A novel dried blood spot LC–MS/MS method for the quantification of apalutamide in mouse whole blood: Application to pharmacokinetic study in mice

A simple, sensitive and rapid assay method has been developed and validated for the estimation of apalutamide on mouse dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The method utilizes liquid extraction of apalutamide from 3 mm punched disks from DBS cards (spiked or study samples). The extracted sample was chromatographed on an Atlantis dC18 column using gradient elution with 0.2% formic acid and acetonitrile at a flow rate of 1.00 mL/min. The total run time was 3.0 min. The MS/MS ion transitions monitored were m/z 478 → 450 for apalutamide and m/z 481 → 453 for the IS (apalutamide-d3 ). Method validation was performed as per regulatory guidelines. The assay was linear in the range of 0.95-2030 ng/mL. The intra- and inter-day precisions were in the ranges of 2.37-8.53 and 6.76-11.5%, respectively. Stability studies showed that apalutamide was stable on DBS cards for one month. This novel method has been applied to analyze the DBS samples of apalutamide obtained from a pharmacokinetic study in mice.