OPTIMIZATION OF PHOTOLUMINESCENCE-FOLLOWING ELECTRON-TRANSFER WITH TRIS(2,2'-BIPYRIDYL)OS(III) AND APPLICATION TO THE DETERMINATION OF DOPAMINE, SEROTONIN AND CARBIDOPA IN BRAIN MICRODIALYSATE

Biogenic monoamines such as dopamine and serotonin play important roles as major neurotransmitters. Simultaneous determination of the concentration changes is thus crucial to understand brain function. We have developed a capillary HPLC combined with a redox-based postcolumn reaction method, the photoluminescence-following electron-transfer (PFET) technique, for the determination of certain neurotransmitters and their metabolites. Tris(2,2'-bipyridine)osmium was the postcolumn oxidant. The spectroscopic properties of the oxidant changed upon a redox reaction. The change was measured using a laser-induced fluorescence setup to quantify electrochemically active analytes from the chromatographic eluent. Spectroscopic and electrochemical properties of the complex, along with its reaction kinetics were studied in detail to optimize the postcolumn reaction efficiency. The complex was capable of oxidizing and detecting catechols. The spectroscopic properties of the complex were not very advantageous, but careful control of the detection system and reaction conditions enabled sensitive detection of monoamine neurotransmitters in rat brain dialysates. The extent of the postcolumn reaction was predicted by the second-order reaction kinetics in fluidic conditions and experimentally verified. Efficient mixing and reaction of the postcolumn reagent was achieved in a radial diffusion mixer without significant band broadening. The optimized system was capable of detecting biogenic monoamines, such as dopamine, serotonin and 3-methoxytyramine, at about 200 pM concentrations in 500 nL samples. Typical analysis time was less than 10 min. We used this method to monitor neurotransmitter concentrations in rat brain dialysates when the rat was given tetrodotoxin or nomifensine. Changes in neurotransmitter concentrations were observed. Another application of this method was detecting carbidopa in rat brain microdialysates, to study the damage in brain tissues by a microdialysis probe. We observed that carbidopa was present in striatal dialysates after the rat was given i.p. injection of carbidopa