Determination of gossypol enantiomers in plasma after administration of racemate using high-performance liquid chromatography with precolumn chemical derivatisation.

ABSTRACT

A high-performance liquid chromatographic assay with precolumn chemical derivatisation was developed for the determination of gossypol enantiomers in plasma, after administration of the racemate. Racemic gossypol acetic acid in plasma was extracted into acetonitrile and analysed using a reversed-phase column and a coulometric detector in the redox mode. To separate the enantiomers, 30 microliters of the chiral derivatising reagent, (R)-(-)-2-amino-1-propanol (50 mg/ml) and 15 microliters of 20% (v/v) acetic acid were added to the acetonitrile layer which was then heated at 60 degrees C for 100 min. The mobile phase used to resolve the derivatised enantiomers was 0.2 M phosphate buffer (pH 3.5)-acetonitrile (3862, v/v). At a flow-rate of 1.5 ml/min, the retention times for derivatised (+)-gossypol and (-)-gossypol were 4.0 and 7.8 min, respectively. Two cancer patients received 10 mg racemic gossypol acetic acid three times a day. In one patient, the racemic, (+)- and (-)-gossypol acetic acid plasma concentrations after 65 days of therapy were 317, 213 and 104 ng/ml, respectively. In the other patient, these values were 362, 210 and 152 ng/ml, respectively, after a week of therapy. This represents, to our knowledge, the first determination of the individual enantiomer levels of gossypol after administration of the racemate.