DETERMINATION OF (+)- AND (-)-GOSSYPOL IN HUMAN PLASMA USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH PRE-COLUMN CHEMICAL DERIVATIZATION

A new assay method for the determination of (+)- and (-)- gossypol using HPLC with pre-column chemical derivatization has been developed. 2, 6-dimethyl-naphthalene was used as internal standard and (+)-2-amino-l-butanol as the chiral derivatizing agent. (+)- and (-)- gossypol derivatives were separated completely on an ODS column. Methanol-isopropanol-water-phosphorie acid (80:20:20:0.1) was used as the mobile phase. The wave length of UV detector was settled at 254 nm. The retention times of (-)- and (+)-gossypol derivatives were 4.2 and 7.7 min, respectively.Acetonitrile-treated protein-free plasma samples were prepared, and (+)-2-amino-l-butanol was added to a final concentration of 2%. The system was kept at 75℃ for 45 min. After solvent demixing by adding sodium chloride, 50～100μl of acetonitrile phase was injected into the column.The linearities with (+)- and (-)-gossypol plasma sample ranged from 0.125 to 2.0μg/ml. The absolute recoveries of (+)-, (-)-gossypol and internal standard were found to be 98.2%, 94.1% and 94.6%, respectively, with within-day and dayto-day variations less than 6% for both (+)- and (-)-gossypol. The sensitivity for detection of (+)- or (-)- gossypol was found to be 0.125μg/ml (S/N 3:1). This method is considered to be suitable for some clinical and experimental studies of gossypol.