Determination of free and total catecholamines in human urine by HPLC with fluorescence detection

A simple and highly sensitive method for the determination of free and total (free + conjugated) catecholamines (norepinephrine, epinephrine and dopamine) in human urine is described which employs HPLC with fluorescence detection. Conjugated catecholamines (sulfate form) are hydrolyzed by a sulfatase‐mediated reaction to the corresponding free amines. After cation exchange chromatography on a Toyopak IC‐SPS cartridge, catecholamines and isoproterenol (internal standard) in urine samples were converted into the corresponding fluorescent compounds by reaction with 1, 2‐diphenylethylenediamine. These compounds were separated within 8 min on a reversed phase column 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 ca 2 fmol per 100 μL injection volume.