Surrogate analyte approach for the determination of endogenous cortisol, cortisone, DHEA, DHEAS in horse hair and sheep wool by LC-HRMS/MS.

Two new quantitative methods for the determination of four stress-related hormones (cortisol, cortisone, dehydroepiandrosterone and dehydroepiandrosterone sulfate) in horse hair and sheep wool by liquid chromatography coupled with hybrid high resolution mass spectrometry were developed and validated. Hormones were extracted overnight from ground samples with methanol after a washing step with the same solvent to remove external contamination. The extract was evaporated and dry residue was solubilized in an acid mixture of methanol and water. The methods were validated according to ICH guidelines for bioanalytical method validation within 5-100 pg/mg with LC-Q Exactive platform (except for DHEA: 100-2000 pg/mg for both matrices and 1-100 pg/mg for DHEAS in mane hair). The validated concentration range was 5-100 pg/mg for wool and 1-100 pg/mg (except for DHEA: 5-100 pg/mg) for mane hair samples with LC-Exploris 120 system. Satisfactory quantitative performances were obtained using isotopic dilution and surrogate analyte approach. After successful validation, the applicability of the methods were tested. The detected concentrations of investigated analytes in real samples collected from animals living in marginal areas were encompassing the interval 1.1-13 and 5.0-30.5 pg/mg in mane hair and wool samples, respectively.