A validated LC-MS/MS method for simultaneous determination of key glucocorticoids in animal hair for applications in conservation biology.

A new method for the determination of main glucocorticoids (cortisol, cortisone, and corticosterone) in hair by liquid chromatography-tandem mass spectrometry was developed. Glucocorticoids were extracted from hair shafts using methanol followed by solid-phase extraction. A validation test was performed using hair from three species of wild mammals with different body size (0.2-800 kg), lifestyle (terrestrial, burrowing and arboreal species), social organization (living in herds or solitary), and different predicted type of hair glucocorticoids: European bison (Bison bonasus), European hamster (Cricetus cricetus), and Eurasian red squirrel (Sciurus vulgaris). Regardless of the species evaluated, the method shows good linearity for all analytes accompanied by satisfactory accuracy (91-114%) and precision (RSD < 13%). Depending on the analyte and hair origin, the calculated limits of quantification were between 0.05 and 1.19 ng/mL, which corresponds to 1.28-31.51 pg/mg. Using cortisol and cortisone as examples, we have demonstrated that measuring multiple glucocorticoids simultaneously provides more comprehensive information than solely concentrating on one, thereby contributing to a more balanced and reliable interpretation of the acquired results. However, the utility of cortisol metabolites as markers of stress response in keratinized tissues should be substantiated by additional experimental studies on targeted animals. We posit that this paper could serve as a crucial catalyst to prompt such experiments.