Interlaboratory Comparison of a Glucagon and Oxyntomodulin Immuno-LC-MS/MS Assay: Implications for Diabetes Research

The quantification of plasma glucagon and oxyntomodulin is important in the assessment of α-cell function, which is impaired in patients with diabetes. We aimed to transfer between laboratories a novel assay that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the sensitive and specific measurement of these highly homologous hormones.

Simultaneous measurement of glucagon and oxyntomodulin used immunoaffinity enrichment and LC-MS/MS. Immunoenrichment used monoclonal antibodies that are available at-cost to researchers (deposited at the Developmental Studies Hybridoma Bank). Pure synthetic glucagon, characterized for purity and concentration, was used as a calibrant and is available to others. A detailed standard operating procedure was shared between 3 laboratories and the performance of the method was evaluated with samples collected from patients with and without diabetes. Method comparison was made with 2 commercially available, FDA-registered glucagon immunoassays.

The method was linear over the normal range (1-20 pM). When measured in duplicate, the median interlaboratory imprecision (%CV) of the measurement of 40 samples was 6.3% (IQR 4.5%) and 14.4% (IQR 12.4%) for glucagon and oxyntomodulin, respectively. Method comparison with commercially available immunoassays demonstrated good (Mercodia, R = 0.92) or fair (Ansh, R = 0.70) agreement. Multivariable linear regression using LC-MS/MS glucagon and oxyntomodulin concentrations to predict immunoassay results indicated significant cross-reactivity of each immunoassay with oxyntomodulin.

We have validated a sensitive and specific assay for glucagon and oxyntomodulin that can be deployed in high-complexity clinical laboratories for research or the care of patients. Commercially available glucagon immunoassays have significant interference from molecules other than glucagon.