Rapid High Sensitivity LC-MS/MS Bioanalytical Method for the Simultaneous Quantification of Gefitinib Based PROTACs – 3 and Gefitinib in Rat Plasma to Support Discovery DMPK Studies

Importance of the Topic

Proteolysis Targeting Chimeras PROTACs are an innovative therapeutic approach that direct the cellular protein degradation machinery toward disease relevant targets. Their large small molecule architecture combines the advantages of small molecule pharmacokinetics with catalytic degradation of proteins. Accurate measurement of PROTAC concentrations in biological matrices is critical to support drug discovery and DMPK profiling.

Objectives and Overview

This study describes the development and validation of a rapid high sensitivity LC MS MS assay for simultaneous quantification of gefitinib based PROTAC-3 and its parent drug gefitinib in rat plasma. Key goals included achieving a subnanogram per milliliter lower limit of detection, a broad dynamic range, and a short analysis time to facilitate preclinical DMPK studies.

Methodology and Instrumentation

Sample Preparation

• Protein precipitation of 20 microliters plasma with 60 microliters acetonitrile containing deuterated gefitinib internal standard
• Centrifugation at 25000 g and transfer to UPLC vial

Chromatography

• Waters ACQUITY UPLC I-Class with HSS T3 C18 column
• Gradient from 5 to 95 percent acetonitrile over two minutes at 600 microliters per minute
• Column temperature 60 degrees Celsius and sample tray at 10 degrees

Mass Spectrometry

• Xevo TQ XS triple quadrupole with positive ESI
• MRM transitions m/z 934.33 to 617.34 for PROTAC-3 and m/z 447.25 to 128.20 for gefitinib
• Optimised cone voltages and collision energies via Intellistart software

Data Processing

• MassLynx for acquisition and TargetLynx for quantification with 1 over x weighting

Main Results and Discussion

The assay achieved a four minute total run time with retention times of 1.40 and 1.86 minutes for gefitinib and PROTAC-3 respectively. The lower limit of detection was 20 picograms per milliliter for PROTAC-3 and 0.05 nanograms per milliliter for gefitinib. Quantitation was linear from 20 picograms to 1000 nanograms per milliliter with correlation coefficients exceeding 0.997. A three day validation demonstrated precision and accuracy within regulatory guidelines and stability over freeze thaw cycles.

Benefits and Practical Applications

• High throughput four minute analysis supports large preclinical DMPK studies
• Exceptional sensitivity allows quantification at picogram levels from small sample volumes
• Simultaneous measurement of parent drug and PROTAC simplifies bioanalysis workflows

Future Trends and Opportunities

Extension of this method to additional PROTAC chemotypes and multiplexed panels will accelerate discovery programs. Integration with high resolution mass spectrometry workflows may enable in depth metabolite profiling. Miniaturized sample preparation and microflow techniques could further improve sensitivity and reduce solvent consumption.

Conclusion

The rapid LC MS MS bioanalytical method provides a robust platform for quantifying gefitinib based PROTAC-3 and gefitinib in rat plasma. Its high sensitivity, wide dynamic range, and short analysis time meet the demands of modern DMPK studies and support the advancement of novel protein degradation therapeutics.

References

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• Békés M Langley DR Crews CM PROTAC Targeted Protein Degraders The Past Is Prologue Nat Rev Drug Discov 2022 21 181200
• McKillop D Hutchison M Partridge EA Bushby N Cooper CM Clarkson-Jones JA Herron W Swaisland HC Metabolic Disposition of Gefitinib in Rat Dog and Man Xenobiotica 2004 34 917934
• Molloy B Mullin L King A Gethings LA Plumb RS Wilson ID The Pharmacometabodynamics of Gefitinib After Intravenous Administration to Mice A Preliminary UPLC IM MS Study Metabolites 2021 11 379
• FDA Bioanalytical Method Validation Guidance for Industry May 2018