title: Development and validation of a GC-C-IRMS method for the confirmation analysis of pseudo-endogenous glucocorticoids in doping control.
abstract: Glucocorticoids are included in the S9 section of the World Anti-doping Agency (WADA) prohibited list international standard. Some among them are pseudo-endogenous steroids, like cortisol and cortisone, which present the same chemical structure as endogenously produced steroids. We are proposing an analytical method based on gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) which allows discrimination between endogenous and synthetic origin of the urinary metabolites of the pseudo-endogenous glucocorticoids. A preliminary purification treatment by high-performance liquid chromatography (HPLC) of the target compounds (TC) (i.e., cortisol, tetrahydrocortisone (THE) 5α-tetrahydrocortisone (aTHE), tetrahydrocortisol (THF), and 5α-tetrahydrocortisol (aTHF)) allows collection of extracts with adequate purity for the subsequent analysis by IRMS. A population of 40 urine samples was analyzed for the TC and for the endogenous reference compounds (ERC: i.e., 11-desoxy-tetrahydrocortisol (THS) or pregnanediol). For each sample, the difference between the delta values of the ERCs and TCs (Δδ values) were calculated and based on that, some decision limits for atypical findings are proposed. The limits are below 3% units except for cortisol. The fit to purpose of the method has been confirmed by the analysis of urine samples collected in two patients under treatment with 25 mg of cortisone acetate (p.o). The samples showed Δδ values higher than 3 for at least 24 h following administration depending on the TC considered. The method can easily be integrated into existing procedures already used for the HPLC purification and IRMS analysis of pseudo-endogenous steroids with androgenic/anabolic activity.
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.
authors.forename: Xavier
authors.initials: X
authors.lastname: de la Torre
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Curcio
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.
authors.forename: Cristiana
authors.initials: C
authors.lastname: Colamonici
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Molaioni
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Cilia
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti 1, 00197, Rome, Italy.|Dipartimento di Medicina Sperimentale, "Sapienza" Università di Roma, Viale Regina Elena 324, 00161, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Botrè
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D003348:Cortisone / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000652:urine; D004300:Doping in Sports; D008401:Gas Chromatography-Mass Spectrometry / Q000592:standards; D005938:Glucocorticoids / Q000008:administration & dosage / Q000652:urine*; D006801:Humans; D006854:Hydrocortisone / Q000652:urine; D016014:Linear Models; D008297:Male; D058573:Performance-Enhancing Substances / Q000008:administration & dosage / Q000652:urine*; D011237:Predictive Value of Tests; D012015:Reference Standards; D065667:Renal Elimination; D015203:Reproducibility of Results; D015813:Substance Abuse Detection / Q000379:methods* / Q000592:standards; D016482:Urinalysis
chemical_list: D005938:Glucocorticoids; D058573:Performance-Enhancing Substances; D003348:Cortisone; D006854:Hydrocortisone
keywords: HPLC sample preparation; IRMS; cortisol, gas chromatography; glucocorticoids; isotope ratio mass spectrometry