Method Development for the Forced Degradation of Next-generation Selective Estrogen Receptor Degrader Imlunestrant

Significance of the topic

Imlunestrant is a next-generation selective estrogen receptor degrader (SERD) approved in 2025 with improved oral bioavailability and brain penetration compared with prior SERDs. Developing a robust, stability-indicating impurity method for imlunestrant is essential for regulatory submissions, stability testing, formulation development, and ensuring patient safety. Forced degradation studies paired with systematic LC method development define degradation pathways and enable sensitive, selective separation of the active pharmaceutical ingredient (API) from degradants and process impurities.

Objectives and study overview

The application note describes development of a liquid chromatographic impurity method for imlunestrant using a forced degradation approach and the Systematic Screening Protocol (SSP). Goals were to: provide good retention and sharp peak shape for the API, resolve imlunestrant from closely eluting degradation products, meet USP 621 criteria for resolution and tailing, and demonstrate spectral/ mass purity of the API peak.

Methodology

The method development followed the SSP in three stages: pH scouting, solvent and stationary-phase screening, and method optimization. A forced degradation mixture was generated by dissolving imlunestrant at 1 mg/mL in 60:40 acetonitrile:water, splitting into acid and base stress conditions (0.1 mL 1 N HCl or 1 N NaOH added to 0.9 mL aliquots) and heating at 70 °C for 4 hours; the two portions were combined to quench and analyzed.

Key chromatographic variables explored:

• pH: low pH (0.1% formic acid) versus high pH (10 mM ammonium hydroxide) assessed on a pH-stable BEH C18 column.
• Organic modifier: acetonitrile and methanol were compared for selectivity and peak shape.
• Stationary phases: several 2.1 x 50 mm columns were screened, including ACQUITY Premier BEH C18, BEH Phenyl, Biphenyl (MaxPeak Premier), CSH Phenyl-Hexyl and CSH C18 to probe pi-pi interactions and mitigate tailing for the basic analyte.
• Optimization: column length, starting percentage organic, column temperature and gradient slope were tuned to improve resolution and cycle time.

Used instrumentation

The work was performed on an ACQUITY Premier QSM system with Column Manager and ACQUITY PDA detector. Mass confirmation/peak purity used the ACQUITY QDa mass detector in positive mode. Data were processed with Empower 3 software.

Operational parameters of the final method (summary):

• Column: ACQUITY Premier BEH C18, 2.1 x 100 mm, 1.7 µm.
• Column temperature: 65 °C; sample temperature: 5 °C; injection volume: 1 µL.
• Mobile phases: A = water with 0.1% formic acid (modifier maintained at 5% D1); B = methanol.
• Flow rate: 0.5 mL/min; gradient from 40% methanol to 95% over 6.86 min; total run time 10.30 min.
• Detection: UV at 260 nm; mass confirmation by QDa (m/z 525 [M+H]+ and 263 [M+2H]2+).

Main results and discussion

pH scouting showed slightly greater retention at high pH, but low-pH conditions (0.1% formic acid) afforded superior peak shape and broader compatibility with stationary phases. Column/solvent screening indicated:

• When acetonitrile was used as organic solvent, BEH C18 offered the best separation among screened phases but some closely eluting degradants remained challenging on other phases.
• When methanol was used, the BEH C18 column provided the most effective separation of the API from nearby degradants; methanol enhanced pi-pi selectivity for aromatic regions and improved resolution in this case.
• Charged Surface Hybrid (CSH) phases were included to mitigate tailing common for basic analytes; however, BEH C18 with formic acid and methanol ultimately gave the best balance of selectivity and peak shape.

Method optimization improved chromatographic efficiency by switching to a 100 mm column, increasing the initial organic content (reducing gradient steepness and cycle time), and elevating temperature to 65 °C. These adjustments sharpened peaks and enhanced separation of closely eluting degradants identified near retention times ~2.6–3.3 min.

Performance metrics with the optimized method:

• Resolution of imlunestrant from the two closest degradants: 8.76 and 4.67, both exceeding USP 621 minimum separation of 2.0.
• Peak tailing factor for imlunestrant: 1.36 (within USP 621 recommended 0.8–1.8 range).
• API peak purity: confirmed by QDa positive scan with consistent extracted ion chromatograms at leading edge, apex and trailing edge, showing only target m/z species (approx. 525 and 263), indicating no co-eluting species.

Key benefits and practical applications

The final method delivers a fast (10.3 min) stability-indicating impurity assay suitable for forced degradation studies and impurity profiling during development and stability testing. Specific practical merits:

• High selectivity and resolution between API and degradants support confident impurity identification and quantification.
• Good peak shape and minimal tailing facilitate robust integration and consistent quantitation in QC environments.
• Compatibility with low-pH mobile phases and hybrid-surface vials minimizes artifacts from analyte–surface interactions.
• Mass-based peak purity assessment (QDa) increases confidence that chromatographic peaks are spectrally pure.

Limitations and considerations

The note focuses on impurity separation and qualitative peak purity; specific quantitative performance characteristics (limits of detection/quantification, method precision and accuracy for individual impurities, and robustness across lots/instruments) were not reported and would be required for full regulatory submission or routine QC transfer. The QDa provides unit-resolution mass confirmation but not high-resolution structural elucidation for unknown degradants.

Future trends and opportunities for application

Anticipated directions and opportunities building on this work include:

• Integration of high-resolution mass spectrometry to identify degradation products and map pathways with greater structural confidence.
• Automated SSP workflows driven by software and machine learning to accelerate column/solvent selection and parameter optimization.
• Broader robustness testing (inter-lab transfer, long-term method ruggedness) and validation for QC implementation.
• Exploration of alternative stationary phases or surface chemistries tailored to minimize metal-surface interactions and further improve peak shape for challenging basic pharmaceuticals.
• Use of orthogonal separations (e.g., HILIC or 2D-LC) when additional selectivity is needed for complex degradation profiles.

Conclusion

Using a systematic screening protocol applied to forced degradation material, an efficient and robust LC-UV (with QDa confirmation) impurity method for imlunestrant was developed. The optimized ACQUITY Premier BEH C18 method with methanol and 0.1% formic acid provides excellent retention, sharp peak shape, and high resolution between the API and degradants while meeting USP 621 criteria. The approach demonstrates how methodical pH, solvent, and stationary-phase screening combined with targeted optimization yields a stability-indicating method suitable for development and regulatory workflows.

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