Post-column terbium complexation and sensitized fluorescence detection for the determination of norepinephrine, epinephrine and dopamine using high-performance liquid chromatography

Terbium sensitized fluorescence was used as a post-column detection system to develop a simple, sensitive and rapid high-performance liquid chromatographic method for the simultaneous determination of catecholamines norepinephrine (NE), epinephrine (E) and dopamine (DA). Catecholamines were separated by an ion-pair reversed-phase chromatography on a BDS-Hypersil analytical column with a mobile phase of methanol and 50 mmol l(-1) acetate buffer (pH 4.7) containing 1.1 mmol l(-1) SOS and 0.11 mmol l-1 EDTA (15 + 85 v/v). Catecholamines and the internal standard (3,4-dihydroxybenzylamine, DHBA) were post-column derivatized by the addition to the eluent of an alkaline solution containing a stoichiometric mixture of terbium(111) chloride and EDTA. Fluorescence detection (lambda(ex) = 300 nm, lambda(em) = 545 nm) is based on the sensitization of terbium ion fluorescence after complexation with catecholamines. The chemical compatibility between the eluent and the post-column reagent was studied and the analytical characteristics of the method were established. Detection limits found were 1.0 x 10(-8), 4.0 x 10(-8) and 7.0 x 10(-8) mol l(-1) for NE, E and DA, respectively. The method has been successfully applied to the determination of catecholamines in urine samples after solid-phase extraction (SPE) pre-treatment. Recoveries from urine spiked with NE (4.0 x 10(-7), 2.0 x 10(-6) and 4.0 x 10(-1) mol l(-1)), E (8.2 x 10(-8), 4.1 X 10(-7) and 8.2 x 10(-7) mol l(-1)) and DA (1.0 x 10(-6), 5.0 x 10(-6) and 1.0 x 10(-5) mol l(-1)) varied from 98 to 100% (mean = 99.3%), from 106 to 107% (mean = 106.3%) and from 98 to 101% (mean = 99.3%), respectively. The between-run precision (relative standard deviation, R.S.D.) for the method for three urine samples at different concentration levels of each catecholamine varied from 3.6 to 7.0%. (C) 2002 Elsevier Science B.V. All rights reserved.