Identification of the aromatase inhibitors anastrozole and exemestane in human urine using liquid chromatography/tandem mass spectrometry

Anastrozole (2,2'-[5-(1H-1,2,4-triazol-1-ylmethyl)-1.3-phenylene]bis(2-methylpropionitrile)) and exemestane (6-methylenandrostan-1,4-diene-3,17-dione) are therapeutically used to treat hormone-sensitive breast cancer in postmenopausal women. For doping purposes they may be used to counteract adverse effects of an extensive abuse of anabolic androgenic steroids (gynaecomastia) and to increase plasma testosterone concentrations. Excretion study urine samples and spot urine samples from women suffering from metastatic breast cancer, being treated with anastrozole or exemestane, were collected and analyzed to develop/optimize a detection system for anastrozole and exemestane to allow the identification of athletes who do not comply with the internationally prohibited use of these cancer drugs. The assay was based on liquid-liquid extraction after enzymatic hydrolysis following liquid chromatography/tandem mass spectrometry (LC/MS/MS). Anastrozole, exemestane and its main metabolite (17-dihydroexemestane) were identified in urine by comparison of mass spectra and retention times with respective reference substances. An assay validation for the analysis of anastrozole and exemestane was performed regarding lower limits of detection (anastrozole: 0.02 ng/mL; exemestane: 3.1 ng/mL; dihydroexemestane: 0.5 ng/mL), interday precisions (6.6-11.1%, 4.9-9.1% and 5.6-8.3% for low [10 ng/mL], medium [50 ng/mL] and high [100 ng/mL] concentration) and recoveries (ranged from 85-97%).