Differential Analysis in Screening Assays for Extractable and Leachable Compounds

Significance of the Topic

The analysis of extractable and leachable compounds in pharmaceutical products is critical for ensuring drug safety and regulatory compliance. Advanced screening methods reduce manual interpretation time and increase confidence in identifying low-level contaminants originating from packaging materials.

Objectives and Study Overview

This study demonstrates a streamlined workflow for untargeted screening of semivolatile extractables and leachables in an ophthalmic drug product (ODP) using an Agilent 7200 GC/Q-TOF system. The goals were to compare stressed and nonstressed samples, confirm identities of detected compounds, and expand coverage with complementary ionization techniques and chemometric software.

Used Instrumentation

• Agilent 7890A gas chromatograph with multimode inlet
• Agilent 7200 GC/Q-TOF mass spectrometer operating in EI and CI modes
• MassHunter Acquisition and Qualitative Analysis software
• Mass Profiler Professional (MPP) for differential analysis
• Custom Personal Compound Databases (PCDLs) for EI and CI

Methodology

Samples of the ODP were subjected to liquid-liquid extraction in n-hexane under normal and heat-stress conditions (60 °C, 24 h). An extractables study was conducted on the empty container rinsed with hexane. Data were acquired in both electron ionization (EI) and methane chemical ionization (CI) modes. EI data were deconvoluted against the NIST14 library, then normalized to an internal standard (triphenyl phosphate) and processed in MPP for fold-change blank subtraction and Venn-diagram visualization. CI data were searched against custom PCDLs and subjected to targeted CI/MS/MS for structural confirmation.

Main Results and Discussion

• EI screening identified over 100 semivolatile compounds; differential analysis highlighted unique and common species across stressed, nonstressed, and extractable samples.
• Venn diagrams revealed eight compounds shared between extractables and stressed leachables, 15 shared between stressed and nonstressed leachables, and six common to all sample types.
• Semi-quantitative estimates using triphenyl phosphate indicated four compounds exceeded the 20 ppm threshold, warranting further toxicological evaluation.
• CI screening confirmed key EI identifications (mass errors <3 ppm) and detected additional E&L compounds not observed in EI mode.
• CI/MS/MS provided unambiguous structural confirmation of challenging analytes, such as ethyl 4-ethoxybenzoate and decylcyclopentane, by assigning fragment formulas and pathways.

Benefits and Practical Applications

This integrated approach accelerates E&L screening by reducing manual spectral assignment, improving sensitivity for low-level species, and offering reliable confirmation through high-resolution CI and MS/MS data. It supports quality control and regulatory submissions by providing comprehensive compound coverage and accurate semiquantitation.

Future Trends and Applications

Emerging efforts will focus on: enhanced soft-ionization reagents, automated spectral libraries augmented with machine learning, real-time chemometric dashboards, and broader integration with predictive risk-assessment models to further streamline E&L workflows.

Conclusion

The combination of GC/Q-TOF in EI and CI modes, chemometric differential analysis, and custom databases offers a robust, high-resolution platform for comprehensive extractable and leachable profiling. This methodology effectively identifies, confirms, and semiquantifies contaminants, supporting safer pharmaceutical packaging.

References

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