Comprehensive Characterization and Impurity Profiling of the GLP-1 Analogue Tirzepatide Using the Xevo™ G3 QTof High Resolution Mass Spectrometer

Importance of the topic

Tirzepatide is a dual GIP/GLP-1 receptor co-agonist with important therapeutic utility for type 2 diabetes and obesity. Thorough chemical characterization and impurity profiling of such lipopeptide therapeutics are essential for ensuring product safety, regulatory compliance, and robust analytical control during development and manufacture. High-resolution LC–MS workflows are particularly valuable to resolve closely related proteoforms, localize modifications, and support stability-indicating method development.

Objectives and overview of the study

This application note reports forced-oxidation stress testing of tirzepatide followed by comprehensive characterization and impurity profiling using the Waters Xevo G3 QTof high-resolution mass spectrometer coupled to an ACQUITY UPLC I-Class system. Key aims were to (1) confirm intact mass and sequence coverage with high mass accuracy, (2) detect and identify oxidative and other degradation products, (3) localize modification sites (oxidation, deamidation) using fragmentation data, and (4) demonstrate ready-made UNIFI Application workflows for streamlined impurity screening and reporting.

Methodology and experimental

Tirzepatide reference solution (0.1 mg/mL in water, 0.1% formic acid) was stressed with 1% (v/v) H2O2 and incubated at 40 °C. Samples were injected at multiple timepoints (0 to 12 h) to monitor the temporal evolution of degradants.

Key LC parameters were: ACQUITY Premier UPLC I-Class system; ACQUITY Premier CSH C18 Peptide column (2.1 × 100 mm, 130 Å, 1.7 µm); column temperature 60 °C; sample temp 40 °C; injection 1 µL; flow 0.4 mL/min; mobile phases water + 0.1% formic acid (A) and ACN + 0.1% formic acid (B).

MS acquisition used the Xevo G3 QTof in positive ion mode (50–2000 m/z), sensitivity mode, 10 Hz scan rate, cone 30 V, source 120 °C, desolvation 350 °C, capillary 2.8 kV, MSE acquisition with low energy 6 V and elevated energy ramp 30–55 V. Data processing and peptide mapping used the UNIFI Application within the waters_connect platform, leveraging BayesSpray deconvolution and built-in peptide mapping workflows.

Used instrumentation

• Waters ACQUITY Premier UPLC I-Class System
• ACQUITY Premier CSH C18 Peptide Column (2.1 × 100 mm, 130 Å, 1.7 µm)
• Waters Xevo G3 QTof Mass Spectrometer
• UNIFI Application and waters_connect software (data processing, peptide mapping, BayesSpray deconvolution)

Main results and discussion

Intact mass and peptide mapping: Tirzepatide was characterized with routine sub-2 ppm mass accuracy; the intact monoisotopic mass matched with 0.3 ppm error and 57 primary fragment ions confirmed. BayesSpray deconvolution, which uses sequence and modification priors, produced clean deconvolved spectra and supported confident species assignment. The K20-linked C20 fatty di-acid modifier and linker were detected and localized via fragment ions.

Impurity profiling and timeline: Forced oxidative stress caused a clear decline of the parent peak (tR ≈ 15.29 min) and emergence of multiple degradants over time. UNIFI workflows enabled automated component detection, mass confirmation, and temporal trend visualization across the time-course.

Isomer separation: Four chromatographically resolved isomeric components corresponding to the Linker + FA20 modified species were identified, each with low mass error (approx. −1.4 to 1.5 ppm). Clear chromatographic resolution of these isomers highlights the importance of optimized peptide stationary phase chemistry for lipopeptides.

Oxidation mapping: Oxidation of the tryptophan residue was a dominant degradation pathway under the applied oxidative conditions. Single, double, and triple oxidized species were identified and characterized with high confidence (mass errors between −0.8 and −2.1 ppm and 40–50 primary fragment matches). Fragmentation (b- and y-ion series) localized the oxidations; for example, triple oxidation was assigned to the tryptophan at b25 based on the N-terminal fragment series.

Deamidation: A deamidation product, putatively localized to Q19, was detected early (present at t = 0), indicating formation during storage or handling rather than as a primary outcome of the oxidative incubation. Fragment ion annotation showed deamidation on b20 and subsequent N-terminal ions, supporting residue-level localization despite the subtle m/z shift associated with deamidation.

Impurity summary and data visualization: The UNIFI peptide mapping workflow and Summary Plot feature allowed rapid compilation of identified components, their retention behavior, temporal trends, fragment coverage, and mass errors, facilitating a concise impurity report for stability assessment.

Benefits and practical applications of the method

• High-confidence identifications: Sub-2 ppm routine mass accuracy and rich fragmentation ensure robust intact mass confirmation and residue localization for lipopeptide therapeutics.
• Targeted deconvolution: BayesSpray improves deconvolution specificity by incorporating sequence and modification priors, producing cleaner intact mass spectra for complex multiply charged peptides.
• Automated workflows: UNIFI Application streamlines screening, annotation, temporal trend analysis, and reporting—accelerating stability- indicating method development and impurity profiling.
• Chromatographic separation: The CSH C18 peptide column provided effective resolution of isomeric lipopeptide forms and related impurities.
• Regulatory and QC relevance: The combined LC–HRMS approach supports forced-degradation studies, stability-indicating method validation, and comparability assessments relevant for generic development and regulatory submissions.

Future trends and potential applications

• Broader adoption of multi-attribute methods (MAM) and automated HRMS-based workflows for routine QC of therapeutic peptides.
• Increased coupling of orthogonal separations (e.g., SEC–MS) with HRMS to simultaneously assess aggregation and primary sequence modifications as critical quality attributes.
• Application of machine learning and advanced spectral libraries to accelerate identification of low-abundance degradants and isomeric species.
• Greater use of targeted deconvolution and prior-informed algorithms (like BayesSpray) for complex proteoform analysis in both innovator and generic product characterization.
• Integration of HRMS workflows into regulatory dossiers for peptide therapeutics, enabling more detailed impurity knowledge and comparability claims for generics.

Conclusion

The study demonstrates that an integrated LC–HRMS workflow based on the Xevo G3 QTof and UNIFI Application provides comprehensive characterization of tirzepatide and a robust impurity profiling capability under forced oxidative stress. High mass accuracy, sequence-aware deconvolution, and rich fragmentation allow confident identification and localization of key degradants (oxidation, deamidation and isomeric forms). These capabilities support stability-indicating method development, quality control, and regulatory submissions for complex lipopeptide therapeutics.

References

1. Alfaris N, et al. GLP-1 Single, Dual, and Triple Receptor Agonists for Treating Type 2 Diabetes and Obesity: A Narrative Review. eClinicalMedicine. 2024;75:102782.
2. Darwish R, Abu-Sharia G, Butler AE. History of Glucagon-Like Peptide-1 Receptor Agonists. Pharmacological Research. 2025;222:108045.
3. Rao PP. Revolutionizing obesity treatment: The Emerging Role of GLP-1 Receptor Agonists and Next-Generation Pharmacotherapies. Obesity Medicine. 2026;60:100686.
4. Xu F, Wang KY, Wang N, Li G, Liu D. Bioactivity of a Modified Human Glucagon-Like Peptide-1. PLoS One. 2017;12(2):e0171601.
5. Peri RV, Anchan H, Jonnalagadda K, Varghese R, Gupta P. Designing GLP-1 delivery: Structural Perspectives and Formulation Approaches for Optimized Therapy. Nutr Diabetes. 2025;15:53.
6. Zhang T, Liu S, He S, Shi L, Ma R. Strategies to Enhance the Therapeutic Efficacy of GLP-1 Receptor Agonists through Structural Modification and Carrier Delivery. Chembiochem. 2025;26:e202400962.
7. Jones K. Tools and Techniques for GLP-1 Analysis. LCGC. 2025.
8. Koza MK, Wuthrich P, Shiner SJ, Lauber MA. Denaturing SEC-MS Analysis of High Molecular Weight Impurities in the GLP-1a Lipopeptides Semaglutide and Tirzepatide. Waters Application Note. 2026.
9. Kim SH, Kim SS, Kim HJ, Park EJ, Na HH. Peptide Mapping Analysis of Synthetic Semaglutide and Liraglutide for Generic Development of Drugs Originating From Recombinant DNA Technology. J Pharm Biomed Anal. 2025;256:116682.
10. Kuril AK, Saravanan K, Subbappa PK. Analytical Considerations for Characterization of Generic Peptide Product: A regulatory insight. Analytical Biochemistry. 2024;694:115633.
11. ICH Q5C, Q6B, Q5E Guidelines. International Conference on Harmonisation.
12. Nauck MA, D'Alessio DA. Tirzepatide, A Dual GIP/GLP-1 Receptor Co-Agonist for the Treatment of Type 2 Diabetes. Cardiovasc Diabetol. 2022;21:169.
13. Gibson K. Investigating the Structure and Physical Stability of GLP1-Like Peptides Using Mass Spectrometry. Cambridge Repository. 2023.
14. Min JS, et al. A Comprehensive Review on the Pharmacokinetics and Drug-Drug Interactions of Approved GLP-1 Receptor Agonists and a Dual GLP-1/GIP Receptor Agonist. Drug Des Devel Ther. 2025;19:3509-3537.