Analysis of Extractable and Leachable (E&L) Compounds Using a Low-Energy EI-Capable High‑Resolution Accurate Mass GC/Q-TOF

Significance of the Topic

The analysis of extractable and leachable (E&L) compounds is essential for ensuring the safety and quality of pharmaceutical and bioprocessing materials. E&L profiling detects organic contaminants released from packaging, single-use systems or device filters, preventing potential product compromise and patient exposure.

Objectives and Study Overview

This study demonstrates the use of a high-resolution accurate mass gas chromatography/quadrupole time-of-flight system (GC/Q-TOF), equipped with a low-energy electron ionization (EI) source, to improve compound identification confidence in E&L investigations. The method evaluation covers differential profiling, library matching, and structural elucidation of unknowns.

Methodology and Instrumentation

Sample preparation: A single-use bioprocessing assembly was extracted under flow-through saline conditions (37 °C, 72 h). Filters were independently extracted with ethanol and a 1:1 water/ethanol mixture; all extracts (except ethanol) were liquid–liquid extracted with dichloromethane, concentrated tenfold, and submitted to GC/Q-TOF analysis.

Used Instrumentation

• Agilent 7250 GC/Q-TOF with low-energy EI (10–15 eV) and standard EI (70 eV)
• Column: DB-5 MS UI, 15 m×0.25 mm×0.25 µm; helium carrier (1.5 mL/min)
• Oven program: 50 °C (5 min) to 320 °C at 10 °C/min (hold 10 min)
• Source temp 200 °C; quadrupole temp 150 °C; transfer line 280 °C; mass range m/z 50–1000
• Retention index calibrated with n-alkane mixture
• Data processing: MassHunter Unknowns Analysis (SureMass), NIST 14 library matching, MassHunter Molecular Structure Correlator for MS/MS, Mass Profiler Professional for differential analysis

Main Results and Discussion

Differential profiling revealed 113 compounds significantly enriched (fold change ≥3, p<0.05) in saline extracts versus blanks. Major identified species include caprolactam, phenol, tri(1,2-propyleneglycol) monomethyl ether, Dowanol isomers, benzoic acid ethyl ester, vanillin, hexanamide, di-tert-butylhydroquinone, ethylparaben, 2,4-di-tert-butylphenol, benzothiazole, dimethyl phthalate and others.

Solvent comparison using a Venn diagram highlighted unique and shared extractables between ethanol and ethanol/water extracts. Low-energy EI enhanced molecular ion visibility, increasing identification confidence for both known and unknown analytes. A workflow combining low-energy EI and accurate mass MS/MS enabled structural elucidation of an unknown benzenemethanol derivative. Moreover, low-energy EI spectra of n-alkanes demonstrated excellent mass accuracy and isotopic fidelity across a growing carbon number range.

Benefits and Practical Applications

• Improved compound identification confidence through preserved molecular ions and high-resolution accurate mass data
• Effective structural elucidation of unknown extractables
• Robust differential analysis facilitating comparison across sample groups
• Application in quality control and safety assessment of pharmaceutical single-use systems

Future Trends and Possibilities

The integration of low-energy EI with high-resolution MS is expected to expand spectral libraries, streamline automated data processing, and enable real-time E&L monitoring. Advancements in chemometric tools and machine learning may further enhance unknown identification and risk evaluation in complex matrices.

Conclusion

Combining low-energy EI with high-resolution accurate mass GC/Q-TOF significantly enhances E&L compound identification by improving molecular ion detection and enabling detailed structural analyses. This approach supports comprehensive safety assessments and quality assurance in biopharmaceutical applications.

Reference

1. Jenke D. Development and Justification of a Risk Evaluation Matrix to Guide Chemical Testing Necessary To Select and Qualify Plastic Components Used in Production Systems for Pharmaceutical Products. PDA J Pharm Sci Technol. 2015;69:677–712.
2. Mire-Sluis A, et al. Extractable and Leachables: Challenges and Strategies in Biopharmaceutical Development. BioProcess Int. 2011 Feb.
3. Agilent SureMass. Agilent Technologies Technical Overview; Publication 5991-8048EN; 2017.