High-performance liquid chromatography with electrochemical detection of gossypol in human plasma.

A sensitive and selective high-performance liquid chromatographic method with electrochemical detection for the determination of gossypol in human plasma is described. Glutathione is used as a protective agent and gossypol dimethyl ether as an internal standard. Acetonitrile-treated protein-free plasma sample is first introduced on to a C18 pre-column for enrichment and clean-up. By using a column-switching technique, gossypol and the internal standard are subjected to further separation on a C8 analytical column, while the major interfering components are eliminated before entering the column. Methanol-0.1 M citrate buffer (pH 3.2) (80:20) is used as the mobile phase. The detector potential on the glassy carbon electrode is maintained at +0.6 V vs. an Ag-AgCl reference electrode. The linearity with human plasma ranged from 5 to 250 ng/ml. The absolute recoveries of gossypol and gossypol dimethyl ether were 91.3 and 97.5%, respectively, with a within-day precision of 2.5% and a day-to-day precision of 3.8%. The limit of detection is 5 ng/ml (signal-to-noise ratio = 3:1). The method is considered to be suitable for the clinical pharmacokinetic studies of gossypol.