Determination of tryptophan, serotonine and melatonin in plants by using HPLC

This thesis deals with the development and optimization of a method for the determination of tryptophan and its metabolites - serotonin and melatonin - in plant material, in grapevine, during one analysis. It uses a high-pressure liquid chromatography. The theoretical part is about tryptophan, its metabolism and basic properties of its metabolites - serotonin and melatonin. Their occurrence in wine is also discussed. Analytical techniques by which these analytes can be determined are also provided. Then information about modern stationary phases, that are suitable for this species, is included. The experimental part consists of optimization of the method, measurement of calibration dependences and measurement of real samples. It is measured by the method of reverse phase chromatography. As first stationary phase it is used a C18 column with core-shell packing, second is a BEH Phenyl column. The mixture of 10 mM acetate buffer (pH = 4.5) and methanol is used as the mobile phase. For detection UV at wavelength 254 nm is used, then for greater sensitivity mass detectionis is used. The basic conditions for the experiment have been set. At the beginning of the analysis, the mobile phase contains 95 % (v/v) buffer and 5 % (v/v) methanol. Then the methanol content is linearly increased to 80 % (v/v) from..