Integrated Single-injection Quan/Qual Characterization of Extractables and Leachables Using a Benchtop Multi- Reflecting Time-of-Flight Mass Spectrometer

Significance of the topic

Extractables and leachables (E&L) present a major analytical challenge for pharmaceutical packaging, medical devices, and drug-delivery systems because low-level migrants can pose toxicological risks. Regulatory and guidance documents increasingly demand rigorous identification above analytical evaluation thresholds and routine quantitation of leachables during product lifecycle management. High-confidence screening together with sensitive targeted quantitation and the ability to retrospectively interrogate archived datasets are therefore essential capabilities for modern E&L laboratories.

Objectives and overview of the study

This application note demonstrates a unified single-injection workflow for concurrent qualitative screening and quantitative leachables analysis using a benchtop multi-reflecting time-of-flight mass spectrometer (Xevo MRT P10). The goal was to run data-independent acquisition (DIA, implemented as MSE) for comprehensive extractables screening at high mass accuracy at the same time as scheduled Time-of-Flight multiple reaction monitoring (Tof MRM) with enhanced duty cycle (EDC) for trace-level targeted quantitation, thereby removing the need for separate qualitative and quantitative injections.

Methodology

A commercial nasal spray was used as a complex aqueous matrix. The product was sampled (neat solution removed), diluted 1:10 in isopropanol and spiked with a Waters E&L system suitability test (SST) mix. Triplicate injections were acquired under mixed-mode conditions combining parallel MSE and scheduled Tof MRM. The workflow included building calibration curves from the SST mix and spiking standards into the nasal spray matrix to evaluate quantitative performance and qualitative identifications.

Used instrumentation

• LC system: ACQUITY Premier with ACQUITY CORTECS C18, 90 Å, 1.6 μm, 2.1 x 100 mm, column temp 50 °C, injection 1 µL, flow 0.3 mL/min.
• Mobile phases: A = water + 1 mM ammonium acetate + 0.1% formic acid; B = methanol. Gradient: 2% B (0.0–0.5 min), ramp to 98% B over 5.5 min, hold 7 min, return to 2% B.
• Mass spectrometer: Waters Xevo MRT P10 (multi-reflecting TOF). Acquisition modes: parallel MSE (DIA; alternating low/high collision energy) and scheduled Tof MRM with enhanced duty cycle (EDC).
• MS conditions: ESI positive and negative, m/z range 50–1200, source temp 120 °C, desolvation temp 550 °C, desolvation gas 800 L/hr, cone gas 50 L/hr, capillary 2.5 kV, cone voltage 40 V, low CE 6 eV, high CE ramp 20–40 eV.
• Software: waters_connect for acquisition, UNIFI Application and MS Quan Application for data processing.

Main results and discussion

The mixed-mode acquisition successfully delivered both sensitive targeted quantitation and high-resolution qualitative data in a single injection. Tof MRM in EDC mode increases ion utilization (approaching near 100% within targeted m/z windows) and improved sensitivity versus conventional TOF acquisition, enabling trace-level quantitation appropriate for E&L thresholds. Propylparaben was quantified in negative ion mode with a linear calibration range from 0.01 ng/mL (S/N ~15) to 100 ng/mL (R2 = 0.99); measured concentration in the spiked matrix averaged 10.04 ng/mL for a nominal 10 ng/mL spike, demonstrating accurate quantitation in a complex matrix.

Concurrent MSE data provided accurate-mass precursor and fragment information across the chromatogram, with mass errors reported in the low- to sub-ppm range (RMS), significantly narrowing candidate formulas and increasing identification confidence. Example identifications included the detection of erucamide with protonated m/z 338.3417 (error ~0.94 ppm) and a diagnostic fragment at m/z 321.3152 (error ~0.34 ppm). Overlaying MRM transitions and MSE extracted ion chromatograms confirmed co-elution and supported identity assignments while delivering independent quantitative traces.

Benefits and practical applications

• Streamlined workflow: simultaneous quan/qual acquisition avoids duplicate injections and reduces sample consumption and instrument time.
• High sensitivity targeted quantitation: Tof MRM with EDC supports low- to sub-ppb/pg levels required for leachables monitoring.
• Comprehensive qualitative coverage: MSE provides full-scan precursor and fragment information for unknown screening and retrospective analysis.
• Regulatory and lifecycle readiness: retrospective interrogation capability supports ongoing risk assessment consistent with evolving guidance (e.g., ICH Q3E draft) and ISO/USP expectations.

Future trends and potential uses

Integrated single-injection quan/qual approaches align with a shift toward lifecycle E&L management, where archived high-resolution datasets must be re-examined as formulations, suppliers, or processes change. Anticipated directions include expanded curated E&L libraries, automated retrospective screening pipelines, tighter integration with toxicological decision frameworks, and broader adoption of targeted HRMS quantitation modes (Tof MRM, HD-MRM) for diverse matrices (biologics, aqueous formulations, device extracts). Further method development could focus on multiplexed scheduling strategies, matrix-matched calibrations, and standardization of performance metrics for LLOQ, mass accuracy, and identification confidence to accelerate regulatory acceptance.

Conclusion

The benchtop Xevo MRT P10 instrument demonstrated that combined MSE and scheduled Tof MRM can deliver high-confidence extractables identification and sensitive leachables quantitation in a single injection. This mixed-mode acquisition reduces analytical burden while preserving the advantages of high-resolution MS for screening and the sensitivity required for targeted quantitation, supporting efficient lifecycle E&L risk management.

References

1. USP-NF/PF, <1664> Assessment of Drug Product Leachables Associated with Pharmaceutical Packaging/Delivery Systems.
2. USP-NF/PF, <1663> Assessment of Extractables Associated with Pharmaceutical Packaging/Delivery Systems.
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4. ISO 10993-18:2020 Biological evaluation of medical devices — Part 18: Chemical characterization of medical device materials within a risk management process.
5. ICH Q3E Guideline for extractables and leachables, draft, August 2025.
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