Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection

We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C18 column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 microliters of the mobile phase. A 10-microliters aliquot of the resulting solution is injected onto a 150 mm x 4.6 mm I.D. column packed with 5-microns octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E1 = +0.25 V and E2 = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90-95% which allows the determination of 0.5 microgram/l analyte.