Precision in Peptide Quantitation:
Developing a Validated LC-MS/MS Method for Semaglutide:
Resolving siRNA Bioanalysis Challenges.
How Resolian helped sponsors overcome analytical barriers to support critical clinical research for this breakthrough GLP-1 therapy.
Semaglutide, a glucagon-like peptide-1 (GLP-1) analogue, has received significant attention for its weight-loss benefits and therapeutic potential in diabetes management.
As clinical research programs expand, reliable and sensitive bioanalytical methods are critical to accurately measure semaglutide concentrations in human plasma.
This ensures sponsors can make confident decisions about efficacy, dosing, and safety.
In this article, we share how Resolian’s bioanalytical team systematically addressed key analytical challenges to deliver a fully validated LC-MS/MS method meeting ICH M10 standards. From overcoming internal standard instability to resolving matrix suppression, our approach demonstrates how technical innovation accelerates the development of next-generation therapeutics.
The Challenges
Developing a robust LC-MS/MS method for semaglutide presented multiple challenges:
- Sensitivity requirements
Detecting concentrations as low as 1 ng/mL required optimized sample preparation and chromatographic conditions. - Matrix effects
Plasma matrix effects (including haemolysed and hyperlipidaemic samples) caused signal suppression outside of acceptable regulatory limits. - Internal standard performance
The SIL internal standard exhibited low recovery (7–22%) and non-specific binding to pipette tips, impacting quantitation accuracy.
The Solution
Resolian’s bioanalytical scientists systematically optimized the method to ensure regulatory compliance and high precision:
- Sample Preparation: Increased MeOH:plasma ratio and sample volume to improve extract cleanliness and boost signal.
- Internal Standard Strategy: Modified internal standard working solution (ISWS) with ammonium bicarbonate and BSA to prevent non-specific binding. Addition of ammonium bicarbonate prior to the protein precipitation step to increase recovery to 100%.
- Chromatographic Adjustment: Switched from ammonium bicarbonate to 1% formic acid mobile phase to eliminate matrix suppression and bring results within acceptable %RE limits.
These refinements allowed for a simple protein precipitation extraction from just 150 µL of plasma, aligning with clinical study sample constraints.
The Results
- Validation Success: Method validated per ICH M10 guidelines for 1–500 ng/mL range
- Stability: Demonstrated 64-day stability at both −20°C and −80°C
- Selectivity: Chromatograms free of interferences across all plasma types
- Matrix Effects: Assay free of matrix effects across all plasma types.
This validated method now supports clinical trials requiring rapid and reliable quantitation of semaglutide.
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