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

Abstract

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.

Long-term, medium-term and acute stress response of urban populations of Eurasian red squirrels affected by different levels of human disturbance

“…In hair samples, cortisol and its metabolite-cortisone were determined using a validated ultra-high performance liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/ MS) method. We decided to follow the concentration of two steroids in the hair because it has beed suggested that measuring multiple glucocorticoids simultaneously may provide more comprehensive information and more reliable interpretation of the acquired results [34]. The method of sample preparation and analysis was described in detail by Sadok et al [34].…”

“…We decided to follow the concentration of two steroids in the hair because it has beed suggested that measuring multiple glucocorticoids simultaneously may provide more comprehensive information and more reliable interpretation of the acquired results [34]. The method of sample preparation and analysis was described in detail by Sadok et al [34]. Briefly, hair samples were washed twice with isopropanol, dried overnight, cut into small pieces into a tube in an amount from 16 to 48 mg, and milled using steel beads.…”

“…The steroids were extracted in the dark with 1 ml of methanol (LC-MS grade) fortified with internal standard (10 μg/ml of cortisol-D 4 ) for 24 hours. The supernatants were further purified by solid-phase extraction using the polymeric reversed phase Strata-X cartridges (30 mg/1 ml) supplied from Phenomenex as described previously (Sadok et al [34]).…”

“…Samples were analyzed using a 1290 infinity UHPLC system coupled to a 6460 triple quadrupole mass spectrometer equipped with the Jet Stream electrospray ion (ESI) source from Agilent Technologies (Santa-Clara, CA, USA) and settings detailed in Sadok et al [34]. The concentrations of steroids were determined in μg/ml from matrix-based calibration plots after signals normalization on the internal standard.…”

“…The analysis of hair and fecal glucocorticoid metabolites is considered less invasive compared to approaches that include blood sampling. For this reason, it has been applied to many wildlife species to monitor their stress responses to various disturbances [8,29,34,35]. Glucocorticoids concentrations in squirrels can depend on different factors like population density [36,37], sex [38][39][40][41][42], season [38,41], body mass [38,40] and condition [37,43].…”