Validated LC‐MS/MS Method for Quantification and Pharmacokinetic Analysis of Talazoparib and Enzalutamide in Rats

The FDA has approved Talazoparib and Enzalutamide for metastatic castration‐resistant prostate cancer (mCRPC) with homologous recombination repair (HRR) gene mutations, based on the TALAPRO‐2 Phase 3 trial. This study developed and validated an LC‐MS/MS method for simultaneous quantification of Talazoparib and Enzalutamide in rat plasma using Apalutamide as the internal standard. Chromatographic separation was achieved with a 70:30 (v/v) acetonitrile–0.1% formic acid system, 10 min runtime, and 1.0 mL/min flow rate. Retention times were 4.13 min (Talazoparib), 5.31 min (Enzalutamide), and 7.89 min (Apalutamide). Detection was performed in positive ionization mode using MRM transitions: m / z 381.35 → 240.56, 465.44 → 305.24, and 478.44 → 285.10, respectively. The method was linear over 0.05–2 ng/mL for Talazoparib and 4–160 ng/mL for Enzalutamide ( r 2 > 0.999). Accuracy and precision were within ±15%–20%, and recovery exceeded 98%. In pharmacokinetic studies on male Wistar rats, Talazoparib showed a C max of 0.88 ng/mL at 3 h and an AUC 0− t of 20 ng·h/mL, while Enzalutamide exhibited a C max of 76.18 ng/mL at 1 h and an AUC 0− t of 1702 ng·h/mL; both had 24 h half‐lives. The validated method enables sensitive, rapid, and reliable bioanalysis for preclinical pharmacokinetic evaluation.