Analytical Workflow for Extractable and Leachable Impurities

Significance of the Topic

Extractables and leachables testing is critical for ensuring the safety and efficacy of pharmaceutical products by assessing potential contaminants migrating from packaging, delivery systems and processing equipment under accelerated and normal usage conditions. It helps mitigate patient risk and maintain drug potency, supporting regulatory compliance and quality assurance.

Objectives and Study Overview

This study demonstrates a comprehensive analytical workflow for the qualitative and quantitative evaluation of extractables and leachables from pharmaceutical-grade O-ring materials. Four differently colored O-ring sets were subjected to multiple extraction media (water, saline, ethanol, surfactant solutions, pH extremes) at 40 °C over a 30-day period to profile volatile, semi-volatile, non-volatile organic compounds and elemental impurities.

Applied Methodology

• Risk assessment following AET (Analytical Evaluation Threshold) principles to prioritize compounds above safety limits.
• Accelerated solvent extractions and static incubations in water, 5M NaCl, 50% and 100% ethanol, 1% polysorbate 80 and acidic/basic solutions.
• LC-MS and GC-MS analyses with full-scan high resolution accurate mass (HRAM) acquisition and MS/MS for structural elucidation.
• ICP-MS elemental analysis under USP 233 and ICH Q3D compliant protocols to quantify trace metals.
• Data processing with deconvolution, library matching (NIST, Wiley, mzCloud), high-resolution filtering and unknown candidate evaluation.

Instrumentation

• Thermo Scientific ASE 350 Accelerated Solvent Extraction system
• Trace 1310 and Trace 1300 Gas Chromatographs coupled to Q Exactive GC Hybrid Quadrupole-Orbitrap and ISQ GC-MS
• Vanquish UHPLC and UltiMate 3000 systems interfaced to Q Exactive Plus Hybrid Quadrupole-Orbitrap LC-MS/MS
• iCAP Q Series ICP-MS and ICP-OES platforms with Qtegra ISDS software
• Software tools: Chromeleon, TraceFinder, Compound Discoverer, MZ Cloud and Mass Frontier for data analysis

Main Results and Discussion

• Identification of a broad range of extractables including Irganox antioxidants, phthalates, diethylene glycol derivatives, phosphine oxides and unknown compounds with elemental compositions determined to <1 ppm mass error.
• Deconvolution of over 2000 peaks and selection of differential features via fold-change relative to controls enabled targeted identification workflows.
• Confidence in compound assignments was enhanced through HRAM scoring and MS/MS fragmentation matching; unknowns with no library hits were proposed by accurate mass and fragmentation patterns.
• Non-volatile and semi-volatile profiles varied by solvent and O-ring material; ethanol extractions yielded the highest number of leachables.
• Elemental analysis revealed only trace levels of metals (Cu, Zn, Pb, Fe, As) below toxicological thresholds in all extraction conditions.

Benefits and Practical Applications

• This integrated platform streamlines the E&L testing workflow from extraction through data reporting, reducing time and increasing confidence in safety assessments.
• High-resolution Orbitrap and robust ICP-MS technologies provide single-scan screening with quantitation capabilities matching triple quadrupole performance.
• Automated data processing and spectral libraries (including a specialized E&L database) support rapid unknown identification and regulatory compliance.
• Applicable to a wide range of single-use systems, packaging materials and pharmaceutical contact components in QC and R&D settings.

Future Trends and Opportunities

• Expansion of curated cloud-based HRAM spectral libraries and real-time spectral sharing to accelerate unknown characterization.
• Enhanced software workflows for de novo structure elucidation (Compound Discoverer v2 and beyond).
• Development of standardized extractables testing protocols and regulatory guidance harmonizing international standards (e.g. USP <1663>/<1664>, BPOG recommendations).
• Integration of AI-driven data analytics and predictive toxicology to refine risk assessments and safety thresholds.

Conclusion

The demonstrated workflow leverages high-resolution GC-MS, LC-MS and ICP-MS technologies combined with advanced data processing to deliver a robust and comprehensive solution for extractables and leachables analysis. It ensures accurate identification, quantitation and risk evaluation of organic and elemental impurities, supporting product safety and regulatory compliance.

References

• Standardized Extractables Testing Protocol. Pharmaceutical Engineering 34 (2014).
• USP <1663> and <1664> General Chapters on E&L Testing.
• ICH Q3D Guideline for Elemental Impurities.