Integrated LC/UV and LC/MS Workflows for Impurity and Degradation Characterization of GLP-1 Agonists

Significance of the topic

The accurate analytical characterization of synthetic peptide therapeutics is critical for product quality, safety, and regulatory compliance. GLP-1 receptor agonists are a rapidly growing class of peptide drugs with increased molecular complexity due to sequence length (typically 30–40 amino acids) and chemical modifications such as fatty-acylation to extend half-life. These features increase hydrophobicity and the tendency for nonspecific interactions, making robust separation and mass confirmation essential to detect low-level impurities, degradation products, and sequence variants that can impact efficacy and safety.

Objectives and study overview

This work evaluates integrated LC/UV and LC/MS workflows for impurity profiling, degradation characterization, and supplier comparison of GLP-1 agonists. The study demonstrates a biocompatible LC front end, ultra-inert high-efficiency peptide column, and both single-quadrupole and Q-TOF mass spectrometers to:

• Resolve closely related peptide species and minor impurities by LC/UV;
• Obtain intact-mass confirmation and deconvoluted spectra by LC/MS;
• Compare purity and impurity profiles across suppliers and under thermal stress for peptides including tirzepatide, retatrutide, liraglutide, and semaglutide.

Used instrumentation

The study used Agilent instrumentation and an ultra-inert LC column:

• Agilent 1290 Infinity II Bio LC system (biocompatible flow paths) for LC/UV and LC/MS separations.
• Agilent InfinityLab Pro iQ Plus (single-quadrupole MS capability) for intact-mass profiling and comparative TICs.
• Agilent 6545XT AdvanceBio LC/Q-TOF MS for high-resolution intact mass measurement and deconvolution.
• Agilent Altura Peptide Plus ultra-inert HPLC column (2.7 µm) with inert-coated stainless steel hardware to reduce nonspecific binding and improve peak shape and sensitivity.

Methodology

The analytical workflow combined optimized LC conditions for biomolecule analysis with mass spectrometric detection:

• Biocompatible LC flow paths to preserve peptide integrity and limit surface adsorption.
• Use of an ultra-inert peptide column to minimize nonspecific peptide–surface interactions and enhance chromatographic efficiency.
• LC/UV detection for quantitative peak shape evaluation and repeatability metrics (peak area RSD, tailing factor).
• LC/Q-TOF acquisition with intact-mass deconvolution to assign molecular weights to main peaks and to identify low-abundance impurities and isomeric forms.
• Thermal stress experiments to induce degradation and evaluate the method’s ability to resolve degradants from the active pharmaceutical ingredient (API).

Main results and discussion

Key findings demonstrate the combined LC/UV and LC/MS approach effectively characterizes GLP-1 agonists:

• Chromatography delivered sharp, well-defined peaks with excellent reproducibility: peak area RSD in the range 0.69–1.22% (n = 5) and tailing factors between 0.83–0.90.
• The Altura Peptide Plus column achieved high separation efficiency, resolving multiple impurity peaks and degradation products from the main API peaks in both control and thermally stressed samples.
• Intact-mass deconvolution by Q-TOF assigned primary masses for main peaks (examples reported include tirzepatide near 4813.5–4813.7 Da and retatrutide near 4731.5–4731.6 Da), while additional low-intensity masses corresponded to impurities, truncated/modified species, and possible isomers.
• Thermal stress accelerated formation of degradants and secondary peaks, clearly separated from the parent peptide, confirming the method’s sensitivity to subtle compositional changes.
• Comparative LC/MS profiling across suppliers revealed clear differences in impurity patterns for tirzepatide and liraglutide, whereas semaglutide displayed a comparatively higher purity profile. These results underscore the utility of intact-mass LC/MS for supplier qualification and lot-to-lot comparability.

Benefits and practical applications

The combined workflow provides multiple practical advantages for peptide therapeutic development and quality control:

• Rapid, high-confidence intact mass confirmation of peptide APIs.
• Sensitive detection and chromatographic resolution of sequence variants, truncations, chemical modifications, and degradation products.
• Reproducible LC/UV metrics suitable for routine QC or stability studies.
• Capability to support supplier qualification, incoming material screening, and comparative quality assessment among vendors.
• Improved sensitivity and peak shape from ultra-inert hardware reduce false negatives due to analyte losses on instrument surfaces.

Future trends and potential applications

Opportunities to extend and enhance the presented workflow include:

• Integration with higher-resolution MS and advanced fragmentation for site-specific identification of modifications and degradants (peptide mapping workflows complementary to intact-mass analysis).
• Automated data processing, AI-assisted deconvolution and impurity annotation to accelerate interpretation for complex peptide profiles.
• Expansion to orthogonal separation modes (e.g., ion-pairing LC, hydrophilic interaction or mixed-mode chromatography) to resolve particularly challenging isomers or hydrophobic acylated species.
• Application to native MS or top-down approaches to capture higher-order structural changes and noncovalent adducts.
• Adoption of standardized LC/MS workflows for regulatory release testing and stability protocols in peptide therapeutics.

Conclusions

The integrated LC/UV and LC/MS workflows, using biocompatible LC hardware and an ultra-inert peptide column, provide robust, reproducible separation and intact-mass confirmation of GLP-1 agonists. The approach resolves minor impurities and degradation products, differentiates supplier-specific impurity patterns, and is suitable for identity verification, impurity profiling, stability testing, and supplier qualification for complex peptide drugs.

References

1. Suresh Babu C. V. Analysis of GLP-1 Agonists Using the Agilent 1290 Infinity II Bio LC System and Altura Ultra Inert HPLC Column. Agilent Technologies application note, publication number 5994-8837EN, 2025.
2. Suresh Babu C. V. Therapeutic Peptide Analysis of GLP-1 Agonists. Agilent Technologies application note, publication number 5994-8951EN, 2026.