Determination of hair cortisol in horses: comparison of immunoassay vs LC-HRMS/MS.

The measure of hair cortisol concentration (HCC) is becoming an emerging approach to monitor mid-/long-term stress in animals, so it is more and more important to develop accurate and reliable methods. In the light of this, the aim of the present study was to compare mane HCCs of 47 horses with different managements, by means of an immunoassay (ELISA) and liquid chromatography coupled to hybrid high-resolution mass spectrometry (LC-HRMS/MS). After the washing step, the ground hair was extracted with methanol. The extract was evaporated and redissolved in two different aqueous solutions, depending on the detection technique. The methods were validated according to EMA guideline for bioanalytical method validation, in the range 2-50 pg mg-1 (ELISA) and 1-100 pg mg-1 (LC-HRMS/MS). Satisfactory quantitative performances were obtained for both of the approaches, but this latter demonstrated better precision. The detected concentrations in real samples were encompassing the range 1.3-8.8 pg mg-1 and 2.0-17.9 pg mg-1 by means of LC-HRMS/MS and ELISA, respectively. Overall, HCCs measured with ELISA technique were 1.6 times higher. The overestimation of immunoassay results might be caused by cross-reactivity phenomena of laboratory reagents and other structurally similar hormones present in the mane.