Analysis of Peptide and Impurities

Importance of the Topic

The analysis of peptide hormones and medium-sized therapeutic molecules is critical in pharmaceutical quality control. These biomolecules often display complex structural features and are prone to degradation or modification. Reliable separation and sensitive detection of active peptides and their impurities ensure drug safety, efficacy, and regulatory compliance.

Objectives and Study Overview

This study demonstrates a rapid reversed-phase LC–MS method for simultaneous separation and detection of somatostatin and related impurities. The primary goals are to achieve high resolution, short analysis time, and robust quantitation suitable for routine QC workflows in drug manufacturing.

Methodology and Instrumentation

The method combines a Shim-pack Scepter C18-120 column with optimized LC and MS parameters:

• HPLC conditions (Nexera X3):
   Column: Shim-pack Scepter C18-120, 50 mm × 2.1 mm I.D., 1.9 µm
   Column temperature: 40 °C
   Mobile phase A: 0.1 % TFA in water
   Mobile phase B: 0.1 % TFA in acetonitrile
   Gradient: 10 % B (0.00–1.50 min) to 80 % B at 5.00 min, then return to 10 % B by 5.01 min and re-equilibrate until 8.00 min
   Flow rate: 0.4 mL/min
• MS conditions (LCMS-2050):
   Ionization: ESI/APCI dual interface, positive mode
   Scan range: m/z 250–2000
   Nebulizing gas: 2.0 L/min, drying gas: 5.0 L/min, heating gas: 7.0 L/min
   DL temperature: 450 °C, desolvation temperature: 200 °C
   Interface voltage: +3.0 kV, Qarray voltage: +20 V

Main Results and Discussion

The optimized method separates somatostatin and its impurities within an 8-minute runtime. Chromatographic peaks exhibit sharp shape and baseline separation, enabling clear identification of low-level impurities. Mass spectral data confirm molecular weights and support impurity profiling. The combined LC–MS approach demonstrates high sensitivity and reproducibility, with consistent retention times across multiple injections.

Benefits and Practical Applications

This LC–MS protocol offers several advantages for pharmaceutical analysis:

• Rapid throughput: full analysis in under 10 minutes
• High resolution: efficient separation of closely eluting peptide variants
• Sensitive detection: low-level impurity monitoring via mass spectrometry
• Robustness: stable retention times and reproducible peak areas for routine QC

Future Trends and Applications

Advancements in high-resolution mass spectrometry and microflow LC are expected to further improve peptide analysis. Integration with automated sample preparation and data processing pipelines will enhance throughput and compliance. Emerging techniques such as ion mobility separation and isotope labeling offer additional dimensions for deeper impurity characterization.

Conclusion

The presented reversed-phase LC–MS method on Shim-pack Scepter C18-120 provides a fast, reliable, and sensitive approach for peptide hormone analysis. Its suitability for quality control ensures accurate monitoring of therapeutic peptides and their impurities, supporting regulatory requirements and manufacturing consistency.

Reference

• Application News 01-00625 (JP, EN), Shimadzu Corporation