Comprehensive workflow for Automated Sample Cleanup and Sensitive Quantitation of Glucagon-like peptide-1 (GLP-1) Analogs from Plasma

Significance of the Topic

GLP-1 receptor agonists such as semaglutide have become essential therapies for obesity and type 2 diabetes. Precise measurement of these peptide drugs in plasma supports dose optimization, safety monitoring and clinical research. Automated sample cleanup improves assay reliability and throughput, enabling sensitive detection across broad concentration ranges.

Objectives and Study Overview

The study aimed to develop and validate an automated LC-MS/MS workflow for semaglutide quantification in human plasma. Key goals included enhancing assay sensitivity, streamlining sample preparation and establishing a robust calibration range. Comparative performance against traditional protein precipitation was also assessed.

Methodology and Instrumentation

• Sample preparation involved protein precipitation with a 1:1 mixture of acetonitrile and methanol spiked with semaglutide and a tirzepatide internal standard.
• Automated cleanup used AssayMAP RP-S cartridges with optimized conditioning, loading, washing and elution steps to maximize peptide recovery.
• Chromatographic separation was performed on an AdvanceBio Peptide Mapping column at 80 ºC using a water/formic acid and acetonitrile/formic acid gradient.
• Detection employed an Agilent 6495D triple quadrupole mass spectrometer operating in MRM mode for semaglutide (1029.4→1238.1) and tirzepatide transitions.

Main Results and Discussion

• AssayMAP cleanup yielded a fivefold improvement in lower limit of quantification compared to protein precipitation alone.
• A linear calibration curve was established from 0.2 to 1000 ng/mL with R² greater than 0.99 using quadratic fit and 1/x² weighting.
• Intra- and inter-day precision and accuracy met regulatory acceptance criteria (±15% bias, CV ≤15%) across low, mid and high QC levels.

Benefits and Practical Applications

Automated sample cleanup reduces hands-on time and variability, supports high-throughput analysis and extends assay sensitivity. The validated method is suitable for pharmacokinetic studies, therapeutic drug monitoring and bioequivalence assessments of GLP-1 analogs.

Future Trends and Applications

• Integration of automation with high-resolution MS to profile peptide metabolites.
• Expansion to other peptide therapeutics and biomarkers in clinical research.
• Adoption in regulated laboratories for routine therapeutic monitoring.

Conclusion

The developed workflow delivers sensitive, precise and reproducible quantitation of semaglutide in plasma. Automated cleanup provides superior assay performance compared to traditional methods, offering a scalable solution for clinical and research laboratories.

References

• Ozempic – semaglutide injection, solution. DailyMed. June 5, 2021.
• Rybelsus – oral semaglutide tablet. DailyMed. June 5, 2021.
• Wegovy – semaglutide injection, solution. December 14, 2021.