Simultaneous determination of gefitinib and its major metabolites in mouse plasma by HPLC-MS/MS and its application
Abstract:
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 6min 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 0.5-100ng/mL for M608236 and 1-200ng/mL for other analytes with the correlation coefficients (r(2))≥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.
Insights
A new high-performance liquid chromatography-tandem mass spectrometry method accurately quantifies gefitinib and its metabolites in non-small cell lung cancer mouse plasma, aiding pharmacokinetic studies.
Area of Science:
- Analytical Chemistry
- Pharmacology
- Oncology
Background:
- Gefitinib (Iressa) is a key oral EGFR tyrosine kinase inhibitor for non-small cell lung cancer (NSCLC) patients with EGFR mutations.
- Understanding gefitinib's pharmacokinetics and metabolism is crucial for optimizing its therapeutic efficacy in NSCLC treatment.
Purpose of the Study:
- To develop and validate a sensitive, rapid, and reliable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method.
- To simultaneously quantify gefitinib and its primary metabolites (M523595, M537194, M387783, M608236) in NSCLC tumor-bearing mouse plasma.
- To apply the validated method for pharmacokinetic studies of gefitinib in mice.
Main Methods:
- Sample preparation involved protein precipitation using acetonitrile with dasatinib as the internal standard.
- Chromatography was performed on an Agilent RRHD SB-C18 column with a 6-minute gradient elution using acetonitrile and water (0.1% formic acid).
- Mass analysis utilized a triple quadrupole mass spectrometer operating in positive multiple reaction monitoring (MRM) mode.
Main Results:
- The method demonstrated excellent linearity with correlation coefficients (r²) ≥ 0.99 across specified calibration ranges.
- High precision (inter- and intra-assay < 15%) and accuracy (92.6%–107.58%) were achieved for all analytes.
- Extraction recoveries ranged from 86% to 105% with no significant matrix effects observed, indicating method robustness.
Conclusions:
- A simple, reproducible, and high-throughput HPLC-MS/MS method was successfully established and validated for quantifying gefitinib and its metabolites.
- This validated method is suitable for application in pharmacokinetic studies of gefitinib in preclinical mouse models of NSCLC.
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