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.