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Test ID: CORTF Cortisol, Free, Serum

Additional Testing Requirements

If multiple specimens are drawn, send separate order for each specimen.

Necessary Information

Include time of draw.

Specimen Required

Container/Tube: Red top

Preferred: Red top

Specimen Volume: 1.25 mL

Collection Instructions: Morning (8 a.m.) specimens are preferred. The 8 a.m. cortisol can be referred to as the a.m. cortisol and can be drawn anywhere between 6 a.m. and 10:30 a.m. in the morning.

Useful For

Assessment of cortisol status in cases where there is known or a suspected abnormality in cortisol-binding proteins or albumin


Assessment of adrenal function in the critically ill or stressed patient, thus preventing unnecessary use of glucocorticoid therapy

Method Name

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Reporting Name

Cortisol, Free, S

Specimen Type

Serum Red

Specimen Minimum Volume

0.75 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
Serum Red Refrigerated (preferred) 28 days
  Ambient  28 days
  Frozen  28 days

Clinical Information

Cortisol, the main glucocorticoid (representing 75%-95% of the plasma corticoids), plays a critical role in glucose metabolism and in the body's response to stress. Both hypercortisolism (Cushing disease) and hypo-cortisolism (Addison disease) can cause disease. Cortisol is also used to treat skin disease, allergic disorders, respiratory system disease, inflammatory disorders, and nephrotic syndrome.


Cortisol levels are regulated by adrenocorticotropic hormone (ACTH), which is synthesized by the pituitary in response to corticotropin releasing hormone (CRH). CRH is released in a cyclic fashion by the hypothalamus, resulting in diurnal peaks (6-8 a.m.) and nadirs (11 p.m.) in plasma ACTH and cortisol levels.


The majority of cortisol circulates bound to corticosteroid-binding globulin (CBG) and albumin. Normally, less than 5% of circulating cortisol is free (unbound). Only free cortisol can access the enzyme transporters in liver, kidney, and other tissues that mediate metabolic and excretory clearance.


Historically, measurements of free cortisol have been achieved from indirect means using a ratio known as the free cortisol index. This measurement takes into account the amount of total cortisol and CBG to give a percentage and ultimately absolute value of free cortisol.  These methods do not take into account the possible variations in albumin levels. These calculations also rely on CBG which can be lowered in critically ill patients despite normal adrenal function. Equilibrium dialysis best serves to separate free from bound cortisol without disrupting the bound fraction.

Reference Values

6-10:30 a.m. Collection: 0.121-1.065 mcg/dL


Cortisol is converted to cortisone in human kidneys and cortisone is less active toward the mineralcorticoid receptor. The conversion of cortisol to cortisone in the kidney is mediated by 11B-hydroxysteroid dehydrogenase isoform-2. Also, cortisol renal clearance will be reduced when there is a deficiency in the cytochrome P450 3A5 (CYP3A5) enzyme as well as a deficiency in P-glycoprotein.


Cortisol binding globulin (CBG) has a low capacity and high affinity for cortisol, whereas albumin has a high capacity and low affinity for binding cortisol. Variations in CBG and serum albumin due to renal or liver disease may have a major impact on free cortisol.


Based on the study by Bancos,(1) normal ranges of free cortisol found in patients without adrenal insufficiency were:

-Free cortisol at baseline: median 0.400 mcg/dL (interquartile range: IQR 2.5-97.5% - 0.110-1.425 mcg/dL)

-Free cortisol at 30 minutes: median 1.355 mcg/dL (IQR 2.5-97.5% - 0.885-2.440 mcg/dL)

-Free cortisol at 60 minutes: median 1.720 mcg/dL (IQR 2.5-97.5% - 1.230-2.930 mcg/dL)


Based on the study by Bancos,(1) the following cutoffs were calculated for exclusion of adrenal insufficiency:

-Free cortisol at baseline*: greater than 0.271 mcg/dL (>271 ng/dL, area under the curve: AUC 0.81)

-Free cortisol at 30 minutes: greater than 0.873 mcg/dL (>873 ng/dL, AUC 0.99)

-Free cortisol at 60 minutes: greater than 1.190 mcg/dL (>1,190 ng/dL, AUC 0.99)

(*Note that baseline free cortisol should not be used to exclude adrenal insufficiency given low performance)


The use of free cortisol in the management of glucocorticoid levels in the stressed patient due to major surgery or trauma requires further studies to establish clinical dosing levels and efficacy.

Clinical Reference

1. Bancos I, Erickson D, Bryant S, et al: Performance of free versus total cortisol following cosyntropin stimulation testing in an outpatient setting. Endocr Pract 2015 Dec;21(12):1353-1363 doi: 10.4158/EP15820

2. Hamrahian AH, Oseni TS, Arafah BM: Measurements of serum free cortisol in critically ill patients. N Engl J Med 2004;350;16:1629-1638

3. Ho JT, Al-Musalhi H, Chapman MJ, et al: Septic shock and sepsis: a comparison of total and free plasma cortisol levels. J Clin Endocrinol Metab 2006;91:105-114

4. le Roux CW, Chapman GA, Kong WM, et al: Free cortisol index is better than serum total cortisol in determining hypothalamic-pituitary-adrenal status in patients undergoing surgery. J Clin Endocrinol Metab 2003;88:2045-2048

5. Huang W, Kalhorn TF, Baillie M, et al: Determination of free and total cortisol in plasma and urine by liquid chromatography-tandem mass spectrometry. Ther Drug Monit 2007;29(2):215-224

Day(s) and Time(s) Performed

Tuesday, Thursday, Friday

Analytic Time

4 days

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.

CPT Code Information


LOINC Code Information

Test ID Test Order Name Order LOINC Value
CORTF Cortisol, Free, S 2145-1


Result ID Test Result Name Result LOINC Value
65423 Cortisol, Free, S 2145-1
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