高速液体クロマトグラフィーと電気化学検出器の組み合せによる血漿カテコールアミンの測定方法の検討

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It is known that changes in the concentration of plasma catecholamine are closely related to the sympathetic nerve system and adrenal function. The determination of plasma catecholamine is, therefore, a very useful way to investigate the disorder of catecholamine synthesis and metabolism and also to diagnose certain diseases. Catecholamine is a substance that exists in body fluids as a very small and labile component. Though several analytical methods of catecholamine are available at present and can be converted to the quantitative determination of plasma catecholamine, nothing has been admitted yet as an established determining method. In this experiment we attempted to test whether or not a combination of high performance liquid chromatography with electrochemical detection could be used as an efficient method of plasma catecholamine determination. The results obtained are as follows : 1) The effect of stabilizing reagents for the recovery of catecholamine : Five different reagents of a) ascorbic acid b) sodium thiosulfate c) EDTA-2Na d) sodium thiosulfate and e) sodium thiosulfate plus EDTA-2Na, were tested, and the reagent of sodium thiosulfate plus EDTA-2Na was concluded to be the most effective stabilizer among them. Moreover at this combination of the reagent, plasma CA was kept stable under - 20°C for 20 days. 2) The effect of protein precipitant : Perchloric acid was employed as a precipitant of the plasma protein. Among four different concentrations of 0.1, 0.5, 1.0 and 2.0N, the 0.5N concentration was disclosed to be the most effective concentration for the recovery of catecholamine in the supernatant. 3) The amount of alumina : Three different amounts of alumina (5, 10 and 50mg) were tested, but no difference was observed between 10 mg and 50mg. pH dependency of catecholamine adsorption on the alumina : Maximum adsorption was observed at pH 8.5 when the experiments were performed with 0.5M tris HC1 buffer. 4) The effects of voltage of electrochemical detector on the accuracy of the determination : Among six different voltages from 0.2 to 1.0 tested, 0.6V was concluded to be the most efficient voltage for the accuracy of determination. 5) DHBA as an internal standard of catecholamine : It was determined that DHBA could be used as an internal standard of the catecholamine. 6) The comparison between the double isotope method and this method : Results from the two different methods correlated quite well, indicating that the method reported in this paper can be used as a reliable quantitative determination of the plasma catecholamine, though the method seems to have several points that should be improved further.
日本内分泌学会の論文

高速液体クロマトグラフィーと電気化学検出器の組み合せによる血漿カテコールアミンの測定方法の検討

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