Sample Preparation Method for Determination of Axitinib in Plasma Using ISOLUTE® SLE+

Significance of the Topic

Axitinib is a potent targeted agent for renal cell carcinoma research emphasizes matching plasma drug levels with clinical outcomes to optimize dosing and reduce toxicity. Reliable measurement in biological matrices supports therapeutic drug monitoring workflows.

Goals and Overview of the Study

The study demonstrates a streamlined sample preparation protocol using supported liquid extraction for selective isolation of axitinib from human plasma. Objectives include minimizing matrix interference and ensuring high recovery for quantitative mass spectrometry analysis.

Methodology and Used Instrumentation

A human plasma aliquot is spiked with an internal standard followed by dilution. Supported liquid extraction is performed on dedicated columns or 96 well plates using diatomaceous earth sorbent. Elution employs methyl tert butyl ether under gravity or gentle vacuum and the extract is dried under nitrogen and reconstituted in a water methanol mixture. Ultrahigh performance liquid chromatography is conducted on a Nexera LC 30AD system with an ACQUITY UPLC BEH C18 column. Detection is by electrospray ionization tandem mass spectrometry on an LCMS 8060 instrument with optimized gas flows and temperature settings.

• UHPLC: Nexera LC-30AD, ACQUITY UPLC BEH C18, gradient elution with ammonium acetate and acetonitrile
• MS: LCMS-8060, ESI positive mode, SRM transitions for axitinib and osimertinib

Main Results and Discussion

Analyte separation reveals two axitinib isomers with the monitored peak at 2.14 minutes used for quantitation. Calibration is linear over 0.01 to 25 ng/mL with r2 above 0.9999. Recovery exceeds 95 percent across low to high concentration ranges and matrix factors indicate negligible ion suppression or enhancement. No endogenous interferences were detected in blank plasma matrices after extraction.

Benefits and Practical Applications of the Method

This approach eliminates emulsions, reduces manual handling and shortens preparation time compared to traditional liquid liquid extraction. High analyte recovery and low matrix effects make it suitable for clinical pharmacokinetic studies and routine therapeutic drug monitoring.

Future Trends and Opportunities

Integration with automated platforms and stable isotope labeled standards could further enhance throughput and quantitative precision. The methodology may extend to other tyrosine kinase inhibitors and support multiplexed assays in personalized oncology.

Conclusion

Supported liquid extraction with subsequent UHPLC MS/MS provides a robust and high-throughput protocol for accurate quantification of axitinib in human plasma, reinforcing its utility in pharmacokinetic and therapeutic monitoring applications.

References

• Biomedical Chromatography 2018;32:e4147