Simultaneous Determination of Total Cortisol and Cortisone in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry: Method Development, Validation and Preliminary Clinical Application

Author(s): Martin Kertys*, Anna Urbanova, Michal Mestanik, Ingrid Tonhajzerova, Juraj Mokry.

Journal Name: Current Pharmaceutical Analysis

Volume 15 , Issue 4 , 2019

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Abstract:

Background: Cortisol as a major glucocorticosteroid product of the adrenal cortex which has been recognized as a stress biomarker in evaluating stress related disorders for a long time. Plasma concentration of cortisol and its metabolite cortisone are usually changed in physiological and psychological tension, anxiety and depression. In order to study these changes properly, we need a sensitive, accurate and reproducible assay for plasma cortisol and cortisone determination.

Objective: The aim of this study was to develop a sensitive and robust method for the determination of total cortisol and cortisone in human plasma using mass spectrometry.

Methods: A fast, sensitive and selective liquid chromatography-tandem mass spectrometry (LCMS/ MS) method was developed, validated, and then the levels of cortisol and cortisone were determined. Plasma samples cleanup procedure was composed of two steps: the first was a protein precipitation with 1 % formic acid in acetonitrile, and the second was an on-line solid phase extraction (SPE). Afterwards, cortisol and cortisone were separated using a C18 ACQUITY UPLC BEHTM column with a gradient elution. The mobile phase A was 0.1 % formic acid in water, the mobile phase B was 0.1 % methanol. For the detection we used a XEVO TQ-S mass spectrometer operating in the ESI positive mode.

Results: The time of analysis was 6.5 minutes and the quantification range was 5-600 ng/mL for cortisol and cortisone, with > 94% recovery for all analytes (cortisol, cortisone and internal standards). The method was validated according to the EMA guideline for bioanalytical method validation.

Conclusion: A simple and sensitive LC-MS/MS method was developed and validated for measurement of cortisol and cortisone in human plasma. Our findings indicate that the proposed analytical method is suitable for routine analysis.

Keywords: Cortisol, cortisone, liquid chromatography, mass spectrometry, human plasma, stress.

[1]
Knutsson, U.; Dahlgren, J.; Marcus, C.; Rosberg, S.; Bronnegard, M.; Stierna, P.; Albertsson-Wikland, K. Circadian cortisol rhythms in healthy boys and girls: relationship with age growth, body composition and pubertal development. J. Clin. Endocrinol. Metab., 1997, 82(2), 536-540.
[2]
Slopen, N.; McLaughlin, K.A.; Shonkoff, J.P. Interventions to improve cortisol regulation in children: a systematic review. Pediatrics, 2014, 133(2), 312-326.
[3]
Mokra, D.; Mokry, J. Glucocorticoids in the treatment of neonatal meconium aspiration syndrome. Eur. J. Pediatr., 2011, 170(12), 1495-1505.
[4]
Hellhammer, D.H.; Wust, S.; Kudielka, B.M. Salivary cortisol as a biomarker in stress research. Psychoneuroendocrinology, 2009, 34(2), 163-171.
[5]
Chrousos, G.P.; Kino, T. Glucocorticoid action networks and complex psychiatric and/or somatic disorders. Stress, 2007, 10(2), 213-219.
[6]
Mizoguchi, K.; Ishige, A.; Takeda, S.; Aburada, M.; Tabira, T. Endogenous glucocorticoids are essential for maintaining prefrontal cortical cognitive function. J. Neurosci., 2004, 24(24), 5492-5499.
[7]
Ferrari, P. The role of 11β-hydroxystreoid dehydrogenase type 2 in human hypertension. Biochim. Biophys. Acta, 2010, 1802(12), 1178-1187.
[8]
Anagnostis, P.; Athyros, G.V.; Tziomalos, K.; Karagiannis, A.; Mikhailidis, P.D. The pathogenetic role of cortisol in the metabolic syndrome: a hypothesis. J. Clin. Endocrinol. Metab., 2009, 94(8), 2692-2701.
[9]
Provost, P.R.; Boucher, E.; Tremblay, Y. Glucocorticoid metabolism in the developing lung: Adrenal-like synthesis patway. J. Steroid Biochem. Mol. Biol., 2013, 138, 72-80.
[10]
Campino, C.; Carvajal, C.A.; Cornejo, J.; San, B.M.; Olivieri, O.; Guidi, G.; Faccini, G.; Pasini, G.; Sateler, J.; Baudrand, R.; Mosso, L.; Owen, G.I.; Kalergis, A.M.; Padilla, O.; Fardella, C.E. 11beta-Hydroxysteroid dehydrogenase type-2 and type-1 (11beta-HSD2 and 11beta-HSD1) and 5beta-reductase activities in the pathogenia of essential hypertension. Endocrine, 2010, 37(1), 106-114.
[11]
Reimondo, G.; Pia, A.; Bovio, S.; Allasino, B.; Daffara, F.; Paccotti, P.; Borretta, G.; Angeli, A.; Terzolo, M. Laboratory differentiation of Cushing’s syndrome. Clin. Chim. Acta, 2008, 388(1-2), 5-14.
[12]
Mostl, E.; Palmer, R. Hormones as indicator of stress. Domest. Anim. Endocrinol., 2002, 23(1-2), 67-74.
[13]
Nozaki, O. Steroid analysis for medical diagnosis. J. Chromatogr. A, 2001, 935(1-2), 267-278.
[14]
Dodd, A.; Ducroq, D.; Neale, S.; Wise, M.; Mitchem, K.; Armston, A.; Barth, J.; El-Farhan, N.; Rees, D.; Evans, C. The effect of serum matrix and gender on cortisol measurement by commonly used immunoassays. Ann. Clin. Biochem., 2014, 51(3), 379-385.
[15]
Zhai, X.; Chen, F.; Zhu, C.; Lu, Y. A simple LC-MS/MS method for the determination of cortisol, cortisone and tetrahydro-metabolites in human urine: assay development, validation and application in depression patients. J. Pharm. Biomed. Anal., 2015, 107, 450-455.
[16]
Allende, F.; Solari, S.; Campino, C.; Carvajal, C.A.; Lagos, C.F.; Vecchiola, A.; Valdivia, C.; Baudrand, R.; Owen, G.I.; Fardella, C.E. LC-MS/MS method for the simultaneous determination of free urinary steroids. Chromatographia, 2014, 77, 637-642.
[17]
Kushnir, M.M.; Rockwood, L.A.; Roberts, L.W.; Yue, B.; Bergquist, J.; Meikle, W.A. Liquid chromatography tandem mass spectrometry for analysis of steroids in clinical laboratories. Clin. Biochem., 2011, 44(1), 77-88.
[18]
van den Ouweland, J.M.; Kema, I.P. The role of liquid chromatography-tandem mass spectrometry in the clinical laboratory. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 2012, 883-884, 18-32.
[19]
El-Farhan, N.; Rees, D.A.; Evans, C. Measuring cortisol in serum, urine and saliva – are our assays good enough? Ann. Clin. Biochem., 2017, 54(3), 308-322.
[20]
Zhou, S.L.; Song, Q.; Tang, Y.; Weng, N.D. Critical review of development, validation, and transfer for high throughput bioanalytical LC-MS/MS methods. Curr. Pharm. Anal., 2005, 1(1), 3-14.
[21]
Li, K.M.; Rivory, L.P.; Clarke, S.J. Solid-phase extraction (SPE) techniques for sample preparation in clinical and pharmaceutical analysis: A brief overview. Curr. Pharm. Anal., 2006, 2(2), 95-102.
[22]
Munaga, S.B.; Valluru, R.K.; Bonga, P.B.R.; Rao, V.S.; Sharma, H.K. Qualification and application of a liquid chromatography-tandem mass spectrometric method for the simultaneous quantification of triflusal and its active metabolite desacetyl triflusal in human plasma using solid phase extraction technique. Curr. Pharm. Anal., 2017, 13(4), 284-397.
[23]
Perogamvros, I.; Owen, L.J.; Newell-Price, J.; Ray, D.W.; Trainer, P.J.; Keevil, B.G. Simultaneous measurement of cortisol and cortisone in human saliva using liquid chromatography-tandem mass spectrometry: application in basal and stimulated conditions. J. Chromatogr. B, 2009, 877(29), 3771-3775.
[24]
Binz, T.M.; Braun, U.; Baumgartner, M.R.; Kraemer, T. Development of an LC-MS/MS method for the determination of endogenous cortisol in hair using (13)C3-labeled cortisol as surrogate analyte. J. Chromatogr. B , 2016, 1033-1034, 65-72.
[25]
Raffaelli, A.; Saba, A.; Vignali, E.; Marcocci, C.; Salvadori, P. Direct determination of the ratio of tetrahydrocortisol+allo-etrahydrocortisol to tetrahydrocortisone in urine by LC-MS-MS. J. Chromatogr. B , 2006, 830(2), 278-285.
[26]
Kushnir, M.M.; Neilson, R.; Roberts, W.L.; Rockwood, A.L. Cortisol and cortisone analysis in serum and plasma by atmospheric pressure photoionization tandem mass spectrometry. Clin. Biochem., 2004, 37(5), 357-362.
[27]
Ray, J.A.; Kushnir, M.M.; Yost, R.A.; Rockwood, A.L.; Meikle, A.W. Perfomance enhancement in the measurement of 5 endogenous steroids by LC-MS/MS combined with differential ion mobility spectrometry. Clin. Chim. Acta, 2015, 438, 330-336.
[28]
Fanelli, F.; Bellunomo, I.; Di Lallo, V.D.; Cuomo, G.; De Iasio, R.; Baccini, M.; Casadio, E.; Cassetta, B.; Vicennati, V.; Gambineri, A.; Grossi, G.; Pasquali, R.; Pagotto, U. Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and references intervals in healthy adults. Steroids, 2011, 76(3), 244-253.
[29]
Lee, S.; Lim, H.; Shin, H.; Kim, S.; Park, J.; Kim, H.; Kim, H.J.; Kim, Y.; Lee, K.; Kim, Y.J. Simultaneous determination of cortisol and cortisone from human serum by liquid chromatography-tandem mass spectrometry. J. Anal. Methods Chem., 2014, 2014, 1-6.
[30]
Walker, B.R. Cortisol-cause and cure for metabolic syndrome? Diabet. Med., 2006, 23(12), 1281-1288.
[31]
European Medicines Agency. 2012.Guideline on Bioanalytical Method Validation., (Available from: http://www.ema.europa.eu/ docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf
[32]
Matuszewski, B.K.; Constanzer, M.L.; Chavez-Eng, C.M. Strategies for the assessement of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem., 2003, 75(13), 3019-3030.
[33]
Gray, G.; Shakerdi, L.; Wallace, A.M. Poor specificity and recovery of urinary free cortisol as determined by the Bayer ADVIA Centaur extraction method. Ann. Clin. Biochem., 2003, 40(5), 563-565.
[34]
El-Farhan, N.; Picket, A.; Ducroq, D.; Bailey, C.; Mitchem, K.; Morgan, N.; Armston, A.; Jones, L.; Evans, C.; Rees, D.A. Method-specific serum cortisol responses to the adrenocorticotrophin test: comapruison of gas chromatography-mass spectrometry and five automated immunoassays. Clin. Endocrinol. (Oxf.), 2013, 78(5), 673-680.
[35]
Kushnir, M.M.; Rockwood, A.L.; Roberts, W.L.; Yue, B.; Berquist, J.; Meikle, A.W. Liquid chromatography tandem mass spectrometry for analysis of steroids in clinical laboratories. Clin. Biochem., 2011, 44(1), 77-88.
[36]
Courtney, C.H.; McAllister, A.S.; McCance, D.R.; Bell, P.M.; Hadden, D.R.; Leslie, H.; Sheridan, B.; Atkinson, A.B. Comparison of one week 0900 h serum cortisol, low and standard dose synacthen test with a 4 to 6 week insulin hypoglycaemia test after pituitary surgery in assessing HPA axis. Clin. Endocrinol. (Oxf.), 2000, 53(4), 431-436.
[37]
Bornstein, S.R.; Allolio, B.; Arlt, W.; Barthel, A.; Don-Wauchope, A.; Hammer, G.D.; Husebye, E.S.; Merke, D.P.; Murad, M.H.; Stratakis, C.A.; Torpy, D.J. Diagnosis and treatment of primary adrenal insufficiency: An endocrine society clinical practice guideline. J. Clin. Endocrinol. Metab., 2016, 101(2), 364-389.
[38]
Antonelli, G.; Artusi, C.; Marinova, M.; Brungolo, L.; Zaninotto, M.; Scaroni, C.; Gatti, R.; Mantero, F.; Plebani, M. Cortisol and cortisone ratio in urine: LC-MS/MS method validation and preliminary clinical application. Clin. Chem. Lab. Med., 2014, 52(2), 213-220.
[39]
Tonhajzerova, I.; Mestanik, M. New perspectives in the model of stress response. Physiol. Res., 2017, 66(2), 173-185.
[40]
Tonhajzerova, I.; Mestanik, M.; Mestanikova, A.; Jurko, A. Respiratory sinus arrhythmia as a non-invasive index of ‘brain-heart’ interaction in stress. Indian J. Med. Res., 2016, 144(6), 815-822.
[41]
Visnovcova, Z.; Mokra, D.; Mikolka, P.; Mestanik, M.; Jurko, A.; Javorka, M.; Calkovska, A.; Tonhajzerova, I. Alterations in vagal-immune pathway in long-lasting mental stress. Adv. Exp. Med. Biol., 2015, 832, 45-50.


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Article Details

VOLUME: 15
ISSUE: 4
Year: 2019
Page: [363 - 370]
Pages: 8
DOI: 10.2174/1573412914666180427094811
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