Determination of Free and Total Catecholamines in Human Erythrocytes, Platelets and Plasma by High Performance Liquid Chromatography with Fluorescence Detection

• Published in: Analytical Sciences Vol. 2; no. 3; pp. 303 - 308
• Main Authors: NOHTA, Hitoshi, MITSUI, Akane, UMEGAE, Yoshihiko, OHKURA, Yosuke
• Format: Journal Article
• Language: English
• Published: Tokyo The Japan Society for Analytical Chemistry 1986; Japan Society for Analytical Chemistry; Japan Science and Technology Agency
• Subjects: 1,2-diphenylethylenediamine; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Catecholamine; erythrocyte; Fundamental and applied biological sciences. Psychology; high performance liquid chromatography; Non peptidic neurotransmitters, polyamines; Other biological molecules; platelet; Assay; Human; Blood cell; Reversed phase chromatography; Fluorogenic reagent; HPLC chromatography; Neurotransmitter; Catecholamine; Ion exchange chromatography; Blood plasma
• ISSN: 0910-6340; 1348-2246
• DOI: 10.2116/analsci.2.303

A simple and highly sensitive method for the determination of free and total (free+conjugated) catecholamines (norepinephrine, epinephrine and dopamine) in human erythrocytes, platelets and plasma is described which employs high performance liquid chromatography with fluorescence detection. Conjugated atecholamines are hydrolyzed by sulfatase-mediated reaction to the corresponding free amines. After cation exchange chromatography on a cartridge of a cation exchanger, Toyopak SP, catecholamines and isoproterenol (internal standard) in a sample are converted into the corresponding fluorescent compounds by reaction with 1, 2-diphenylethylenediamine. These compounds are separated on a reversed-phase column, TSK-gel ODS-120T, with isocratic elution using a mixture of water, methanol and acetonitrile containing a Tris•hydrochloric acid buffer (pH 7.0). The detection limit for each catecholamine is a. fmol/100-μl injection volume.