Analysis of GLP-1 Agonists Using the Agilent 1290 Infinity II Bio LC System and Altura Ultra Inert HPLC Column

Significance of the Topic

Glucagon-like peptide-1 (GLP-1) receptor agonists represent a vital class of therapeutic peptides used to regulate blood glucose levels. Their structural complexity and hydrophobic modifications demand highly selective and reproducible analytical methods. Accurate profiling of impurities and degradation products is critical to ensure safety and efficacy in biopharmaceutical development.

Study Objectives and Overview

This application brief evaluates the performance of the Agilent 1290 Infinity II Bio LC System paired with an Altura Peptide Plus Ultra Inert HPLC column. The focus is on analyzing two GLP-1 agonists—tirzepatide and retatrutide—under both control and forced-degradation conditions, demonstrating chromatographic efficiency, reproducibility, and mass spectrometric confirmation.

Methodology and Instrumentation

A biocompatible LC flow path was used to minimize nonspecific peptide interactions. Key conditions include:

• Column: Altura Peptide Plus, 2.1 × 150 mm, 2.7 μm, Ultra Inert hardware
• Mobile Phases: A = 0.1% formic acid in water; B = 0.1% formic acid in acetonitrile
• Gradient: 80:20 to 10:90 (A:B) over 25 minutes; reequilibration to 80:20 until 30 minutes
• Flow Rate and Temperature: 0.4 mL/min at 55 °C
• Detection: UV at 214, 220, 280 nm
• MS: Agilent 6545XT AdvanceBio LC/Q-TOF, positive AJS ESI; drying gas 325 °C/13 L/min; sheath gas 350 °C/12 L/min; capillary 4000 V; reference mass 922.009798; m/z range 100–1700

Main Results and Discussion

The system delivered high-efficiency separations for both peptides. UV chromatograms showed sharp, symmetric peaks with area RSDs of 0.69–1.22% and tailing factors of 0.83–0.90 (n = 5). Thermal stress at 85 °C for 3 hours produced distinct degradation peaks, demonstrating the column’s sensitivity to subtle peptide modifications. LC-MS analysis confirmed main peptide masses (4813.68 Da for tirzepatide, 4731.54 Da for retatrutide) and resolved low-intensity impurity and isomer peaks, highlighting excellent resolution and identification capabilities.

Benefits and Practical Applications

The combination of an inert biocompatible LC system and Ultra Inert column technology offers:

• Enhanced recovery and reproducibility for hydrophobic peptides
• Clear separation of impurities and degradation products for quality control
• Robust performance under forced-degradation studies
• Comprehensive characterization via coupled UV and high-resolution MS detection

Future Trends and Opportunities

Advancements may include expanded Ultra Inert stationary phases for broader peptide classes, integration with automated high-throughput workflows, and application of machine learning to interpret complex peptide impurity profiles. Adapting this platform for multi-modification peptides and next-generation biologics will further strengthen biopharmaceutical analytics.

Conclusion

The Agilent 1290 Infinity II Bio LC System coupled with an Altura Peptide Plus Ultra Inert column provides a powerful, reliable solution for the analysis of GLP-1 receptor agonists. Its biocompatible flow path and inert hardware ensure high chromatographic performance, reproducibility, and in-depth impurity profiling essential for peptide drug development.

References

• Müller T. D.; et al. Glucagon-Like Peptide 1 (GLP-1). Mol. Metab. 2019, 30, 72–130.