Walk-up Mass Confirmation of Glucagon-like Peptides (GLP-1) Using the Xevo™ G3 QTof Combined with RemoteAnalyzer® Open Access Software

Significance of the Topic

Rapid and reliable mass confirmation of therapeutic peptides is essential across research, development and quality control in peptide drug programs. GLP-1 receptor agonists (GLP-1 RAs) such as semaglutide, tirzepatide and liraglutide are high-molecular-weight peptide therapeutics that require high-resolution mass spectrometry (HRMS) to verify identity, detect synthesis byproducts, and confirm purification quality. Walk-up, open-access workflows that combine fast UPLC separations with automated HRMS acquisition and deconvolution reduce turnaround time and enable non-expert users to obtain actionable, publication-grade mass confirmation data.

Objectives and Study Overview

The application note demonstrates a walk-up platform for rapid, automated mass confirmation of three GLP-1 analogues (semaglutide, tirzepatide, liraglutide). The workflow couples a ballistic 1.5 minute UPLC method with a Xevo G3 QTof mass spectrometer under the control of RemoteAnalyzer open-access software. Data acquisition and automated deconvolution/ reporting are performed using waters_connect integrated with the Intact Mass Application, employing BayesSpray deconvolution in an untargeted analysis mode that nevertheless includes expected peptide modifications.

Methodology

Sample preparation and injection:

• Individual peptide solutions prepared at 0.1 mg/mL in 80:20 water:methanol with 0.1% formic acid.
• Autosampler kept at 6 °C; injection volume 0.5 µL into UPLC.

Chromatography:

• Waters ACQUITY Premier UPLC I-Class System with ACQUITY Premier BEH C18 1.7 µm, 2.1 × 50 mm column.
• Column temperature 60 °C; ballistic UPLC gradient designed for ~1.5 minute runtime for high throughput.

Mass spectrometry:

• Waters Xevo G3 QTof operated in positive ion mode, sensitivity acquisition, mass range 50–2000 m/z, 10 Hz scan rate.
• Source temperature 120 °C, desolvation 350 °C, capillary 2.8 kV, cone voltage 30 V.
• MSE fragmentation used with fixed low energy 6 V and elevated energy ramp 30–55 V to capture both intact and fragment information.

Data processing and workflow:

• RemoteAnalyzer provides browser-based walk-up sample submission (single vial, 48-vial or 96-well plate compatible) and integrates with waters_connect for instrument control and Intact Mass App processing.
• BayesSpray deconvolution in Intact Mass leverages prior information (sequence and expected modifications) to convert multiply charged raw spectra into accurate zero-charge monoisotopic masses, producing cleaner spectra than generic MaxEnt approaches for targeted peptide confirmation.
• Automated PDF reports (UV-aligned TIC and deconvolved spectra) are emailed to designated recipients; raw data remain accessible for further UNIFI processing if required.

Used Instrumentation

• ACQUITY Premier UPLC I-Class System (Waters)
• ACQUITY Premier BEH C18 column, 1.7 µm, 2.1 × 50 mm
• Xevo G3 QTof Mass Spectrometer (Waters)
• RemoteAnalyzer (SpectralWorks) open-access software
• waters_connect and Intact Mass Application for acquisition and processing
• UNIFI for storage and optional downstream interrogation

Main Results and Discussion

The workflow successfully analyzed all three peptides in under five minutes per sample and delivered automated Intact Mass App reports by email. BayesSpray deconvolution produced high-quality zero-charge monoisotopic mass assignments with excellent mass measurement accuracy: semaglutide 4111.119 (0.9 ppm), tirzepatide 4810.527 (0.4 ppm), and liraglutide 3748.9510 (1.2 ppm). Raw mass spectra showed predominant multiply charged ion envelopes (examples: [M+4H]4+ and [M+3H]3+) that were cleanly deconvolved to single masses. The combined use of a sub-2 ppm-capable QTof, short ballistic gradient, and targeted deconvolution allowed rapid, confident confirmation of peptide identity suitable for iterative synthesis and screening decisions.

Benefits and Practical Applications

• High throughput: short ballistic UPLC runtime and small injection volumes enable rapid processing of single samples or batches (48-vial/96-well formats).
• Accessibility: browser-based RemoteAnalyzer allows non-experts to submit samples and receive condensed, interpretable reports without deep MS expertise.
• Automated reporting: integrated processing and email delivery accelerate decision cycles in synthesis and purification workflows.
• Data quality: BayesSpray-driven deconvolution provides cleaner, more accurate monoisotopic masses when prior sequence/modification information is available, producing sub-2 ppm performance in this set of GLP-1 analogues.
• Flexibility: raw data remain accessible for further analysis in UNIFI or downstream applications (e.g., degradant profiling, fragment analysis).

Future Trends and Potential Uses

The presented platform exemplifies a broader trend toward automated, open-access HRMS for peptide and biologics screening. Likely future developments include:

• Tighter integration of informatics for real-time decision support and LIMS connectivity.
• Enhanced deconvolution and AI-assisted interpretation to handle heterogeneous modification patterns and low-abundance variants.
• Expanded walk-up capability across additional instrument types and multi-omics workflows to support parallel screening (e.g., intact proteins, ADCs, peptide libraries).
• Higher throughput via multiplexing, shorter columns, and more aggressive gradient optimization while retaining mass accuracy for routine QC and early-stage discovery.

Conclusion

The combination of RemoteAnalyzer open-access software, waters_connect with the Intact Mass App, a Xevo G3 QTof, and a ballistic UPLC method provides a practical, walk-up solution for fast and accurate mass confirmation of GLP-1 analogues. The approach delivers sub-2 ppm-level monoisotopic mass accuracy, automated reporting, and retained raw data for follow-up analysis—supporting rapid decision-making in peptide synthesis, purification screening, and early drug development.

References

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2. Badgujar D, Bawake S, Sharma N. Identification and characterization of major degradation products of synthetic liraglutide using liquid chromatography-high resolution mass spectrometry. J Pept Sci. 2025;31(1):e3652.
3. Jones K. Tools and Techniques for GLP-1 Analysis. Chromatography Online. June 9, 2025.
4. Fontana A, et al. Automated open-access liquid chromatography high resolution mass spectrometry to support drug discovery projects. J Pharm Biomed Anal. 2020;178:112908.