Improved solid-phase extraction and liquid chromatography with electrochemical detection of urinary catecholamines and 5-S-L-cysteinyl-L-dopa

Abstract

We describe a rapid, precise, accurate liquid chromatographic procedure for determining urinary catecholamines and 5-S-L-cysteinyl-L-dopa. The catecholamines (norepinephrine, epinephrine, and dopamine) and 5-S-L-cysteinyl-L-dopa are extracted from 1.0 ml of urine together with internal standards, by using a Bond-Elut strong cation-exchange (SCX) and an affinity phenylboronic acid (PBA) extraction column in series. The eluate obtained from PBA column is then chromatographed on a reversed-phase C18 column with a mobile phase containing pentane- and heptanesulfonate as ion-pair reagents. The detection is achieved with an amperometric detector set at an oxidation potential of +0.55 V. The chromatography is complete is less than 8 min for catecholamines and less than 5 min for cysteinyldopa. The method can measure less than 2 micrograms/l for catecholamines and 5 micrograms/l for cysteinyldopa. Analytical recoveries of catecholamines and cysteinyldopa added to urine pool ranged from 90-107%. Between run coefficient of variation ranged from 4.7 to 8%. None of the drugs and catecholamines metabolites tested interfered with the assay.