title: A high-throughput mass spectrometry assay to simultaneously measure intact insulin and C-peptide.
abstract: BACKGROUND
  Measurements of fasting levels of insulin and C-peptide are useful in documenting insulin resistance and may help predict development of diabetes mellitus. However, the specific insulin and C-peptide levels associated with specific degrees of insulin resistance have not been defined, owing to marked variability among immunoassays and lack of standardization. Herein, we describe a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for intact insulin and C-peptide.
  
  
  METHODS
  Insulin and C-peptide were enriched from patient sera using monoclonal antibodies immobilized on magnetic beads and processed on a robotic liquid handler. Eluted peptides were analyzed by LC-MS/MS. Bovine insulin and a stable isotopically-labeled (13C/15N) C-peptide were utilized as internal standards.
  
  
  RESULTS
  The assay had an analytical measurement range of 3 to 320 μIU/ml (18 to 1920 pmol/l) for insulin and 0.11 to 27.2 ng/ml (36 to 9006 pmol/l) for C-peptide. Intra- and inter-day assay variation was less than 11% for both peptides. Of the 5 insulin analogs commonly prescribed to treat diabetes, only the recombinant drug insulin lispro caused significant interference for the determination of endogenous insulin. There were no observed interferences for C-peptide.
  
  
  CONCLUSIONS
  We developed and validated a high-throughput, quantitative, multiplexed LC-MS/MS assay for intact insulin and C-peptide.
authors.affiliation: Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Taylor
authors.affiliation: Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States. Electronic address: Nigel.J.Clarke@questdiagnostics.com.
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Clarke
authors.affiliation: Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, United States.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McPhaul
mesh_terms: D002096:C-Peptide / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods; D057166:High-Throughput Screening Assays; D006801:Humans; D007328:Insulin / Q000097:blood*; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D002096:C-Peptide; D007328:Insulin
keywords: C-peptide; Diabetes; Immunocapture; Insulin; LC–MS/MS