Determination of the new aromatase inhibitor exemestane in biological fluids by automated high-performance liquid chromatography followed by radioimmunoassay

Abstract A procedure for the determination of exemestane, a new aromatase inhibitor, in biological fluids is described in this paper. Exemestane is extracted from human plasma and urine by solid-phase and liquid-liquid extraction, respectively. The test compound is then isolated from endogenous steroids, its metabolites and/or degradation products by HPLC. The exemestane-containing fraction is collected and its exemestane content measured by radioimmunoassay (RIA). The automated HPLC system allowed a high specificity and reproducibility of retention times, and eliminated almost all manual operations. The RIA allowed the accurate and precise measurement of 12 pg of exemestane/ml in plasma (inter- and intra-assay RSD=17.7 and 13.4%, respectively) and 25 pg/ml in urine (inter- and intra-assay RSD=14.5% and 8.7%, respectively). The recovery of the whole procedure was evaluated by comparison of the RIA calibration curve obtained in plasma or urine (after extraction and HPLC) with that obtained directly in RIA buffer (without extraction and HPLC). The calibration curves were practically superimposable, indicating that the recovery of the whole procedure was excellent. The method was validated in terms of reproducibility, recovery and precision in the range 10–500 pg of exemestane/ml of plasma and 20–1000 pg/ml of urine. Finally the plasma levels of exemestane in a postmenopausal healthy volunteer treated daily for 7 days with oral exemestane at a dose of 1 mg (the lowest dose administered in clinical trials) were monitored using the method here described. Exemestane was detectable in all plasma samples collected (up to 24 h after drug intake). Therefore the analytical method described here should be sufficiently sensitive and specific for the determination of exemestane in plasma and urine from clinical trials in which therapeutic doses of the drug (10–25 mg/day) are administered.