Validation of an LC-MS/MS method for simultaneous quantitation of enzalutamide, N-desmethylenzalutamide, apalutamide, darolutamide and ORM-15341 in mice plasma and its application to a mice pharmacokinetic study

A sensitive and rapid LC-MS/MS method was developed and validated for the simultaneous quantitation of enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), apalutamide, darolu[amide and ORM-15341 (active metabolite of darolutamide) in mice plasma as per regulatory guidelines. The analytes and the internal standard (I.S.: apalutamide-d(3)) were extracted from 50 mu L mice plasma by simple protein precipitation using acetonitrile, followed by chromatographic separation using an Atlantis C-18, column with an isocratic mobile (0.2% formic acid:acetonitrile; 30:70, v/v) at a flow rate of 0.8 mL/min within 2.5 min. Detection and quantitation was done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 465 -> 209, 451 -> 195, 478 -> 450, 399 -> 178, 397 -> 194 and 481 -> 453 for enzalutamide, N-desmethylenzalutamide, apalutamide, darolutamide, ORM-15341 and the I.S. respectively. The calibration curves were linear from 1.07 to 2000 ng/mL with r(2) >= 0.99 for all the analytes. The intra-and inter-batch accuracy and precision (% CV) across quality controls varied from 88.5-111% and 1.13-13.1, 85.4-106% and 3.15-14.3, respectively for all the analytes. All the analytes were found to be stable under different conditions. The method was applied to an intravenous pharmacokinetic study in mice. (C) 2018 Elsevier B.V. All rights reserved.