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Study of selected mushroom toxins of Amanita muscaria.
Adámek, Michal ; Bosáková, Zuzana (advisor) ; Hložek, Tomáš (referee)
The presented bachelor's thesis was focused on the development of an HPLC-MS/MS method for the separation and eventual quantification of the main toxins of Amanita muscaria (fly agaric). After optimization of tandem mass detection conditions, MRM transitions were found for muscarine (174 → 57), ibotenic acid (159 → 113), muscimol (115 → 98) and cycloserine (103 → 75), which was used as an internal standard. Measurements were performed on Luna Omega 1.6 µm polar C18 column in RP-HPLC mode with an optimized mobile phase composition of 2:98 (v/v) acetonitrile/deionized water with the addition of 0.1% formic acid in both components and Ascentis Express RP- Amide in HILIC mode with an optimized mobile phase composed of 70:30 (v/v) methanol/deionized water with the addition of 0.5% formic acid in both components. Calibration curves with a range of 0.01 to 10 µg/ml for RP-HPLC and 0.01 to 25 µg/ml for HILIC were measured under optimized conditions. Urine and blood serum solutions of a patient with Amanita muscaria poisoning, including diluted versions of these solutions, were measured. Ibotenic acid and muscimol were found in the patient's urine, muscarine, ibotenic acid and muscimol in the blood serum. The RP-HPLC method gave narrower peaks with lower retentions and lower noise levels. The studied toxins...
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Determination of antiepileptics in breast milk with chromatographic methods
Ernestová, Viktoria ; Hložek, Tomáš (advisor) ; Kubíčková, Anna (referee)
The present thesis is devoted to the development of a new method for the determination of a selected group of antiepileptic drugs (brivaracetam, eslicarbazepine, felbamate, gabapentin, lacosamide, perampanel, pregabalin, rufinamide) in human breast milk. The rationale for identifying antiepileptic drugs in this rather complex matrix is to assess and optimize the use of these drugs in breastfeeding mothers to avoid unnecessary and risky exposure of infants. The aforementioned drugs have various undesirable side effects that can have a negative impact not only on the mother but also on the breastfed infant. The protein precipitation method using acetonitrile/methanol mixture was used for sample preparation. A method based on liquid chromatography with tandem mass detection was developed and tested for the determination of the concentrations of these drugs. In the course of testing, different chromatographic columns with different stationary phases were tested to achieve optimal separation conditions and experimental set-ups for the full range of antiepileptic drugs tested.The whole methodology will be subsequently validated according to the general guidelines for clinical and toxicological method validation according to international standards and its use in routine laboratory practice will be intended.
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Optimization of conditions for derivatization of fatty acids and perfluorinated organic acids with methylchloroformate
Hložek, Tomáš ; Čabala, Radomír (advisor) ; Feltl, Ladislav (referee)
Statistical methods designed for optimization were applied for selection of optimal experimental conditions of derivatization process for perfluorinated acids, PFA (C8, C10 ,C12), fatty acids, FA (C8, C12, C14) and their mixtures. The calibration curves and the extraction efficiences for system acetonitrile/heptane were measured for all studied acids. The Plackett-Burman screening was performed for obtaining the significant experimental parameters from their original series and significant ones were remitted to optimization via central composite design, which takes a second order effects and mutual interactions in between parameters into account. Optimal conditions of derivatization were found for PFA, FA and their mixtures. The principle of the search of optimal selective derivatization conditions for one group of acids in the presence of the other is based on the search of optimum on the response surface where the dependent variable is either sum or difference of acid group responses normalized to common internal standard. It was found that FA influence total response more than PFA and that in the case of demand for selective derivatization of PFA in the presence of FA in the sample, the most significant parameter is the volume of methylchloroformate added. The complete selectivity of...
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Disposition and metabolism of cannabinoids
Hložek, Tomáš
This thesis describes in the form of a commentary on own original publications research on the problems of cannabinoids, ie. phytocannabinoids and some synthetic cannabinoids, their pharmacokinetics and effects. The work consists of four thematic areas: the pharmacokinetics of delta-9- tetrahydrocannabinol (THC) and cannabidiol (CBD) in rats, depending on the route of administration; THC concentration time profile in humans (after inhalation) and implications for transport safety; the pharmacokinetic profile of synthetic cannabinoids in rats; extraction and determination of phytocannabinoids in plant material. The first part of the thesis was to determine pharmacokinetic profiles of THC, CBD and combination thereof (1:1 weight ratio) in rats with respect to administration common in humans, i.e. inhalation, oral and subcutaneous administration. THC, its metabolites (11-hydroxy-tetrahydrocannabinol, 11-OH-THC; 11-nor-delta-9- carboxytetrahydrocannabinol, THCOOH) and CBD concentrations in serum and brains of animals were monitored at the 24 hours experimental interval during the study. Except for inhalation administration, co-administration of CBD inhibited THC metabolism (after both oral and subcutaneous), resulting in an increase in THC concentrations in both serum and brain of the rats relative to...
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Disposition and metabolism of cannabinoids
Hložek, Tomáš
This thesis describes in the form of a commentary on own original publications research on the problems of cannabinoids, ie. phytocannabinoids and some synthetic cannabinoids, their pharmacokinetics and effects. The work consists of four thematic areas: the pharmacokinetics of delta-9- tetrahydrocannabinol (THC) and cannabidiol (CBD) in rats, depending on the route of administration; THC concentration time profile in humans (after inhalation) and implications for transport safety; the pharmacokinetic profile of synthetic cannabinoids in rats; extraction and determination of phytocannabinoids in plant material. The first part of the thesis was to determine pharmacokinetic profiles of THC, CBD and combination thereof (1:1 weight ratio) in rats with respect to administration common in humans, i.e. inhalation, oral and subcutaneous administration. THC, its metabolites (11-hydroxy-tetrahydrocannabinol, 11-OH-THC; 11-nor-delta-9- carboxytetrahydrocannabinol, THCOOH) and CBD concentrations in serum and brains of animals were monitored at the 24 hours experimental interval during the study. Except for inhalation administration, co-administration of CBD inhibited THC metabolism (after both oral and subcutaneous), resulting in an increase in THC concentrations in both serum and brain of the rats relative to...
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Disposition and metabolism of cannabinoids.
Hložek, Tomáš ; Balíková, Marie (advisor) ; Miovský, Michal (referee) ; Šustková, Magdaléna (referee)
This thesis describes in the form of a commentary on own original publications research on the problems of cannabinoids, ie. phytocannabinoids and some synthetic cannabinoids, their pharmacokinetics and effects. The work consists of four thematic areas: the pharmacokinetics of delta-9- tetrahydrocannabinol (THC) and cannabidiol (CBD) in rats, depending on the route of administration; THC concentration time profile in humans (after inhalation) and implications for transport safety; the pharmacokinetic profile of synthetic cannabinoids in rats; extraction and determination of phytocannabinoids in plant material. The first part of the thesis was to determine pharmacokinetic profiles of THC, CBD and combination thereof (1:1 weight ratio) in rats with respect to administration common in humans, i.e. inhalation, oral and subcutaneous administration. THC, its metabolites (11-hydroxy-tetrahydrocannabinol, 11-OH-THC; 11-nor-delta-9- carboxytetrahydrocannabinol, THCOOH) and CBD concentrations in serum and brains of animals were monitored at the 24 hours experimental interval during the study. Except for inhalation administration, co-administration of CBD inhibited THC metabolism (after both oral and subcutaneous), resulting in an increase in THC concentrations in both serum and brain of the rats relative to...
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Optimization of conditions for derivatization of fatty acids and perfluorinated organic acids with methylchloroformate
Hložek, Tomáš ; Feltl, Ladislav (referee) ; Čabala, Radomír (advisor)
Statistical methods designed for optimization were applied for selection of optimal experimental conditions of derivatization process for perfluorinated acids, PFA (C8, C10 ,C12), fatty acids, FA (C8, C12, C14) and their mixtures. The calibration curves and the extraction efficiences for system acetonitrile/heptane were measured for all studied acids. The Plackett-Burman screening was performed for obtaining the significant experimental parameters from their original series and significant ones were remitted to optimization via central composite design, which takes a second order effects and mutual interactions in between parameters into account. Optimal conditions of derivatization were found for PFA, FA and their mixtures. The principle of the search of optimal selective derivatization conditions for one group of acids in the presence of the other is based on the search of optimum on the response surface where the dependent variable is either sum or difference of acid group responses normalized to common internal standard. It was found that FA influence total response more than PFA and that in the case of demand for selective derivatization of PFA in the presence of FA in the sample, the most significant parameter is the volume of methylchloroformate added. The complete selectivity of...
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