ИСТИНА

To establish the methods for catecholamines and their metabolites determination, we used two promising approaches. One of them includes the formation of the intensely fluorescent complexes of the analytes with lanthanoid ions; another one consists in obtaining the strongly fluorescent derivatives of catecholamines and their metabolites with aromatic amines in the presence of the enzyme – horseradish peroxidase. In the first case we used the system based on the intensively fluorescent ternary complex of analytes with europium and oxytetracycline. Such system provides the enhancement of fluorescence of catecholamines up to 200-400-fold in comparison with their intrinsic fluorescence and moves the maximum of emission batochromically. In the second system, multiplex determination of catecholamines and their metabolites simultaneously in one sample was achieved due to directed regulation of wavelengths of excitation and emission (in the ranges 300 – 356 and 425 – 480 nm, respectively) and application of two derivatization agents - meso-1,2-diphenylethylenediamine and benzylamine. Besides, first-order derivative fluorescence spectroscopy was applied for the resolution of their spectra. The conditions for the impregnation of the components of the indicator reactions of both above-mentioned systems to the polymer structures of chitosan and collagen gels in 96-well microplates were optimized. With the application of the developed sensor matrix the solid-phase indicator systems were established for highly sensitive, selective, and reproducible determination of individual markers of neuromediator exchange (dopamine, noradrenaline, normetanephrine, serotonin, homovanillic, and vanillylmandelic acids). The proposed procedures provide rapid registration of the analytical signal of a large number of samples at low concentrations of analytes: 1 fM – 1 nM). For instance, it is possible to determine dopamine in the concentration range 0.5 – 1.0 pМ with the detection limit 0.2 pМ (RSD, % = 4.3).