Determination of the Androgen Receptor Inhibitor Enzalutamide and Its Metabolites in Animal Plasma and Brain Homogenates Using LC-MS/MS and Its Application to Pharmacokinetic Studies

To facilitate development of the androgen receptor inhibitor, enzalutamide, we developed and validated methods for simultaneous determination of enzalutamide, its carboxylic acid metabolite (M1), and N-desmethyl enzalutamide (M2) in plasma from mice, rats, and dogs, and brain from mice. Following addition of stable isotope-labeled internal standards, plasma and brain samples (0.05 and 0.3 mL, respectively) were mixed with 0.1% formic acid and extracted with tert-butyl methyl ether, and then injected into a liquid chromatography-tandem mass spectrometry system. The eluent was monitored in positive electrospray ionization mode. To bracket the concentrations of the drug and metabolites in study samples, calibration curves were constructed from 20 to 50000 ng/mL for plasma and 10 to 25000 ng/g for brain. Validation data demonstrated that these methods were selective, reproducible, and accurate. Using these methods, brain-to-plasma concentration ratios in mice were determined to be 0.72 for enzalutamide, 0.048 for M1, and 1.4 for M2.