LC-MS/MS-ESI method for simultaneous quantification of darolutamide and its active metabolite, ORM-15341 in mice plasma and its application to a pharmacokinetic study.

A sensitive and rapid LC-MS/MS method was developed and validated for the simultaneous quantitation of darolutamide and its active metabolite i.e. ORM-15341 in 50μL mice plasma using bicalutamide as an internal standard (I.S.) as per regulatory guidelines. Sample processing was accomplished through liquid-liquid extraction. Chromatographic separation was achieved using an Atlantis C18 column with an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (35:65, v/v) at a flow rate of 0.8mL/min within 2.5min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 397→202, 395→202 and 429→255 for darolutamide, ORM-15341 and I.S, respectively in the negative ionization mode. The calibration curve was linear from 0.61-1097ng/mL for both darolutamide and ORM-15341. The intra- and inter-day precisions were in the range of 1.34-13.8 and 4.85-12.9 and 3.91-13.7 and 6.54-14.2%, for darolutamide and ORM-15341, respectively. Darolutamide and ORM-15341 were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice.