Introduction and Objective: The quantification of plasma glucagon and oxytomodulin is important in the assessment of alpha-cell function, which is impaired in patients with diabetes. We aimed to develop, validate, and 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.Methods: Simultaneous measurement of glucagon and oxyntomodulin used immunoaffinity enrichment and LC-MS/MS. Immunoenrichment used novel free-for-research monoclonal antibodies (deposited at the Iowa Hybridoma Bank). Calibration of glucagon was achieved with a pure standard (characterized for purity and concentration, available to others). A detailed standard operating procedure was developed and shared between three laboratories and the performance of the method was evaluated with samples collected from fasting patients with and without diabetes. Method comparison was made with two commercially available, FDA-registered glucagon immunoassays (i.e., Mercodia and Ansh).Results: The method was linear at all three sites 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 the immunoassay results indicated significant cross-reactivity of each immunoassay with oxyntomodulin.Conclusion: We have developed and validated a sensitive and specific high-throughput assay for glucagon and oxyntodomulin that can be easily 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.
J.O. Becker: None. A. Moradian: None. T. Lin: None. S. Sechi: Stock/Shareholder; Current; Pfizer Inc., Merck & Co., Inc., GE Healthcare Systems, Organon, Viatris Inc. M. MacCoss: Consultant; Current; Thermo Fisher Scientific, Alnylam Pharmaceuticals, Inc. Advisory Panel; Current; Talus Biosciences, Profound Therapeutics. Research Support; Current; Waters, Sciex, Bruker, Agilent, Eisai Co., Ltd., Novartis Pharmaceuticals Corporation. W. Qian: None. J.E. van Eyk: None. A.N. Hoofnagle: Research Support; Current; Waters, Inc.
National Institutes of Health (1U01DK137097)
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