A FULLY AUTOMATED HPLC METHOD FOR THE DETERMINATION OF CATECHOLAMINESIN BIOLOGICAL SAMPLES UTILIZING ETHYLENEDIAMINE CONDENSATION AND PEROXYOXALATE CHEMILUMINESCENCE DETECTION

A fully automated in-line extraction reversed-phase high-performance l iquid chromatography (HPLC) method with chemiluminescence detection wa s developed for the analysis of human and Tat plasma catecholamines (C As), norepinephrine (NE), epinephrine (E) and dopamine (DA). N-Methyld opamine (N-MeDA) was used as an internal standard. The method involves collection of plasma samples, which are first diluted with a sample d ilution buffer containing N-MeDA, and in-line extraction of CAs using a carboxylic acid small resin precolumn (SERUMOUT-CEX). This pre-extra ction process was coupled with an HPLC system including reversed-phase mode separation on an analytical column (TSK gel ODS-80Ts), fluorogen ic derivatization with ethylenediamine (ED) and finally postcolumn per oxyoxalate chemiluminescence reaction detection using bis tro-2-(3,6,9 -trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) and hydrogen peroxide. T he optimized mobile phase compositions, flow rates, operation timing f or the adsorption and desorption of CAs in the precolumn, the separati on in the analytical column and the optimum fluorogenic and chemilumin ogenic reaction conditions were investigated.The detection limit for a ll the CAs was about 1 fmol (signal-to-noise ratio is 2). Excellent li nearity of the calibration curves for CAs was observed in the range fr om 5 to 500 fmol for each CA using the internal standard. The relative standard deviations of the method for determining NE (183 fmol), E (2 3.6 fmol) and DA (6.1 fmol) in 50 muL of human plasma (n = 3) were 2.8 , 2.7 and 3.1%, respectively, for the within-day assay and 5.0, 3.8 an d 4.0%, respectively, for the between-day assay. The method was applic able to the determination of CAs in 25-50 muL of human or rat plasma.