Quantification of Fecal Glucocorticoid Metabolites in Shelter Cats Using LC/MS/MS: Potential Biomarkers of Stress Associated with Shelter Admission

Cats can experience physiological stress during transitions to unfamiliar environments, such as shelter admission. To enable non-invasive stress assessment in cats, fecal glucocorticoid metabolites were quantified by liquid chromatography–tandem mass spectrometry (LC/MS/MS) with Girard’s reagent T derivatization. Five analytes were targeted: three glucocorticoids (cortisol, cortisone, and corticosterone) and their fecal metabolites, 11β-hydroxyetiocholanolone and 11-oxoetiocholanolone. Thirty-five cats housed at an animal welfare center were classified into a shorter-admission group (<2 weeks after admission, n = 16) or a longer-admission group (≥2 weeks, n = 19). The three parent glucocorticoids showed no detectable peak in any sample, whereas both metabolites were detected in 28 of the 35 samples and were strongly correlated in both the shorter- and longer-admission groups (ρ = 0.9316 and 0.9699, respectively). Both metabolites were significantly higher in the shorter-admission group (p = 0.0036 and 0.0029, respectively), whereas no significant differences were detected by sex or neuter status. In an incubation experiment, concentration changes within 48 h (≤0.12 ng/mg) were smaller than the between-group differences (1.70–2.16 ng/mg). These findings suggest that both metabolites are potential biomarkers of stress associated with shelter admission and that their measurement may provide a practical, non-invasive approach for monitoring stress in shelter cats.