Quantification of glucagon and oxyntomodulin by protein precipitation-immunoaffinity enrichment-LC-MS/MS.

INTRODUCTION: Glucagon and oxyntomodulin are peptide hormones differentially released from proglucagon that function in regulating blood glucose. Their overlapping amino acid sequences make the development of specific immunoassays difficult, but the specificity of liquid chromatography-tandem mass spectrometry can be used to distinguish the peptides. 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 both analytes. METHODS: Bulk plasma proteins were precipitated in ethanol/ammonium hydroxide. Analytes were enriched using monoclonal antibodies generated in-house and analyzed using liquid chromatography-tandem mass spectrometry. A glucagon calibration material was sourced commercially and characterized for purity and concentration by high-performance liquid chromatography-ultraviolet detection and amino acid analysis. Single-point calibration was used to minimize between-day variability. RESULTS: The novel antibodies performed acceptably (peptide recovery 45-59 %). The assay was precise (<13 %CV) and linear over the range of 1.3-14.7 pM and 1.1-13.7 pM for glucagon and oxyntomodulin, respectively. The glucagon calibration material concentration was determined to be 1.596 mg/g. Tube-type studies supported the use of protease inhibitor tubes at the time of blood draw. Patients with type 1 diabetes had lower concentrations of glucagon when maintained on an insulin pump, but not with injectable insulin. CONCLUSION: We have validated a method with a highly detailed standard operating procedure. We have characterized calibration materials to help maintain accuracy and achieve between-day and between-laboratory harmonization. The assay will be beneficial in better understanding α-cell health and glycemic control in diabetes and other diseases.