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

Abstract

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(III) chloride and EDTA. Fluorescence detection ( λ ex=300 nm, λ 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 1.0×10 −8, 4.0×10 −8 and 7.0×10 −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 (4.0×10 −7, 2.0×10 −6 and 4.0×10 −6 mol l −1), E (8.2×10 −8, 4.1×10 −7 and 8.2×10 −7 mol l −1) and DA (1.0×10 −6, 5.0×10 −6 and 1.0×10 −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%.