Advancing anti-obesity drug development with robust quantitation of GLP-1 analogs using human plasma

Importance of the topic

Glucagon-like peptide-1 (GLP-1) receptor agonists such as semaglutide and liraglutide have become central to therapeutic strategies for type 2 diabetes and obesity. Accurate, sensitive, and reproducible quantitation of these peptide drugs in human plasma is essential for pharmacokinetic, bioequivalence, safety, and drug‑development studies. Robust LC‑MS/MS workflows that address peptide adsorption, matrix effects, and throughput constraints therefore play a critical role in supporting anti‑obesity drug development and clinical monitoring.

Goals and overview of the study

The application note describes a complete LC‑SRM (selected reaction monitoring) triple‑quadrupole mass spectrometry workflow for quantifying semaglutide and liraglutide in human plasma. Primary aims were to:

• Develop a reproducible sample preparation (SPE) method for plasma extraction of the two GLP‑1 analogs.
• Establish chromatographic and mass spectrometric conditions providing high sensitivity and minimal carryover.
• Demonstrate a compliance‑ready data acquisition and processing solution.

Methodology

Sample preparation: 200 µL human plasma aliquots were spiked with standards (semaglutide or liraglutide) and subjected to protein precipitation by adding 200 µL cold acetone, vortexing, and centrifugation. Approximately 300 µL supernatant was processed by mixed‑mode strong anion exchange (SAX) SPE using SOLAµ 2 mg 96‑well plates. The SPE protocol included methanol and 5% ammonia conditioning/washes, vacuum drying, and elution with 75 µL of 70:30 acetonitrile:methanol. Eluates were diluted with 125 µL water prior to analysis. Liraglutide and semaglutide were used reciprocally as internal standards (IS) at 10 ng/mL. Recovery evaluation used two sample types (experimental and theoretical) at concentrations 0.05, 0.1, and 0.2 ng/mL to calculate % recovery from peak area ratios.

Chromatography: Separation employed a Hypersil GOLD Peptide column (2.1 × 50 mm, 1.9 µm) on a Vanquish Horizon UHPLC. Mobile phases were 0.4% formic acid in water (A) and 0.1% difluoroacetic acid in acetonitrile (B). The method used 0.25 mL/min flow, 20 µL injections, a 60 °C column temperature, and a gradient returning to initial conditions by 10 min. Needle wash and a divert window (2.5–6.0 min) were used to limit source contamination.

Mass spectrometry: Detection was performed on a Thermo Scientific TSQ Certis triple quadrupole in SRM mode. Source and scan settings included positive ionization (≈4100 V), ion transfer tube ~200 °C, vaporizer ~225 °C, sheath/aux gases defined, Q1/Q3 resolution ~1.2 FWHM, collision gas ~1.5 mTorr and cycle time 0.6 s. Representative SRM scheme: liraglutide precursor m/z 938.8 with the quantifier fragment at m/z 1064.0; semaglutide precursor m/z 1029.4 with the quantifier fragment at m/z 1238.4. Quantifier ions were paired with two confirming transitions for identity confirmation.

Used instrumentation

• Thermo Scientific Vanquish Horizon UHPLC system
• Hypersil GOLD Peptide column (2.1 × 50 mm, 1.9 µm)
• Thermo Scientific TSQ Certis Triple Quadrupole Mass Spectrometer
• Thermo Scientific SOLAµ SAX 96‑well SPE plates (2 mg)
• Chromeleon software v7.4 for instrument control, acquisition and reporting
• General consumables: Eppendorf tubes, low‑protein‑bind 96‑well plates

Main results and discussion

Sensitivity and dynamic range: The LC‑SRM assay achieved an LLOQ of 0.05 ng/mL for both semaglutide and liraglutide and a linear dynamic range from 0.05 to 100 ng/mL (≈3.5 orders of magnitude). Precision and accuracy met typical bioanalytical acceptance: precision generally ≤15% RSD and accuracy within ±20% at the LLOQ and tighter limits across higher concentrations.

Recovery: By optimizing the SPE elution volume and eliminating a drying step, average extraction recoveries were reproducible (approximately 60–70% reported across low‑level concentrations), with demonstrated consistency across tested concentrations (0.05–0.2 ng/mL).

Carryover: Despite the propensity of peptide therapeutics to adsorb to system surfaces, the described chromatographic conditions and needle wash strategy limited carryover to below 15% of the LLOQ, typically <10% in the reported experiments. This low carryover supports high‑throughput sample sequences without excessive additional wash cycles.

Throughput and robustness: The sample preparation workflow is compatible with 96‑well automation and completes in under an hour per batch. Reducing elution volume and removing evaporation improved reproducibility and simplified workflow for routine bioanalysis.

Benefits and practical applications of the method

• High sensitivity and broad linear range suitable for pharmacokinetic studies, therapeutic monitoring, and preclinical/clinical development of GLP‑1 analogs.
• Reproducible SPE extraction with acceptable recovery and compatibility with high‑throughput formats.
• Minimal carryover and low adsorption losses due to low‑adsorption column choice and optimized wash/divert strategy.
• Compliance‑ready data handling through Chromeleon software, facilitating regulatory documentation and audit trails.

Future trends and opportunities

Potential directions to extend and adapt this workflow include:

• Integration of immunocapture or affinity enrichment to further improve sensitivity and selectivity for low‑abundance peptides or metabolites.
• Microflow or nanoflow LC approaches to enhance sensitivity where sample volume is limited.
• Implementation of automated positive‑pressure SPE workstations to increase throughput and reduce operator variability.
• Extension of the assay to additional GLP‑1 analogs, metabolites and conjugates, and incorporation into multiplexed peptide panels for comprehensive PK/PD profiling.
• Method translation and validation according to regulatory bioanalytical guidelines for use in clinical trials and therapeutic drug monitoring.

Conclusion

The application note documents a complete, practical LC‑SRM workflow that delivers sensitive and reproducible quantitation of semaglutide and liraglutide in human plasma. Key strengths are the robust SAX‑based SPE extraction compatible with 96‑well automation, use of a low‑adsorption peptide column to minimize carryover, and a TSQ triple‑quadrupole SRM method achieving 0.05 ng/mL LLOQ with reliable precision and accuracy across a large dynamic range. The approach is well suited to support anti‑obesity drug development, pharmacokinetic studies, and laboratory workflows requiring both throughput and regulatory compliance.

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.