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1 Department of Laboratory Medicine & Pathology, Mayo Clinic and Foundation, Rochester, MN 55905.
aAddress correspondence to this author at: Hilton 730, Department of Laboratory Medicine & Pathology, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905. Fax 507-284-9758; e-mail singh.ravinder{at}mayo.edu.
Background: Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce analytical interference, improve accuracy, and shorten the analysis time, we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for urinary cortisol and cortisone.
Methods: We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d4 to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride, washed, and dried, and 10 µL of the reconstituted extract was injected onto a reversed-phase C18 column and analyzed using a tandem mass spectrometer operating in the positive mode.
Results: Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7–828 nmol/L (0.25–30 µg/dL). Interassay CVs were 7.3–16% for mean concentrations of 6–726 nmol/L (0.2–26.3 µg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 µg/dL). Recovery of cortisol and cortisone added to urine was 97–123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 µg cortisol/24 h (r2 = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x - 12.1 µg cortisol/24 h (r2 = 0.67; n = 99).
Conclusion: The sensitivity and specificity of the LC-MS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.
The following articles in journals at HighWire Press have cited this article:
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  S. K. Baid, D. Rubino, N. Sinaii, S. Ramsey, A. Frank, and L. K. Nieman Specificity of Screening Tests for Cushing's Syndrome in an Overweight and Obese Population J. Clin. Endocrinol. Metab., October 1, 2009; 94(10): 3857 - 3864. [Abstract] [Full Text] [PDF]
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 M Madhavan, B A Borlaug, A Lerman, C S Rihal, and A Prasad Stress hormone and circulating biomarker profile of apical ballooning syndrome (Takotsubo cardiomyopathy): insights into the clinical significance of B-type natriuretic peptide and troponin levels Heart, September 1, 2009; 95(17): 1436 - 1441. [Abstract] [Full Text] [PDF]
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 L. Wood, D. H Ducroq, H. L Fraser, S. Gillingwater, C. Evans, A. J Pickett, D. W Rees, R. John, and A. Turkes Measurement of urinary free cortisol by tandem mass spectrometry and comparison with results obtained by gas chromatography-mass spectrometry and two commercial immunoassays Ann Clin Biochem, July 1, 2008; 45(4): 380 - 388. [Abstract] [Full Text] [PDF]
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 T. Remer, T. Dimitriou, and C. Maser-Gluth Renal Net Acid Excretion and Plasma Leptin Are Associated with Potentially Bioactive Free Glucocorticoids in Healthy Lean Women J. Nutr., February 1, 2008; 138(2): 426S - 430S. [Abstract] [Full Text] [PDF]
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 S. Kidambi, H. Raff, and J. W Findling Limitations of nocturnal salivary cortisol and urine free cortisol in the diagnosis of mild Cushing's syndrome Eur. J. Endocrinol., December 1, 2007; 157(6): 725 - 731. [Abstract] [Full Text] [PDF]
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 T. Remer and C. Maser-Gluth Simultaneous Measurements of Urinary Free Cortisol and Cortisone for the Assessment of Functional Glucocorticoid Activity Clin. Chem., October 1, 2007; 53(10): 1870 - 1871. [Full Text] [PDF]
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D. Erickson, N. Natt, T. Nippoldt, W. F. Young Jr., P. C. Carpenter, T. Petterson, and T. Christianson Dexamethasone-Suppressed Corticotropin-Releasing Hormone Stimulation Test for Diagnosis of Mild Hypercortisolism J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 2972 - 2976. [Abstract] [Full Text] [PDF]
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S. K. Baid, N. Sinaii, M. Wade, D. Rubino, and L. K. Nieman Radioimmunoassay and Tandem Mass Spectrometry Measurement of Bedtime Salivary Cortisol Levels: A Comparison of Assays to Establish Hypercortisolism J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3102 - 3107. [Abstract] [Full Text] [PDF]
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 K. Usa, R. J. Singh, B. C. Netzel, Y. Liu, H. Raff, and M. Liang Renal interstitial corticosterone and 11-dehydrocorticosterone in conscious rats Am J Physiol Renal Physiol, July 1, 2007; 293(1): F186 - F192. [Abstract] [Full Text] [PDF]
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 S. Kidambi, J. M. Kotchen, C. E. Grim, H. Raff, J. Mao, R. J. Singh, and T. A. Kotchen Association of Adrenal Steroids With Hypertension and the Metabolic Syndrome in Blacks Hypertension, March 1, 2007; 49(3): 704 - 711. [Abstract] [Full Text] [PDF]
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 R. Basu, D. S. Edgerton, R. J. Singh, A. Cherrington, and R. A. Rizza Splanchnic Cortisol Production in Dogs Occurs Primarily in the Liver: Evidence for Substantial Hepatic Specific 11{beta} Hydroxysteroid Dehydrogenase Type 1 Activity Diabetes, November 1, 2006; 55(11): 3013 - 3019. [Abstract] [Full Text] [PDF]
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J. W. Findling and H. Raff Cushing's Syndrome: Important Issues in Diagnosis and Management J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 3746 - 3753. [Abstract] [Full Text] [PDF]
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Y. Wang, D. Zhong, J. P. Fawcett, and J. Gu Autosampler Programming for Improved Sample Throughput in Liquid Chromatography/Mass Spectrometry Clin. Chem., November 1, 2005; 51(11): 2216 - 2218. [Full Text] [PDF]
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R. L. Taylor, S. K. Grebe, and R. J. Singh Quantitative, Highly Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for Detection of Synthetic Corticosteroids Clin. Chem., December 1, 2004; 50(12): 2345 - 2352. [Abstract] [Full Text] [PDF]
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M. Fenske How Much "Urinary Free Cortisol" Is Really Cortisol during Water Diuresis in Healthy Individuals? Clin. Chem., June 1, 2004; 50(6): 1102 - 1104. [Full Text] [PDF]
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R. E. Nelson, S. K. Grebe, D. J. O'Kane, and R. J. Singh Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Measurement of Estradiol and Estrone in Human Plasma Clin. Chem., February 1, 2004; 50(2): 373 - 384. [Abstract] [Full Text] [PDF]
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A. W. Meikle, J. Findling, M. M. Kushnir, A. L. Rockwood, G. J. Nelson, and A. H. Terry Pseudo-Cushing Syndrome Caused by Fenofibrate Interference with Urinary Cortisol Assayed by High-Performance Liquid Chromatography J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3521 - 3524. [Abstract] [Full Text] [PDF]
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B. E. Pearson Murphy Commercial Radioimmunoassays Do Not Measure Urinary Free Cortisol Accurately and Should Not Be Used for Physiological Studies J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 4003 - 4003. [Full Text] [PDF]
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M. M. Kushnir, A. L. Rockwood, G. J. Nelson, A. H. Terry, and A. W. Meikle Liquid Chromatography-Tandem Mass Spectrometry Analysis of Urinary Free Cortisol Clin. Chem., June 1, 2003; 49(6): 965 - 967. [Full Text] [PDF]
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