Determination of 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in human plasma by a simple and rapid high-performance liquid chromatography assay

In order to study the pharmacodynamic effects of drugs on dopamine and serotonin metabolism, a reversed-phase HPLC assay coupled with electrochemical detection (ECD) for measuring plasma concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) was developed, The system was operated isocratically using a mobile phase of aqueous 0.03 M KH2PO4 buffer containing 0.15 mM EDTA in methanol (8.75:1.25), with a final pH of 4.0. The flow rate was set at 1.5 mL/min, and potentials at +450 mV. Using a signal to noise ratio of >3, the minimum detection limit assessed by direct on-column injection of a standard solution for DOPAC and 5-HIAA was <1 pg. The assays were linear from basal concentrations (1-10 ng/mL) to 100 ng/mL. The intra- and interassay variations were <10% and <20%, respectively.