Advertisement

Journal of Endocrinological Investigation

, Volume 41, Issue 3, pp 315–323 | Cite as

Diagnostic performance of a newly developed salivary cortisol and cortisone measurement using an LC–MS/MS method with simple and rapid sample preparation

  • K. Mészáros
  • G. Karvaly
  • Z. Márta
  • B. Magda
  • J. Tőke
  • N. Szücs
  • M. Tóth
  • K. Rácz
  • A. PatócsEmail author
Original Article

Abstract

Background

Late-night salivary cortisol level is one of the first-line tests recommended by the Endocrine Society for the diagnosis of endogenous hypercortisolism. Most routine laboratories measure cortisol levels using immunoassay tests which fail to determine low cortisol levels accurately due to the numerous interfering substances. A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method with simple and rapid sample preparation was developed for the simultaneous measurement of cortisol and cortisone and its performance in the diagnosis of endogenous hypercortisolism was evaluated.

Methods

324 late-night saliva samples were analyzed from which 272 samples were derived from patients with a suspected diagnosis of endogenous hypercortisolism. Salivary cortisol levels were assayed using an electrochemiluminescent immunoassay (ECLIA, Cortisol II, Roche), and simultaneous measurement of cortisol and cortisone was performed using an LC–MS/MS method.

Results

A strong correlation between cortisol results measured using ECLIA and LC–MS/MS (r 2 = 0.892) was demonstrated. Receiver operating characteristics (ROC) analysis showed good diagnostic performance of cortisol and cortisone levels assayed using LC–MS/MS method and for cortisol measured using ECLIA.

Conclusions

Late-night salivary cortisol and cortisone are useful parameters for the diagnosis of hypercortisolism. Using samples obtained from patients where the diagnosis of hypercortisolism is extremely challenging cut-off values for midnight salivary cortisol and cortisone measured by LC–MS/MS method were established.

Keywords

LC–MS/MS ECLIA Hypercortisolism Late-night saliva Cortisol Cortisone 

Abbreviations

CS

Cushing’s syndrome

LC–MS/MS

Liquid chromatography–tandem mass spectrometry

ECLIA

Electrochemiluminescent immunoassay

ROC

Receiver operating characteristics

HPA

Hypothalamic–pituitary–adrenal

LNSC

Late-night salivary cortisol

UFC

Urine free cortisol

CBG

Cortisol binding globulin

ESI

Electrospray ionization

SPE

Solid-phase extraction

MRM

Multiple reaction monitoring

DP

Declustering potential

CE

Collision energy

CXP

Cell exit potential

AUC

Areas under curve

HSD11β

11-Beta-hydroxysteroid dehydrogenase

Notes

Acknowledgements

The authors receive financial support from Hungarian Academy of Sciences “Lendulet 2013” Grant (AP) and Bionics Innovation Center (AP, KR, GK). The authors are grateful to Professor Barna Vasarhelyi (Department of Laboratory Medicine, Semmelweis University) for useful comments and critical review of the manuscript.

Author contributions

Study design: AP, KM, MT, KR; performing LC–MS/MS measurements: KM, GK, ZM, BM; evaluation of results: AP, KM, GK; clinical evaluation of patients: MT, KR, NS, JT; writing the manuscript: KM, AP; all authors reviewed and agreed with the submission of manuscript.

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest to declare.

Ethical approval

The study protocol was approved by the National Scientific and Ethical Committee, Medical Research Council of Hungary (TUKEB, ETT), and was executed according to the Declaration of Helsinki principles.

Informed consent

Informed consent was obtained from all patients involved in the study.

References

  1. 1.
    Guaraldi F, Salvatori R (2012) Cushing Syndrome: maybe not so uncommon of an endocrine disease. J Am Board Fam Med 25:199–208CrossRefPubMedGoogle Scholar
  2. 2.
    Catargi B, Rigalleau V, Poussin A et al (2003) Occult Cushing’s syndrome in type-2 diabetes. J Clin Endocrinol Metab 88:5808–5813CrossRefPubMedGoogle Scholar
  3. 3.
    Tiryakioglu O, Ugurlu S, Yalin S et al (2010) Screening for Cushing’s syndrome in obese patients. Clinics 65:9–13CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Chiodini I, Mascia M, Muscarella S et al (2007) Subclinical hypercortisolism among outpatients referred for osteoporosis. Ann Intern Med 147:541–548CrossRefPubMedGoogle Scholar
  5. 5.
    Omura M, Saito J, Yamaguchi K, Kakuta Y, Nishikawa T (2004) Prospective study on the prevalence of secondary hypertension among hypertensive patients visiting a general outpatient clinic in Japan. Hypertens Res 27:193–202CrossRefPubMedGoogle Scholar
  6. 6.
    Nieman L, Biller B, Findling J et al (2008) The diagnosis of Cushing’s Syndrome: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 3:1526–1540CrossRefGoogle Scholar
  7. 7.
    Elias P, Martinez E, Barone B, Mermejo L, Castro M, Moreira A (2014) Late-night salivary cortisol has a better performance than urinary free cortisol in the diagnosis of Cushing’s syndrome. J Clin Endocrinol Metab 9:2045–2051CrossRefGoogle Scholar
  8. 8.
    Ceccato F, Barbot M, Zilio M et al (2015) Screening tests for Cushing’s Syndrome: urinary free cortisol role measured by LC-MS/MS. J Clin Endocrinol Metab 100:3856–3861CrossRefPubMedGoogle Scholar
  9. 9.
    Bou Khalil R, Baudry C, Guignat L et al (2011) Sequential hormonal changes in 21 patients with recurrent Cushing’s disease after successful pituitary surgery. Eur J Endocrinol 165:729–737CrossRefPubMedGoogle Scholar
  10. 10.
    Beko G, Varga I, Glaz E et al (2010) Cutoff values of midnight salivary cortisol for the diagnosis of overt hypercortisolism are highly influenced by methods. Clin Chim Acta 11:364–367CrossRefGoogle Scholar
  11. 11.
    Perogramvos I, Keevil B, Ray D, Trainer P (2010) Salivary cortisone is a potential biomarker for serum free cortisol. J Clin Endocrinol Metab 95:4951–4958CrossRefGoogle Scholar
  12. 12.
    Raff H, Singh R (2012) Measurement of late-night salivary cortisol and cortisone by LC-MS/MS to assess preanalytical sample contamination with topical hydrocortisone. Clin Chem 58:947–948CrossRefPubMedGoogle Scholar
  13. 13.
    Del Corral P, Schurman RC, Kinza MJ, Kordick CA, Rusch JL, Nadolski JB (2016) Salivary but not plasma cortisone tracks the plasma cortisol response to exercise: effect of time of day. J Endocrinol Invest 39:315–322CrossRefPubMedGoogle Scholar
  14. 14.
    Handelsman D, Wartofsky L (2013) Requirement for mass spectrometry sex steroid assays in the Journal of Clinical Endocrinology and Metabolism. J Clin Endocrinol Metab 98:3971–3973CrossRefPubMedGoogle Scholar
  15. 15.
    Cuzzola A, Petri A, Mazzini F, Salvadori S (2009) Application of hyphenated mass spectrometry techniques for the analysis of urinary free glucocorticoids. Rapid Commun Mass Spectrom 23:2975–2982CrossRefPubMedGoogle Scholar
  16. 16.
    Methlie P, Hustad S, Kellman R et al (2013) Multisteroid LC–MS/MS assay for glucocorticoids and androgens and its application in Addison’s disease. Endocr Connect 2:125–136CrossRefPubMedCentralGoogle Scholar
  17. 17.
    Nieman L, Biller B, Findling J et al (2008) The diagnosis of Cushing’s Syndrome: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 93:1526–1540CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Szappanos Á, Tőke J, Lippai D et al (2010) Bone turnover in patients with endogenous Cushing’s syndrome before and after successful treatment. Osteoporos Int 21:637–645CrossRefPubMedGoogle Scholar
  19. 19.
    Szappanos Á, Patócs A, Tőke J et al (2009) BclI polymorphism of the glucocorticoid receptor gene is associated with decreased bone mineral density in patients with endogenous hypercortisolism. Clin Endocrinol 71:636–643CrossRefGoogle Scholar
  20. 20.
    Elecsys Cortisol II (2017) Roche Diagnostics. https://dialog1.roche.com/hu/hu/elabdoc. Accessed 11 Jan 2017
  21. 21.
    Guidance for industry—bioanalytical method validation. Food and Drug Administration. http://www.fda.gov/downloads/Drugs/Guidance/ucm070107.pdf. Accessed 11 Jan 2017
  22. 22.
    Magnisali P, Chalioti M-B, Livadaraa T et al (2011) Simultaneous quantification of 17-OH progesterone, 11-deoxycortisol, 4-androstenedione, cortisol and cortisone in newborn blood spots using liquid chromatography–tandem mass spectrometry. J Chromatogr B 879:1565–1572CrossRefGoogle Scholar
  23. 23.
    AbuRuz S, Millership J, Heaney L, McElnay J (2003) Simple liquid chromatography method for the rapid simultaneous determination of prednisolone and cortisol in plasma and urine using hydrophilic lipophilic balanced solid phase extraction cartridges. J Chromatogr B 798:193–201CrossRefGoogle Scholar
  24. 24.
    Newell-Price J, Trainer P, Besser M, Grossman A (1998) The diagnosis and differential diagnosis of Cushing’s syndrome and pseudo-Cushing’s states. Endocr Rev 19:647–672PubMedGoogle Scholar
  25. 25.
    Meinardi J, Wolffenbuttel B, Dullaart R (2007) Cyclic Cushing’s syndrome: a clinical challenge. Eur J Endocrinol 157:245–254CrossRefPubMedGoogle Scholar
  26. 26.
    Androulakis I, Kaltsas G, Chrousos MD (2017) Pseudo-Cushing’s states. National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/books/NBK279081/. Accessed 11 Jan 2017
  27. 27.
    Antonelli G, Ceccato F, Artusi C, Marinova M, Plebani M (2015) Salivary cortisol and cortisone by LC-MS/MS: validation, reference intervals and diagnostic accuracy in Cushing’s syndrome. Clin Chim Acta 451:247–251CrossRefPubMedGoogle Scholar
  28. 28.
    Ceccato F, Barbot M, Zilio M et al (2013) Performance of salivary cortisol in the diagnosis of Cushing’s syndrome, adrenal incidentaloma, and adrenal insufficiency. Eur J Endocrinol 169:31–36CrossRefPubMedGoogle Scholar
  29. 29.
    Løvås K, Thorsen TE, Husebye ES (2006) Saliva cortisol measurement: simple and reliable assessment of the glucocorticoid replacement therapy in Addison’s disease. J Endocrinol Invest 29:727–731CrossRefPubMedGoogle Scholar
  30. 30.
    Friso S, Pizzolo F, Choi SW et al (2008) Epigenetic control of 11 beta-hydroxysteroid dehydrogenase 2 gene promoter is related to human hypertension. Atherosclerosis 199:323–327CrossRefPubMedGoogle Scholar
  31. 31.
    Mongia A, Vecker R, George M et al (2012) Role of 11βHSD type 2 enzyme activity in essential hypertension and children with chronic kidney disease (CKD). J Clin Endocrinol Metab 97:3622–3629CrossRefPubMedGoogle Scholar
  32. 32.
    Ferrari P, Krozowski Z (2000) Role of the 11b-hydroxysteroid dehydrogenase type 2 in blood pressure regulation. Kidney Int 57:1374–1381CrossRefPubMedGoogle Scholar
  33. 33.
    Lutz SZ, Peter A, Machicao F et al (2016) Genetic variation in the 11β-hydroxysteroid-dehydrogenase 1 gene determines NAFLD and visceral obesity. J Clin Endocrinol Metab 101:4743–4751CrossRefPubMedGoogle Scholar
  34. 34.
    Stimson R, Walker B (2013) The role and regulation of 11β-hydroxysteroid dehydrogenase type 1 in obesity and the metabolic syndrome. Horm Mol Biol Clin Invest 15:37–48Google Scholar

Copyright information

© Italian Society of Endocrinology (SIE) 2017

Authors and Affiliations

  • K. Mészáros
    • 1
    • 2
    • 3
  • G. Karvaly
    • 1
    • 3
  • Z. Márta
    • 4
  • B. Magda
    • 4
  • J. Tőke
    • 5
  • N. Szücs
    • 5
  • M. Tóth
    • 5
  • K. Rácz
    • 5
    • 6
  • A. Patócs
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Laboratory Medicine, MTA-SE Lendulet Research GroupHungarian Academy of Sciences, Semmelweis UniversityBudapestHungary
  2. 2.“Lendület” Hereditary Endocrine Tumours Research Group, HAS-SEBudapestHungary
  3. 3.Bionics Innovation CenterBudapestHungary
  4. 4.MS Metabolomics Research Group, HASBudapestHungary
  5. 5.2nd Department of MedicineSemmelweis UniversityBudapestHungary
  6. 6.Molecular Medicine Research Group, HAS-SEBudapestHungary

Personalised recommendations