Simultaneous determination of gefitinib and its major metabolites in mouse plasma by HPLC–MS/MS and its application to a pharmacokinetics study

• A simple and rapid method for determination of gefitinib and its four major metabolites in mouse plasma was developed and evaluated.
• It was the first time to simultaneous determination of gefitinib and its four major metabolites in mouse plasma by a validated method.
• The method is applicable to the pharmacokinetics of gefitinib in the mouse.
• The PK profiles of gefitinib major metabolites in mouse plasma were firstly studied and compared.

Gefitinib (Iressa) is the first oral EGFR tyrosine kinase inhibitor and it brings benefits to non-small cell lung cancer patients with EGFR mutation. In this study, a simple, rapid and credible high performance liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous quantification of gefitinib and its main metabolites M523595, M537194, M387783 and M608236 in NSCLC tumor-bearing mouse plasma. Sample extraction was done by protein precipitation using acetonitrile containing dasatinib as the internal standard. The chromatography run time was 6 min using an Agilent RRHD SB-C18 column with a gradient of acetonitrile and water (0.1% formic acid, v/v). The mass analysis was performed by a triple quadrupole mass spectrometry in positive multiple reaction monitoring mode. The calibration range was 0.5–100 ng/mL for M608236 and 1–200 ng/mL for other analytes with the correlation coefficients (r2) ≥ 0.99. For quality control samples, inter- and intra-assay precision was less than 15% and accuracies ranged from 92.6% to 107.58% for all analytes. The extraction recoveries were in the range of 86–105% and no significant matrix effect was observed. This simple and reproducible high-throughput method was successfully applied to the pharmacokinetic study of gefitinib and its major metabolites in mouse.