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Development of a UHPLC-MS/MS method suitable for the determination of cabozantinib in blood serum
Kleinová, Jana ; Kozlík, Petr (advisor) ; Hraníček, Jakub (referee)
The aim of this Bachelor thesis was to develop a UHPLC-MS/MS method for the determination of Cabozantinib in blood serum. This method was used to monitor the release of the active substance in a model organism - the rat. First, the mass spectrometer setup was optimized, where MRM transitions for Cabozantinib and isotope-labelled Cabozantinib-D4 were found. For Cabozantinib, an MRM transition of 502.2 → 323.1 was found with optimal energy levels of Q1 = -26.0 V, CE = -40.0 V and Q2 = -22.0 V. For Cabozantinib-D4, the MRM transition of 506.2 → 327.0 was found with optimal energy levels being Q1 = -26.0 V, CE = -39.0 V and Q2 = -22 V. The method setup parameters were as follows: The chromatography column used was a Poroshell 120 EC-C18, 2.1x50 mm, 1.9 um, (Agilent Technologies). The mobile phase consisted of acetonitrile (B) and formic acid (A), the flow rate of the mobile phase was 0.350 ml/min, the analysis time was 5.5 min and the injection volume was 1 μl at gradient elution (time: 0; 0.5; 2.0; 3.0; 3.5; 4.0; 5.5 min, B: 10; 10; 60; 100; 10; 10 %). The method was linear (weighted regression 1/x2 ) with a regression coefficient equal to 0.9978, showing an excellent linearity of the method. The precision (relative standard deviation) of the method was within 14 %. The accuracy (relative error) was...
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Validation of LC-MS method and monitoring of pharmaceuticals in water samples.
Molnárová, Lucia ; Bosáková, Zuzana (advisor) ; Kozlík, Petr (referee)
Occurrence, accumulation and subsequent fate of pharmaceuticals in environment currently represent a very actual topic. Worldwide, thousands of tons of pharmaceutical substances are consumed every year. A large portion of pharmaceuticals is, in unchanged or metabolized forms, disposed via sewage systems and wastewater treatment plants. Considering the fact that wastewater treatment processes are not able to completely eliminate all active substances or their metabolites, pharmaceuticals are systematically washing out into the water system and increasingly contaminate the ground and surface waters. The problematics of continuous control and progressive elimination of pharmaceutical residues from environment are still not completely solved. Thus, the development and availability of accurate and fast commercial analyses are highly desired. The aim of this diploma thesis was the optimization and validation of multi-residue UHPLC- MS/MS analytical method designated for the determination of 52 pharmaceuticals in drinking and waste waters. The work was carried out in laboratories of ALS Czech Republic. An analytical method was subsequently used for monitoring of pharmaceuticals in both drinking and waste waters, as well as for the determination of efficiency of removing these compounds within the...
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Development of a UHPLC-MS/MS method for determination of rivaroxaban in rat serum
Plášilová, Denisa ; Kozlík, Petr (advisor) ; Hraníček, Jakub (referee)
This bachelor thesis is focused on development of a specific UHPLC-MS/MS method for the determination of rivaroxaban in rat serum. At first, the setting of the mass spectrometer was optimized. Suitable MRM transitions were found for rivaroxaban and its isotopically labeled internal standard. For rivaroxaban was found the MRM transition 436.1 → 145.05 with optimal energy levels Q1 = -12.0 V; CE = -30.0 V; Q2 = -27.0 V. For rivaroxaban D4 was found the MRM transition 440.1 → 145.0 with optimal energy levels Q1 = -22.0 V; CE = -31.0 V; Q2 = -25.0 V. The ion source setting parameters were as follows: nebulizing gas flow 3 l/min; heating gas flow 10 l/min; interface temperature 300 řC; desolvation temperature 526 řC; DL temperature 250 řC; heat block temperature 400 řC; drying gas flow 10 l/min. The optimal chromatographic method was as follows: A Poroshell 120 SB AQ, 100 × 2.1 mm, 2.6 µm (Agilent) chromatographic column; the mobile phase consisted of acetonitrile with the addition of 0.1% formic acid (A) and distilled water with the addition of 0.1% formic acid (B); flow rate of the mobile phase 0.5 ml/min; gradient elution (time: 0-1-2-3.5-4-6.5 min; A: 20-20-80-80-20-20 % v/v); autosampler temperature 15 řC; column temperature 40 řC; time of analysis 6.5 minutes; injection volume 2 µl. The optimized...
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Study of the amount of antioxidants in extracts from Melissa officinalis
Bartoš, Karel ; Kubíčková, Anna (advisor) ; Kozlík, Petr (referee)
This diploma thesis deals with the determination of selected antioxidants, namely rosmarinic acid, caffeic acid, cinnamic acid and t-4-hydroxycinnamic acid in aqueous and ethanolic extracts from Melissa officinalis. The aim of the work was to find out whether the above- mentioned antioxidants were present in the extracts prepared at home. A new UHPLC method with UV and MS detection was developed to monitor the content of rosmarinic acid, caffeic acid, cinnamic acid and t-4-hydroxycinnamic acid in the extracts. A BEH C18 column (2,1 mm × 100 mm, particle size 1,7 µm) was selected for separation, the mobile phase consisted of methanol (component A) and 0.1% aqueous formic acid solution at pH 2.6 (component B), the ratio of components being changed according to the gradient program. The method was validated and repeatability, limits of detection and limits of quantification, linearity, yield and robustness were determined after optimization. Analysis of 20 aqueous and 20 ethanolic extracts was performer differing in extraction time, type of solvent and lighting conditions. Based on the comparison of retention time and MS detection, the presence of 3 of 4 selected phenolic acids was confirmed, namely rosmarinic acid, caffeic acid and cinnamic acid in ethanolic extracts. Unfortunately, the presence of...
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Validation of LC-MS method and monitoring of pharmaceuticals in water samples.
Molnárová, Lucia ; Bosáková, Zuzana (advisor) ; Kozlík, Petr (referee)
Occurrence, accumulation and subsequent fate of pharmaceuticals in environment currently represent a very actual topic. Worldwide, thousands of tons of pharmaceutical substances are consumed every year. A large portion of pharmaceuticals is, in unchanged or metabolized forms, disposed via sewage systems and wastewater treatment plants. Considering the fact that wastewater treatment processes are not able to completely eliminate all active substances or their metabolites, pharmaceuticals are systematically washing out into the water system and increasingly contaminate the ground and surface waters. The problematics of continuous control and progressive elimination of pharmaceutical residues from environment are still not completely solved. Thus, the development and availability of accurate and fast commercial analyses are highly desired. The aim of this diploma thesis was the optimization and validation of multi-residue UHPLC- MS/MS analytical method designated for the determination of 52 pharmaceuticals in drinking and waste waters. The work was carried out in laboratories of ALS Czech Republic. An analytical method was subsequently used for monitoring of pharmaceuticals in both drinking and waste waters, as well as for the determination of efficiency of removing these compounds within the...
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DEVELOPMENT OF A UHPLC-MS/MS METHOD FOR NILOTINIB DETERMINATION IN RAT SERUM
Černá, Kateřina ; Kozlík, Petr (advisor) ; Křížek, Tomáš (referee)
This bachelor thesis aimed to develop a UHPLC-MS/MS method for the determination of nilotinib in rat serum. The developed UHPLC-MS/MS method was used to monitor the pharmacokinetic release of the active substance in a rat model organism in a project focused on the formulation of a tablet containing nilotinib with a slower release than before. The optimal conditions of the method were as follows. Chromatographic column Acquity UPLC BEH PHENYL 100x2.1 mm, 1.7 μm from Waters. The mobile phase consisted of methanol and distilled water, both with the addition of 0.1% formic acid using gradient elution. The flow rate of the mobile phase was 0.3 mL/min, the temperature in an autosampler 15 řC, the column temperature 40 řC, the analysis time 6.5 minutes, and the injection volume 2 μl. The MRM transition monitored for nilotinib was: 530.2 → 289.10 (Q1 = -26 V; CE = -31 V; Q3 = -20 V) and for nilotinib D6: 536.2 → 295.15 (Q1 = -26 V; CE = -31 V; Q3 = -14 V). The setting of the ion source was as follows: nebulizing gas flow 3 L/min; drying gas flow 10 L/min; source temperature 300 řC; desolvation capillary temperature 250 řC. The method was partially validated. The coefficient of determination 1.0000 shows the excellent linearity of the method. The accuracy, expressed as a relative error, was up to 20 %. The...
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Development of UHPLC-MS/MS screening method for the determination of benzodiazepines in urine samples
Havelková, Lucie ; Bosáková, Zuzana (advisor) ; Kozlík, Petr (referee)
The aim of this diploma thesis was the development of a screening method for analysis of 17 benzodiazepines in urine samples using ultra high-performance liquid chromatography with tandem mass spectrometric detection. The partial task was to optimize the conditions for the enzymatic hydrolysis of benzodiazepine glucuronides present in urine using design of experiments (DOE). The optimized chromatographic system consisted of a Zorbax Eclipse Plus Phenyl-Hexyl RRHD column (100 × 2.1 mm, 1.8 μm) and mobile phase consisting of water with 0.1 % acetic acid (component A) and acetonitrile with 0.1 % acetic acid (component B) in various ratios according to the gradient program. Flow rate was 0.2 ml/min, column temperature was 40 řC, and total analysis time was 12 min. Calibration curves for all analytes were measured under optimized conditions in methanol and urine. After optimal detection conditions for oxazepam-glucuronide were found, oxazepam glucuronide was hydrolysed using β-glucuronidase from the abalone to confirm the functionality of the enzyme within the pilot experiment. Optimization of enzymatic hydrolysis conditions via 27 experiments proposed by program Minitab 16 using the Box-Behnken design will be realized later.
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Analysis of Historical Pharmaceutical Preparations of Naloxone, Adrenaline and Ephedrine.
Nováková, Lucie ; Nesměrák, Karel (advisor) ; Červený, Václav (referee)
The aim of the thesis was to analyze the historical pharmaceutical preparations, including the determination of the active substance and identify theirs possible degradation products. A historical pharmaceutical preparation of naloxone was analyzed by mass spectrometry. Historical pharmaceutical preparations of adrenaline and ephedrine were analyzed by UHPLC-MS and were quantified using a calibration curve. In the historical injection solution of naloxone, "NARCAN", dated around 1980, there were no significant degradation products and the measured mass and UV spectrum was consistent with the spectrum of naloxone. The analyzed sample of naloxone was stable even after 35 years of storage. In the analyzed historical injection solution of adrenaline, "Adrenalin Hydrochlor., Dr. Heisler" (dated between 1917 and 1938) was determined 5.26 ± 0.11 % of the declared amount of adrenaline. In the measured spectras were noticeable degradation products, which have not been described in the literature yet and their identification was beyond the scope of this paper. The analyzed sample of adrenaline was almost completely degraded during about ninety years. The stability test carried out with four standard solutions of adrenaline proved influence of oxygen, light, temperature and time on the degradation of adrenaline. In...
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