Development and Validation of a UPLC-MS/MS Method for the Determination of Apalutamide in Human Plasma and Its...

A UPLC-MS/MS method was developed for determining apalutamide (APT) in human plasma, applied to a bioequivalence study of two tablet formulations. Plasma samples were pretreated by protein precipitation with acetonitrile using APT-13C-d₃ as an internal standard. Separation was on a Waters BEH C18 column with gradient elution (0.1% formic acid in water/acetonitrile) at a flow rate of 0.50 mL/min. ESI (+) and MRM transitions were employed, with m/z 478.1 → 221.2 for APT and m/z 482.2 → 225.3 for APT-13C-d₃. A crossover bioequivalence study (single-dose, two-period) was conducted under fasting or fed conditions. A good linear relationship was observed over the concentration range of 6.00~1200.00 ng/mL. The intrabatch and interbatch accuracy ranged from 95.06% to 104.22% (RSD ≤ 5.05%). The recovery was not less than 102.36%, and no significant matrix effect was observed for APT and APT-13C-d₃. In both fed and fasting trials, the 90% CI for the geometric mean ratios of C_max and AUC_0-72 between the test and reference formulations fell within the range of 80.00%~125.00%. This method is simple, rapid, and accurate, making it suitable for the determination of APT concentration in human plasma. Additionally, the test and reference APT tablets were demonstrated to be bioequivalent.