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

[ Vol. 15 , Issue. 4 ]

Author(s):

Martin Kertys*, Anna Urbanova, Michal Mestanik, Ingrid Tonhajzerova and Juraj Mokry   Pages 363 - 370 ( 8 )

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.

Affiliation:

Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Bratislava, Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Bratislava, Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Bratislava, Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Bratislava, Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Bratislava

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