A High-Throughput Mass Spectrometry Multiplexed Assay to Measure Insulin and C-Peptide

Program: Abstracts - Orals, Poster Previews, and Posters
Session: THR 586-595-Diabetes Genetics & Epidemiology
Thursday, March 5, 2015: 1:00 PM-3:00 PM
Hall D-F, Diabetes (San Diego Convention Center)

Poster Board THR-592
Steven W. Taylor, PhD1, Richard E Reitz, MD1, Nigel J. Clarke, PhD2 and Michael John McPhaul, MD3
1Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, 2Quest Diagnostics Nichols Inst, San Juan Capistrano, CA, 3Medical Director, Endocrinology, San Juan Capistrano, CA
The incidence of type 2 diabetes mellitus has increased markedly in the United States and worldwide in the past 2 decades. This increase is traceable largely to the dramatic increase in the prevalence of obesity and insulin resistance in developed nations.

Elevated fasting insulin levels have been shown to reflect the presence of insulin resistance and to predict future development of diabetes mellitus. However, the clinical measurement of insulin levels has been largely relegated to the realm of specialized testing employed primarily in research and epidemiological studies examining insulin resistance, prediabetes, and early Type 2 diabetes. Although insulin measurements have a direct relation to more sophisticated assessments of insulin resistance, researchers have not yet defined specific insulin levels that are associated with specific degrees of insulin resistance. This is at least in part to the marked variability among available assays.  C-peptide measurements are less variable than insulin assays, but also suffer from a lack of standardization.

To address these aspects, we developed a multiplexed method to measure insulin and C-peptide using an LC tandem mass spectrometry assay. The assay involves enrichment of the peptides from patient sera using 2 different monoclonal antibodies immobilized on magnetic beads and processing on a robotic liquid handler.  Eluted peptides are directly analyzed by LC-MS/MS.  The assay has a clinical reportable range from 2.5 to 320 µIU/mL for insulin and 0.11 to 27.2 ng/mL for C-peptide.   Intra- and inter-day assay variation is less than 11% for both peptides. Of the 5 insulin analogues commonly prescribed for diabetes treatment, only the recombinant human insulin drug Humalog® (insulin lispro) causes significant interference for the determination of endogenous insulin. There were no observed interferences for C-peptide.

In summary, we have developed a multiplexed method to measure insulin and C-peptide using an LC tandem mass spectrometry assay. This approach offers a highly accurate and reproducible tool to measure insulin and C-peptide. Applying this tool to clinical samples will permit the standardization of both measurements using physicochemical properties of the analytes. This in turn will facilitate comparison of results obtained in different studies and to highly characterized standard reference materials.

Disclosure: SWT: Researcher, Quest Diagnostics. RER: Coinvestigator, Quest Diagnostics. NJC: Coinvestigator, Quest Diagnostics. MJM: Principal Investigator, Quest Diagnostics.

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