Bioanalysis
The high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay has been developed and fully validated to support the bioequivalence study. Gefitinib was obtained from European Directorate for the Quality of Medicines with purity of 99.5%. Gefitinib-d3 (Internal standard, IS, purity: 98%; expiration date: May 6, 2021) was purchased from Toronto Research Chemicals. Chromatography was performed at 40℃ using ultrafast liquid chromatography system (UFLC 20AD, Shimadzu, Japan) equipped with Luna HILIC column (5µm, 100×2 mm, Phenomenex). Mobile phase for gradient elution composed of water (A, 0.4% formic acid, pH=3.2) and acetonitrile (B) delivered at a flow rate of 0.4 mL/min. The whole analytical time was 5 min. QTRAP 5500 mass spectrometer (SCIEX) equipped with an positive electrospray ionization (ESI) source was adopted. Multiple reaction monitoring (MRM) transitions were at m/z 447.3→128.0 for gefitinib, 453.2→127.9 for IS, respectively. Plasma sample were prepared using liquid-liquid extraction using acetonitrile: water (1:1, v/v ). The linearity range of HPLC-MS/MS was 1-1000 ng/mL with lower limit of quantification of 1 ng/mL and with good linearity of r 2 =0.999. The results of bioanalytical method validation meet the criteria of FDA Guidelines, such as accuracy and precision, matrix effect, stability, linearity, recovery, incurred sample reanalysis.