Pyrotinib is a novel irreversible tyrosine kinase inhibitor developed for the 
treatment of human epidermal growth factor receptor 2 (HER2)-positive breast 
cancer. The results of phase I clinical trial demonstrated that pyrotinib was 
well tolerated and exhibited potent antitumor activity. As a promising 
therapeutic agent for HER2-positive breast cancer, it is of great importance to 
investigate the biotransformation of pyrotinib in humans and identify the major 
enzymes involved in its metabolism during its early stage of development for 
safety consideration. For this purpose, a robust analytical method based on 
ultra-performance liquid chromatography/quadrupole time-of-flight mass 
spectrometry (UPLC/Q-TOF MS) was established to characterize the metabolites of 
pyrotinib in human plasma, feces, and urine, and identify the primary enzymes 
responsible for its metabolism. As a result, a total of 24 metabolites were 
identified, including 16 phase I metabolites resulting from dealkylation, 
oxidation, dehydrogenation, and carbonylation, and 8 phase II metabolites 
originating from cysteine and N-acetylcysteine conjugation. Pyrotinib was 
absorbed into blood by 1h, reached its peak level at 4h, and afterwards 
underwent slow elimination. The principal metabolites detected in humans (M1, 
M2, and M5) were products resulting from O-depicoline and pyrrolidine lactam 
formation, whose structures have been confirmed by the synthetic references. In 
addition, fecal clearance was the major route of excretion for pyrotinib. 
Further phenotyping experiment proved that CYP3A4 was the most active enzyme 
responsible for the biotransformation of pyrotinib, implying the vital necessity 
of the assessment of the potential CYP3A-mediated drug-drug interactions in 
humans. Taken together, this study provided valuable metabolic data to explicate 
the dynamic process of pyrotinib in humans, and important reference basis for 
its safety evaluation and rational clinical application. The results will also 
benefit the assessment of the contributions to the overall activity or toxicity 
from the key metabolites.