Determination of the monoamine oxidase B inhibitor Ro 19-6327 in plasma by high-performance liquid chromatography using precolumn derivatization with fluorescamine and fluorescence detection

Abstract

A specific high-performance liquid chromatographic (HPLC) method using precolumn derivatization and fluorescence detection was developed for the determination of the monoamine oxidase B inhibitor Ro 19-6327 in human plasma. After extraction of the basified plasma with tert-butyl methyl ether—1-butanol (8:2, v/v) and back-extraction into dilute phosphoric acid, the solution was neutralized with phosphate buffer and the drug derivatized with fluorescamine. The derivative was chromatographed on a reversed-phase C 8 column, using phosphate buffer—acetonitrile (68:32, v/v) as mobile phase, and fluorescence detection (excitation 370 nm, emission 485 nm). The limit of quantification was 1 ng/ml using 1 ml of plasma. The recovery was 79% in the range 5–200 ng/ml and the inter-assay precision was 3.1–7.9% in the range 2–500 ng/ml. The compound proved to be stable in human plasma. Moderate instability was found in rat plasma and, surprisingly, severe instability in dog plasma. Measures for handling unstable dog plasma samples are described. An HPLC method with UV detection was used for the analysis of dog and rat plasma samples, which is also described briefly. The fluorescence method, which was five times more sensitive than the UV method, was successfully applied to a human tolerance study.