Rationale Pyrotinib is an irreversible EGFR/HER2 inhibitor that has shown antitumor activity and tolerance in the treatment of breast cancer. Studies focused on its metabolic pathways and major metabolites are insufficient. In the evaluation of drug safety and therapeutic use, metabolite characterization is critical. The metabolism of pyrotinib in vitro was studied utilizing rat, dog and human hepatocytes in this study. Methods Pyrotinib (10 μM) was incubated with hepatocytes in Williams' E medium. The metabolites were examined and profiled using ultrahigh‐performance liquid chromatography coupled with quadrupole/orbitrap high‐resolution mass spectrometry. The metabolite structures were deduced by comparing their precise molecular weights, fragment ions and retention times with those of the parent drug. Results A total of 16 metabolites, including 6 novel ones, were discovered and structurally described under the present conditions. Oxidation, demethylation, dehydrogenation, O‐dealkylation and glutathione (GSH) conjugation were all involved in the metabolism of pyrotinib in hepatocytes. The most predominant metabolic route was identified as GSH conjugation (M5). Conclusions This study generated valuable metabolite profiles of pyrotinib in several species, which will aid in the understanding of the drug's disposition in various species and in evaluating the contribution of metabolites to overall effectiveness and toxicity of pyrotinib.

The aim of the present study was to develop a liquid chromatography/electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) method for the determination of olaparib in rat plasma. The plasma samples were processed using one‐step protein precipitation with acetonitrile and then separated on Waters Acquity BEH C18 column (50 × 2.1 mm, particle size 1.7 μm) using water containing 0.1% formic acid and acetonitrile as mobile phase with optimized gradient elution. Mass spectrometric detection was carried out by selective reaction monitoring mode via positive ESI mode with precursor‐to‐product transitions of m/z 435.3 > 367.1 and m/z 443.1 > 375.2 for olaparib and 2H8‐olaparib (internal standard). The method was linear over the concentration range 0.1–2000 ng/ml with correlation coefficient >0.9987. The lower limit of quantitation was 0.1 ng/ml. The method showed excellent accuracy and precision, negligible matrix effect and high extraction recovery. The validated method was subsequently utilized to determine the concentration of olaparib in rat plasma and further applied to the pharmacokinetic study of olaparib in rat plasma. Our results demonstrated that olaparib showed gender‐dependent pharmacokinetics in rats. Compared with that in males, olaparib showed high plasma exposure, long half‐life, low clearance and high bioavailability in females.