Multiplexed Quantification of Proglucagon-Derived Peptides by Immunoaffinity Enrichment and Tandem Mass Spectrometry after a Meal Tolerance Test

Clin Chem. 2016 Jan;62(1):227-35. doi: 10.1373/clinchem.2015.244251. Epub 2015 Oct 1.


Background: Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g., ELISA, RIA), which often suffer from issues of selectivity.

Methods: We developed a multiplexed assay for measuring PGDPs including GLP-1 (7-36) amide, GLP-1 (9-36) amide, glucagon, and oxyntomodulin by mass spectrometry and used this assay to examine the effect of a meal tolerance test on circulating concentrations of these hormones. Participants fasted overnight and were either given a meal (n = 8) or continued to fast (n = 4), with multiple blood collections over the course of 3 h. Plasma samples were analyzed by microflow immunoaffinity (IA)-LC-MS/MS with an isotope dilution strategy.

Results: Assay performance characteristics were examined and established during analytical validation for all peptides. Intra- and interassay imprecision were found to be 2.2%-10.7% and 6.8%-22.5%, respectively. Spike recovery was >76%, and dilution linearity was established up to a 16-fold dilution. Immediately after the meal tolerance test, GLP-1 and oxyntomodulin concentrations increased and had an almost identical temporal relationship, and glucagon concentrations increased with a slight delay.

Conclusions: IA-LC-MS/MS was used for the simultaneous and selective measurement of PGDPs. This work includes the first indication of the physiological concentrations and modulation of oxyntomodulin after a meal.

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid
  • Fasting*
  • Glucagon / blood*
  • Glucagon / immunology
  • Glucagon-Like Peptide 1 / blood*
  • Glucagon-Like Peptide 1 / immunology
  • Humans
  • Immunoassay*
  • Mice
  • Mice, Inbred BALB C
  • Oxyntomodulin / blood*
  • Oxyntomodulin / immunology
  • Tandem Mass Spectrometry*


  • Oxyntomodulin
  • Glucagon-Like Peptide 1
  • Glucagon