Determination of plasma levels of homovanillic acid (HVA), as an index of dopaminergic system function, by HPLC with amperometric detection and a new SPE procedure

Homovanillic acid (HVA, 4-hydroxy-3-methoxyphenylacetic acid) is one of the main metabolites of dopamine and can thus be used as an index of dopamine metabolism which is involved in reward systems, emotional responses, personality trait expression and psychopathological phenomena. The changes occurring in the levels of HVA can be of great importance for the diagnosis of behavioural disorders. Therefore, it is fundamental that reliable analytical methods are available for the determination of homovanillic acid in human plasma. A HPLC method with amperometric detection is currently under development for this purpose and preliminary results seem to be quite promising. Homovanillic acid is analysed on a C8 column (250×4.6 mm I.D., 5 µm) with a mobile phase composed of MeOH and a pH 4.8 citrate buffer (10:90) also containing octanesulfonic acid and EDTA. Electrochemical detection is carried out using an Antec Decade amperometric detector (working electrode: glassy carbon; reference electrode: Ag/AgCl; auxiliary electrode: stainless steel) set at + 0.800 V. Solid-phase extraction (SPE) on strong anion exchange (SAX) cartridges was chosen for sample purification and pre-concentration. After conditioning, 500 µL of plasma are loaded onto the cartridge, followed by suitable washing and elution, obtaining a final sample volume of 250 µL (1:2 pre-concentration). Assays are currently underway to validate the method and to apply it to plasma samples from "at risk" adolescents for studies regarding neuroendocrine responses.