Apalutamide was sourced from Selleck Biotechnology Ltd. (Kanagawa, Japan), while N-desmethylapalutamide was obtained from MedChemExpress Ltd. (Illinois, USA). Enzalutamide, which was used as the internal standard (IS), was acquired from Funakoshi Co., Ltd. (Tokyo, Japan).

Stock solutions of apalutamide and N-desmethylapalutamide (300 µg/mL each) were prepared in methanol. These stock solutions were diluted to concentrations of 5, 10, 25, 50, 100, and 150 µg/mL using methanol to give the corresponding standard solutions. Enzalutamide was dissolved in acetonitrile to give a concentration of 4 µg/mL.

Calibration standards were also established at concentrations of 0.5, 1, 2.5, 5, 10, and 20 µg/mL for apalutamide and N-desmethylapalutamide by adding an arbitrary amount of drug-free plasma. Following preparation, an aliquot (100 µL) of each calibration standard and the IS solution (200 µL) was mixed in a microtube, vortexed for 10 s, and then centrifuged at 15,000 × g and 4 °C for 10 min. The resulting supernatant (150 µL) was diluted by adding 20 mM acetate buffer (pH 5.0, 100 µL) in a fresh microtube. After centrifugation at 10,000 g and 4 °C for 2 min, an aliquot (50 µL) of the supernatant was injected into the HPLC system.

The HPLC system (Shimadzu Corporation, Japan) was equipped with a Kinetex 2.6 μm C18 column (100 mm length × 2.1 mm inner diameter, Phenomenex Inc., Torrance, CA, USA) and a UV-visible detector set to 254 nm (Shimadzu Corporation, Japan). The mobile phase consisted of 20 mM acetate buffer (pH 5.0) and acetonitrile in a 60:40 ratio. The flow rate was maintained at 0.3 mL/min and the column temperature was 40 °C. A detection time of 10 min was employed.

The analytical method was validated for linearity, recovery rate, intraday variability, interday variability, and long-term stability in accordance with Food and Drug Association (FDA) guidelines for validation of analytical procedures for drug concentration [10]. The equations for relative standard deviation (RSD %; i.e., the precision) and relative error (RE %; i.e., the accuracy) were as follows:

$$\:RSD=\frac\times\:100$$

$$\:RE=\frac\times\:100$$

The mean RSD and RE values at each concentration were within ± 15% of the theoretical concentration across all levels, except at the lower limit of quantitation, where they were within ± 20%. To assess the stabilities of apalutamide and N-desmethylapalutamide, calibration standards in drug-free plasma were stored at − 20 and − 80 °C for 2 and 4 weeks. The ratio of the spiked values was calculated using the following equation:

$$\begin\:Ratio\:of\:spiked\:values = \hfill \\\,\,\,\,\,\,\frac}} \times \:100 \hfill \\ \end $$

A ratio within ± 15% of the theoretical value at each concentration level was used as a criterion.

The Institutional Review Board of Dokkyo Medical University Saitama Medical Center approved the study protocol (approval number: 24063). Written informed consent was obtained from patients treated with apalutamide. Prior to the subsequent dose, blood samples were collected at times of 0, 2, and 4 weeks after apalutamide administration. The patient received levodopa/carbidopa hydrate, safinamide mesylate, and atorvastatin.