Sensitive LC-MS method for the quantitative analysis of semaglutide and liraglutide in human plasma

Importance of the Topic

This work addresses the critical need for highly sensitive and accurate quantification of two clinically important GLP-1 analogs, semaglutide and liraglutide, in human plasma. Reliable measurements at low concentrations support pharmacokinetic investigations, therapeutic drug monitoring, and safety assessments in patients with type 2 diabetes and obesity.

Study Aims and Overview

The primary objective was to develop and validate an LC-MS method capable of detecting semaglutide and liraglutide down to 0.1 ng/mL in plasma. The study demonstrates the analytical performance in terms of linear dynamic range, precision, accuracy, and carryover behavior, using each peptide as an internal standard for the other.

Methodology and Instrumentation

Sample Preparation:

• Stock solutions (1 mg/mL) in methanol; calibration standards from 1 to 1000 ng/mL.
• Protein precipitation with cold acetone, followed by centrifugation.
• SPE µ-elution plate for cleanup; 200 µL plasma spiked with peptides and internal standard at 10 ng/mL.

Chromatography and Mass Spectrometry:

• UHPLC on Hypersil GOLD Peptide column (2.1 × 50 mm, 1.9 µm) at 60 °C.
• Gradient elution with 0.4% formic acid in water (A) and 0.1% difluoroacetic acid in acetonitrile (B), 35–95% B over 8 min.
• TSQ Altis Plus triple quadrupole MS in SRM mode; optimized source temperatures, gas flows, and collision energies for quantifier and qualifier ions.

Main Results and Discussion

The method achieved a limit of quantitation of 0.1 ng/mL for both peptides. Calibration was linear over three orders of magnitude (0.1–100 ng/mL), with:

• Precision ≤ 10% RSD across all levels.
• Accuracy within ± 20% of nominal values.
• Carryover below 10% of the LOQ for semaglutide and under 14% for liraglutide after a 100 ng/mL injection.

Chromatographic retention times were consistent (approximately 3.4 min), and the embedded “shark teeth” wash cycles minimized residual signal without extending run times significantly.

Benefits and Practical Applications

This robust LC-MS approach enables:

• Precise pharmacokinetic profiling of GLP-1 analogs in clinical trials and routine monitoring.
• Sensitive detection of low-level drug exposure to optimize dosing.
• Compliance with bioanalytical regulatory guidelines for accuracy, precision, and carryover.

Future Trends and Opportunities

Emerging areas to enhance and extend this work include:

• Automation of sample preparation to increase throughput.
• Multiplexed assays for simultaneous analysis of additional peptide therapeutics.
• Integration of high-resolution mass spectrometry for improved selectivity.
• Application to real-world clinical studies and personalized dosing strategies.

Conclusion

The presented LC-MS method provides a sensitive, accurate, and efficient solution for quantifying semaglutide and liraglutide in human plasma. Its validated performance supports reliable drug quantitation in pharmacokinetic and therapeutic monitoring applications.

Instrumentation Used

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific TSQ Altis Plus triple quadrupole mass spectrometer
• Hypersil GOLD Peptide column (2.1 × 50 mm, 1.9 µm)
• SPE µ-elution plate for sample cleanup
• Thermo Scientific Chromeleon CDS software (v7.3.2)

References

1. Drucker DJ, Habener JF, Holst JJ. Discovery, characterization, and clinical development of the glucagon-like peptides. Journal of Clinical Investigation. 2017;127(12):4217–4227.
2. Nauck MA, Meier JJ. Incretin hormones: Their role in health and disease. Diabetes, Obesity and Metabolism. 2018;20(S1):5–21.