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

Abstract

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.