Determination of homovanillic acid (HVA) in human plasma by HPLC with coulometric detection and a new SPE procedure

A sensitive high-performance liquid chromatographic method has been developed for the determination of homovanillic acid (HVA), the main metabolite of dopamine, in human plasma. Analyses were carried out on a reversed-phase column (C8, 250×4.6 mm I.D., 5 μm) using a mobile phase composed of 10% methanol and 90% aqueous citrate buffer, containing octanesulfonic acid and EDTA at pH 4.8. Coulometric detection was used, setting the guard cell at +0.100 V, the first analytical cell at -0.200 V and the second analytical cell at +0.500 V. A careful solid-phase extraction procedure, based on strong anion exchange (SAX) cartridges (100 mg, 1 mL), was implemented for the pre-treatment of plasma samples. Extraction yield was satisfactory, the mean value being 98.0 %. The calibration curve was linear over the concentration range of 0.2-25.0 ng/mL of homovanillic acid. The limit of quantitation (LOQ) was 0.2 ng/mL and the limit of detection (LOD) was 0.1 ng/mL. The method was successfully applied to plasma samples of former alcohol, cocaine and heroin addicts. Results were satisfactory in terms of precision and accuracy. Hence the method is suitable for the determination of homovanillic acid in human plasma.