Simultaneous Automated Determination of Catecholamines, Serotonin, and Their Metabolites in Brain Tissue by HPLC and Electrochemical Detection

Abstract

A robust procedure with minimal tissue sample pretreatment, based on reversed-phase, ion-pair high performance liquid chromatography (HPLC) combined with electrochemical detection has been developed for the automated and simultaneous determination of the biogenic amines norepinephrine (NE), epinephrine (E), dopamine (DA), 5-hydroxytryptamine (5-HT) and their metabolites 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), 3,4-dihydroxyacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (H I A A) in as little as 0.5 mg brain tissue. The chromatographic conditions remain stable for at least 8 weeks. In particular small amounts of samples which do not allow reexamination are routinely automatically analysed without close supervision. The sample pretreatment comprises only homogenization of the tissue samples in the HPLC eluent and centrifugation. The supernatant can be directly injected into the HPLC system without further purification. Moreover, optimal concentrations of octane sulfonic acid (230 μM) and methanol (6%; v/v) in the recirculating eluent, a reversed-phase analytical column filled with 3 μm Nucleosil 100 C₁₈ material, constant column temperature (22 ± 1 °C) and an electrochemical detector with a dual electrode permitted baseline resolution without interference with the pharmacological dose of alpha-methyl-p-tyrosine frequently involved in catecholamine turnover studies as well as the determination of catecholamines, serotonin and their metabolites in femtomolar quantities.