Validation of a chiral LC-MS/MS-ESI method for the simultaneous quantification of darolutamide diastereomers in mouse plasma and its application to a stereoselective pharmacokinetic study in mice.

A simple, selective and reliable LC-MS/MS method was validated for simultaneous quantitation of darolutamide diastereomers in 50 μL mouse plasma using warfarin as an internal standard (IS) as per 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 in multiple reaction monitoring mode following the transitions m/z 397 → 202 and 307 → 250 for darolutamide diastereomers and the IS, respectively, in the negative ionization mode. The linearity range was 100-2400 ng/mL for each diastereomer. The intra- and inter-day precisions were in the ranges of 1.78-4.20 and 4.34-14.6, and 3.63-4.74 and 4.78-5.15 for diastereomer-1 and diastereomer-2, respectively. Both diastereomers were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice. Following oral administration of darolutamide at 10 mg/kg, maximum concentration in plasma was 4189 and 726 ng/mL for diastereomer-1 and diastereomer-2, respectively. The terminal half-life was found to be ~0.50 h for both the diastereomers. The AUC(0-t) was found to be 18,961 ng*h/mL for diastereomer-1 and 1340 ng*h/mL diastereomer-2.