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A fully automated HPLC method for the determination of catecholamines in biological samples utilizing ethylenediamine condensation and peroxyoxalate chemiluminescence detection
Author(s) -
Prados Pablo,
Higashidate Sakae,
Imai Kazuhiro
Publication year - 1994
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130080102
Subject(s) - chemistry , peroxyoxalate , chromatography , chemiluminescence , detection limit , high performance liquid chromatography , derivatization , extraction (chemistry) , solid phase extraction , ethylenediamine , oxalate , analytical chemistry (journal) , inorganic chemistry , organic chemistry
A fully automated in‐line extraction reversed‐phase high‐performance liquid chromatography (HPLC) method with chemiluminescence detection was developed for the analysis of human and rat plasma catecholamines (CAs), norepinephrine (NE), epinephrine (E) and dopamine (DA). N ‐Methyldopamine ( N ‐MeDA) was used as an internal standard. The method involves collection of plasma samples, which are first diluted with a sample dilution buffer containing N ‐MeDA, and in‐line extraction of CAs using a carboxylic acid small resin precolumn (SERUMOUT‐CEX). This pre‐extraction process was coupled with an HPLC system including reversed‐phase mode separation on an analytical column (TSK gel ODS‐80Ts), fluorogenic derivatization with ethylenediamine (ED) and finally postcolumn peroxyoxalate chemiluminescence reaction detection using bis [4‐nitro‐2‐(3,6,9‐trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) and hydrogen peroxide. The optimized mobile phase compositions, flow rates, operation timing for the adsorption and desorption of CAs in the precolumn, the separation in the analytical column and the optimum fluorogenic and chemiluminogenic reaction conditions were investigated. The detection limit for all the CAs was about 1 fmol (signal‐to‐noise ratio is 2). Excellent linearity of the calibration curves for CAs was observed in the range from 5 to 500 fmol for each CA using the internal standard. The relative standard deviations of the method for determining NE (183 fmol), E (23.6 fmol) and DA (6.1 fmol) in 50 μL of human plasma ( n = 3) were 2.8, 2.7 and 3.1%, respectively, for the within‐day assay and 5.0, 3.8 and 4.0%, respectively, for the between‐day assay. The method was applicable to the determination of CAs in 25–50 μL of human or rat plasma.