Analysis of biologically active compounds using high performance separation methods

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...