Determination of the catechol-O-methyltransferase inhibitor Ro 40-7592 in human plasma by high-performance liquid chromatography with coulometric detection

Abstract

A sensitive and specific high-performance liquid chromatographic method has been developed to measure the catechol-O-methyl-transferase (COMT) inhibitor 3,4-dihydroxy-4′-methyl-5-nitrobenzophenone (Ro 40-7592) in human plasma. The compound and the internal standard were extracted from plasma at pH 2 with n-butyl chloride-ethyl acetate (95:5, v/v). The extract was chromatographed on a reversed-phase column (Hypersil ODS, 5 μm) using a mixture of phosphate buffer (0.05 M, pH 2), methanol and tetrahydrofuran (45:55:5, v/v/v) as the mobile phase. Long-retained components were removed from the system by means of a simple column-switching system. Quantification of the catechol-O-methyltransferase inhibitor was performed by means of coulometric detection (0.15 V). The limit of quantification was about 1 ng/ml, using a 1-ml specimen of plasma. The recovery from human plasma was > 88%. The mean inter-assay precision was 5.3% in the range 2.5–1000 ng/ml. Linearity of the standard curve was obtained in the concentration range 2.5–500 ng/ml. The catechol-O-methyltransferase inhibitor was stable in human plasma when stored for six months at −20°C and for 24 h at room temperature. The practicability of the new method was demonstrated by the analysis of more than 400 plasma samples from a tolerance study performed in human volunteers.