A Rapid UPLC™-MS/MS Method for the Quantification of PROTACs-3-Gefitinib and Urinary Metabolites Following Subcutaneous Administration to Male Rats

Importance of the Topic

Proteolysis Targeting Chimeras (PROTACs) represent an emerging class of therapeutic agents that leverage the ubiquitin–proteasome system to achieve selective degradation of disease-related proteins. Unlike conventional small-molecule inhibitors, PROTACs remove the entire protein, offering advantages such as reduced resistance development, complete loss of protein function and the ability to target previously intractable proteins. Accurate quantification of PROTACs and their metabolites in biological matrices is essential for drug discovery, pharmacokinetic profiling and safety assessment.

Study Objectives and Overview

This application note describes the development and validation of a rapid UPLC-MS/MS assay for simultaneous measurement of a PROTAC based on gefitinib (PROTAC-3-gefitinib) and its primary urinary metabolites following a 10 mg/kg subcutaneous dose in male rats. Key goals included achieving high sensitivity, broad dynamic range and robust chromatographic separation to support drug metabolism and pharmacokinetic (DMPK) studies.

Methodology and Instrumentation

Four male Sprague-Dawley rats received a single subcutaneous injection of PROTAC-3-gefitinib (10 mg/kg) formulated in DMSO/PEG/Cremophor/saline. Urine was collected over intervals from 0–24 h, stored at –20 °C and thawed prior to analysis. Calibration standards (1–1000 ng/mL) and QC samples (3, 75, 800 ng/mL) were prepared by spiking blank rat urine. Sample cleanup involved protein precipitation with cold acetonitrile:methanol (4:1) containing a deuterated gefitinib internal standard.

Used Instrumentation

• ACQUITY Premier UPLC System with HSS T3 1.8 µm, 2.1×100 mm column at 60 °C
• Xevo TQ Absolute Tandem Quadrupole Mass Spectrometer operated in positive-ion ESI MRM mode
• MassLynx 4.2 for data acquisition
• TargetLynx XS for data processing and quantification

Key Results and Discussion

The method achieved linear calibration for PROTAC-3-gefitinib over 1–1000 ng/mL with r²=0.9987 and a negligible intercept. Chromatographic resolution allowed clear separation of the parent compound and six major metabolites arising from amide hydrolysis, N-dealkylation and phase II conjugation. Sensitivity was sufficient to measure low-level metabolites (M6, M8) alongside higher abundant species (M1, M4, M7, M9) in a single run without sample dilution. Urinary elimination profiles revealed incomplete clearance of the parent compound by 24 h and identified O-glucuronide, sulfate and cleavage metabolites as the predominant excreted species.

Benefits and Practical Applications

• High sensitivity and wide dynamic range permit quantification of both low- and high-abundance analytes in one batch
• Rapid UPLC separation (≤4 min) enhances throughput for DMPK screening
• Reduced non-specific binding ensures reproducible peak shapes and accurate quantification
• Minimal sample volume supports micro-sampling and the 3Rs (replacement, reduction, refinement)

Future Trends and Potential Applications

The presented approach can be extended to other PROTAC scaffolds and biological matrices. Future advances may include integration with high-resolution mass spectrometry for structural elucidation of novel metabolites, automation of sample preparation, and miniaturized platforms for longitudinal micro-sampling in preclinical and clinical studies.

Conclusion

A fast and robust UPLC-MS/MS method was established for the simultaneous quantification of PROTAC-3-gefitinib and its metabolites in rat urine. The high sensitivity, broad dynamic range and excellent chromatographic separation provided reliable measurement of parent drug and low-level metabolites, facilitating comprehensive DMPK profiling of PROTAC drug candidates.

References

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