Abstract
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.
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