Extractable analysis of rubber stoppers for pharmaceutical applications

Importance of the Topic

Extractables and leachables (E&L) analysis plays a critical role in ensuring the safety and performance of pharmaceutical packaging materials. Rubber stoppers can release small molecules that may compromise product integrity or pose toxicological risks. High-resolution accurate-mass (HRAM) UHPLC-MS workflows provide the sensitivity, mass accuracy, and structural detail needed for comprehensive nonvolatile extractable profiling.

Objectives and Study Overview

This study aimed to establish a robust, high-throughput workflow for untargeted extractable analysis of rubber stoppers used in pharmaceutical applications. Key goals included:

• Comparing isopropanol (IPA) and dichloromethane (DCM) extraction under ISO 10993-12 conditions.
• Acquiring HRAM Full Scan and data-dependent MS2 (ddMS2) data with fast polarity switching on an Orbitrap Exploris 120.
• Implementing automated compound identification using Thermo Scientific Compound Discoverer 3.2 with multiple database and spectral library searches.

Methodology and Instrumentation

Rubber stoppers were extracted with IPA and DCM at 50 °C for 72 hours. Extracts were analyzed directly by UHPLC-UV-HRAM-MS/MS.

• Chromatography: Vanquish Horizon UHPLC, Hypersil GOLD C18 (2.1 × 100 mm, 1.9 µm), 0.4 mL/min, 50 °C. Mobile phases: water with 10 mM ammonium acetate (A) and methanol (B); gradient from 5 % to 99 % B over 20 min.
• Mass spectrometry: Orbitrap Exploris 120 with OptaMax NG ESI source, positive/negative polarity switching, full scan (m/z 125–1250) at 60 000 resolution, top 4 ddMS2 at 15 000 resolution, EASY-IC internal calibration.
• Data processing: Compound Discoverer 3.2 using node-based workflows for unknown identification, parallel searches against mzVault E&L, mzCloud, NIST HRMS, ChemSpider, and custom E&L databases.

Main Results and Discussion

The combined UHPLC-Orbitrap method achieved a ~1 s duty cycle for four scan events, delivering sub-ppm mass accuracy and rich HCD fragmentation data.

• Known extractables identified included plasticizers (e.g., epoxidized soybean oil), antioxidants and their degradation products (Irganox 1010, Irgafos 168), fatty acid esters (methyl ricinoleate, glyceryl ricinoleate), erucamide, and PEG monoethers.
• IPA and DCM extracts produced similar chromatographic profiles; DCM yielded higher signal intensities, while certain polar compounds (e.g., C21H40O3, C20H34O3) appeared predominantly in IPA extracts.
• Automated spectral library matching (mzCloud mirror plots) and database search provided rapid confirmation of known compounds.
• Unknowns were elucidated by Structure Proposals and FISh Scoring, supported by mzLogic fragment matching to refine ChemSpider candidates and achieve >95 % coverage scores.
• The Partially Reprocessing feature enabled rapid re-annotation of compound classes based on shared fragment ions, enhancing coverage and confidence.

Benefits and Practical Applications

This workflow offers:

• High throughput and efficiency for routine E&L screening in QA/QC and regulatory environments.
• Comprehensive identification of both known and unknown small molecules using HRAM data.
• Flexible data mining and reporting tools to customize outputs and support toxicological risk assessments.

Future Trends and Opportunities

Emerging developments may include:

• Expanded high-resolution spectral libraries and machine learning-driven fragment prediction to streamline unknown identification.
• Integration of automation and cloud-based platforms for remote data processing and collaborative review.
• Stricter regulatory requirements for detailed extractable characterization, driving adoption of advanced HRAM workflows.

Conclusion

The combination of UHPLC with the Orbitrap Exploris 120 and Compound Discoverer 3.2 enables an efficient, reliable platform for nonvolatile extractable analysis of rubber stoppers. Fast polarity switching HRAM acquisition and advanced software tools deliver confident identification of both known and novel compounds, meeting increasingly stringent regulatory demands.

References

• FDA Guidance and USP <1663>: Container Closure Systems for Packaging Human Drugs and Biologics; Assessment of Extractables Associated with Pharmaceutical Packaging.
• ISO 10993-12 and ISO 10993-18: Biological Evaluation of Medical Devices – Sample Preparation and Reference Materials.