title: Development and validation of a high-resolution mass-spectrometry-based method to study the long-term stability of natural and synthetic glucocorticoids in faeces.
abstract: Faecal glucocorticoid analysis is a powerful non-invasive tool for the study of the animal endocrine status and stress physiology, which is mainly carried out by immunoassays, characterised by some limitations. In this study, an ultra high-performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry (U-HPLC-HRMS) method was developed to confirm the presence of glucocorticoids in bovine faeces during a long-term stability study. Because of the complex nature of faeces, an appropriate extraction and purification procedure was developed. To this extent, a Plackett-Burman experimental design was successfully applied to determine the key conditions for optimal extraction of glucocorticoids from faeces. The targeted analysis, including natural and synthetic glucocorticoids, was successfully validated according to CD 2002/657/EC. Decision limits and detection capabilities for prednisolone, prednisone, methylprednisolone and the metabolites 20α-dihydroprednisolone and 20β-dihydroprednisolone ranged, respectively, from 0.15 to 2.95 μg kg(-1) and from 0.40 to 5.20 μg kg(-1). Limits of detection and limits of quantification for the natural glucocorticoids dihydrocortisone, cortisol and cortisone ranged, respectively, from 0.55 to 2.10 μg kg(-1) and from 0.70 to 5.00 μg kg(-1). The stability study of glucocorticoids in faecal matrix demonstrated that lyophilising the faeces, storage at -80°C, and aerobic conditions were optimal for preservation and able to significantly (p < 0.05) limit degradation up to 10 weeks.
authors.affiliation: Ghent University, Faculty of Veterinary Medicine, Department of Veterinary Public Health & Food Safety, Laboratory of Chemical Analysis, Salisburylaan 133, B-9820 Merelbeke, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: De Clercq
authors.affiliation: Ghent University, Faculty of Veterinary Medicine, Department of Veterinary Public Health & Food Safety, Laboratory of Chemical Analysis, Salisburylaan 133, B-9820 Merelbeke, Belgium.
authors.forename: Julie
authors.initials: J
authors.lastname: Vanden Bussche
authors.affiliation: Ghent University, Faculty of Veterinary Medicine, Department of Pharmacology, Toxicology and Biochemistry, Laboratory of Pharmacology and Toxicology, Salisburylaan 133, B-9820 Merelbeke, Belgium.
authors.forename: Siska
authors.initials: S
authors.lastname: Croubels
authors.affiliation: CER Groupe, Département Santé, Rue du Point du Jour 8, B-6900 Marloie, Belgium.
authors.forename: Philippe
authors.initials: P
authors.lastname: Delahaut
authors.affiliation: Ghent University, Faculty of Veterinary Medicine, Department of Veterinary Public Health & Food Safety, Laboratory of Chemical Analysis, Salisburylaan 133, B-9820 Merelbeke, Belgium. Electronic address: Lynn.Vanhaecke@ugent.be.
authors.forename: Lynn
authors.initials: L
authors.lastname: Vanhaecke
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005243:Feces / Q000737:chemistry*; D005938:Glucocorticoids / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D006854:Hydrocortisone / Q000032:analysis; D013058:Mass Spectrometry / Q000379:methods*; D011239:Prednisolone / Q000032:analysis
chemical_list: D005938:Glucocorticoids; D011239:Prednisolone; D006854:Hydrocortisone
keywords: Faeces; Plackett–Burman; glucocorticoids; stability study; ultra-high performance liquid chromatography-orbitrap-high resolution mass spectrometry