Characterization and Quantification of Glucagon Like Peptide-1 Agonists and their Impurities Using Liquid Chromatography/Mass Spectrometry (LC/MS)

Significance of the topic

GLP-1 agonists represent a critical class of peptide-based therapeutics for diabetes and obesity management. Impurity profiling during development and manufacturing is essential to safeguard safety and efficacy. Advanced LC-MS techniques enable sensitive detection and characterization of oxidative and other degradation products that can affect drug performance.

Objectives and study overview

This study aimed to characterize and quantify impurity profiles of three GLP-1 receptor agonists—liraglutide, semaglutide, and tirzepatide—under forced oxidative and pH stress. A secondary goal was to develop a rapid MRM-based quantitation method for both unmodified and mono-oxidized tirzepatide using triple quadrupole LC-MS.

Methodology and instrumentation

Liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC/Q-TOF MS) was employed for high-resolution impurity characterization. Key instrumentation included the Agilent 1290 Infinity II Bio LC system and Agilent 6545XT AdvanceBio LC/Q-TOF MS. Peptide standards were prepared in acetonitrile, subjected to oxidative stress with varying concentrations of H2O2, and incubated overnight.

Quantitative analysis utilized an Agilent 1290 Infinity II Bio LC system paired with a 6495D triple quadrupole mass spectrometer. MRM transitions were optimized for tirzepatide and its oxidized form. Calibration curves ranged from 0.025 to 250 ng/mL for native and 0.25 to 250 ng/mL for mono-oxidized tirzepatide.

Main results and discussion

• TIC profiles revealed multiple oxidation products with increasing H2O2 levels, confirming formation of mono-, di-, and tri-oxidized species.
• Deconvoluted spectra indicated mass shifts of +16 Da, +32 Da, and +48 Da correlating to successive oxidation events.
• Charge state distributions and MS/MS fragmentation further localized oxidation sites, including tryptophan modification.
• MRM quantitation exhibited linearity over four orders of magnitude for native and three orders for oxidized tirzepatide, with R2 > 0.996.
• Limits of quantitation were 0.025 ng/mL (native) and 0.25 ng/mL (mono-oxidized), precision (%RSD) < 6%, and accuracy between 81 % and 115 %.

Benefits and practical applications

• Comprehensive impurity screening ensures therapeutic peptide quality and compliance with regulatory standards.
• High-resolution LC/Q-TOF MS combined with MRM-based quantitation supports both structural elucidation and routine monitoring.
• The MassHunter BioConfirm software streamlines data analysis for peptide modifications and impurity identification.

Future trends and opportunities

Advances may include real-time monitoring of peptide stability, integration of ion mobility for improved isomer separation, and machine learning-driven data analysis. Expanding these methodologies to novel peptide therapeutics will enhance drug development and quality control.

Conclusion

The combination of LC/Q-TOF MS and LC/triple quadrupole MS offers a robust workflow for detailed impurity profiling and precise quantitation of GLP-1 agonists. The demonstrated sensitivity and dynamic range meet stringent requirements for biopharmaceutical analysis.

References

• Characterization of Forced Degradation Impurities of Glucagon-Like Peptide-1 Agonists by LC/Q-TOF Mass Spectrometry. Agilent application note 5994-7992EN.
• Quantification of Glucagon-Like Peptide-1 Agonist Tirzepatide Using an Agilent 6495D Triple Quadrupole LC/MS System. Agilent application note 5994-7794EN.