Column Selection for RPLC-UV Impurity Analysis of Fatty Acid Modified GLP-1 Receptor Agonists

Significance of the topic

Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are increasingly used for type 2 diabetes and weight management. Fatty-acid modifications enhance their pharmacokinetics but introduce structural complexity that demands robust impurity screening. Reverse-phase liquid chromatography with ultraviolet detection (RPLC-UV) remains a frontline tool for quality control. Tailored column selection is critical to resolve low-level contaminants that could affect safety and efficacy.

Objectives and study overview

This study evaluates four reversed-phase column chemistries for RPLC-UV impurity analysis of two fatty-acid modified GLP-1 RAs, liraglutide and semaglutide. The aim is to identify optimal stationary phases and chromatographic conditions for high-resolution separation of peptide variants and to demonstrate mass spectrometric (MS) identification of trace impurities down to 0.1 % relative abundance.

Methodology and applied instrumentation

Sample preparation involved reconstitution of research-grade liraglutide and expired semaglutide at 0.5 mg/mL. Chromatographic runs were performed on an ACQUITY UPLC I-Class PLUS system. Five columns were screened:

• CORTECS Premier C18+ (1.6 µm, 2.1 × 150 mm)
• CORTECS Premier C18+ (2.7 µm, 2.1 × 150 mm)
• ACQUITY Premier Peptide CSH C18 (130 Å, 1.7 µm, 2.1 × 150 mm)
• ACQUITY Premier CSH Phenyl-Hexyl (1.7 µm, 2.1 × 150 mm)
• ACQUITY Premier BEH C8 (1.7 µm, 2.1 × 150 mm)

Chromatographic conditions included a 60 °C column temperature, 6 °C sample tray, 6 µL injection, and a shallow gradient of 0.1 % TFA in water (A) to 0.1 % TFA in acetonitrile (B). MS detection used positive‐mode ESI, 50–2000 m/z, 1.2 kV capillary voltage, 350 °C desolvation, and a 1 Hz scan rate.

Main results and discussion

Column performance differed by analyte. For liraglutide, CORTECS C18+ and Peptide CSH C18 provided the best resolution of isomeric and oxidized variants. For semaglutide, CORTECS C18+ and CSH Phenyl-Hexyl excelled, with Phenyl-Hexyl showing enhanced separation after gradient optimization to avoid coelution with phenol. Scaling from 1.6 µm to 2.7 µm particles reduced resolution but retained acceptable impurity detail, supporting method transfer to conventional HPLC. Despite TFA’s ion-suppression effect, MS spectra of low-abundance impurities (0.1 %) were obtained. Substituting formic acid for TFA increased MS sensitivity at the expense of some UV baseline noise and selectivity shifts.

Benefits and practical applications

• Direct comparison of multiple reversed-phase chemistries for lipopeptide impurity analysis
• Guidance for column selection tailored to fatty-acid modified peptide structures
• Demonstration of MS identification of trace impurities under ion-suppressive conditions
• Pathway for method scalability between UHPLC and HPLC platforms

Future trends and potential applications

• Investigation of alternative mobile phase additives for balanced UV and MS performance
• Extension of method principles to other therapeutic lipopeptides and peptide conjugates
• Integration of high-resolution MS and tandem MS for structural elucidation of novel variants
• Development of green solvents and sustainable chromatographic practices

Conclusion

No single column universally separates all fatty-acid modified GLP-1 RAs. Systematic screening identifies optimal stationary phases for each analyte. The presented RPLC-UV/MS workflows enable sensitive detection and characterization of peptide impurities, offering a robust foundation for method development and quality control in biotherapeutic analysis.

Reference

1. Watanabe JH, Kown J, Nan B, Reikes A. Trends in Glucagon-like Peptide 1 Receptor Agonist use, 2014 to 2022. Journal of the American Pharmacists Association. 2024;64:133–138.
2. Clements BR, Rainville P. Development of Separation Methods for GLP-1 Synthetic Peptides Utilizing a Systematic Protocol and MaxPeak High Performance Surface Technology. Application note 720008267. Waters Corporation; 2024.
3. Knudsen LB, Lau K. The Discovery and Development of Liraglutide and Semaglutide. Frontiers in Endocrinology. 2019;10:155.
4. Koza SM, Chambers EE. Selecting a Reversed-Phase Column for the Peptide Mapping Analysis of a Biotherapeutic Protein. Application note 720005924. Waters Corporation; 2017.