Screening To Quantitation: DDA Of Extractables To Determine Markers Of Interest For Quantitation Using Tof MRM With A Multi-Reflecting Time-of-Flight Mass Spectrometer

Significance of the topic

The safety assessment of pharmaceutical packaging and medical devices requires sensitive and selective analytical workflows to detect extractables and leachables (E&L) at low concentrations. High-resolution mass spectrometry (HRMS) approaches that combine broad screening with targeted quantitation improve confidence in identifying potential leachables and enable robust trace-level measurement to support regulatory compliance and risk assessment.

Objectives and study overview

This study evaluated a two-step workflow: (1) use of data-dependent acquisition (DDA) on a multi-reflecting time-of-flight (MRT) MS to generate clean, compound-specific MS/MS fragments for extractables screening; (2) curation of DDA-derived fragment ions into targeted TOF multiple reaction monitoring (Tof MRM) transitions for sensitive quantitation. The workflow was tested using a standard extractable mix (SST) and a nasal spray device matrix spiked with Cyasorb 2908 to demonstrate identification specificity, mass accuracy and quantitative performance.

Methodology and instrumentation

• Screening strategy: DDA was employed to obtain high-specificity MS/MS spectra. Survey scans were collected at 10/20 Hz (m/z 50–1200). MS/MS scans were 20/50 Hz, selecting the top 10 ions for fragmentation when intensity exceeded 20,000 cps; switching back occurred below 10,000 cps or after 3 s. Collision energy was ramped 10–75 V across the m/z range.
• Targeted quantitation: DDA-derived fragment ions were assembled into Tof MRM transitions and optimized. Tof MRM runs used an Enhanced Duty Cycle (EDC) synchronization to trap and release target ions in phase with the pusher, maximizing ion use and sensitivity.
• Liquid chromatography: ACQUITY Premier system with ACQUITY CORTECS C18 (1.6 µm, 2.1 × 100 mm). Mobile phases: water + 1 mM ammonium acetate + 0.1% formic acid (A) and methanol (B). Flow rate 0.3 mL/min, column 50 °C, injection 1 µL, 15 min gradient.
• Mass spectrometry: Waters Xevo MRT (MRT = multi-reflecting TOF) operated in positive ESI. Acquisition mass range m/z 50–1200; source/desolvation 120 °C/550 °C; desolvation/cone gas 800/50 L/hr. Collision energy used low (6 eV) and high (20–60 eV) settings depending on method.
• Data processing: waters_connect platform with UNIFI and MS Quan applications for data acquisition, processing and quantitation.

Results and discussion

• Identification and mass accuracy: All compounds in the SST mix were detected across DDA and DIA modes. Mass accuracy root mean square (RMS) across DDA injections and analytes was ≤1 ppm, supporting high-confidence assignments.
• MS/MS specificity: DDA spectra showed a substantial reduction in fragment peaks not attributed to the precursor compared with DIA. For Cyasorb 2908 at RT 9.08 min, DDA reduced extraneous peaks by ~33% in the standard and ~48% in spiked matrix, improving spectral clarity and facilitating selection of diagnostic fragments for targeted work.
• Tof MRM sensitivity and quantitative performance: Calibration for Cyasorb 2908 was linear from 0.01 ng/mL (S/N ~17) to 100 ng/mL (R2 = 0.9947). The spiked nasal solution measured 9.39 ng/mL versus an expected 10 ng/mL, demonstrating accurate quantitation in a real matrix. Signal-to-noise and percent deviations across levels indicated reliable low-level detection and acceptable accuracy across the calibration range.
• Mechanistic advantage of EDC: The Enhanced Duty Cycle in Tof MRM increases ion utilization toward ~100% for selected m/z ranges by temporal trapping/release synchronized to the TOF pusher, yielding improved sensitivity relative to conventional TOF acquisition modes.

Key figures and tables summarized

• Figure comparisons: MS/MS spectra comparison (DIA vs DDA) illustrated the decreased background fragment complexity in DDA, particularly in matrix, simplifying fragment selection for Tof MRM transitions.
• Calibration and quantitation: Calibration curve for Cyasorb 2908 demonstrated low ng/mL LLOQ (0.01 ng/mL) with good linearity and S/N, and measured concentrations in spiked samples closely matched expected values.

Benefits and practical applications

• Improved specificity: DDA produces cleaner, precursor-specific MS/MS spectra compared to DIA, reducing ambiguous fragment assignments and facilitating robust transition selection for targeted assays.
• High sensitivity targeted quantitation: Tof MRM with EDC on a high-resolution TOF enables trace-level quantitation suitable for regulatory E&L thresholds.
• Streamlined workflow: The two-step approach—qualitative DDA screening followed by targeted Tof MRM quantitation—balances broad detection capability with sensitive, high-confidence measurement of markers of interest.
• Regulatory relevance: The demonstrated sensitivity, mass accuracy and matrix performance align with needs for compliance with USP and ISO guidance for leachables assessment.

Instrumentation used

• Waters ACQUITY Premier UHPLC system with ACQUITY CORTECS C18 column (1.6 µm, 2.1 × 100 mm).
• Waters Xevo MRT mass spectrometer (multi-reflecting TOF) operating in DDA and Tof MRM modes.
• Data management on waters_connect platform using UNIFI and MS Quan applications.

Future trends and potential applications

• Automation and informatics: Integration of automated fragment selection, transition generation and library matching will speed conversion from screening to targeted assays and reduce manual curation effort.
• Expanded TOF MRM use: Adoption of TOF MRM with EDC for other trace analyses (small molecules, degradants, peptides) can broaden high-resolution targeted workflows across pharmaceutical and biological matrices.
• Hybrid acquisition strategies: Combining DDA, DIA and targeted TOF MRM in adaptive workflows will enhance coverage while preserving quantitation sensitivity.
• Regulatory harmonization: Continued validation of HRMS-targeted workflows against regulatory criteria will support wider acceptance for E&L testing and quality control.

Conclusions

The study demonstrates a practical, effective pipeline for extractables screening and targeted quantitation: DDA on a multi-reflecting TOF provides high-specificity MS/MS spectra for marker selection, and Tof MRM with EDC delivers sensitive, accurate quantitation down to low pg–ng/mL levels in matrix. This workflow addresses key analytical challenges in E&L programs by combining confident identification with trace-level measurement capability on a single high-resolution platform.

References

1. USP-NF/PF, Assessment of Drug Product Leachables Associated with Pharmaceutical Packaging/Delivery Systems (General Chapter <1664>).
2. ISO 10993-18:2020. Biological evaluation of medical devices — Part 18: Chemical characterization of medical device materials within a risk management process.
3. Stevens D., Cabovska B., Bailey A. Detection and Identification of Extractable Compounds from Polymers. Waters Application Note 720004211. January 2012.
4. 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.
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 720008972, October 2025.