A Rapid UPLC-MS/MS Discovery Bioanalytical Method for the Quantification of Gefitinib and Four of Its Major Metabolites in Mouse Plasma

Importance of Topic

The understanding of pharmacokinetics and concentration–response relationships is fundamental in drug discovery and development. Early and accurate measurement of drug and metabolite levels in biological matrices enables informed candidate selection, optimization of bioavailability, and prediction of human exposure. Ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) has emerged as a gold-standard analytical platform, offering specificity, sensitivity, and high throughput essential for screening large numbers of compounds.

Objectives and Study Overview

This application note describes the rapid development and validation of a UPLC-MS/MS method for simultaneous quantification of gefitinib and its four primary metabolites in mouse plasma. The aim was to establish a simple sample preparation protocol, achieve chromatographic separation of analytes sharing product ions, and demonstrate quantification over a wide concentration range following intravenous (IV) and oral (PO) administration.

Methodology and Instrumentation

Mouse plasma samples were spiked with authentic gefitinib and O-desmethyl gefitinib standards (15–7 500 ng/mL) and quality controls (30, 1 000, 6 000 ng/mL). A protein precipitation step using four-fold volume of methanol containing a d6-labeled internal standard was employed. The supernatant was diluted 1:50 in 50:50 methanol:water prior to injection.

Used Instrumentation

• ACQUITY UPLC I-Class PLUS System
• Xevo TQ-S micro Triple Quadrupole Mass Spectrometer
• ACQUITY UPLC BEH C18 Column (2.1 × 100 mm, 1.7 µm)

Chromatographic conditions included a 60 °C column temperature, 650 µL/min flow, 0.1% formic acid in water/10 mM ammonium acetate (A) and 0.1% formic acid in acetonitrile (B), with a 5–50% B linear gradient over 2.9 minutes and total run time of 5.2 minutes. Electrospray ionization in positive mode was used with multiple reaction monitoring transitions for gefitinib, O-desmethyl gefitinib, and three additional metabolites.

Main Results and Discussion

The method achieved baseline resolution of gefitinib and O-desmethyl metabolite eluting at 1.88 min and 1.76 min respectively, crucial for distinguishing shared product ions (m/z 128.23). Other metabolites eluted between 1.16 min and 2.45 min. Calibration curves were linear (r2 ≥ 0.9994) over 15–7 500 ng/mL. Limits of quantification were 15 ng/mL for gefitinib and its O-desmethyl metabolite. Intra- and inter-batch precision (%CV) ranged from 3.8% to 7.8% for gefitinib and 2.9% to 9.9% for the O-desmethyl metabolite. Quantification of three minor metabolites was performed against the gefitinib calibration curve due to lack of authentic standards.

After a 10 mg/kg IV dose, peak plasma concentrations reached 4 960 ng/mL for gefitinib, 280 ng/mL for O-desmethyl, and 230 ng/mL for M605211 at one hour. A 50 mg/kg oral dose yielded 8 600 ng/mL for gefitinib, 1 100 ng/mL for O-desmethyl, 33.9 ng/mL for M387783, and 613 ng/mL for M605211. Secondary concentration maxima observed at four hours suggest enterohepatic recirculation. Derived pharmacokinetic parameters included clearance of 14.4 mL/min/kg, volume of distribution (Vz) of 3.2 L/kg, half-life of 2.6 h (3.8 h PO), and bioavailability of 0.76.

Benefits and Practical Application of the Method

• Rapid analysis (5.2 min per injection) suitable for high-throughput screening
• Minimal sample volume (10 µL mouse plasma) and simple protein precipitation
• Simultaneous quantification of parent drug and four metabolites
• Wide dynamic range (15–7 500 ng/mL) with excellent linearity and precision
• Robust separation of analytes sharing fragmentation pathways

Future Trends and Opportunities

Advances may include integration with high-resolution MS for metabolite profiling, further miniaturization using micro-sampling techniques, automation of sample preparation, and application of artificial intelligence for method optimization. Translational studies bridging animal and human pharmacokinetics will benefit from such rapid, multiplexed assays.

Conclusion

A UPLC-MS/MS assay for gefitinib and its major metabolites in mouse plasma has been successfully developed and validated. The method offers rapid throughput, simplicity, and robust performance, supporting efficient pharmacokinetic evaluation in drug discovery.

Reference

1. McKillop D, et al. Tumor penetration of gefitinib. Molecular Cancer Therapeutics. 2005;4(4).
2. Zhang Q, et al. Effect of weekly or daily dosing regimen of gefitinib in mouse models of lung cancer. Oncotarget. 2017;8(42):72447–72456.
3. McKillop D, et al. Pharmacokinetics of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, in rat and dog. Xenobiotica. 2004;34(10):901–915.