title: Application of multivariate statistics to the Steroidal Module of the Athlete Biological Passport: A proof of concept study.
abstract: The Technical Document TD2014EAAS was drafted by the World Anti-Doping Agency (WADA) in order to fight the spread of endogenous anabolic androgenic steroids (EAAS) misuse in several sport disciplines. In particular, adoption of the so-called Athlete Biological Passport (ABP) - Steroidal Module allowed control laboratories to identify anomalous EAAS concentrations within the athletes' physiological urinary steroidal profile. Gas chromatography (GC) combined with mass spectrometry (MS), indicated by WADA as an appropriate technique to detect urinary EAAS, was utilized in the present study to develop and fully-validate an analytical method for the determination of all EAAS markers specified in TD2014EAAS, plus two further markers hypothetically useful to reveal microbial degradation of the sample. In particular, testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol, dehydroepiandrosterone, 5α-dihydrotestosterone, were included in the analytical method. Afterwards, the multi-parametric feature of ABP profile was exploited to develop a robust approach for the detection of EAAS misuse, based on multivariate statistical analysis. In particular, Principal Component Analysis (PCA) was combined with Hotelling T(2) tests to explore the EAAS data obtained from 60 sequential urine samples collected from six volunteers, in comparison with a reference population of single urine samples collected from 96 volunteers. The new approach proved capable of identifying anomalous results, including (i) the recognition of samples extraneous to each of the individual urine series and (ii) the discrimination of the urine samples collected from individuals to whom "endogenous" steroids had been administrated with respect to the rest of the samples population. The proof-of-concept results presented in this study will need further extension and validation on a population of sport professionals.
authors.affiliation: Centro Regionale Antidoping e di Tossicologia "A. Bertinaria", Regione Gonzole 10/1, 10043 Orbassano, Torino, Italy; Dipartimento di Chimica, Università degli Studi di Torino, Via P. Giuria 7, 10125 Torino, Italy. Electronic address: ealladio@unito.it.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Alladio
authors.affiliation: Dipartimento di Chimica, Università degli Studi di Torino, Via P. Giuria 7, 10125 Torino, Italy. Electronic address: roberto.caruso@edu.unito.it.
authors.forename: Roberto
authors.initials: R
authors.lastname: Caruso
authors.affiliation: Centro Regionale Antidoping e di Tossicologia "A. Bertinaria", Regione Gonzole 10/1, 10043 Orbassano, Torino, Italy. Electronic address: enrico.gerace@antidoping.piemonte.it.
authors.forename: Enrico
authors.initials: E
authors.lastname: Gerace
authors.affiliation: Dipartimento di Chimica, Università degli Studi di Torino, Via P. Giuria 7, 10125 Torino, Italy. Electronic address: eleonora.amante@edu.unito.it.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Amante
authors.affiliation: Centro Regionale Antidoping e di Tossicologia "A. Bertinaria", Regione Gonzole 10/1, 10043 Orbassano, Torino, Italy. Electronic address: alberto.salomone@antidoping.piemonte.it.
authors.forename: Alberto
authors.initials: A
authors.lastname: Salomone
authors.affiliation: Centro Regionale Antidoping e di Tossicologia "A. Bertinaria", Regione Gonzole 10/1, 10043 Orbassano, Torino, Italy; Dipartimento di Chimica, Università degli Studi di Torino, Via P. Giuria 7, 10125 Torino, Italy. Electronic address: marco.vincenti@unito.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Vincenti
mesh_terms: D045930:Anabolic Agents / Q000652:urine; D056352:Athletes; D008192:Deception; D004300:Doping in Sports; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D057230:Limit of Detection; D015999:Multivariate Analysis; D025341:Principal Component Analysis; D015203:Reproducibility of Results; D015813:Substance Abuse Detection / Q000379:methods*; D013739:Testosterone / Q000031:analogs & derivatives / Q000652:urine
chemical_list: D045930:Anabolic Agents; D013739:Testosterone
keywords: Athlete Biological Passport; Endogenous anabolic androgenic steroids; Gas chromatography–mass spectrometry; Hotelling's T(2) test; Multivariate statistics; Principal components analysis