(T1330-02-07) Quantitative Bioanalysis of Large Peptides and Proteins: Case Study of GLP-1 Receptor Agonist Semaglutide in Plasma

Purpose: The development of peptide drugs necessitates efficient bioanalytical assays for accurate measurement in biologicals. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is preferred technology for its specificity and sensitivity. However, LC-MS/MS assays for peptides such as high-profile diabetes and weight loss GLP-1 receptor agonist Semaglutide, pose significant challenges in non-specific binding and MRM optimization. This study presents a streamlined approach for optimized LC-MS/MS quantification of Semaglutide in plasma. A Quantitative bioanalysis assay has been developed utilizing the integrated peptide MRM optimisation tool, bespoke sample extraction, and a selective chromatography system which minimizes non-specific binding issues. Through this efficient workflow and verification study a robust and sensitive method suitable for clinical bioanalysis studies is demonstrated

Methods: We present the development and performance of a highly sensitive and robust analytical method for accurate quantification of Semaglutide in human plasma using tandem quadrupole mass spectrometer coupled to UHPLC system. Peptide MRMs were optimized via an interface between the Mass Spectrometry software, MassLynx, and in-silico Peptide and protein MRM optimization tool Skyline (Macoss Lab software). Samples were prepared for analysis using mixed-mode solid-phase extraction media. The LC-MS/MS method was further optimized for sensitive mass detection using on board optimisation tools. The chromatographic separation was optimized for maximum recovery and minimum surface interactions using the ACQUITY Premier Peptide BEH C18 column. The final method was validated across 3 sites for robustness and reproducibility.

Results: The efficiency of method development was enhanced over traditional methods through the use of the seamless interface between Masslynx and Skyline, allowing for an in silico guided approach to the faster optimization of Semaglutide peptide MRMs. The method’s performance was verified and confirmed through its’ excellent selectivity, sensitivity, and reproducibility afforded by the ACQUITY premier UPLC with Xevo TQ Absolute tandem mass spectrometer system. Chromatography peak shape and peak resolutions were excellent throughout the analysis and the negative performance effects of non-specific binding commonly experienced during chromatography separation of peptides were not observed in the assay, demonstrating the impact of minimizing metal surface interactions through use of the of the Acquity Premier UPLC and Columns. The non binding surface of the chromatography system reduced to time needed to passivate the LC-MS system and resulted in a lower LoD. Assay linearity was assessed over the clinically relevant concentration range (0.1 ng/mL to 20 ng/mL yielding an excellent correlation coefficient of < 0.99. Precision and Accuracy evaluated using Quality Control (QC) samples across 3 different concentrations demonstrated satisfactory results, with % Accuracy and Coefficient of Variation (CV) falling within an acceptable range (+/- 15%). The bioanalytical method was subject to cross validation in three separate laboratories using identical instrumentation. The results showed excellent agreement both in terms of linearity and sensitivity. Thus suggesting that this assay can be employed for multisite studies.

Conclusion: A sensitive and selective assay for the quantification of semaglutide in human plasma has been successfully developed using the UHPLC-MS/MS and SPE. The assay provides analytical sensitivity down to 0.2 ng/mL for semaglutide. Excellent linearity was observed across the calibration range of 0.2 – 100 ng/mL and no analyte carryover or interference was observed in the blank plasma samples. Repeatability of the method was < 4% at the LLQC level and accuracy of quantification was >90%, well within acceptable limits for a discovery “fit for purpose” bioanalytical assay. The MassLynx–Skyline Interface was used to simplify the MS MRM method development process allowing for greater than 60 combinations of precursor ion, product ion, collision energy and cone voltages to be rapidly evaluated and the optimal combination selected.

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3. Overgaard RV, Delff PH, Petri KCC, Anderson TW, Flint A, Ingwersen SH. Population Pharmacokinetics of Semaglutide for Type 2 Diabetes. Diabetes Ther. 2019 Apr;10(2):649-662. doi: 10.1007/s13300-019-0581-y.
4. Tanna N, Dunning C & Molloy BJ. MassLynx-Skyline Interface (MSI) – A New Automated Tool to Streamline MRM Method Development and Optimization for Large Molecule Quantification. Waters Application Note. 720006813, April 2020
5. M. DeLano, T. H. Walter, M. A. Lauber, M. Gilar, M. C. Jung, J. M. Nguyen, et al. Using Hybrid Organic–Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC. Anal. Chem. 2021 (93) 14, 5773-57. DOI: 10.1021/acs.analchem.0c05203

Acknowledgements: Samantha Ferris and Robert Plumb are employees of Waters Corp. The authors reported there is no funding associated with the work described in this article.


Semaglutide MRM method development


Semaglutide LC-MS method