Untargeted Metabolomics in Doping Control: Detection of New Markers of Testosterone Misuse by Ultrahigh Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

Raro, Montse; Ibáñez, María; Gil, Rubén; Fabregat, Andreu; Tudela, Eva; Deventer, Koen; Ventura, Rosa; Segura, Jordi; Marcos, Josep; Kotronoulas, Aristotelis; Joglar, Jesús; Farré, Magı́; Yang, Sheng; Xing, Yanyi; Eenoo, Peter Van; Pitarch, Elena; Hernández, Félix; Sancho, Juan V.; Pozo, Óscar J.

doi:10.1021/acs.analchem.5b02254

Cited by 40 publications

(46 citation statements)

References 31 publications

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“…The use of the LC-MS/MS allows for the direct analysis of intact phase II metabolites [17][18][19], avoiding the time consuming and laborious steps of enzymatic hydrolysis and derivatization needed for the GC-MS analysis. Additionally, the coupling of LC with versatile and sensitive MS analyzers allowed for the reevaluation of the T metabolome [20][21][22] and led to the identification of many unreported metabolites of endogenous steroids [20,[23][24][25][26].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of two glucuronides resistant to enzymatic hydrolysis as markers of testosterone oral administration

Kotronoulas

Gómez-Gómez

Segura

et al. 2017

The Journal of Steroid Biochemistry and Molecular Biology

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Testosterone (T) has traditionally been the most commonly reported doping agent by doping control laboratories. The screening of T misuse is performed by the quantification of six endogenous androgenic steroids and the ratio T/E included in the Athlete Biological Passport (ABP). The inclusion of additional metabolites can improve the screening capabilities of ABP. In this study, the potential of 3α-glucuronide-6β-hydroxyandrosterone (6OH-Andros3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio3G) as markers of T oral administration was evaluated. These glucuronides have been shown to be resistant to enzymatic hydrolysis and their quantification by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was reported as the only way to obtain feasible results. Urine samples were collected from five volunteers before and after the oral administration of 40mg of T undecanoate and were analyzed by a LC-MS/MS method recently developed. Concentration of 6OH-Andros3G and 6OH-Etio3G compounds and those of the glucuronides of T (TG), epitestosterone (EG), androsterone and etiocholanolone were established and different concentration ratios were calculated. The detection windows (DWs) for the T administration obtained by each selected ratio were compared to the one of TG/EG. The results showed that four out of the nine tested markers presented DWs much larger for all volunteers than those obtained by the World Anti-Doping Agency established T/E marker or other alternative markers. The 6OH-Andros3G/EG, 6OH-Etio3G/EG, 6OH-Andros3G/TG and 6OH-Etio3G/TG markers were able to identify the T abuse up to 96h after the administration, extending our detection capability for the misuse up to 84h more than the classic marker. The importance of these markers was also highlighted by their prolonged capacity to detect the T misuse in the case of one volunteer whose TG/EG barely exceeded his individual threshold. As a consequence, the four markers presented in this study seem to have an exceptional potential as biomarkers of T oral administration.

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Section: Introductionmentioning

confidence: 99%

Evaluation of two glucuronides resistant to enzymatic hydrolysis as markers of testosterone oral administration

Kotronoulas

Gómez-Gómez

Segura

et al. 2017

The Journal of Steroid Biochemistry and Molecular Biology

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“…1‐CPG was synthesized and purified as previously described . The β‐glucuronidase preparation (from Escherichia coli type K12) was purchased from Roche Diagnostics (Mannheim, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Thus, several T metabolites conjugated with either cysteine or with glucuronic acid but resistant to enzymatic hydrolysis have demonstrated their suitability for the detection of the oral administration of testosterone. Additionally, in a previous study conducted by our laboratory, an untargeted metabolomics analysis revealed the presence of a novel marker for the determination of T cypionate administration. The structure of this marker was confirmed to be 1‐cyclopentenoylglycine (1‐CPG), that likely originates from the metabolism of cypionic acid released after hydrolysis of the administered T ester .…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, in a previous study conducted by our laboratory, an untargeted metabolomics analysis revealed the presence of a novel marker for the determination of T cypionate administration. The structure of this marker was confirmed to be 1‐cyclopentenoylglycine (1‐CPG), that likely originates from the metabolism of cypionic acid released after hydrolysis of the administered T ester . The urinary response of 1‐CPG was found to clearly increase after the administration of T cypionate opening an alternative for the screening of this drug.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part II: Intramuscular administration

Kotronoulas

Gómez-Gómez

Fabregat

et al. 2017

Drug Testing and Analysis

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In the fight against doping, the introduction of alternative markers to the steroid profile can be considered as an effective approach to improve the screening capabilities for the detection of testosterone (T) misuse. The aim of this study was to evaluate the potential of several T metabolites (cysteinyl conjugated and glucuronoconjugated resistant to enzymatic hydrolysis) to detect both the transdermal and the intramuscular administration of T. In Part I of the study, we studied the potential of these metabolites for the detection of T transdermal administration. Results revealed that resistant glucuronides can be a suitable complement to the current steroid profile. In this, Part II, dedicated to the intramuscular administration, we studied the potential of cysteinyl conjugated, resistant glucuronoconjugated and 1-cyclopentenoylglycine (1-CPG) for the detection of a single intramuscular injection of T cypionate. Possible differences in the excretion profile of all markers were explored between individuals with low basal (n=6) and medium basal (n=6) values of the testosterone/epitestosterone ratio (T/E). The results showed that all tested markers presented low intra-individual stability in basal conditions. Despite this, all glucuronoconjugated markers and 1-CPG, but not the cysteinyl conjugated markers, provided detection windows that were similar or longer than those obtained by markers currently included in the steroid profile. Based on the results obtained from the 2 parts of this study and from previously reported data, the potential applicability and the limitations of including these markers in the steroid profile are discussed.

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“…Nevertheless, knowing the limits of targeted approach [21] and the confirmed potential of untargeted approach [22], the present study aimed to assess the global biological effect of stanozolol-treatment by means of chemometric models, after generating and comparing horse urine LC-HRMS fingerprints collected from control and stanozololtreated horses. Furthermore, by the means of metabolomics approach, the possibility to extend the detection window of stanozolol is considered.…”

Section: Generic Namementioning

confidence: 99%

Urine Fingerprints of Stanozolol Treated Horses by Liquid Chromatography High Resolution Mass Spectrometry

Stojiljkovic¹,

Bubanj²,

Đorđević³

et al. 2017

J Sports Med Dop Stud

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The current detection methods for stanozolol are all based on targeted approach. The present study aimed to assess the global biological effect of stanozolol-treatment by means of chemometric models, after generating and comparing horse urine LC-HRMS fingerprints collected from control and stanozolol-treated horses. The animal study was conducted according to an ethically approved protocol at two different places in France: Chamberet and Coye la Forêt. The total duration of the animal phase was seven months and only females were selected to partake. The sixteen mares in this study were not actively racing horses, but were in good physical condition. SIMCA-P+ software and R free software environment were used for multivariate data analysis. Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures-Discriminant Analysis (OPLS-DA) were applied to build some descriptive and predictive models. The analyzed horse urine fingerprints based on the 220 features selected after suppression of confounding factors show changes in metabolic states after chronic stanozolol treatment. This proof of concept study confirms the power of untargeted approach in doping control since the changes are present over seven months after anabolic administration.

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Untargeted Metabolomics in Doping Control: Detection of New Markers of Testosterone Misuse by Ultrahigh Performance Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

Cited by 40 publications

References 31 publications

Evaluation of two glucuronides resistant to enzymatic hydrolysis as markers of testosterone oral administration

Evaluation of two glucuronides resistant to enzymatic hydrolysis as markers of testosterone oral administration

Evaluation of markers out of the steroid profile for the screening of testosterone misuse. Part II: Intramuscular administration

Urine Fingerprints of Stanozolol Treated Horses by Liquid Chromatography High Resolution Mass Spectrometry

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