From Extractables Screening to Targeted Leachables Quantitation Using DDA and Tof- MRM on the Xevo™ MRT Mass Spectrometer

Importance of the topic

The identification and quantitation of extractables and leachables (E&L) from pharmaceutical packaging and medical devices are critical for patient safety and regulatory compliance. Low-level migrants originating from polymers, additives, and degradation products can pose toxicological risks; therefore sensitive and selective analytical workflows are required to detect, identify and quantify these species at or below analytical evaluation thresholds. Integrating comprehensive screening and targeted quantitation on a single high-resolution platform simplifies laboratory workflows and improves data consistency.

Objectives and overview

This application note demonstrates a two-step analytical strategy on the Xevo MRT Mass Spectrometer: (1) use data-dependent acquisition (DDA) to generate highly specific MS/MS spectra and select characteristic fragment ions for target selection; (2) build and apply a targeted time-of-flight multiple reaction monitoring (Tof-MRM) assay, employing enhanced duty cycle (EDC) trapping, to achieve highly sensitive quantitation of leachables. The workflow was applied to a commercial nasal spray matrix spiked with a standardized E&L system suitability test (SST) mix to evaluate identification specificity, calibration linearity, and quantitative accuracy.

Methodology and experimental design

• Sample: Commercial nasal spray solution diluted 1:10 with isopropanol and spiked with Waters E&L SST mix; injections performed in triplicate.
• Screening approach: DDA survey scans (10/20 Hz) over m/z 50–1200 with MS/MS (20/50 Hz) of the top 10 ions; trigger threshold >20,000 cps; back-switch below 10,000 cps or 3 s timeout; collision energy ramp 10–75 eV across m/z range. DDA used to isolate precursor-specific fragment spectra for marker selection.
• Targeted quantitation: Tof-MRM methods constructed from curated DDA fragments; utilized EDC synchronization to trap and release product ions aligned with the TOF pusher timing, maximizing ion utilization and sensitivity. Scheduled acquisition windows were used to optimize duty cycle.
• Calibration and data handling: Calibration curves were acquired alongside the spiked matrix; standard addition was applied where true blanks were unreliable. Data acquisition and processing used waters_connect platform with UNIFI and MS Quan Applications.

Used instrumentation

• LC: ACQUITY Premier System with CORTECS C18, 90 Å, 1.6 µm, 2.1 × 100 mm column; column temp 50 °C; injection volume 1 µL; flow 0.3 mL/min. Mobile phases: A = water + 1 mM ammonium acetate + 0.1% formic acid; B = methanol. Gradient: 2% B (0–0.5 min) to 98% B over 5.5 min, hold 7 min, re-equilibrate to 2% B.
• MS: Xevo MRT Mass Spectrometer operated in ESI+ / ESI-; source 120 °C; desolvation 550 °C; desolvation gas 800 L/hr; cone gas 50 L/hr; capillary 2.5 kV; cone voltage 40 V; collision energy ramp 10–75 eV.
• Software: waters_connect acquisition platform with UNIFI and MS Quan Applications for processing, calibration, and standard addition workflows.

Main results and discussion

• DDA versus MSE specificity: DDA produced cleaner, precursor-specific MS/MS spectra by substantially reducing fragment peaks not attributable to the selected precursor. For Cyasorb 2908, DDA reduced the number of spectral peaks by ~33% in standard comparison and by ~48% when standards were spiked into matrix relative to MSE (data-independent acquisition).
• Fragment selection: Compound-specific fragments from DDA spectra were selected and optimized for Tof-MRM transitions, improving transition specificity for quantitation.
• Tof-MRM sensitivity and linearity: Using EDC-enhanced Tof-MRM, Cyasorb 2908 produced a linear calibration from 0.01 ng/mL to 100 ng/mL with R2 = 0.999. The lowest calibrator (0.01 ng/mL) showed S/N ≈ 19. The measured concentration of Cyasorb 2908 in the spiked nasal solution was 9.39 ng/mL (target 10 ng/mL), demonstrating quantitation within ~6% of expected value.
• Standard addition for challenging blanks: For analytes prone to background contamination (e.g., Irganox 1076), the MS Quan standard addition tool was used to account for non-zero background. The calculated concentration for Irganox 1076 was 10.7 ng/mL (target 10 ng/mL), showing the approach yields accurate results despite blank contamination.

Benefits and practical applications

• Unified platform: The Xevo MRT enables both high-confidence extractables characterization (HRMS) and highly sensitive targeted leachables quantitation (Tof-MRM) on a single instrument, simplifying lab workflows and reducing inter-instrument variability.
• Improved transition selection: DDA provides precursor-specific MS/MS spectra that reduce background fragment interferences, enabling selection of cleaner transitions for targeted assays.
• Enhanced sensitivity with HRMS: EDC-enabled Tof-MRM approaches near-complete ion utilization over defined m/z windows, improving sensitivity to levels comparable with triple quadrupole systems while retaining high mass accuracy and resolving power.
• Robust quantitation strategies: Combination of scheduled Tof-MRM, calibration curves, and standard addition workflows supports accurate quantitation even in matrices where true blanks are difficult to obtain.

Future trends and potential applications

• Broader adoption of HRMS-based targeted quantitation: As MRT and TOF-based MRM modes continue to demonstrate sensitivity and dynamic range comparable to triple quadrupoles, more laboratories may consolidate screening and quantitation workflows onto HRMS platforms.
• Automated transition discovery: Integration of automated DDA-derived fragment selection into routine method development pipelines could accelerate generation of robust targeted assays for new E&L compounds.
• Advanced data processing: Enhanced informatics (spectral deconvolution, transition scoring, and background correction) will further improve quantitation confidence in complex matrices and low-level analytes.
• Regulatory alignment: Continued validation studies demonstrating equivalence to established triple quadrupole methods will support regulatory acceptance of HRMS-based Tof-MRM for leachables quantitation.

Conclusion

Combining DDA-based fragment discovery with Tof-MRM quantitation on the Xevo MRT Mass Spectrometer provides a compact and effective workflow for E&L analysis. DDA yields cleaner, precursor-specific MS/MS spectra that facilitate selection of diagnostic product ions, while EDC-enhanced Tof-MRM delivers high sensitivity and excellent linearity for targeted quantitation. The approach produced accurate measurements in a spiked nasal spray matrix (results within ~10% of target values) and addresses practical challenges such as non-zero blanks via standard addition. Overall, the platform supports integrated extractables characterization and leachables quantitation with strong analytical performance.

References

1. USP-NF/PF. Assessment of Drug Product Leachables Associated with Pharmaceutical Packaging/Delivery Systems, General Chapter <1664>.
2. USP-NF/PF. Assessment of Extractables Associated with Pharmaceutical Packaging/Delivery Systems, General Chapter <1663>.
3. Norwood D., et al. Considerations for Extractables and Leachables in Pharmaceutical Research. Pharmaceutical Research, 2008, 25, 727–739.
4. ISO 10993-18:2020. Biological evaluation of medical devices — Part 18: Chemical characterization of medical device materials within a risk management process.
5. 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, October 2025.
6. Tomcyzk N., Wallace A., Richardson K., Grzyb A., Wildgoose J. Targeted High Resolution Quantification with Tof-MRM and HD-MRM. Waters Application Brief, June 2013.
7. Sanig R., Kirk J., Gethings L., Lock R. Increased Identification Confidence for Extractables Screening Using the Xevo MRT Mass Spectrometer. Waters Application Note, August 2025.