Amino Acid Composition Test of Semaglutide and Liraglutide Using an Agilent 1260 Infinity II Prime Bio LC

Significance of the topic

Quality control of synthetic peptide therapeutics relies on accurate determination of their amino acid composition. Confirming the presence and quantity of each amino acid, including non-natural or modified residues, is essential to ensure batch consistency, efficacy, and safety. Amino acid analysis also complements peptide mapping by resolving isobaric residues, such as leucine and isoleucine, that mass spectrometry cannot distinguish.

Objectives and Study Overview

The application note describes the development and validation of a reversed-phase liquid chromatography method for quantifying the amino acid composition of two GLP-1 analogues, semaglutide and liraglutide. Pre-column derivatization with ortho-phthalaldehyde (OPA) and fluorescence detection are used to achieve high sensitivity. Key aims include:

• Hydrolyze peptides into constituent amino acids under controlled acidic conditions.
• Derivatize free amino acids immediately prior to injection using an automated autosampler workflow.
• Separate and quantify 15 proteinogenic amino acids and the non-natural 2-aminoisobutyric acid (Aib).
• Demonstrate method linearity, specificity, and recovery for routine quality testing.

Used Methodology and Instrumentation

Hydrolysis: 24-hour incubation of peptides with 6 M HCl under vacuum and nitrogen purge at 110 °C to cleave peptide bonds. Neutralization by dilution and pH adjustment precedes analysis.
Derivatization: Automated pre-column reaction with OPA in borate buffer, performed within the Agilent autosampler. This minimizes manual handling and preserves reagent stability.
Chromatography and Detection:

• Agilent 1260 Infinity II Prime Bio LC system
• Agilent AdvanceBio AAA column (4.6 × 100 mm, 2.7 µm)
• Mobile phases: phosphate-borate buffer (A) and organic mix (ACN/MeOH/H₂O, 45:45:10, B)
• Fluorescence detector: excitation 340 nm, emission 450 nm
• Agilent OpenLab CDS software, version 2.7

Main Results and Discussion

Calibration curves for 15 amino acids showed R² > 0.999 across 10–250 µM (Aib 1–125 µM). Fluorescence detection outperformed UV for sensitivity. Glutamine converted to glutamic acid during hydrolysis; tryptophan was lost under acidic conditions. Semaglutide and liraglutide analyses confirmed expected amino acid profiles, including Aib in semaglutide. Recoveries ranged from 40 % to 111 %, reflecting partial losses from side-chain modifications and hydrolysis inefficiencies. The method clearly resolved isoleucine and leucine, overcoming a common limitation of MS-based peptide mapping.

Benefits and Practical Applications

• Rapid, high-resolution quantification of amino acid composition for peptide drug characterization
• Automated derivatization reduces human error and improves reproducibility
• Ability to quantify non-standard residues and distinguish isobaric amino acids
• Compliance with pharmacopeial guidelines for peptide analysis

Future Trends and Potential Applications

Improvements in hydrolysis protocols may enhance recovery of labile residues. Integration with mass spectrometry detection could extend analysis to undetected amino acids, such as tryptophan. Expanding the method to higher-throughput platforms and applying it to bioconjugates or antibody fragments represent further opportunities.

Conclusion

The Agilent AdvanceBio AAA LC solution, coupled with OPA derivatization and automated sample preparation, provides a reliable and sensitive approach to amino acid composition analysis of synthetic peptides. The method delivers clear separation of critical residues, including isoleucine and leucine, and quantifies modified amino acids, supporting robust quality control of peptide therapeutics.

References

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