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Analysis of biologically active compounds using high performance separation methods
Adamusová, Hana ; Bosáková, Zuzana (advisor) ; Jelínek, Ivan (referee) ; Tůma, Petr (referee)
In the first part of this doctoral thesis, a new analytical HPLC-MS/MS method for monitoring of concentration changes of 17β-estradiol (βE2) during in vitro mouse sperm capacitation was developed. Capacitation was performed for three initial concentrations of βE2 (200, 20 and 2 μg/L). For all the concentrations a similar trend for the total unbound βE2 was observed. In general, the βE2 concentration decreased to reach its minimum and then increased again. The position of the minimum differed for the individual tested βE2 concentrations. Experimentally obtained results were subjected to the kinetic analysis. The curves fitted through the experimentally determined points displayed an autocatalytic character. For the agreement between the curves obtained by fitting through the experimental points and the theoretical calculated curves, it is necessary to assume that the first step is adsorption of βE2 onto the surface of the sperm controlled by Langmuir isotherm. The kinetic study was also used to study the effects of fluorides and aluminium fluoride complexes on the capacitation of mouse sperm. The experimental points were in very good agreement with the shape of the theoretical curves and this fact verifies the mechanism of the mouse sperm capacitation kinetics. In the second part of this work, two...
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Analysis of biologically active compounds using high performance separation methods
Adamusová, Hana ; Bosáková, Zuzana (advisor) ; Jelínek, Ivan (referee) ; Tůma, Petr (referee)
In the first part of this doctoral thesis, a new analytical HPLC-MS/MS method for monitoring of concentration changes of 17β-estradiol (βE2) during in vitro mouse sperm capacitation was developed. Capacitation was performed for three initial concentrations of βE2 (200, 20 and 2 μg/L). For all the concentrations a similar trend for the total unbound βE2 was observed. In general, the βE2 concentration decreased to reach its minimum and then increased again. The position of the minimum differed for the individual tested βE2 concentrations. Experimentally obtained results were subjected to the kinetic analysis. The curves fitted through the experimentally determined points displayed an autocatalytic character. For the agreement between the curves obtained by fitting through the experimental points and the theoretical calculated curves, it is necessary to assume that the first step is adsorption of βE2 onto the surface of the sperm controlled by Langmuir isotherm. The kinetic study was also used to study the effects of fluorides and aluminium fluoride complexes on the capacitation of mouse sperm. The experimental points were in very good agreement with the shape of the theoretical curves and this fact verifies the mechanism of the mouse sperm capacitation kinetics. In the second part of this work, two...
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Optimization of derivatization conditions of selected amino acids with FMOC-Cl
Chytrová, Marie ; Bosáková, Zuzana (advisor) ; Adamusová, Hana (referee)
Chiral HPLC method (mobile phase methanol/0.5% triethylammonium acetate buffer, pH 6.0 40/60 (v/v), teicoplanin based chiral stationary phase, fluorescence detection - - ex 254 nm, em 314 nm) was used for studying a recovery of derivatization procedure for D/L-alanin, D/L-valin, D/L-leucin and D/L-isoleucin. 9-Fluorenylmethyl chloroformate (FMOC-Cl) was used as a derivatization agent. The influence of derivatization temperature (22, 40 and 60 řC) and pH value of 0.2 M NaHCO3 (8.5 - 9.5) on derivatization recovery was monitored in terms of D-enantiomer peak areas for the individual amino acid. The optimal derivatization conditions were found to be pH 8.5 and temperature 40 řC. The developed method was applied to analysis of the real sample, food supplement BCCA Extreme Pure, containing L-isoleucin, L-leucin and L-valin. The sample was D-enantiomers free. Key words: chiral HPLC, teicoplanin, amino acids, derivatization, 9-fluorenylmethyl chloroformate (FMOC-Cl).
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Study of influence of 17β-estradiol and 17α-ethynylestradiol on capacitation of mouse sperm in vitro.
Adamusová, Hana ; Bosáková, Zuzana (advisor) ; Pacáková, Věra (referee)
Environmental estrogens (natural and synthetic) belong to a group of contaminants called endocrine disrupting chemicals (EDCs). These compounds interfere with a function of endocrine system. They can bind to natural estrogen receptors or block synthesis of endogenous hormones. 17β-Estradiol (BE2) is a natural endocrine disruptor produced more by women than men. Also a synthetic 17α-ethynylestradiol (EE2) belongs to EDCs. It is used as a main part of birth control pills. Together with other endocrine disruptors EE2 is probably responsible for feminization of male animals living in effluents of waste water treatment plants. The aim of this thesis was to develop a method for determining free 17β-estradiol and 17α-ethynylestradiol available to capacitating sperm. In order to determine a status of BE2 during mouse sperm capacitation in vitro, a high performance liquid chromatography (HPLC) method with tandem mass spectrometry (MS/MS) detection was used. A reversed-phase separation mode using a SunFire C18 column with a simple mobile phase composed of acetonitrile and water at the ratio 40/60 (v/v) containing 0,1% formic acid in both components was applied. Under the optimized separation conditions, calibration curves for 17β-estradiol and 17α-ethynylestradiol were measured in the concentration range of 1-250...
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