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, darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma as per regulatory guidelines. The analytes and the internal standard (I.S.: apalutamide-d₃) were extracted from 50 μL mice plasma by simple protein precipitation using acetonitrile, followed by chromatographic separation using an Atlantis C₁₈ 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 r² ≥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.

Apalutamide; Darolutamide; Enzalutamide; LC–MS/MS; Method validation; Mice plasma; N-desmethylenzalutamide; ORM-15341; Pharmacokinetics.