Impurity Profiling of GLP-1 Receptor Agonists by HILIC-MS

Importance of the Topic

GLP-1 receptor agonists represent a key class of biotherapeutic peptides for managing type 2 diabetes and obesity. Ensuring the safety and efficacy of these agents requires comprehensive impurity profiling, as structural variants can arise during synthesis or storage and may impact drug performance and regulatory compliance.

Objectives and Study Overview

This application note demonstrates an orthogonal hydrophilic interaction liquid chromatography–mass spectrometry (HILIC-MS) method for profiling impurities in Exenatide, Semaglutide, and Tirzepatide. The goals are to achieve robust separation of product-related variants and to confirm their identities using a compact single quadrupole MS system.

Methodology and Instrumentation

Samples of Exenatide, Semaglutide, and Tirzepatide were prepared in DMSO or acetonitrile/water, with a forced oxidation step applied to Exenatide. Separation employed a deactivated stainless-steel HILIC-Z column and an Agilent 1290 Infinity III Bio LC System coupled to an Agilent InfinityLab LC/MSD iQ single quadrupole MS. The mobile phase comprised ammonium formate buffer (pH 3) and acetonitrile with a gradient from 90 % to 45 % organic over 20 minutes at 0.4 mL/min. UV detection at 280 nm and positive-mode electrospray MS (m/z 500–1450) were used to monitor eluting peptides and impurities while minimizing nonspecific adsorption.

Main Results and Discussion

The HILIC method achieved clear resolution of the three therapeutic peptides and their low-level impurities. Key findings include:

• Tirzepatide impurity at 1.1 % identified as a tyrosine insertion (+163 Da) by extracted ion chromatography (EIC) and MS.
• Semaglutide impurities at 0.2–0.4 % comprising a stereoisomeric variant, an N-terminal histidine cyclization (+12 Da), and a truncated form (–222 Da).
• Exenatide variants including methionine oxidation (+16 Da), glycine addition (+57 Da), leucine/isoleucine insertion (+113 Da), and N-terminal histidine deletion (–137 Da); forced oxidation produced separable R and S sulfoxide diastereomers.

Use of a single quadrupole MS enabled detection of coeluting or tailing impurities that would be challenging to quantify by UV alone.

Benefits and Practical Applications

The described HILIC-MS platform offers an orthogonal separation to reversed-phase LC, enhancing impurity coverage in peptide therapeutics. The biocompatible hardware prevents metal-induced adsorption, and the stackable single quadrupole MS provides accessible mass confirmation. This approach supports both routine quality control and forced degradation studies.

Future Trends and Applications

Further refinements could include targeted gradient adjustments to resolve stereoisomeric sulfoxides, integration into two-dimensional LC workflows for comprehensive profiling, and coupling with higher-resolution MS or data-driven algorithms for automated impurity identification. Ongoing development of novel HILIC stationary phases may improve selectivity for challenging peptide variants.

Conclusion

The presented HILIC-MS method is a versatile and robust solution for impurity profiling of GLP-1 receptor agonists. Its orthogonal retention mechanism and MS confirmation capability make it a valuable tool for ensuring peptide drug quality and regulatory compliance.

Used Instrumentation

• Agilent 1290 Infinity III Bio High-Speed Pump and Multisampler with Thermostat
• Agilent 1290 Infinity III Multicolumn Thermostat with Bio Heat Exchanger
• Agilent 1290 Infinity III Diode Array Detector (280 nm)
• Agilent InfinityLab LC/MSD iQ single quadrupole mass detector

References

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