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The method of determining catecholamines has been studied, and such sensitive methods as radioenzymatic assay and gas chromatography are used for their measurement, but these techniques are not widespread because of their complexity. In this study, we developed a high pressure liquid chromatographic method with a fluorescent reaction system for the determination of plasma, urine and tissue catecholamine concentration. The eluates from plasma, urine and tissue were chromatographied on a Hitachi #3011C Gel (2.6 x 250mm) and reacted to the mixed solution of 0.1M KH2PO4, 0.2M K2HPO4 and 0.0075% K3Fe(CN)6 (the first solution), followed by the mixture of 0.1% ascorbic acid and 5N NaOH. A stock solution (0.1mg/ml of norepinephrine, 0.1mg/ml of epinephrine and 2.0mg/ml of dopamine) was stored in 0.2N CH3COOH at 4 degrees C, and was diluted at 1/5,000-1/40,000 when needed. Recovery of norephinephrine after extraction was 54.3-58.9% and that of epinephrine was 49.2-55.8%. The accuracy was acceptable and sensitivity was 30-50pg. The mean plasma norepinephrine concentrations (p-NE) were 105.6 +/- 6.8pg/ml in normal subjects and 157.7 +/- 74.5pg/ml in essential hypertensives (EH). The mean epinephrine concentrations (p-E) were 30.8 +/- 16.3pg/ml in normals and 20.0 +/- 11.9pg/ml in EH. PNE increased after standing or after furosemide plus 2-hour standing, but p-E did not increase as much as p-NE. Catecholamine concentration in the rat brain and adrenal medullary tissue could be determined by the use of this method.

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A Simple and Rapid Assay for the Determination of Plasma Urine and Tissue Catecholamine Concentrations by High Pressure Liquid Chromatography with Fluorescent Reaction