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

A sensitive, specific and rapid analytical method for the quantitation of e xemestane (EXE) in human plasma has been developed. EXE, 6-methylen-androst a-1,4-diene-3,17-dione, is an orally active irreversible steroidal aromatas e inhibitor used for the therapy of metastatic postmenopausal breast cancer , with estrogen-dependent pathological conditions. The method involves extr action of EXE from human plasma by solid phase extraction using C2 endcappe d sorbent in the 96 well plate format (50 mg/2 mi). After conditioning of t he sorbent with 1 ml of acetonitrile (x2) the plates were rinsed with 1 ml of water (sl). The prepared samples (0.5 ml plasma, spiked with [C-13(3)] E XE as internal standard (IS) and diluted with 0.5 mi water) were loaded and drawn through the plate with a minimum of vacuum. The plates were then was hed with 1 ml acetonitrile:water (10:90) followed by a drying step for 30 m in at full vacuum. Elution was by 0.15 ml of 0.1% trifluoracetic acid in ac etonitrile (x2) under a minimum of vacuum. Aliquots of 80 mu l were finally injected into the LC-MS-MS system. A Zorbax SE C8 column (4.6 x 150 mm, 5 mu m) was used to perform the chromatographic separation; the mobile phase was 100% acetonitrile. RIS detection used the heated nebulizer interface, w ith 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 conc entration range 0.05-25 ng ml(-1). (C) 2000 Elsevier Science B.V. All right s reserved.