Highly selective and automated online SPE LC–MS3 method for determination of cortisol and cortisone in human hair as biomarker for stress related diseases

• Determination of cortisol and cortisone in human hair at very low concentrations.
• Development &validation of anonline SPE–LC–MS3 method with minimal matrix effects.
• Thirty-three healthy volunteers with an age range of 4–63 years were evaluated.
• We found a positive correlation of cortisol with age.
• Cortisol-to-cortisone ratio in dyed and natural hair was significantly different.

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 50 µL of sample leading to a LOQ of 2 pg mg−1. Good linear responses were observed in the range 2–200 pg mg−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 3 cm hair samples (close to scalp) of healthy volunteers with an age range of 4–63. Average concentrations in hair were 12.7±14 pg mg−1 and 41.6±42 pg mg−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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