ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2012) 29 P1476

Comparison of immunoassay and mass spectrometry measurement of cortisol during ACTH1-24 stimulation tests

M Burt1,2, B Mangelsdorf1, M Whiting2,3, A Rogers2, J Ho1,2, W Inder4 & M Doogue1,2


1Repatriation General Hospital, Adelaide, South Australia, Australia; 2Flinders University, Adelaide, South Australia, Australia; 3Flinders Medical Centre, Adelaide, South Australia, Australia; 4Princess Alexandra Hospital, Brisbane, Queensland, Australia.


Measurement of plasma cortisol by immunoassay after ACTH1–24 stimulation is used to assess the hypothalamic–pituitary–adrenal (HPA) axis. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) has greater specificity and equilibrium dialysis allows measurement of free plasma cortisol. We investigated whether measuring cortisol by LC–MS/MS improves the sensitivity of ACTH1–24 stimulation testing in pituitary patients. We studied 60 controls (34 female, age 61±12 years, BMI 27.7±5.6 kg/m2) and 21 patients with pituitary disease in whom HPA sufficiency (n=8) or deficiency (n=13) had been defined by insulin tolerance test (peak cortisol cut-off 550 nmol/l) or morning cortisol (deficient ≤100 nmol/l, sufficient ≥500 nmol/l). Subjects received 1 μg ACTH1–24 intravenously and 250 μg ACTH1–24 intramuscularly about 7 days apart. Total and free (following equilibrium dialysis) plasma cortisol were measured by in-house LC–MS/MS assay and total plasma cortisol by immunoassay (Elecsys 2010, Roche Diagnostics). The lower limits of the 95% confidence intervals derived from normal subjects were used to define the pituitary patients HPA status. The 30 min cortisol during the 1 μg ACTH1–24 stimulation test and the 30 and 60 min cortisols during the 250 μg ACTH1–24 stimulation tests were equally concordant with previous HPA axis assessment. Measurements of total cortisol by immunoassay were concordant with previous HPA axis assessment in 19/21 and 20/21 patients using the 1 and 250 μg ACTH1–24 tests respectively. The sensitivities of total and free cortisol by LC–MS/MS were similar to those derived from the immunoassay (Table). We conclude that measurement of total or free plasma cortisol by LC–MS/MS after ACTH1–24 stimulation provides similar results to immunoassay in pituitary patients.

Table 1
0 min 30 min 60 min
1 μg ACTH1–24 stimulation test
Total cortisol (immunoassay, nmol/l)a199–763441–919317–864
Concordance (n, (%))b17 (81)19 (90)17 (81)
Total cortisol (LC–MS/MS, nmol/l)a213–933497–1125327–1067
Concordance (n, (%))b16 (76)20 (95)16 (76)
Free cortisol (LC–MS/MS, nmol/l)a4.0–45.831.6–94.615.3–62.5
Concordance (n, (%))b16 (76)20 (21)18 (86)
250 μg ACTH1–24 stimulation test
Total cortisol (immunoassay, nmol/l)a176–703492–978606–1025
Concordance (n, (%))b16 (76) 20 (95) 20 (95)
Total cortisol (LC–MS/MS, nmol/l)a192–824524–1153630–1233
Concordance (n, (%))b16 (76)19 (90)19 (90)
Free cortisol (LC–MS/MS, nmol/l)3.6–36.132.1–101.842.4–119.4
Concordance (n, (%))b16 (76)20 (95)20 (95)
a95% confidence intervals derived from normal subjects.
bWith previous HPA axis assessment in patients with pituitary disease

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This work was supported, however funding details unavailable.

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