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, exemplified by semaglutide, demands highly sensitive and specific bioanalytical assays. Accurate quantification at sub-ng/mL levels is essential for defining pharmacokinetic profiles and supporting drug development and clinical monitoring.

Objectives and Article Overview

This study presents a streamlined, automated LC-MS/MS workflow for quantifying semaglutide in human plasma. It demonstrates method development using software-driven optimization of multiple reaction monitoring (MRM) transitions, combined with high-performance sample preparation and chromatography technologies.

Methodology

Sample preparation employed protein precipitation with methanol containing an internal standard, followed by Oasis MAX mixed-mode SPE µElution plates for cleanup and concentration. Chromatographic separation used a reversed-phase gradient on a C18 peptide column at 65 °C. MRM transition optimization was automated via waters_connect software, which screened multiple charge states, cone voltages, and collision energies to identify the most sensitive precursor-product ion pair.

Used Instrumentation

• Xevo TQ Absolute triple quadrupole mass spectrometer with ESI+ mode
• ACQUITY Premier UPLC System with MaxPeak HPS surface technology
• ACQUITY Premier Peptide CSH C18 column (2.1×50 mm, 1.7 µm)
• Oasis MAX µElution 96-well SPE plates and QuanRecovery sample plates
• waters_connect for Quantitation Software v2.3 with MRM Optimization tool

Results and Discussion

Automated MRM optimization evaluated 25 precursor-product ion and collision energy combinations, selecting the [M+4H]4+ → specific fragment transition. Semaglutide eluted at 2.18 min with symmetric peaks and minimal carryover. Calibration was linear (R2 = 0.9985) over 0.2–100 ng/mL with a lower limit of quantification of 0.2 ng/mL (S/N >5). Quality control replicates showed accuracy >90% and precision (%CV) <10.6%, meeting fit-for-purpose bioanalytical criteria.

Benefits and Practical Applications

• High sensitivity enables accurate PK profiling of low-abundance peptides
• Automated MRM optimization accelerates method development and reduces variability
• Advanced surface technologies minimize non-specific binding, improving recovery and peak shape
• Workflow is adaptable to other therapeutic peptides and proteins in complex matrices

Future Trends and Potential Applications

Emerging informatics tools and machine learning could further automate transition selection and method refinement. Continued advancements in surface coatings and microflow LC may enhance sensitivity and throughput. The integration of multiplexed assays will support simultaneous quantification of multiple biologics in clinical and preclinical studies.

Conclusion

This application note demonstrates a robust, fully optimized LC-MS/MS workflow for sub-ng/mL quantification of semaglutide in plasma. The combination of automated MRM optimization, high-performance surfaces, and mixed-mode SPE delivers precise, accurate, and efficient bioanalysis suited for peptide therapeutics development.

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