Determination of femtomole concentrations of catecholamines by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

Abstract

A highly sensitive method for determination of the plasma catecholamines, norepinephrine (NE), epinephrine (E) and dopamine (DA) is described. The method consists of the extraction of the catecholamines, using 3,4-dihydroxybenzylamine as internal standard, from plasma with alumina (5 mg), followed by a reversed-phase column separation, on-column fluorogenic derivatization with ethylenediamine (ED) and post-column peroxyoxalate chemiluminescent reaction detection utilizing bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO) and hydrogen peroxide. In order to optimize the reaction conditions for high-performance liquid chromatography to obtain highly sensitive detection, the effects of changing reagent compositions on the chemiluminescence yield were investigated. The following are the optimized conditions. Eluent, a mixture of 50 mmol l^–1 potassium acetate (pH 3.20)–50 mmol l^–1 potassium phosphate (pH 3.20)–acetonitrile (90.15. + 4.85 + 3v/v/v) containing 1 mmol l^–1 sodium hexanesulfonate (40 °C) and flow rate, 0.5 ml min^–1. Fluorogenic reagent solution, 105 mmol l^–1 ED and 175 mmol l^–1 imidazole in acetonitrile–ethanol (90 + 10 v/v) and flow rate, 0.25 mmol l^–1. Reaction coil (15 m × 0.5 mm i.d.) heated at 80 °C. Chemiluminogenic reagent solution, 0.25 mmol l^–1 TDPO, 150 mmol l^–1 hydrogen peroxide and 110 mmol l^–1 trifluoroacetic acid in dioxane–ethyl acetate (50 : 50 v/v) and flow rate, 1.4 ml min^–1. The detection limits for all the catecholamines were 1 fmol (signal-to-noise ratio at 2). The standard deviations of the method for the determination of NE, E and DA added to rat plasma (2.5 nM) were 3, 3 and 4%, respectively. The concentrations of rat plasma catecholamines measured by the proposed method were NE 1.21, E 0.12 and DA 0.63 pmol ml^–1 and were lower than those obtained previously by radioenzymic-paper chromatography.