Pharmacokinetics, Bioavailability, and Metabolism of Zongertinib, a Novel HER2‐Selective Tyrosine Kinase Inhibitor, in Rat by Liquid Chromatography Hyphenated With Electrospray Ionization Tandem Mass Spectrometry

Zongertinib, a selective human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor, has been approved by the US Food and Drug Administration for the treatment of non-small cell lung cancer. This study aimed to develop a sensitive and cost-effective LC-MS/MS method for quantifying zongertinib in rat plasma. Following protein precipitation with acetonitrile, samples were analyzed on an ACQUITY BEH C₁₈ column using a gradient of 0.1% formic acid and acetonitrile at 40°C within a 2-min run time. The assay demonstrated excellent linearity from 1 to 1000 ng/mL (r > 0.995). All validation parameters-including precision, accuracy, matrix effect, recovery, and stability-met accepted criteria for bioanalytical quantification. The validated method was successfully applied to a pharmacokinetic study in rats. Additionally, metabolites in rat plasma were investigated using LC-Orbitrap-HRMS. Three metabolites were identified and structurally characterized based on accurate mass and fragmentation patterns, revealing metabolic pathways such as oxygenation, demethylation, and epoxide hydrolysis. This is the first report on the method validation for the measurement of zongertinib in biological matrices, which enables clinical development of zongertinib and can be applied for clinical pharmacokinetics and therapeutic drug monitoring in future clinical practice.