this thesis. In spite of it new metod for determination and separation of three carotenoids was developed. This method used isocratic elution at column Zorbax SB C18 (50 x 4,6 mm, 2 μm), UV detection at 450 nm, mobile phase of methylene chloride:hexan:acetonitrile (2,5:2,5:95), injected volume 1 μl and flow rate at 1 ml/min.
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Application of HILIC method for the determination of stability of ascorbic acid
Pavlovičová, Soňa ; Nováková, Lucie (advisor) ; Šatínský, Dalibor (referee)
Author: Soňa Pavlovičová Title: The application of HILIC method in the stability evaluation of ascorbic acid Language: czech Keywords: ascorbic acid, HILIC, stability, stabilizating agents Ascorbic acid, vitamin C, is a very polar compound, and is therefore difficult to be analysed by RP-HPLC. For these reason HILIC method (Hydrophilic Interaction Liquid Chromatography) was developed and the optimal conditions for the analysis were determined. The analysis occured on the ZIC-HILIC column (150 x 2.1, 3.5 μm), the mobile phase consisted from acetonitrile and 50 mM ammonium acetate buffer pH 6.8. The method used UV detection at 268 nm. Under these conditions the analysis take approximately 4 minutes. Chlorogenic acid was used for quatification. Various stability factors as temperature, concentration, the presence of oxygen and pH of the solution were studied. This work also solved the troubles with stabilizating agents and their influence on stability of ascorbic acid. Optimal conditions were obtained, when the temperature was decreased to 4řC and 10 mM oxalic acid was used for stabilization. Good results were also obtained with 5 % o-phosphoric acid as stabilizating agent. The method was validated and it met pharmacopea requirements.
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Verapamil determination using sequential injection analysis with chemiluminescence detection
Homolová, Zuzana ; Sklenářová, Hana (advisor) ; Šatínský, Dalibor (referee)
The verapamil was determined by the technique of sequential injection analysis (SIA) with chemiluminescence (CL) detection. The CL was emitted during the oxidation of the analyte by permanganate in aqueous sulphuric acid in the presence of CL enhancer polyphosphate (PFS). Concentration, order, volume of reagents and flow rate were optimised. Also influence of solvents 60% methanol and 60% ethanol was proved. The optimum order, volumes and concentrations of reagents for aqueous solution of verapamil were: 30 μl of 10 mM KMnO4, 50 μl of 2% PFS, 50 μl of 1 mM verapamil, 30 μl of 1 M H2SO4 and for 60% ethanolic solution of verapamil were: 20 μl of 10 mM KMnO4, 30 μl of 2% PFS, 50 μl of 1 mM verapamil, 30 μl of 2 M H2SO4, 30 μl of 2% PFS and 30 μl/s flow rate. The transient CL signal was recorded at the wavelength 390 nm. Calibration curves relating the intensity of CL (peak heights) to the concentration of the analyte were curvilinear with rectilinear sections in the range 510-5 - 510-3 M for aqueous solution and 110-5 - 110-3 M for 60% ethanolic solution of verapamil. The limits of detection were 210-5 M for aqueous solution and 110-5 M for 60% ethanolic solution. Repeatability of peak heights (RSD, n = 10) ranged between 4.43 % (110-4 M) and 1.83 % (110-3 M) for aqueous solution, and...
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Optimization of chromatographic separation for the determination of 4-aminophenol and active substances in Valetol
Daňková, Jana ; Šatínský, Dalibor (advisor) ; Sklenářová, Hana (referee)
This thesis deals with the optimization of separation conditions determine paracetamol, caffeine, propifenazone and 4-aminophenol in Proprietary Medicinal Products Valetol. As an analytical method was chosen HPLC with UV detection mainly for the following reasons: many other tablets contain excipients which are from the sample during analysis impossible, and separated themselves determination of active substances to monitor the stability of active substances The work was conceived as original, and the emphasis is on the greatest possible simplicity and less time consuming and optimized separation while maintaining high accuracy and reliability of the method. Optimal conditions suitable for the final validation of the separation were found on a column of C-18 RP-amide. Total analysis time does not exceed 13 minutes with equilibration after gradient elution of the column to its original condition. All substances are separated with a resolution greater than 1.5. The peaks of substances are symmetric and do not show significant peak tailing besides AMF. The zero line is despite the gradient elution stable.
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The analysis of statines by gas chromatography
Pavlovičová, Soňa ; Šatínský, Dalibor (advisor) ; Pospíšilová, Marie (referee)
Gas chromatography analysis of statins Soňa Poláková (roz. Pavlovičová) Abstract A new GC method for determination of statins after derivatizacis using FID detector was developed. During the experiments the following results were achieved. Optimum separation results in the analysis of statins achieved in the following temperature gradient: one minute at 250 ř C, the temperature is further increased at 10 ř C / min to a final temperature of 320 ř C with other isothermal sections. A flame ionization detector was kept at the temperature 320 ř C. The optimum derivatization reagent was chosen BSTFA + TMCS for simvastatin, and N- tert-butyl (dimethylsilyl) trifluoroacetamide for atorvastatin. Addition of anhydrous potassium carbonate was obtained in alkaline environment, which allowed the derivatization. The best temperature for the derivatization of both drugs was obtained at 90 ř C. Time that is necessary to be run derivatization is 60 minutes with simvastatin and atorvastatin for 30 minutes. Quantification was performed in both cases using mefenamic acid as an internal standard, which was also derivatized, in the same derivatization agent. IS derivatization proceeded at 60 ř C for 60 minutes. The greatest yield response was achieved using half the amount of derivatization reagent than the amount of drug. Due...
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Separation and Determination of Betamethasone and chloramphenicol in multicomponent pharmaceutical preparations using a monolithic columns coupled to sequential injection system
Salabová, Markéta ; Šatínský, Dalibor (advisor) ; Chocholouš, Petr (referee)
SEPARATION AND DETERMINATION OF BETAMETHASONE AND CHLORAMPHENICOL IN MULTICOMPONENT PHARMACEUTICAL PREPARATIONS USING A MONOLITHIC COLUMNS COUPLED TO SEQUENTIAL INJECTION SYSTEM Markéta Salabová Abstract A new separation method based on a reversed-phase sequential injection chromatography (SIC) technique was used for simultaneous determination of chloramphenicol and betamethasone in pharmaceutical eye drops in this contribution. Short monolithic column coupled with a sequential injection analysis (SIA) system enabled separation of two compounds in one step. A Chromolith Flash RP-18e, 25-4.6 mm column with 5 mm precolumn (Merck, Germany) and a FIAlab 3000 system (USA) with a 6-port selection valve and 5 ml syringe were used for sequential injection chromatographic separations in study. The mobile phase used was acetonitrile-water (30:80, v/v), flow rate 0.48 ml min-1 ; UV detection was at two wavelengths 241 nm and 278 nm, (absorption maximum of betamethasone and chloramphenicol). The basic validation parameters showed good results: linearity of determination for both compounds including internal standard (propylparaben) > 0.999; repeatability of determination (RSD) in the range 0.8 - 1.7% at two different concentration levels, and detection limits in the range 0.5 - 1.0 µg ml-1 . The chromatographic...
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Analytical methods for the quantitative determination of statins in biological samples based on HPLC and GS
Kotlíková, Olga ; Šatínský, Dalibor (advisor) ; Nováková, Lucie (referee)
Analytical methods for the quantitative determination of statins in biological samples based on HPLC and GC Olga Kotlíková Abstract The aim of the present study was to review published analytical methods for the quantitative determinations of presently avalaible four 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors ( statins ), atorvastatin, simvastatin, lovastatin and fluvastatin, for therapeutic drug monitoring purpose. Almost all assay reviewed are based on either high-performance liquid chromatography (HPLC) or gas chromatography (GC). HPLC is the most popular method of the two. Most often a mass detector and a UV detector, in case of fluvastatin a fluorescence detector have been used. Moreover, some derivatization procedures have been used to decrease sensitivity in some of the assay. HPLC-mass spectrometry (MS) assays offer improved sensitivity and selectivity but are not available for most laboratories. Besides, study on the stability of statins existing in solution with their lactone equilibrium products in a pH- dependent manner is included. This review shows that most method allow quantitative determination of statins in plasma or serum, in the ng/ml range, therefore they are suitable for therapeutic drug monitoring purpose of this category drugs. But still there is neccessity to...
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Derivatization method for determination of cholesterol in biological material by gas chromatography
Krausová, Martina ; Solich, Petr (advisor) ; Šatínský, Dalibor (referee)
Název práce : Derivatizační metoda pro stanovení cholesterolu v biologickém materiálu metodou plynové chromatografie Diplomant : Martina Krausová Abstrakt Analysed matters mostly occur in complicated mixtures in biological material. It is necessary to separate individual analits. Gas chromatography can be used for this purpose. It is sensitive analytical method with great separative effect. This method is applied with substance which can be turned into vapour by heating them. Substance that dissolves when heated must be put through derivation reaction at the rise of volatile derivatives. One of these derivative methods is described in this diploma work. This reaction is acylation of cholesterol into acyl - cholesterol which can be determined thanks to it's volatility by gas chromatography. V biologickém materiálu se analyzované látky většinou vyskytují ve složitých směsích. Jednotlivé analyty je nutné oddělit. K tomuto účelu lze využít plynovou chromatografii. Je citlivá analytická metoda s velkou separační účinností. Uplatňuje se u látek, které lze zahřátím převést na páry. U látek, které se při zahřátí rozkládají, je nutné je před analýzou podrobit derivatizační reakci za vzniku těkavých derivátů. Jedna z těchto derivatizačních metod je popsána v této diplomové práci. Touto reakcí je acylace cholesterolu...
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