HILIC Analysis of GLP-1 Receptor Agonists

Importance of the topic

Hydrophilic interaction liquid chromatography (HILIC) offers a powerful approach for comprehensive analysis of GLP-1 receptor agonist therapies used in diabetes and obesity management. Simultaneous measurement of the peptide active ingredient, related impurities and diverse formulation excipients in a single LC run enhances quality control and streamlines workflow in pharmaceutical development and manufacturing.

Study objectives and overview

This application note demonstrates a generic HILIC method combining diode array detection (DAD) and evaporative light scattering detection (ELSD) on a low-adsorption, biocompatible LC flow path. The study evaluates the simultaneous analysis of four marketed GLP-1 agonists (Exenatide, Liraglutide, Semaglutide, Tirzepatide), formulation components and product-related impurities. It also examines the impact of stainless-steel surfaces versus a deactivated flow path on chromatographic performance.

Methodology and instrumentation

A 2.1×150 mm, 2.7 µm HILIC-Z column (deactivated stainless-steel hardware) was run on an Agilent 1290 Infinity III Bio LC System. Key parameters:

• Mobile phase A: 100 mM ammonium formate, pH 3; B: ACN
• Gradient: 90 % B at 0–1 min to 45 % B at 19 min, then re-equilibration
• Flow rate: 0.4 mL/min, column temperature: 40 °C, autosampler: 10 °C
• Injection volume: 1 µL, needle wash with 50/50 acetonitrile:water
• DAD at 280 nm (reference 360 nm), ELSD at 50 °C evaporator and nebulizer, 1.0 SLM nitrogen, 80 Hz data rate

Used instrumentation

• Agilent 1290 Infinity III Bio High-Speed Pump and Multisampler with sample thermostat
• Agilent 1290 Infinity III Bio Multicolumn Thermostat with Quick Connect Bio heat exchanger
• Agilent 1290 Infinity III Diode Array Detector with Max-Light Cartridge cell
• Agilent 1290 Infinity III Evaporative Light Scattering Detector

Key results and discussion

• HILIC-ELSD separated common excipients (glycerol, mannitol, sucrose, NaCl, phosphate, zinc) with comparable detector response after concentration adjustment.
• HILIC-DAD resolved Exenatide, Liraglutide, Semaglutide and Tirzepatide based on amino acid composition and fatty acid conjugation.
• Simultaneous DAD and ELSD detection allowed quantification of peptide, impurities, preservatives and buffering components in one run.
• Deactivated stainless-steel flow path markedly improved peak shape, sensitivity and recovery of phosphate and peptide peaks compared to a standard stainless-steel setup.
• Oxidative product-related impurities of Exenatide were clearly resolved on the low-adsorption system but remained masked on the stainless-steel path.

Benefits and practical applications

• Single-run profiling of active peptide, impurities and formulation constituents reduces analysis time and resource use.
• Biocompatible, low-adsorption flow path minimizes metal-peptide interactions, improving accuracy and peak shape.
• Method is adaptable for routine QC, stability testing and impurity monitoring in GLP-1 agonist development.

Future trends and opportunities

Further optimization of HILIC stationary phases and integration with mass spectrometry could expand sensitivity and specificity. Emerging bioinert materials and online sample preparation modules may enable high-throughput workflows. The approach can be extended to other peptide-based biologics and complex formulations in industrial and regulatory laboratories.

Conclusion

A robust HILIC-DAD-ELSD method on a deactivated, low-adsorption LC system enables comprehensive quality assessment of GLP-1 receptor agonist drug products. The combination of simultaneous multi-detector read-outs and bioinert hardware delivers reliable peptide and excipient analysis in a single chromatographic run.

References

1. Yu M et al. Battle of GLP-1 Delivery Technologies. Adv Drug Deliv Rev 2018, 130, 113–130.
2. McCalley DV. Understanding and Manipulating HILIC Separations. J Chromatogr A 2017, 1523, 49–71.
3. Agilent Technologies. Analysis of Metals, Halides, and Inorganic Ions Using HILIC. 2017, publication 5991-8602EN.
4. Guimaraes GJ, Bartlett MG. Managing Nonspecific Adsorption in LC Hardware: A Review. Anal Chim Acta 2023, 1250, 340994.