A rapid HPLC-ED method for the analysis of homovanillic acid (HVA) in human plasma

Homovanillic acid (HVA, 4-hydroxy-3-methoxyphenylacetic acid) is one of the main metabolites of dopamine; for this reason, the monitoring of its levels in human plasma can be a useful research tool for assessing physiological and pathological conditions. In fact, HVA concentrations in human blood can be used as markers for neuroblastoma, and may be the expression of dopamine metabolism which is involved in reward systems, emotional responses, personality traits and psychopathology. The changes occurring in the levels of HVA could be of great importance for the diagnosis of behavioural disorders, therefore, it is important to have at disposal reliable analytical methods for the determination of HVA in human plasma. A rapid and feasible HPLC method has been developed for this purpose. Chromatographic analysis is carried out on a C8 reversed-phase column, using a mobile phase composed of acetonitrile, methanol and aqueous phosphate buffer. Coulometric detection has been used to obtain the highest sensitivity, and is carried out as follows: conditioning cell set at +100 mV; analytical cell set at detector 1 = -200 mV and detector 2 = +500 mV. Under these experimental conditions HVA is detected as a neat oxidation peak with a retention time of 6.2 minutes. The application of the method to plasma samples requires a careful sample pre-treatment step, which is carried out by means of a solid-phase extraction procedure. A 500-µL aliquot of plasma is loaded onto a C8 (50 mg, 1 mL) cartridge after addition of pH 2.5 phosphate buffer; the analyte is then eluted with the mobile phase. Preliminary assays with this procedure provided satisfactory extraction yield values, as well as good sample purification from matrix interference. The method seems thus to be promising for a sensitive and selective determination of HVA in human plasma.