Higher Throughput Intact Mass Confirmation and Impurity Screening of GLP-1 Analogues Using Multi-Reflecting TOF Technology and INTACT Mass 1.9 Application

Importance of the topic

Accurate intact-mass characterization of GLP-1 analogue peptides is critical across discovery, process development and quality control to ensure molecular identity, batch consistency and regulatory compliance. GLP-1 therapeutics are chemically diverse and prone to sequence truncations, chemical modifications (e.g., lipidation), oxidation and deamidation; this creates complex chromatographic and spectral profiles that demand high resolving power, mass accuracy and automated data handling to achieve high-throughput, defensible results.

Objectives and overview of the study

This application note demonstrates a platform workflow that combines a high-resolution multi-reflecting time-of-flight mass spectrometer with an automated intact-mass application to deliver rapid main-species confirmation, automated purity assessment and targeted impurity annotation for GLP-1 analogues. The goals were to (1) develop a one-minute reversed-phase LC–MS method suitable for plate-based screening; (2) obtain sub-ppm monoisotopic mass accuracy and sufficient sensitivity to detect low abundance impurities; and (3) automate processing and reporting to reduce manual review and enable review-by-exception.

Methodology and experimental design

Key elements of the workflow:

• Sample preparation: direct dilution of commercial GLP-1 analogues (tirzepatide, semaglutide, liraglutide) into water with 0.1% formic acid to concentrations spanning 0.1 to 0.001 mg/mL; no cleanup step required.
• Chromatography: a rapid RPLC platform method (1-minute total run) using a sub-2 µm CSH C18 column operated at elevated temperature and high flow rate to preserve separation of main species and closely related impurities under accelerated conditions.
• Mass spectrometry: data acquired on a multi-reflecting TOF instrument using MSE (data-independent acquisition) to collect intact mass (MS1) and optional fragment information (MS2-like) in a single run.
• Data processing: automated deconvolution using BayesSpray (monoisotopic output) and the waters_connect INTACT Mass 1.9 Application to perform peak detection, adduct consolidation, targeted impurity annotation, delta-mass reporting and standardized PDF reporting per injection.

Used instrumentation

• LC system: Waters ACQUITY Premier UPLC with Binary Solvent Management and Tunable UV detector at 214 nm.
• Column: ACQUITY Premier CSH C18, 130 Å, 1.7 µm, 2.1 × 50 mm; column temperature 60 °C; sample temperature 6 °C; injection volume 1 µL; flow 0.8 mL/min; mobile phases water/ACN, each with 0.1% formic acid.
• MS: Waters Xevo MRT Mass Spectrometer (multi-reflecting TOF) running positive polarity, mass range 50–2000 m/z, 10 Hz scan rate, source/desolvation temperatures and capillary voltages optimized for peptide analysis; MSE collision energy ramp used for concurrent fragmentation data.
• Software: waters_connect INTACT Mass 1.9 Application with sample submission app v2.7.0 and BayesSpray deconvolution algorithms.

Main results and discussion

High-resolution performance and automated processing delivered the following outcomes:

• Mass accuracy and resolution: the Xevo MRT produced ~100,000 FWHM resolving power and consistent sub-ppm monoisotopic mass accuracy across peptide standards; representative tirzepatide data showed ~0.1 ppm mass error.
• Sensitivity and dynamic range: targeted impurity assignment was possible down to ~0.03% relative abundance versus the main species, demonstrating both sensitivity and quantitative capability for low-level variants.
• Deconvolution and adduct handling: BayesSpray monoisotopic deconvolution combined with adduct consolidation logic prevented artifactual inflation of impurity percentages by merging adduct signals into the main component.
• Impurity discrimination: high mass accuracy supported confident assignment among near-isobaric substitution variants (for example, a +Pro, des‑Aib variant in tirzepatide was assigned based on lowest ppm error while alternate candidates were excluded).
• Throughput and robustness: the one-minute LC–MS platform method enabled batch, plate-based analysis with concurrent acquisition and parallel processing, allowing results to be available in just over one minute per sample and enabling near-real-time decision making.
• Review efficiency: the INTACT Mass Application provided plate-level dashboards with pass/warning/fail color coding and rule-based flagging that substantially reduced manual review time by focusing analyst attention on outliers.

Benefits and practical applications

This integrated workflow offers several practical advantages for peptide drug development and QC:

• High-throughput screening: rapid per-sample turnaround supports large sample sets generated during process optimization, formulation screening and stability studies.
• Reduced manual workload: automated peak detection, deconvolution, impurity annotation and report generation minimize operator-dependent variability and spreadsheet-driven review.
• Regulatory readiness: standardized, audit-ready PDF reports and consistent purity algorithms facilitate traceable documentation for submissions and QC release.
• Flexible interrogation: MSE acquisition allows retrospective structural interrogation without re-running samples, supporting follow-up characterization when needed.

Future trends and potential uses

Emerging and likely developments building on this approach include:

• Broader adoption of high-resolution multi-reflecting TOF analyzers for intact mass QC of larger and more heavily modified peptides and small proteins as instrument throughput improves.
• Deeper integration of AI/ML models into deconvolution and impurity annotation to support increasingly complex modification patterns and low-abundance species detection.
• Expanded use of plate-based workflows and parallel processing to enable higher sample throughput in regulated QC laboratories, including automated LIMS integration for sample metadata and results traceability.
• Further development of orthogonal, rapid fragmentation strategies and hybrid acquisition modes to increase confidence in sequence-level assignments without loss of throughput.

Conclusion

The combination of multi-reflecting TOF mass spectrometry, rapid UPLC separations and an automated intact-mass application provides a practical, high-throughput platform for GLP-1 analogue characterization. The system delivers sub-ppm mass accuracy, high resolving power and sensitivity to low-level impurities while minimizing manual review through automated deconvolution, adduct management and standardized reporting. This approach supports rapid screening and QC activities, improves data consistency, and strengthens the defensibility of impurity assignments for peptide therapeutics.

References

1. Fox J, Denbigh L, Berger SJ, Pittman N. Accelerating GLP‑1 Development with High‑Throughput LC‑MS Using the BioAccord LC‑MS System and the INTACT Mass Application. Waters Application Note. 2025.
2. Ahmad S, Singh N, Pargaonkar A, Vig D, Knierman M. LC/MS-Based Characterization of GLP-1 Therapeutic Peptide Liraglutide and Its Impurities. Agilent Application Note. 2023.
3. Ranbaduge N, Shion H, Yu YQ. Streamlined LC‑MS Analysis of Stress‑Induced Impurities of a Synthetic Peptide Using the BioAccord System and the waters_connect INTACT Mass Application. Waters Application Note. 2022.
4. Waters Corporation. Xevo MRT Mass Spectrometer – Product Information and Technical Overview. Waters. 2024.