Method-specific serum cortisol responses to the adrenocorticotrophin test: comparison of gas chromatography-mass spectrometry and five automated immunoassays

Clin Endocrinol (Oxf). 2013 May;78(5):673-80. doi: 10.1111/cen.12039.


Objective: The serum cortisol response to the adrenocorticotrophin (ACTH) test is known to vary significantly by assay, but lower reference limits (LRL) for this response have not been established by the reference gas chromatography-mass spectrometry (GC-MS) method or modern immunoassays. We aimed to compare the normal cortisol response to ACTH stimulation using GC-MS with five widely used immunoassays.

Design, patients and measurements: An ACTH test (250 μg iv ACTH1-24 ) was undertaken in 165 healthy volunteers (age, 20-66 years; 105 women, 24 of whom were taking an oestrogen-containing oral contraceptive pill [OCP]). Serum cortisol was measured using GC-MS, Advia Centaur (Siemens), Architect (Abbott), Modular Analytics E170 (Roche), Immulite 2000 (Siemens) and Access (Beckman) automated immunoassays. The estimated LRL for the 30 min cortisol response to ACTH was derived from the 2·5th percentile of log-transformed concentrations.

Results: The GC-MS-measured cortisol response was normally distributed in males but not females, with no significant gender difference in baseline or post-ACTH cortisol concentration. Immunoassays were positively biased relative to GC-MS, except in samples from women on the OCP, who showed a consistent negative bias. The LRL for cortisol was method-specific [GC-MS: 420 nm; Architect: 430 nm; Centaur: 446 nm; Access 459 nm; Immulite (2000) 474 nm] and, for the E170, also gender-specific (female: 524 nm; male 574 nm). A separate LRL is necessary for women on the OCP.

Conclusions: Normal cortisol responses to the ACTH test are influenced significantly by assay and oestrogen treatment. We recommend the use of separate reference limits in premenopausal women on the OCP and warn users that cortisol measurements in this subgroup are subject to assay interference.

MeSH terms

  • Adrenocorticotropic Hormone / blood*
  • Adult
  • Aged
  • Female
  • Gas Chromatography-Mass Spectrometry / methods*
  • Humans
  • Hydrocortisone / blood*
  • Immunoassay / methods*
  • Male
  • Middle Aged
  • Reproducibility of Results
  • Young Adult


  • Adrenocorticotropic Hormone
  • Hydrocortisone