Highly selective and automated online SPE LC–MS3 method for determination of cortisol and cortisone in human hair as biomarker for stress related diseases
2015
Quinete, Natalia | Bertram, Jens | Reska, Marcus | Lang, Jessica | Kraus, Thomas
Hair analysis has been increasingly used to establish long-term biomarkers of exposure to both endogenous and exogenous substances, with a special emphasis on steroidal hormones. Hair cortisol and cortisone have been associated to physiological and psychological strains, anxiety and depression. Hair is a very complex matrix, which might jeopardize analyte detection at low concentrations. A new, highly selective and sensitive method based on fragments of second order, MS³ (MS/MS/MS), was developed and validated for the analysis of hair cortisol and cortisone. An online solid phase extraction was performed on a C8 restricted access material (RAM) phase following by separation on a reversed-phase C18 column using methanol and 0.02% ammonium hydroxide as mobile phase. The developed method required minimal sample preparation and the injection of only 50µL of sample leading to a LOQ of 2pgmg⁻¹. Good linear responses were observed in the range 2–200pgmg⁻¹ (R²>0.99) and extraction recoveries ranged between 77–125% and 70–123% for cortisol and cortisone, respectively. Intra- and inter-assay coefficients of variation were between 1.4 and 14%. In order to evaluate the applicability of the method, preliminary tests (N=33) were conducted in 3cm hair samples (close to scalp) of healthy volunteers with an age range of 4–63. Average concentrations in hair were 12.7±14pgmg⁻¹ and 41.6±42pgmg⁻¹ for cortisol and cortisone, respectively. Further investigations on cortisol and cortisone as biomarkers for chronic psychological strain will be assessed as a next step.
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