Determination of brequinar in rat plasma by direct deproteinization and reversed-phase high-performance liquid...

A direct deproteinization method for the determination of brequinar in rat plasma by high-performance liquid chromatography (HPLC) has been developed. This assay avoids the use of dichloromethane, a known carcinogen, in an existing extraction method. Acetonitrile was used to denature plasma proteins and the supernatant was injected onto the HPLC column. Chromatographic separation of brequinar was conducted on a Biophase octyl column using a mixture of acetonitrile and 0.1 M phosphoric acid (50:50, v/v) as the mobile phase and detection of brequinar was by UV absorbance at 254 nm. The method has been validated in rat plasma over the concentration range of 0.05-50.00 micrograms/ml which is adequate for the determination of pharmacokinetics of brequinar in animals.