The Short Cosyntropin Test Revisited: New Normal Reference Range Using LC-MS/MS

J Clin Endocrinol Metab. 2018 Apr 1;103(4):1696-1703. doi: 10.1210/jc.2017-02602.


Background: The cosyntropin test is used to diagnose adrenal insufficiency (AI) and nonclassical congenital adrenal hyperplasia (NCCAH). Current cutoffs for cortisol and 17-hydroxyprogesterone (17-OHP) are derived from nonstandardized immunoassays. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers direct measurement of steroids, prompting the need to re-establish normal ranges.

Objective: The goal of this study was to define cutoff values for cortisol and 17-OHP in serum by LC-MS/MS 30 and 60 minutes after intravenous administration of 250 µg tetracosactide acetate to healthy volunteers and to compare the results with LC-MS/MS with routine immunoassays.

Methods: Cosyntropin testing was performed in healthy subjects (n = 138) and in patients referred for evaluation of adrenocortical function (n = 94). Steroids were assayed by LC-MS/MS and compared with two immunoassays used in routine diagnostics (Immulite and Roche platforms). The cutoff level for cortisol was defined as the 2.5% percentile in healthy subjects not using oral estrogens (n = 121) and for 17-OHP as the 97.5% percentile.

Results: Cortisol cutoff levels for LC-MS/MS were 412 and 485 nmol/L at 30 and 60 minutes, respectively. Applying the new cutoffs, 13 of 60 (22%) subjects who had AI according to conventional criteria now had a normal test result. For 17-OHP, the cutoff levels were 8.9 and 9.0 nmol/L at 30 and 60 minutes, respectively.

Conclusions: LC-MS/MS provides cutoff levels for cortisol and 17-OHP after cosyntropin stimulation that are lower than those based on immunoassays, possibly because cross-reactivity between steroid intermediates and cortisol is eliminated. This reduces the number of false-positive tests for AI and false-negative tests for NCCAH.

Trial registration: NCT00218660.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 17-alpha-Hydroxyprogesterone / blood*
  • Adolescent
  • Adrenal Hyperplasia, Congenital / blood
  • Adrenal Hyperplasia, Congenital / diagnosis*
  • Adrenal Insufficiency / blood
  • Adrenal Insufficiency / diagnosis*
  • Adult
  • Aged
  • Aged, 80 and over
  • Chromatography, Liquid
  • Cosyntropin*
  • Female
  • Humans
  • Hydrocortisone / blood*
  • Male
  • Middle Aged
  • Reference Values
  • Tandem Mass Spectrometry
  • Young Adult


  • Cosyntropin
  • 17-alpha-Hydroxyprogesterone
  • Hydrocortisone

Associated data