Postcolumn derivatization of catecholamines with 2-cyanoacetamide for fluorimetric monitoring in high-performance...

Catecholamines in the eluates of high-performance liquid chromatography were reacted with 2-cyanoacetamide in borate buffer in flow analysis mode, and the intensity of fluorescence developed was recorded. Under the optimum conditions (column: Hitachi 3011 C, 25 cm X 2.6 mm I.D., 45 degrees C; eluent: 0.05 M KH2PO4 containing 0.05% H3PO4, 0.60 ml/min; reagent solution: a mixture of 1% 2-cyanoacetamide, 0.50 ml/min, and a 0.60 M H3BO3--KOH buffer, 1.0 ml/min; size of reaction coil: 5 m X 0.5 mm I.D.; reaction temperature: 100 degrees C; wavelengths for detection: 383 nm for excitation and 486 nm for emission), this method allowed simultaneous determination of 5-500 pmol catecholamines with high reproducibility. The lower limits of detection (signal-to-noise ratio = 2) for epinephrine, norepinephrine and dopamine were 0.28, 0.11 and 0.098 pmol, respectively. Some applications of the analysis of urinary and serum catecholamines are also presented.