Determination of free catecholamines in human urine by direct injection of urine into a liquid chromatographic column-switching system with fluorimetric detection

Abstract

An ion-exchange chromatographic method combined with ion exclusion was developed for the determination of free catecholamines in human urine. Catecholamines were separated by ion exclusion from most acidic and neutral impurities by filtration through an anion-exchange column with a hydrophilic matrix (Asahipak ES-502N) and the excluded catecholamines were separated by ion-exchange chromatography on a column of weakly acidic ion exchanger with a hydrophilic matrix (Asahipak ES-502C), connected in series to the Asahipak ES-502N column with a four-way automatic valve. A sodium succinate-borate buffer of pH 6.7 (0.035 mol of succinic acid, 0.0075 mol of borate and 0.5 mmol of ethylenediaminetetraacetate were dissolved in 1 kg of water and the pH of the solution was adjusted to 6.7 with sodium hydroxide) was used as the mobile phase, and the temperature of both columns was kept at 30°C. The catecholamines in the eluate were determined fluorimetrically by post-column derivatization with glycylglycine. A diluted urine sample was injected directly onto the first column. The first column was back-flushed with the mobile phase for 52.5 min after the elution of the catecholamines from the first to the second column. Then the columns were washed with the mobile phase for 10 min in the normal direction before the next sample was injected into the first column. Samples could be analysed every 70 min and 5 pmol/ml of epinephrine, 5 pmol/ml of norepinephrine and 25 pmol/ml of dopamine in human urine could be determined.