title: Non-targeted LC-MS based metabolomics analysis of the urinary steroidal profile.
abstract: The urinary steroidal fraction has been extensively explored as non-invasive alternative to monitor pathological conditions as well as to unveil the illicit intake of pseudo-endogenous anabolic steroids in sport. However, the majority of previous approaches involved the a priori selection of potentially relevant target analytes. Here we describe the non-targeted analysis of the urinary steroidal profiles. The workflow includes minimal sample pretreatment and normalization according to the specific gravity of urine, a 20 min reverse phase ultra-performance liquid chromatographic separation hyphenated to electrospray time-of-flight mass spectrometry. As initial validation, we analyzed a set of quality control urines spiked with glucurono- and sulfo-conjugated steroids at physiological ranges. We then applied the method for the analysis of samples collected after single transdermal administration of testosterone in hypogonadal men. The method allowed profiling of approximately three thousand metabolic features, including steroids of clinical and forensic relevance. It successfully identified metabolic pathways mostly responsible for groups clustering even in the context of high inter-individual variability and allowed the detection of currently unknown metabolic features correlating with testosterone administration. These outcomes set the stage for future studies aimed at implementing currently monitored urinary steroidal markers both in clinical and forensic analysis.
authors.affiliation: Institute of Molecular Systems Biology, ETH Zurich, Switzerland; Department of Chemistry and Pharmaceutical Technologies, Sapienza University of Rome, Italy; Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Italy.
authors.forename: Amelia
authors.initials: A
authors.lastname: Palermo
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Italy; Department of Experimental Medicine, Sapienza University of Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Botrè
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Italy.
authors.forename: Xavier
authors.initials: X
authors.lastname: de la Torre
authors.affiliation: Institute of Molecular Systems Biology, ETH Zurich, Switzerland. Electronic address: nzamboni@ethz.ch.
authors.forename: Nicola
authors.initials: N
authors.lastname: Zamboni
mesh_terms: D000328:Adult; D000368:Aged; D045930:Anabolic Agents / Q000652:urine*; D002853:Chromatography, Liquid; D004300:Doping in Sports; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D055432:Metabolomics; D008875:Middle Aged; D013256:Steroids / Q000652:urine*
chemical_list: D045930:Anabolic Agents; D013256:Steroids
keywords: Anabolic androgenic steroids; Glucocorticoids; Liquid chromatography; Mass spectrometry; Non-targeted metabolomics; Testosterone gel; Urinary steroidal profile