Use of isotope ratio mass spectrometry to detect doping with oral testosterone undecanoate: inter-individual variability of 13C/12C ratio.
ABSTRACT The metabolic effect of multiple oral testosterone undecanoate (TU) doses over 4 weeks was assessed in seven voluntary men. The protocol was designed to detect accumulation of the substance by choosing the appropriate spot urines collections time and to study the urinary clearance of the substance after weeks of treatment. Urines were analysed by a new GC/C/isotope ratio mass spectrometry (IRMS) method to establish the delta(13)C-values of testosterone metabolites (androsterone and etiocholanolone) together with an endogenous reference compound (16(5alpha)-androsten-3alpha-ol). The significant differences in inter-individual metabolism following TU intake was illustrated by large variations in delta(13)C-values of both T metabolites (maximum Deltadelta(13)C-values = 5.5 per thousand), as well as by very stable longitudinal T/E profiles and carbon isotopic ratios in the first hours following administration. According to T/E ratios and delta(13)C-values, the washout period after 80 mg TU intake was less than 48 h for all subjects and no accumulation phenomenon was observed upon chronic oral administration.
Article: Testosterone and doping control.[show abstract] [hide abstract]
ABSTRACT: Anabolic steroids are synthetic derivatives of testosterone, modified to enhance its anabolic actions (promotion of protein synthesis and muscle growth). They have numerous side effects, and are on the International Olympic Committee's list of banned substances. Gas chromatography-mass spectrometry allows identification and characterisation of steroids and their metabolites in the urine but may not distinguish between pharmaceutical and natural testosterone. Indirect methods to detect doping include determination of the testosterone/epitestosterone glucuronide ratio with suitable cut-off values. Direct evidence may be obtained with a method based on the determination of the carbon isotope ratio of the urinary steroids. This paper aims to give an overview of the use of anabolic-androgenic steroids in sport and methods used in anti-doping laboratories for their detection in urine, with special emphasis on doping with testosterone. Review of the recent literature of anabolic steroid testing, athletic use, and adverse effects of anabolic-androgenic steroids. Procedures used for detection of doping with endogenous steroids are outlined. The World Anti-Doping Agency provided a guide in August 2004 to ensure that laboratories can report, in a uniform way, the presence of abnormal profiles of urinary steroids resulting from the administration of testosterone or its precursors, androstenediol, androstenedione, dehydroepiandrosterone or a testosterone metabolite, dihydrotestosterone, or a masking agent, epitestosterone. Technology developed for detection of testosterone in urine samples appears suitable when the substance has been administered intramuscularly. Oral administration leads to rapid pharmacokinetics, so urine samples need to be collected in the initial hours after intake. Thus there is a need to find specific biomarkers in urine or plasma to enable detection of long term oral administration of testosterone.British journal of sports medicine 08/2006; 40 Suppl 1:i21-4. · 2.55 Impact Factor
Article: Detection of testosterone administration based on the carbon isotope ratio profiling of endogenous steroids: international reference populations of professional soccer players.[show abstract] [hide abstract]
ABSTRACT: The determination of the carbon isotope ratio in androgen metabolites has been previously shown to be a reliable, direct method to detect testosterone misuse in the context of antidoping testing. Here, the variability in the 13C/12C ratios in urinary steroids in a widely heterogeneous cohort of professional soccer players residing in different countries (Argentina, Italy, Japan, South Africa, Switzerland and Uganda) is examined. Carbon isotope ratios of selected androgens in urine specimens were determined using gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS). Urinary steroids in Italian and Swiss populations were found to be enriched in 13C relative to other groups, reflecting higher consumption of C3 plants in these two countries. Importantly, detection criteria based on the difference in the carbon isotope ratio of androsterone and pregnanediol for each population were found to be well below the established threshold value for positive cases. The results obtained with the tested diet groups highlight the importance of adapting the criteria if one wishes to increase the sensitivity of exogenous testosterone detection. In addition, confirmatory tests might be rendered more efficient by combining isotope ratio mass spectrometry with refined interpretation criteria for positivity and subject-based profiling of steroids.British journal of sports medicine 07/2009; 43(13):1041-4. · 2.55 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Anti-doping authorities have high expectations of the athlete steroidal passport (ASP) for anabolic-androgenic steroids misuse detection. However, it is still limited to the monitoring of known well-established compounds and might greatly benefit from the discovery of new relevant biomarkers candidates. In this context, steroidomics opens the way to the untargeted simultaneous evaluation of a high number of compounds. Analytical platforms associating the performance of ultra-high pressure liquid chromatography (UHPLC) and the high mass-resolving power of quadrupole time-of-flight (QTOF) mass spectrometers are particularly adapted for such purpose. An untargeted steroidomic approach was proposed to analyse urine samples from a clinical trial for the discovery of relevant biomarkers of testosterone undecanoate oral intake. Automatic peak detection was performed and a filter of reference steroid metabolites mass-to-charge ratio (m/z) values was applied to the raw data to ensure the selection of a subset of steroid-related features. Chemometric tools were applied for the filtering and the analysis of UHPLC-QTOF-MS(E) data. Time kinetics could be assessed with N-way projections to latent structures discriminant analysis (N-PLS-DA) and a detection window was confirmed. Orthogonal projections to latent structures discriminant analysis (O-PLS-DA) classification models were evaluated in a second step to assess the predictive power of both known metabolites and unknown compounds. A shared and unique structure plot (SUS-plot) analysis was performed to select the most promising unknown candidates and receiver operating characteristic (ROC) curves were computed to assess specificity criteria applied in routine doping control. This approach underlined the pertinence to monitor both glucuronide and sulphate steroid conjugates and include them in the athletes passport, while promising biomarkers were also highlighted.Forensic science international 08/2011; 213(1-3):85-94. · 2.10 Impact Factor
V – CONCLUSIONS
An interesting feature of this study is displayed by two of the seven subjects for
whom the excretion occurs in the first hours after administration. Thus, it is
conceivable that testing of these individuals in doping control would lead likely
to negative results when using common urinary makers. In order to increase the
sensitivity of TU detection, further investigations on this group of individuals are
needed for identification of specific biomarkers of this class of doping agent.
Use of isotope ratio mass spectrometry to detect doping with ora
Use of isotope ratio mass spectrometry to detect doping with oral testosterone undecanoate:
Inter- -individual variability of individual variability of 13
l testosterone undecanoate:
Norbert Baume1, Christophe Saudan1, Aurélien Desmarchelier1, Emmanuel Strahm1, Pierre-Edouard Sottas1,
Carlo Bagutti2, Michel Cauderay3, Yorck Olaf Schumacher4, Patrice Mangin1and Martial Saugy1
1Laboratoire suisse d’Analyse du Dopage, Institut Universitaire de Médecine Légale, Rue du Bugnon 21, 1005 Lausanne, Switzerland
2Centre Médical Vidy-Med, Rte de Chavannes 11, 1000 Lausanne 23, Switzerland
3Hôpital Riviera, Site du Samaritain, Bd. Paderewski 3, 1800 Vevey, Switzerland
4Abtlg. Sportmedizin, Medizinische Universitätsklinik Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany
III – PROTOCOL OF THE STUDY
The four weeks study consisted in the oral intake of testosterone undecanoate pills (80 mg testosterone undecanoate and 115 mg mannitol) on Monday,
Wednesday and Friday between 6.30 am and 8 am. One sport urine (U1) was collected on the first day of the protocol, prior to any medication. Subsequently,
spot urines were collected on day 24 before intake of the eleventh pill (U2), and after 4 (U3), 8 (U4) and 24 hours (U5). Finally, a spot urine (U6) was obtained 10
days after the last administration (day 39). A maximum standard deviation of 30 min may be considered for the collection time of all spot urines.
Four months after this protocol, the same subjects were self-administered a single dose of testosterone undecanoate (80 mg testosterone undecanoate and 115
mg mannitol) on the morning. All urine samples were collected during 24 hours after intake (excretion study).
IV – RESULTS AND DISCUSSION
1. TIME PROFILE OF T/E RATIO
Time course in hours of urinary T/E ratio (upper panels, u ) and δ13C-values of androsterone (l), etiocholanolone (n) and
androstenol (?) of seven subjects (S1–S7) throughout the protocol study. The two x axis breaks are shown by paired oblique
Urinary T/E ratio of subject
S1 (n), S3 (?), S4 (l) and
mean values (× × × ×) (± SEM) of
S2, S5, S6 and S7 following
a single oral dose of TU
2. TIME PROFILE OF δ δ δ δ13C-VALUES
T/E ratios of the seven Caucasian individuals
have basal values in the range of 0.06 to 5.09. The
values remain very similar for urine sample U2
collected after three weeks of treatment and 48
hours after last TU intake (P > 0.5). In the course
of the study, the T/E ratios show important inter-
individual variability upon ingestion of the
substance. Excepted for S1, the values of all
subjects in samples collected after 4 hours are
significantly higher than their respective basal
values. Eight hours after administration of the
substance (U4), this ratio is still significantly
higher for subjects S2, S4, S6 and S7.
The excretion studies confirm the pharmaco-
individuals. Excepted for the T/E profile of subject
S1 who shows almost no variation, the excretion
peak occurs at 4 hours for the other subjects. The
T/E ratio return to basal values at about 15 hours
after a single dose administration for most of the
individuals. Surprisingly, the pharmacokinetics of
TU in subject S4 is slower with respect to the
other subjects (excretion time of about 24 hours).
This trend was more pronounced during the
multidose medication where the T/E ratio in urine
U5 was significantly higher than the basal values.
These findings put forth an intra-individual
variability in the testosterone metabolism.
each of the seven
VI – REFERENCES
1. WADA technical document: reporting and evaluation guidance for testosterone, epitestosterone, T/E
ratio and other endogenous steroids; 2004. http://www.wada-ama.org
2. Davidson DW, O’Carroll R, Bancroft J. Increasing circulating androgens with oral testosterone
undecanoate in eugonadal men. J Steroids Biochem Mol Biol 1987;26:713-5.
3. Peng SH, Segura J, Farré M, González JC, de la Torre X. Plasma and urinary markers of oral
testosterone undecanoate misuse, Steroids 2002;67:39-50.
4. Saudan C, Baume N, Mangin P, Saugy M. Urinary analysis of 16(5α)-andorsten-3α-ol by gas
chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis.
J Chromatogr B 2004;810:157-64.
I – INTRODUCTION
Detection of doping with endogenous steroids still remains a difficult issue in sport. Monitoring indirect urinary and serum markers such as testosterone
glucuronide/epitestosterone glucuronide (T/E) and testosterone glucuronide/luteinizing hormone ratios or serum testosterone/17a-hydroxyprogesterone ratio
have been used to discriminate exogenous administration of testosterone from endogenous origin. Since 2004, the World Anti-Doping Agency (WADA) imposed
the submission of the sample to isotope ratio mass spectrometry (IRMS) analysis for determination of the13C/12C ratio of selected steroids, if the urinary T/E
ratio value is equal or greater than 4.0 as well as for altered steroid profiles.
Oral application of testosterone undecanoate (TU) has been found to raise serum testosterone levels in healthy and hypogonadal men. Clearance of orally
administered TU is fast, and hence drug testing analysis of the urinary concentration can only be performed in the first hours after administration.
Although long-term effect of intramuscularly administration of T on urinary profile of androgens has been investigated, little is known about the response of
urinary markers used in doping controls after chronic oral TU administrations.
II – AIM OF THE STUDY
The purpose is to examine the urinary profile of 7 subjects who received an oral dose of 80 mg TU three times a week during a period of one month. Carbon
isotopic ratio of androsterone, etiocholanolone and 5α-androst-16-en-3α-ol (androstenol as endogenous reference) together with T/E ratio are employed as
parameters for a discussion about variability of inter-individual androgen metabolism and diagnostic criteria in doping control.
Typical GC-C-IRMS m/z 44 mass chromatograms of
fractions F1 (androsterone and etiocholanolone acetates)
and F2 (androstenol acetate) spiked with an internal
standard (IS, 5α-androstan-3β-ol acetate). The square-
topped peaks represent pulses of CO2reference gas.
Similarly to what found for T/E ratios, the δ13C-
values of the steroids in urine sample U2 are not
significantly different from those determined in U1
(P > 0.5). Accumulation phenomenon caused by
body storage and slow elimination in the urine
was therefore not observed in all subjects.
Conversely, the data tend to show that the
washout period is less than 48 hours for all
The results obtained with some individuals (S2,
S4 and S6) tend
androsterone and etiocholanolone contained in
urines U3 and U4 originates almost completely
from the conversion
contribution of endogenous steroids metabolism.
Global statistical analysis shows significance (P <
0.02) with respect to
androsterone and etiocholanolone in urines U3
As observed on the urinary T/E profiles, an
important inter-individual variability in the
metabolic clearance of TU was put forth with the
δ13C-values of the seven male volunteers.
The stability of the δ13C-values of the subject S3
during the multidose medication study may be
explained by fast metabolization and excretion of
to demonstrate that
of TU, with limited
basal values for
Our study shows that about 60 % of the samples
collected during the first hours after oral intake
of 80 mg TU (7 urines U3 and U4) would have
been reported as indicative of administration of
an endogenous steroid. If complementary specific
analyses would have been conducted, a misuse of
TU for both subjects with stable steroid profiles
could have been revealed.
3. IMPLICATION IN ANTI-DOPING ANALYSES