Impurity Profiling of Tirzepatide Under Stress Conditions

Importance of the topic

The rise of GLP-1 receptor agonists such as tirzepatide in treating type-2 diabetes and obesity has intensified the need for reliable analytical methods. Peptide drugs are prone to degradation via oxidation, deamidation and backbone cleavage, which can affect their safety and efficacy. Accurate impurity profiling under stress conditions is therefore critical for ensuring product quality and regulatory compliance.

Objectives and Study Overview

This study evaluates the performance of a single-quadrupole LC/MS system (Agilent InfinityLab Pro iQ Plus) for detecting low-level tirzepatide impurities formed under different pH (5, 7 and 9) and storage durations (up to 7 days at 5 °C). The goal was to demonstrate a streamlined, cost-effective workflow suitable for routine QC/QA environments.

Methodology and Instrumentation

• Sample Preparation: Tirzepatide stock solution (2 mg/mL) in 15 % acetonitrile/water was diluted to 50 ng/µL in buffers at pH 5, 7 and 9. Samples were stored at 5 °C in the autosampler and analyzed after 1, 3 and 7 days without additional cleanup.
• Chromatography: Agilent 1290 Infinity II Bio LC with ZORBAX RRHD 300 Å StableBond C18 column (2.1×150 mm, 1.8 µm). Gradient elution from 20 % to 80 % acetonitrile (+0.1 % formic acid) over 15 min, flow rate 0.4 mL/min, column at 60 °C, 1 µL injection.
• Mass Spectrometry: Agilent InfinityLab Pro iQ Plus single quadrupole MS in positive ESI mode; m/z range 500–2 500, fragmentor 95 V, source temperatures 300 °C (drying) and 250 °C (sheath gas).
• Data Analysis: Agilent OpenLab CDS 2.8 with integrated spectral deconvolution optimized for multiply charged peptides.

Main Results and Discussion

The system reliably detected native tirzepatide (4 813.1 Da) and two oxidation products (+32 Da and +4.4 Da) at retention times 7.57 and 7.71 min, as well as an unknown degradant at ~4 689.6 Da. Charge states +3, +4 and +5 were clearly resolved. Oxidation increased over time, especially at pH 5, with relative impurity levels below 2 % detectable even after one week.

Benefits and Practical Applications

The Pro iQ Plus LC/MS workflow offers high sensitivity for trace impurities, broad dynamic range, integrated deconvolution and minimal method complexity. Its cost-effectiveness and ease of use make it ideal for routine pharmaceutical QC/QA laboratories.

Future Trends and Possibilities

Further developments may include MS/MS for structural elucidation of unknown degradants, enhanced automation of data processing, extension to other peptide therapeutics and method transfer across regulatory environments.

Conclusion

This work demonstrates that a single-quadrupole LC/MS platform can efficiently profile tirzepatide impurities under stress conditions, ensuring robust quality control and safeguarding therapeutic performance.

Reference

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