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(III) chloride and EDTA. Fluorescence detection (λex=300 nm, λem=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%.