RP-HPLC-UV Method for Simultaneous Quantification of Second Generation Non-Steroidal Antiandrogens Along with their Active Metabolites in Mice Plasma: Application to a Pharmacokinetic Study.

Abstract

A simple, specific and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the quantitation of second generation antiandrogens and their active metabolites namely apalutamide, enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma. The method involves extraction of apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 along with internal standard (IS) from 100 µL mice plasma through a simple protein precipitation process. The chromatographic analysis was performed on a Waters Alliance HPLC system using a gradient mobile phase (comprising 10 mM ammonium acetate and acetonitrile in a flow-gradient) and X-Terra Phenyl column. The UV detection wave length was set at λmax 250 nm. Apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 and the IS eluted at 13.6, 11.4, 9.68, 6.11, 6.93 and 4.69 min, respectively with a total run time of 15 min. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 209 - 5215 ng/mL (r 2=0.998). The intra- and inter-day precisions were in the range of 0.56-13.5 and 1.04-13.9%, respectively. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.