Analysis of gossypol by high performance liquid chromatography

Several high performance liquid chromatographic methods for the analysis of gossypol in different kinds of sample were developed. (1) Pure gossypol: separation on C18 column with MeOH/H2O/CHCl3 (70:30:40) containing 0.1% H3PO4 as mobile phase or on SO3H column with MeOH/citrate buffer (pH 6.3) (55:45) as mobile phase was recommended. With these systems, minute amounts of contaminants difficult to separate by other HPLC systems could be determined. (2) Plant material: acetone was selected as the extraction solvent. After evaporation of acetone from the extract, the residue was redissolved in 1% HOAc in CHCl3. An aliquot of this solution was chromatographed and quantified by peak area method. The mean recovery of pure gossypol added to plant material was 91.1 +/- 1.1% (S.D.). (3) Plasma sample: a HPLC method with electrochemical detector was developed. A plasma sample with glutathione as protective agent and gossypol dimethyl ether as internal standard was introduced on to a C18 pre-column. By using column-switching technique, a certain part of the eluate containing gossypol and gossypol dimethyl ether was subjected to a C8 analytical column for further separation. MeOH/citrate buffer (pH 3.2) (80:20) was used as the mobile phase. The optimum potential for detection was +0.6 V vs. Ag-AgCl. The assay sensitivity was 5 ng/ml. This method is sensitive and selective, suitable for clinical pharmacokinetic studies.