Comprehensive Workflow for the Quantification of Peptides and Proteins in Plasma: Semaglutide a Case Study

Significance of the Topic

The rise of peptide and protein therapeutics has created a demand for ultra-sensitive bioanalytical methods to monitor low-level drug concentrations in plasma. Semaglutide, a GLP-1 receptor agonist used in diabetes and obesity management, circulates in the low nmol/L range and requires robust quantification across its pharmacokinetic elimination phase.

Objectives and Study Overview

This study demonstrates a streamlined LC-MS/MS workflow for quantifying semaglutide in human plasma. The approach integrates automated MRM transition selection, high-performance chromatography, and optimized sample cleanup to achieve sub-ng/mL sensitivity and reliable pharmacokinetic profiling.

Methodology and Instrumentation

Sample Preparation

• Calibration standards and QC samples prepared in human plasma (0.2 to 100 ng/mL).
• Protein precipitation with methanol containing liraglutide internal standard followed by Oasis MAX mixed-mode SPE extraction.
• Final extracts collected in QuanRecovery plates to minimize peptide loss.

Chromatography and Mass Spectrometry

• ACQUITY Premier UPLC equipped with peptide CSH C18 column at 65 °C and MaxPeak high performance surfaces.
• Xevo TQ Absolute triple quadrupole mass spectrometer operated in positive ESI mode.
• Automated MRM method development via MassLynx v4.2 integrated with Skyline interface.

Major Findings and Discussion

The in silico-assisted MSI tool generated over 60 precursor-product ion and collision energy combinations, selecting the [M + 4H]4+ → major fragment transition for semaglutide quantitation. The optimized UPLC method delivered sharp, reproducible peaks at a retention time of 2.18 minutes with no detectable carryover. Calibration was linear (R2 = 0.9985) from 0.2 to 100 ng/mL, with a lower limit of quantification of 0.2 ng/mL and signal-to-noise above 5:1. QC precision (%CV < 10.6) and accuracy (> 90%) met acceptance criteria for fit-for-purpose bioanalytical assays.

Benefits and Practical Applications

• Enables accurate quantification of semaglutide during the elimination phase at sub-ng/mL levels.
• Automated MRM development reduces method development time and manual errors.
• High performance surfaces and recovery plates minimize non-specific binding, improving sensitivity and peak performance.
• Adaptable workflow for other peptide and protein therapeutics in drug discovery and clinical research.

Future Trends and Opportunities

Continued integration of in silico tools and advanced surface chemistries will further accelerate peptide assay development. Expanding this workflow to diverse peptide classes and larger biomolecules can support comprehensive pharmacokinetic and biomarker studies. Automation improvements and cloud-based informatics may enhance throughput and data sharing in regulated environments.

Conclusion

The presented UPLC-MS/MS workflow achieves high sensitivity and robustness for semaglutide quantification in human plasma. By combining automated MRM optimization, optimized sample cleanup, and high performance instrumentation, the method meets stringent requirements for modern peptide bioanalysis and offers a template for future therapeutic peptide assays.

References

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