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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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Development and validation of HPLC method for separation and determination of active substances in pharmaceutical preparation Valetol
Ullrichová, Jana ; Šatínský, Dalibor (advisor) ; Matysová, Ludmila (referee)
Development and validation of HPLC method for separation and determination of active substances in pharmaceutical preparation Valetol Jana Ullrichová Abstract A method for the determination of paracetamol, propyphenazone and caffeine by the high- performance liquid chromatographic method with ultraviolet detection has been developed and validated. The analysis was performed at the room temperature in an isocratic mode on the reversed phase ODS Hypersil 5 µm C-18 column (250x4,6mm). A mobile phase (water : 2-propanol : diethylamine : methanol (50 + 15 + 3 + 32) adjusted to pH 7.5 by means of phosphoric acid) was suitable for the separation and the determination of paracetamol, caffeine and propyphenazone. UV detection was applied at 273 nm. Injected volume was 5 µl, flow rate 0,5 ml.min-1 . The developed method is sensitive and selective and can be applied for the routine studies of pharmaceuticals in the tablet form.
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Using of sequential injection chromatography method for separation and determination of salicylic acid and triamcinolone acetonide in pharmaceutical preparation Triamcinolon-IVAX
Holík, Pavel ; Šatínský, Dalibor (advisor) ; Solich, Petr (referee)
Using of sequential injection chromatography method for separation and determination of salicylic acid and triamcinolone acetonide in pharmaceutical preparation Triamcinolon-IVAX Pavel Holík Abstract A novel and fast simultaneous determination of triamcinolone acetonide (TCA) and salicylic acid (SA) in topical pharmaceutical formulations by sequential injection chromatography (SIC) as an alternative to classical high performance liquid chromatography (HPLC) has been developed. A recently introduced Onyx™ monolithic C18 (50 mm × 4,6 mm, Phenomenex® ) with 5 mm monolithic precolumn were used for the first time for creating sequential injection chromatography system based on a FIAlab® 3000 with a six-port selection valve and 5,0 ml syringe pump in study. The mobile phase used was acetonitrile/water (35:65, v/v), pH 3.3 adjusted with acetic acid at flow rate 15 µl.s-1 . UV detection provided by fibre-optic DAD detector was set up at 240 nm. Propylparaben was chosen as suitable internal standard (IS). There is only simple pre-adjustment of the sample of topical solution (dilution with mobile phase) so the analysis is not uselessly elongated. Parameters of the method showed good linearity in wide range, correlation coefficient >0,999; system precision (relative standard deviation, R.S.D.) in the range 0,45-1,95%...
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Determination of bopindolol by the technique of sequential injection analysis with chemiluminescence detection
Holá, Petra ; Polášek, Miroslav (advisor) ; Šatínský, Dalibor (referee)
The oxidation of bopindolol [ 4-(2-benzoyloxy-3-tert-butylaminopropyl)-2-methylindole (I)], by permanganate in the medium of aqueous H2SO4 is accompanied by the emission of chemiluminescence (CL) radiation. The CL signal is enhanced by hexametaphosphate. This CL reaction was used for devising automated sequential injection analysis (SIA) assay of I in pharmaceutical preparations. The PC-controlled SIA setup consisted of a Cavro XL 3000 2.5- ml syringe pump, Vici-Valco 10-port selection valve and Spectra-Physics FS970 flow- through fluorescence detector equipped with a lab-made CL detection module. The net CL signal of I increased by a factor of 3 (compared to purely aqueous test solution of I injected) if the test solution contained 60% (v/v) of methanol. Optimal order, concentrations and volumes of aspirated zones of reactants were: 61l of 80mM Na hexametaphosphate, 60 l of I in 60% (v/v) methanol, 40 l of 30mM H2SO4 and 9 l of 0.5mM KMnO4. Calibration curve relating the intensity of CL (peak height) to the concentration of I was linear in the range 1 - 8 M I; the limit of detection (S/N = 3) was 0.2 M I. The sample throughput was 100 h-1 . The repeatability of the peak heights was characterised by RSD 1.8% for 15 replicate injections of 2 M I. The SIA-CL method was used for the assay of I...
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