Characterization of Synthetic Peptide Drug and Impurities using High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography/Mass Spectrometry (LC/MS)

Significance of the topic

Peptide-based therapies such as Semaglutide and Liraglutide have revolutionized treatment of diabetes and obesity.
Reliable assessment of impurities, degradation products and aggregates is essential to guarantee drug safety and minimize immunogenic risks.

Objectives and overview of the study

This work demonstrates a streamlined workflow for peptide API confirmation and impurity and aggregate profiling using single quadrupole LC–MS and two-dimensional liquid chromatography.
Additional focus includes UV purity testing optimization and amino acid composition analysis via derivatization.

Methodology and instrumention

Forced degradation of peptides under heat, acidic, basic and oxidative conditions generated impurity profiles.
One-dimensional separations were performed with trifluoroacetic acid (TFA) and difluoroacetic acid (DFA) mobile phases to optimize chromatographic resolution and MS compatibility.
Heart-cutting 2D-LC enabled MS-friendly analysis of low-level impurities and aggregate species.
Acid hydrolysis and OPA derivatization were applied to quantify common and non-natural amino acid residues.

Used instrumentation

• Agilent 1260 Infinity II Bio Prime LC
• Agilent 1290 Infinity II Bio 2D-LC with Poroshell CS-C18 column
• Agilent InfinityLab LC/MSD iQ and LC/MSD XT single quadrupole MS detectors
• AdvanceBio Peptide mapping and Poroshell columns for 1D and 2D separations
• OpenLab CDS software for spectral deconvolution

Key results and discussion

Optimization of TFA concentration (0.05–0.5%) revealed 0.4% TFA for UV purity testing and 0.1% formic acid for MS-friendly 2D analysis as optimal conditions.
Single quadrupole MS confirmed intact masses of APIs and forced degradation products.
2D-LC distinguished reversible and irreversible peptide aggregates based on size exclusion and MS deconvolution.
OPA derivatization enabled determination of 16 amino acid residues including 2-aminoisobutyric acid and aminoethylethanolamine.

Benefits and practical applications

• Rapid confirmation of peptide API identity and purity
• Comprehensive impurity and aggregate profiling for quality control
• High sensitivity MS detection without extensive sample preparation
• Amino acid composition analysis supporting formulation and stability studies

Future trends and potential applications

Adoption of high-resolution MS and automation to enhance sensitivity and throughput.
Integration of real-time monitoring with microfluidic LC-MS platforms.
Expansion of methods to other peptide therapeutics and complex biologics.

Conclusion

Combined single quadrupole LC–MS and 2D-LC approaches provide a robust, adaptable workflow for detailed characterization of peptide drugs, impurities and aggregates, supporting regulatory compliance and accelerating development.

Reference

1. Chae-Young Ryu. Efficient Method Optimization of Semaglutide Analysis Using an Agilent 1260 Infinity II Bio Prime LC System and Blend Assist. Agilent Technologies application note 5994-7414EN, 2024.
2. Chae-Young Ryu. Rapid Confirmation of GLP-1 Analog Using Agilent InfinityLab LC/MSD iQ. Agilent Technologies application note 5994-7415EN, 2024.
3. Chae-Young Ryu. Peptide Drug Stability Analysis Using Agilent InfinityLab LC/MSD and OpenLab CDS Deconvolution. Agilent Technologies application note 5994-7500EN, 2024.
4. Chae-Young Ryu. Confirmation of Peptide-Related Impurity Intact Mass Using Agilent 1290 Infinity II Bio 2D-LC and InfinityLab LC/MSD XT. Agilent Technologies application note 5994-7654EN, 2024.
5. Chae-Young Ryu. Amino Acid Composition of Peptide Drug Using Agilent 1260 Infinity II Prime LC. Agilent Technologies application note 5994-7749EN, 2024.
6. Chae-Young Ryu. Comprehensive Aggregate Profiling of Liraglutide and Semaglutide Using 1290 Infinity II Bio 2D-LC and InfinityLab LC/MSD XT. Agilent Technologies application note 5994-7740EN, 2024.