Validated Chiral LC-ESI-MS/MS Method for the Simultaneous Quantification of Darolutamide Diastereomers and Its Active Metabolite in Mice Plasma: Application to a Pharmacokinetic Study.

A simple, selective and reliable LC-MS/MS method was developed and validated for the simultaneous quantitation of darolutamide diastereomers (diastereomer-1 and diastereomer-2) and its active metabolite i. e. ORM-15341 in mice plasma using warfarin as an internal standard (IS) as per the regulatory guidelines. Plasma samples were extracted by liquid-liquid extraction and the chromatographic separation was achieved on a Chiralpak IA column with an isocratic mobile phase 5 mM ammonium acetate:absolute alcohol (20:80, v/v) at a flow rate of 1.0 mL/min. Detection and quantitation was done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: 397→202, 395→202 and 307→250 for darolutamide diastereomers, ORM-15341 and the IS, respectively in the negative ionization mode. The calibration curves were linear (r>0.992) in the range of 100-2400 ng/mL for all the analytes. The intra- and inter-day precisions were in the range of 1.25-10.2 and 1.58-12.3; 2.85-5.68 and 1.85-9.58; 2.34-12.1 and 2.58-7.38 for diastereomer-1, diastereomer-2 and ORM-15341, respectively. Both diastereomers and ORM-15341 were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice.