LC–MS–MS determination of exemestane in human plasma with heated nebulizer interface following solid-phase extraction in the 96 well plate format

A sensitive, specific and rapid analytical method for the quantitation of exemestane (EXE) in human plasma has been developed. EXE, 6-methylen-androsta-1,4-diene-3,17-dione, is an orally active irreversible steroidal aromatase inhibitor used for the therapy of metastatic postmenopausal breast cancer, with estrogen-dependent pathological conditions. The method involves extraction of EXE from human plasma by solid phase extraction using C2 endcapped sorbent in the 96 well plate format (50 mg/2 ml). After conditioning of the sorbent with 1 ml of acetonitrile ( x2) the plates were rinsed with 1 ml of water ( x2). The prepared samples (0.5 ml plasma, spiked with [ 13C 3] EXE as internal standard (IS) and diluted with 0.5 ml water) were loaded and drawn through the plate with a minimum of vacuum. The plates were then washed with 1 ml acetonitrile:water (10:90) followed by a drying step for 30 min at full vacuum. Elution was by 0.15 ml of 0.1% trifluoracetic acid in acetonitrile ( x2) under a minimum of vacuum. Aliquots of 80 μl were finally injected into the LC–MS–MS system. A Zorbax SB C8 column (4.6×150 mm, 5 μm) was used to perform the chromatographic separation; the mobile phase was 100% acetonitrile. MS detection used the heated nebulizer interface, with multiple reaction monitoring (MRM) (297→121 m/ z for EXE and 300→123 m/ z for IS) operated in positive ion mode. A weighed linear regression analysis (weighing factor 1/ x 2) was used to calculate EXE concentration in standard and unknown samples. The method was fully validated in the concentration range 0.05–25 ng ml −1.