Quantification of Semaglutide in Human Plasma Using a Benchtop Multi-reflecting Time-of-Flight Mass Spectrometer

Importance of the Topic

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have become indispensable in managing type 2 diabetes and obesity. Their complex peptide structures and low endogenous levels in plasma demand highly sensitive and selective analytical methods for accurate quantification, impurity profiling and supporting rapid drug development and quality control.

Aims and Study Overview

This work presents a high-throughput liquid chromatography–high resolution mass spectrometry (LC-HRMS) approach for quantifying semaglutide in human plasma. By implementing a ballistic chromatographic gradient on a benchtop multi-reflecting time-of-flight (MRT) mass spectrometer, the total run time is reduced to under 3 minutes while maintaining excellent analytical performance.

Methods and Instrumentation

• Sample Preparation: Protein precipitation of human plasma (1:3 plasma:acetonitrile/methanol, v/v) followed by addition of 5% formic acid. Semaglutide stock prepared in methanol/formic acid; liraglutide used as internal standard.
• Calibration Range: 0.05–500 ng/mL in solvent; 5–500 ng/mL in acidified plasma extracts.
• Instrumentation:
  • LC: Waters ACQUITY Premier System with Peptide CSH C18 column (1.7 µm, 2.1×150 mm) at 65 °C, 0.8 mL/min flow, 2 µL injection, 2.5 min gradient.
  • MS: Xevo MRT Mass Spectrometer, ESI positive mode, full scan m/z 50–1200 at 20 Hz, lock mass leucine enkephalin, capillary 0.9 kV, cone 40 V, desolvation 550 °C.
• Data Processing: waters_connect platform with UNIFI application.

Main Results and Discussion

• Sensitivity and Linearity: Limit of detection at 0.05 ng/mL in solvent and 5 ng/mL in plasma; linear dynamic range spanning four orders of magnitude (0.05–500 ng/mL), R² > 0.99.
• Selectivity and Carry-Over: No significant interferences in plasma matrix; carry-over below 0.1% after injecting the highest standard.
• Throughput and Solvent Economy: Total chromatographic runtime of 2.5 minutes—fourfold faster than a 10-minute method—while halving organic solvent consumption and reducing energy per sample.
• Stability: Semaglutide and internal standard responses remained within 5% and 15% variation, respectively, over 96 hours at 4 °C.

Benefits and Practical Applications

This method delivers rapid, cost-effective and reliable quantification of semaglutide in routine bioanalysis, supporting high sample throughput in clinical and regulatory laboratories. The reduced runtime and solvent usage lower operating costs and environmental impact.

Future Trends and Potential Applications

• Expansion to other GLP-1RA and peptide therapeutics using similar ballistic LC-HRMS strategies.
• Integration of qualitative HRMS scans with quantitative workflows for simultaneous impurity profiling.
• Adoption of greener chromatography metrics and automation to further enhance lab sustainability and productivity.

Conclusion

The ballistic LC-MRT-TOF MS method offers exceptional sensitivity, selectivity and stability for semaglutide quantification in human plasma. Its high-throughput capability, solvent reduction and robust performance make it an ideal workflow for both discovery and regulated environments.

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