MSACL 2024 Abstract

Introduction
Glucagon and oxyntomodulin are peptide hormones differentially released from proglucagon and function in regulating blood glucose concentrations. Their overlapping amino acid sequences make the development of specific immunoassays difficult, but with adequate antibodies for immunoaffinity enrichment, the specificity of liquid chromatography-tandem mass spectrometry can be used to subsequently distinguish each peptide from one another. We aimed to develop a sensitive and specific mass spectrometric assay that uses non-proprietary reagents and normal-flow liquid chromatography in the simultaneous quantification of glucagon and oxyntomodulin.

Methods
Bulk plasma proteins were precipitated in ethanol/ammonium hydroxide. Glucagon, oxyntomodulin, and their isotope-labeled internal standards were then enriched from the reconstituted supernatant using non-proprietary monoclonal antibodies generated in-house and analyzed using liquid chromatography-tandem mass spectrometry. A purified primary glucagon calibration material was sourced commercially and carefully characterized by liquid chromatography-mass spectrometry, HPLC-UV, and amino acid analysis. This primary glucagon calibrator and purified commercially available oxyntomodulin were used to generate a secondary single point calibration material for assay calibration.

Results
Glucagon and oxyntomodulin, with this method and freely available monoclonal antibodies can be quantified down to about 1 pM in 200µL of plasma using a Waters Acquity HPLC and TQ-S mass spectrometer.

Conclusion
We have validated a method with a highly detailed, implementable SOP. We have characterized a reference material that could help maintain accuracy within our lab over time and could help other labs achieve between-assay and between-laboratory harmonization. Additionally, the monoclonal antibodies developed during this work are available to other labs for research use through the Iowa Developmental Studies Hybridoma Bank.