Rapid Mixed-Mode SPE Method Development of Tyrosine Kinase Inhibitor Oncology Pharmaceuticals Oasis™ Method Development Plate

Significance of the Topic

This application note addresses the critical role of mixed mode solid phase extraction in bioanalytical workflows for small molecule tyrosine kinase inhibitors in plasma matrices. Mixed mode SPE combines reversed phase and ion exchange mechanisms to improve selectivity and recovery when traditional methods are insufficient, accelerating method development and ensuring robust quantitation for oncology therapeutics.

Study Objectives and Overview

The study aimed to streamline sample preparation by screening four mixed mode SPE sorbents (MCX, WAX, WCX, MAX) on an Oasis µElution Method Development Plate. Multiple blockbuster tyrosine kinase inhibitor pharmaceuticals were evaluated for extraction efficiency and matrix effects from plasma. An optimized MCX sorbent protocol was then validated by rapid UPLC tandem mass spectrometry to demonstrate linear and accurate bioanalytical quantitation.

Applied Methodology and Instrumentation

A rapid mixed mode SPE screening protocol used acid-pretreated plasma, sample loading, a formic acid wash, and dual elution steps on a 2 by 4 sorbent plate. Final extraction employed an Oasis MCX 96-well µElution plate with acidified plasma, wash solution, and methanol elution. Chromatographic analysis utilized a three minute UPLC gradient on an ACQUITY UPLC CSH C18 column at 45 C with a flow rate of 0.7 mL per minute. Mass spectrometric detection was performed on a Xevo TQ-XS triple quadrupole with electrospray ionization in multiple reaction monitoring mode. Data acquisition and quantitation were carried out with MassLynx and TargetLynx software.

Used Instrumentation

• Waters ACQUITY I-Class Plus UPLC System
• ACQUITY UPLC CSH C18 Column 2.1 mm x 30 mm, 1.7 µm, 130 Å
• Xevo TQ-XS Triple Quadrupole Mass Spectrometer
• MassLynx v4.1 and TargetLynx Software

Main Results and Discussion

In sorbent screening the MCX sorbent achieved analyte recoveries above 70 percent and matrix effects below 15 percent for most tyrosine kinase inhibitors. One compound with strong plasma protein binding showed lower recovery, suggesting a need for enhanced pretreatment. The final LC-MS/MS assay delivered calibration curve linearity with R squared above 0.99, lower limit of quantification at 0.91 ng per mL, and accuracy within fifteen percent across the dynamic range. The three minute UPLC method enabled efficient separation and high throughput.

Benefits and Practical Applications

• Rapid screening of four mixed mode sorbents in a single plate reduces method development time
• High recovery and low matrix interference enhance assay sensitivity and selectivity
• Three minute UPLC runs support high sample throughput
• µElution format eliminates evaporation steps and streamlines workflow

Future Trends and Opportunities

Future developments could extend this approach to additional therapeutic classes and more complex biological matrices. Integration with automation and laboratory information management systems will improve reproducibility and throughput. Optimized sample pretreatment strategies may enhance recovery of strongly protein bound analytes. Advances in sorbent chemistries and microelution technologies will continue to refine bioanalytical sample preparation.

Conclusion

The mixed mode SPE method development plate and optimized MCX protocol provide a fast and robust workflow for extracting tyrosine kinase inhibitors from plasma. Coupled with rapid UPLC-MS/MS analysis, this approach yields high recovery, selectivity, and reproducibility, enabling reliable bioanalytical quantitation in oncology research and clinical studies.

References

1. Williams RE and Leatherwood HM Top 200 Small Molecule Drugs by Retail Sales in 2023 Poster, accessed June 2024
2. DrugBank Online Database entry DB00480, accessed June 2024
3. Chambers E Diehl D Mazzeo J Simple and Fast SPE UPLC MS MS Method for an Allergy Tablet Mixture of Acids and Bases Application note
4. Danaceau JP Trudeau ME Automated Bioanalytical Sample Preparation Strategy for LC MS Quantification of Apixaban Application note 720007946 July 2023
5. Rahim F VanTran Trudeau ME Bioanalytical Sample Extraction for Lenalidomide from Plasma Using MCX SPE Application note 720008140 December 2023