Analysis of a Synthetic Peptide and Its Impurities

Importance of the Topic

The reliable separation and identification of synthetic peptides and their related impurities is critical in biopharmaceutical research and quality control. Impurities can arise from synthesis, post‐synthetic modifications, or degradation during storage. Effective analytical methods that are compatible with both UV and mass spectrometric detection provide accurate impurity profiling and sequence confirmation for therapeutic peptides.

Objectives and Study Overview

This study evaluates the performance of an Agilent AdvanceBio Peptide Plus column in combination with formic acid–based mobile phases to separate bivalirudin, a 20‐residue synthetic peptide, and its major impurities. The goal was to develop a method that maintains high chromatographic resolution while enabling seamless transfer between LC/UV and LC/MS analyses.

Methodology

The mobile phase consisted of 0.1 % formic acid in water (A) and acetonitrile (B), using a linear gradient from 17 % to 37 % B over 22 minutes, with a final ramp to 95 % B. The column dimensions were 2.1 × 150 mm and operated at 60 °C with a flow rate of 0.4 mL/min. Injection volumes were 5 μL for UV and 1 μL for MS detection. Aged bivalirudin trifluoroacetate was prepared at 1 mg/mL in 0.1 % formic acid.

Used Instrumentation

• Agilent 1290 Infinity II HPLC: binary pump, autosampler, thermostatted column compartment, diode array detector
• Agilent 6545XT AdvanceBio LC/Q-TOF MS with Dual Jet Stream source
• Data processing: OpenLab 2.2 CDS (UV) and MassHunter BioConfirm B.08.00 (MS/MS)

Main Results and Discussion

The FA‐compatible method achieved baseline separation of bivalirudin and five major impurities. LC/MS deconvolution and MS/MS fragmentation revealed:

• Peak 1: –129 Da deletion corresponding to loss of Glu residue, localized by b₁₅ and y₄ fragments
• Peak 2: Main product, monoisotopic mass 2178.99 Da
• Peak 3: –57 Da deletion indicative of missing Gly
• Peak 4: –18 Da consistent with dehydration (loss of H₂O)
• Peak 5: +1 Da deamidation of Asn to Asp at position 9, confirmed by y₅ and b₁₅ ions

The hybrid positively charged surface of the AdvanceBio Peptide Plus phase improved peak shape and resolution with formic acid, overcoming the limitations of traditional C18 columns.

Benefits and Practical Applications

• Enhanced mass spectrometric sensitivity without TFA suppression
• Robust peak shapes and high resolution using MS-compatible mobile phases
• Seamless transfer between UV and MS workflows for rapid impurity profiling

Future Trends and Opportunities

Advancements in hybrid and core-shell phases will further improve peptide separations with MS compatibility. Integration of high-throughput LC/MS/MS screening and automated data analysis will accelerate impurity identification. The approach may extend to larger peptides, peptide‐drug conjugates, and complex biotherapeutics.

Conclusion

The Agilent AdvanceBio Peptide Plus column with formic acid mobile phases provides a versatile and MS-friendly method for separating synthetic peptides and characterizing impurities. The approach delivers reliable sequence confirmation and impurity identification with minimal method transfer effort.

References

• Eggen I et al. Control Strategies for Synthetic Therapeutic Peptide APIs Part III: Manufacturing Process Considerations. Pharm Technol. 2014;38(5).