Gold Standard Test for Measuring Cortisol Levels
The 24-hour urinary free cortisol (UFC) measurement using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the gold standard test for measuring cortisol levels, particularly when diagnosing Cushing's syndrome. 1, 2
Primary Testing Methods for Cortisol Assessment
24-hour Urinary Free Cortisol (UFC)
- Measures bioavailable cortisol excreted over a full day
- Advantages:
- Independent of cortisol-binding globulin (CBG) changes
- Not affected by time-of-day fluctuations
- Reflects overall cortisol production
- High specificity (100%) when using LC-MS/MS methodology 3
- Collection protocol:
- At least 2-3 separate 24-hour collections recommended to account for intra-patient variability 1
- Complete collection is essential for accuracy
Late Night Salivary Cortisol (LNSC)
- Based on loss of normal circadian rhythm in Cushing's syndrome
- Advantages:
- Collection protocol:
- Sampling at usual bedtime rather than fixed midnight time
- At least 2-3 samples recommended
Overnight Dexamethasone Suppression Test (DST)
- Assesses impaired glucocorticoid feedback
- Protocol:
- 1mg dexamethasone given between 11:00 PM and midnight
- Serum cortisol measured at 8:00 AM the following morning
- Normal response: cortisol <1.8 μg/dL (50 nmol/L) 1
- Advantages:
- High sensitivity (>90%)
- Strong negative predictive value
Comparative Performance of Testing Methods
| Test | Sensitivity | Specificity | Key Advantages | Key Limitations |
|---|---|---|---|---|
| 24-hr UFC (LC-MS/MS) | 89-100% | 100% | Gold standard; measures bioavailable cortisol | Requires complete collection; affected by renal function |
| LNSC | 95-100% | 95-100% | Most specific; convenient | Not useful for shift workers; affected by oral contamination |
| DST | >90% | Lower than LNSC | Strong negative predictive value | Affected by medications, CBG levels, absorption issues |
Important Considerations and Pitfalls
Factors Affecting UFC Accuracy
- Patient-related factors:
- Renal impairment (creatinine clearance <60mL/min)
- Very high or low urinary volume
- Sex, BMI, age, and sodium intake can influence levels 1
- Incomplete collection leads to falsely low results
Factors Affecting DST Accuracy
- False positives may occur with:
- Rapid absorption/malabsorption of dexamethasone
- CYP3A4 inducers (phenobarbital, carbamazepine)
- Increased CBG levels (oral estrogens, pregnancy) 1
- False negatives may occur with:
- Medications inhibiting dexamethasone metabolism (fluoxetine, cimetidine)
- Decreased CBG levels (nephrotic syndrome) 1
Factors Affecting LNSC Accuracy
- False positives may occur with:
- Topical hydrocortisone contamination
- Disrupted day/night cycle (shift workers) 1
Special Populations
Children and Adolescents
- In children with suspected Cushing's syndrome:
Adrenal Incidentalomas
- 1mg DST is the preferred screening test for autonomous cortisol secretion 1
- Cortisol values <1.8 μg/dL exclude dysregulated cortisol production 1
Modern Analytical Methods
LC-MS/MS offers superior specificity and sensitivity compared to immunoassays for cortisol measurement across all sample types (serum, urine, saliva) 2, 4. This method is particularly valuable for UFC measurement as it:
- Avoids cross-reactivity with cortisol metabolites
- Provides more accurate quantification
- Allows simultaneous measurement of cortisol and cortisone 2
In conclusion, while multiple tests are available for cortisol assessment, 24-hour UFC collection with LC-MS/MS analysis remains the gold standard for measuring cortisol levels, particularly in the diagnosis of Cushing's syndrome, due to its high specificity and ability to measure bioavailable cortisol independent of binding protein variations.