Comprehensive extractables and leachables characterization through an integrated single injection Quan/Qual analysis using a benchtop multi-reflecting time-of-flight mass spectrometer

Significance of the topic

Comprehensive and reliable characterization of extractables and leachables (E&L) from pharmaceutical packaging and medical devices is critical for patient safety and regulatory compliance. Analytical workflows must both detect and identify unknown extractables above toxicological evaluation thresholds and quantify trace-level leachables in finished drug products. The integration of high-sensitivity targeted quantitation with confident untargeted screening in a single injection addresses major throughput and data-consistency challenges in E&L analysis.

Objectives and overview of the study

This work evaluates a combined acquisition strategy on a benchtop multi-reflecting time-of-flight mass spectrometer (MRT MS) that runs a time-of-flight multiple reaction monitoring (Tof MRM) mode in parallel with data-independent acquisition (MSE). The goals were to demonstrate: (1) simultaneous high-sensitivity quantitation of targeted leachables, (2) concurrent retrospective screening and confident identification of extractables via high-accuracy precursor and fragment data, and (3) applicability in a complex matrix (nasal spray spiked with an E&L standard mix).

Methodology

Sample sets and design:

• Standard E&L mixture (E&L SST) containing typical extractable compounds was analyzed neat and spiked into a nasal spray solution to simulate a complex matrix.
• Standard addition was used to correct for compounds present in blanks, demonstrated for Irganox 1076.

Acquisition strategy:

• Dual-mode acquisition in a single injection combining Tof MRM (targeted quantitation) and MSE (DIA-style qualitative acquisition supplying precursor and fragment ions across the run).
• Tof MRM employed an Enhanced Duty Cycle (EDC) approach to trap and time-release target ions, increasing ion utilization across specific m/z windows and improving sensitivity.
• MSE alternated low- and high-collision-energy scans to provide simultaneous precursor and fragment ion data for library searching and structural confirmation.

Chromatography and general LC-MS settings:

• LC: ACQUITY Premier system with ACQUITY CORTECS C18, 90 Å (1.6 μm, 2.1 x 100 mm); flow 0.3 mL/min; column temp 50 °C; injection 1 μL; 15 min gradient.
• MS: Xevo MRT P10 MRT MS, acquisition m/z 50–1200; MSE scan speed 10 Hz; MRM dwell times optimized per transition; ESI+ 2.5 kV / ESI- 1.5 kV; source temp 120 °C; desolvation 550 °C; desolvation gas 800 L/hr; cone gas 50 L/hr; collision energies low 6 eV, high 20–60 eV.
• Data handling: waters_connect platform with UNIFI and MS Quan applications for acquisition and processing. Screening used the Waters E&L library.

Used Instrumentation

• Waters Xevo Premier System (MRT P10 mass spectrometer).
• ACQUITY Premier UHPLC system and ACQUITY CORTECS C18 column (2.1 x 100 mm, 1.6 μm, 90 Å).
• Software: waters_connect platform, UNIFI Application, MS Quan application.

Key results and discussion

Sensitivity and quantitation:

• Optimized Tof MRM transitions delivered high sensitivity for trace-level leachables. Example: propylparaben calibration in negative ion mode was linear from 0.01 ng/mL (S/N ≈ 23) to 100 ng/mL with R2 = 0.99.
• Replicate spiked measurements returned an average concentration of 10.04 ng/mL for a nominal 10 ng/mL spike, indicating accurate quantitation at the low-ng/mL level.
• For a compound present in blanks (Irganox 1076), a standard-addition approach yielded a calculated spiked concentration of ~10.7 ng/mL vs. the actual 10 ng/mL, illustrating effective correction for background contamination.

Comparative performance of MRM vs MSE:

• MRM transitions showed marked sensitivity gains compared to extracted ion chromatograms (XIC) from MSE acquired in the same injection, enabling trace quantitation while preserving the qualitative DIA dataset.

Identification confidence from MSE data:

• MSE data screened against an E&L library provided confident identifications with mass accuracies reported as low- to sub-ppm RMS, reducing candidate space and increasing ID confidence.
• Example putative ID: protonated m/z 338.3417 assigned to erucamide with mass error 0.94 ppm and a fragment at m/z 321.3152 with 0.34 ppm error.

Calibration and recovery summary (table summary):

• Propylparaben calibration covered multiple matrix and solvent injections from 0.01 to 100 ng/mL. Measured concentrations of spiked samples clustered around the expected 10 ng/mL with deviations generally within ±15% across calibration points, and signal-to-noise ratios increased consistently with concentration.

Benefits and practical applications

• Single-injection mixed-mode acquisition reduces sample throughput bottlenecks by providing concurrent quantitative and qualitative data, minimizing instrument time and sample consumption.
• High sensitivity Tof MRM enables quantitation at low- to sub-ng/mL levels needed for leachables assessments and for meeting analytical evaluation thresholds.
• MSE DIA offers retrospective screening capability: unknowns discovered post-acquisition can be interrogated without re-analysis because precursor and fragment ion data are already available.
• High mass accuracy (low- to sub-ppm) narrows candidate lists, improving confidence for library matching and structural elucidation—valuable for regulatory submissions and toxicological assessment.

Future trends and opportunities for application

• Broader adoption of combined targeted/untargeted single-injection workflows in regulated E&L testing and QA/QC labs to increase throughput and data completeness.
• Expansion of high-quality E&L spectral libraries and integration with machine-learning approaches to accelerate unknown identification and reduce manual interpretation time.
• Further optimization of duty-cycle and ion-utilization strategies to extend sensitivity across wider mass ranges and more complex matrices (biological or highly formulated drug products).
• Regulatory harmonization recognizing dual-mode HRMS workflows, encouraging standardized validation approaches for simultaneous quant/qual acquisition.

Conclusion

The study demonstrates that a benchtop MRT time-of-flight mass spectrometer can perform integrated Tof MRM quantitation and MSE-based qualitative screening in a single injection. This approach combines high sensitivity for trace-level leachables with high-accuracy precursor/fragment information for confident extractables identification, streamlining E&L workflows and enabling retrospective interrogation of complex samples without re-acquisition.

References

1. USP-NF/PF. Assessment of Drug Product Leachables Associated with Pharmaceutical Packaging/Delivery Systems, USP General Chapter <1664>.
2. International Organization for Standardization. ISO 10993-18:2020. Biological evaluation of medical devices — Part 18: Chemical characterization of medical device materials within a risk management process.
3. Sanig R., Kirk J., Gethings L., Lock R. Increased Identification Confidence for Extractables Screening Using the Xevo MRT Mass Spectrometer. Waters Application Note 720008970. August 2025.
4. Daly M., Gethings L., Hughes C., Lock R., Syed N. Tof MRM for the Quantification of Peptide Biomarkers in Human Glioblastoma with the Xevo MRT Mass Spectrometer. Waters Application Note 720008972. October 2025.
5. Stevens D., Cabovska B., Bailey A. Detection and Identification of Extractable Compounds from Polymers. Waters Application Note 720004211. January 2012.