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Complementary information concerning the suspected interindividual transmission of GW1516, a substance prohibited in sport, through intimate contact: a case report

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Purpose Inadvertent and/or unknowing exposure to drugs and drug residues has been frequently debated in situations of so-called adverse analytical finding (AAF) in the context of sports drug testing programs. Transfer of drug residues via unprotected intercourse is a conceivable scenario but scientific data and authentic case reports are scarce. Herein, investigations into two AAFs with the peroxisome proliferator-activated receptor delta (PPARδ) agonist GW1516 are reported and discussed. Methods To probe for a contamination scenario involving sexual intercourse, two assays were used to determine semenogelin in human urine, with one employing an immunochromatographic lateral flow approach and another based on liquid chromatography–tandem mass spectrometry. Further, drug-residue testing using patients’ ejaculate was conducted by utilizing liquid chromatography in conjunction with a triple quadrupole mass spectrometer, followed by re-analysis of suspect samples (i.e., samples indicating the presence of relevant compounds) using high resolution/high mass accuracy mass spectrometry. Results In one case, but not the other, the possibility of intimate contact as the source of the AAF was confirmed after a thorough investigation of potential contamination scenarios. Subsequent research revealed analytical evidence for the presence of seminal fluid in one of the female athlete’s doping control urine samples, and the analysis of clinical ejaculate specimens provided first data on an authentic concentration level of GW1516 and its metabolites in human seminal fluid. Conclusions The combined facts substantiate the possibility of an AAF caused by unprotected sexual intercourse and the plausibility of the case-related arguments.
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Forensic Toxicology
https://doi.org/10.1007/s11419-024-00689-x
CASE REPORT
Complementary information concerningthesuspected interindividual
transmission ofGW1516, asubstance prohibited insport,
throughintimate contact: acase report
J.Breuer1 · A.M.Garzinsky1· A.Thomas1· E.Nieschlag2· S.Kliesch2· M.Fedoruk3· H.Geyer1,4· M.Thevis1,4
Received: 14 February 2024 / Accepted: 14 April 2024
© The Author(s) 2024
Abstract
Purpose Inadvertent and/or unknowing exposure to drugs and drug residues has been frequently debated insituations of
so-called adverse analytical finding (AAF) in the context of sports drug testing programs. Transfer of drug residues via unpro-
tected intercourse is a conceivable scenario but scientific data and authentic case reports are scarce. Herein, investigations into
two AAFs with the peroxisome proliferator-activated receptor delta (PPARδ) agonist GW1516 are reported and discussed.
Methods To probe for a contamination scenario involving sexual intercourse, two assays were used to determine semenogelin
in human urine, with one employing an immunochromatographic lateral flow approach and another based on liquid chro-
matography–tandem mass spectrometry. Further, drug-residue testing using patients’ ejaculate was conducted by utilizing
liquid chromatography in conjunction with a triple quadrupole mass spectrometer, followed by re-analysis of suspect samples
(i.e., samples indicating the presence of relevant compounds) using high resolution/high mass accuracy mass spectrometry.
Results In one case, but not the other, the possibility of intimate contact as the source of the AAF was confirmed after a
thorough investigation of potential contamination scenarios. Subsequent research revealed analytical evidence for the presence
of seminal fluid in one of the female athlete’s doping control urine samples, and the analysis of clinical ejaculate specimens
provided first data on an authentic concentration level of GW1516 and its metabolites in human seminal fluid.
Conclusions The combined facts substantiate the possibility of an AAF caused by unprotected sexual intercourse and the
plausibility of the case-related arguments.
Keywords Seminal fluid· Exposome· Sports· Doping· GW1516
Introduction
A central topic of anti-doping research has been the growing
necessity of distinguishing scenarios of intentional doping
from situations commonly referred to as contamination or
unknowing exposure. A major challenge in that context is
the complex environmental influence, which can affect an
athlete’s career considerably more serious than the general
population due to their participation in sports drug testing
programs. Prohibited substances can be transferred in trace
amounts to athletes, which can then lead to an adverse ana-
lytical finding (AAF) [1]. The detection of minute concen-
trations of doping agents has been of utmost importance to
doping controls, and the evolution of analytical techniques
has offered increasing sensitivities that allow for the required
analytical retrospectivity in sports drug testing. However,
distinguishing between intentional drug use (i.e., doping)
committed some time ago and trace contamination has
become more and more difficult. One frequently discussed
scenario of drug exposure in the context of AAFs is the
transmission of substances through intimate contact such as
sexual intercourse, which was accepted as early as 2004 as
a possible contamination scenario [2].
Non-threshold doping substances (prohibited in- and out-
of-competition) classified in the World Anti-Doping Agency
* M. Thevis
thevis@dshs-koeln.de
1 Institute ofBiochemistry, Center forPreventive Doping
Research, German Sport University Cologne, Am Sportpark
Müngersdorf 6, 50933Cologne, Germany
2 University Hospital Muenster (UKM), Muenster, Germany
3 Science andResearch, USADA, ColoradoSprings, USA
4 European Monitoring Center forEmerging Doping Agents
(EuMoCEDA), Cologne, Bonn, Germany
Forensic Toxicology
(WADA) Prohibited List without a so-called minimum
reporting limit (MRL) are of particular concern as their
detection and confirmation results in an AAF regardless of
their concentration [3].
One example is GW1516 (also known as GW501516
or Cardarine), a peroxisome proliferator-activated recep-
tor delta (PPARδ) agonist [4, 5]. The PPARδ in skeletal
muscle cells has distinct roles in the regulation of lipid,
carbohydrate, and energy homeostasis. Studies have shown
that the AMPK-PPARδ pathway can be influenced by orally
administered drugs such as GW1516 to improve exercise
adaptation or even increase athletic endurance performance
without training [6]. GW1516 was discussed as an emerging
doping agent in 2008 and first explicitly mentioned in the
WADA Prohibited List in 2009 as a hormone and metabolic
modulator [7]. Clinical trials with GW1516 were discontin-
ued when animal studies demonstrated carcinogenic effects
[8].
However, even though GW1516 is not commercially
available as an approved drug, 139 AAFs involving GW1516
have been reported to WADA since 2009. The number of
cases varies widely, from a minimum of 1 (2012) to a maxi-
mum of 31 (2016) cases per year. Overall, GW1516 AAFs
account for 1–12% of cases in substance class S4 [9].
There are several reports describing the possibility of
doping control samples containing prohibited substances
because of intimate contact [1013], but little (if any) infor-
mation has been presented on analytical options in support
of follow-up investigations and result management. In the
herein presented two cases, the combined information from
routine doping controls, follow-up analyses, and new data
on drug levels in seminal fluid provided a dataset plausi-
bly and convincingly supporting the claimed scenario of
contamination.
Case reports
A urine sample from a female athlete that returned low
but detectable amounts of the metabolites of GW1516
(GW1516 sulfoxide estimated at 4pg/mL and GW1516
sulfone estimated at 5pg/mL) and LGD-4033 metabolite
was reported as an AAF. The doping control sample was
taken out-of-competition, and the athlete claimed to have
had unprotected sexual intercourse on the day of sample
collection. Based on a variety of factors, the athlete did
not face any period of ineligibility. A urine sample from
a second female athlete that returned low but detectable
amounts of the metabolites of letrozole (bis(4-cyanophe-
nyl)methanol) and GW1516 sulfoxide estimated at 14pg/
mL and GW1516 sulfone estimated at 5pg/mL. The dop-
ing control sample was taken out-of-competition, and the
athlete claimed to have had unprotected sexual intercourse
on the day of sample collection. Investigations revealed
that her partner had taken oral solutions of letrozole and
GW1516 on a daily basis for a period of 2 to 3weeks.
Based on credible testimony, the athlete did not face any
period of ineligibility.
Material andmethods
Chemicals andreagents
Acetonitrile (ACN), methanol and formic acid were obtained
from VWR chemicals (Langenfeld, Germany). Ammonium
acetate and acetic acid were obtained from Merck (Darm-
stadt, Germany). Ultrapure water was received from a Barn-
stead GenPure xCAD Plus from Thermo Scientific (Bremen,
Germany). The Rapid Stain Identification (RSID™)-semen
field test was obtained from Independent Forensics (Lom-
bard, IL). The seminal fluid samples were collected as part
of routine andrological examinations at Muenster University
Hospital (Germany) and consent was obtained for further
research purposes.
Probing forthepresence ofsemenogelin bylateral
flow immunochromatographic analysis
To test for the presence of semenogelin, a lateral flow immu-
nochromatographic analysis was performed as an initial-
testing procedure as described by Breuer etal., 2021 [13].
In brief, 20µL of urine was diluted with 80µL RSID™
Universal Buffer and transferred on the lateral flow immune
strip. Positive and negative controls were prepared and ana-
lyzed as recommended. Ten minutes after application, the
result (presence or absence of semen) was photographically
documented.
Probing forthepresence ofsemenogelin I byliquid
chromatography–tandem mass spectrometry (LC–
MS/MS)
The LC–MS/MS analysis was performed as described by
Breuer etal., 2021 [13]. Briefly, after preparation of the
urine samples by solid-phase extraction, tryptic diges-
tion was performed. The analysis for the presence of three
specific peptides of semenogelin I was conducted using a
Vanquish™ UHPLC system coupled to a Thermo Scien-
tific Orbitrap Exploris™ 480 mass spectrometer (Thermo
Scientific, Bremen, Germany). Characteristics of peptides
of semenogelin and the internal standard (ISTD) used for
the LC–MS/MS assay are listed in Table1.
Forensic Toxicology
Analysis ofGW1516 inseminal fluid: sample
preparation andextraction procedure
To 100µL of seminal fluid, 5μL of an ISTD working solu-
tion was added and mixed with 100µL acetic acid(1%). To
precipitate proteins, 800µL of ice-cold ACN was added,
and the sample was vortexed for 20s and cooled for 20min
at 4°C. Following centrifugation (15min, 14,000 × g), the
pellet was discarded and the supernatant was evaporated
to dryness in a vacuum centrifuge. The dried sample was
reconstituted in 100μL of H2O/ACN (80/20, v/v).
Analysis ofGW1516 inseminal fluid: LC–MS/
MS instrumentation andanalytical conditions:
initial‑testing procedure
LC–MS/MS analysis of seminal fluid was conducted using
an Aquity I-Class ultra-performance liquid chromato-
graph (UPLC) coupled to a Xevo Triple Quadrupole-XS
mass spectrometer (TQ-MS), both from Waters (Esch-
born, Germany). Seminal fluid samples prepared for analy-
sis were injected onto a Poroshell C-8 analytical column
(50 × 3.0mm, 2.7 μm particle size; Agilent, Waldbronn,
Germany) employing the eluents A (0.1% formic acid in
water) and B (0.1% formic acid in ACN) at a flow rate of
0.4 mL/min. The method started at 5% B, increasing to 15%
B from 1 to 2min, followed by an increase to 55% B from
2 to 11min. B was then increased to 100% from 11 to 12
min, followed by equilibration at 1% B from 12 to 15 min.
Before the next injection, the needle was washed for 1 min
while B was increased from 1% to 5%. The compounds were
introduced into the mass spectrometer by electrospray ioni-
zation and were detected by time-based multiple-reaction
monitoring experiments. The data were processed using
TargetLynx™ (Waters, Eschborn, Germany) after analysis.
Analysis ofGW1516 inseminal fluid: Liquid
chromatography–high resolution mass
spectrometry (LC–HRMS) instrumentation
andanalytical conditions: confirmation procedure
A LC–HRMS method was developed for the analysis of
GW1516 and its metabolites. The LC–HRMS system used
was a Vanquish HPLC system coupled to a Thermo Scien-
tific Orbitrap Exploris™ 480, both from Thermo Scientific
(Bremen, Germany). For chromatography, a Poroshell 120
EC-C18 analytical column (50 × 3.0mm, 2.7μm particle
size; Agilent, Waldbronn, Germany) was used, connected to
an EC 4/3 Nucleoshell RP 18 Plus guard column (4 × 3mm,
5μm particle size) from Macherey–Nagel (Düren, Ger-
many). The gradient with a run time of 15min and a flow
rate of 0.4mL/min was performed using water containing
0.1% acetic acid as solvent A and ACN containing 0.1%
acetic acid as solvent B. The method was started with 0%
B, increasing to 90% B in 11min. Subsequently, 100% B
was held for 1min followed by re-equilibration at 0% B for
3min. The injection volume was 5µL. Measurements were
conducted in positive ionization mode with an ionization
voltage of 3kV and a transfer tube temperature of 320°C.
A resolution of 60,000full width at half maximum (FWHM)
was selected for the full-scan and 45,000 FWHM for HRMS/
MS experiments, and the full-scan was performed in a range
of m/z 100–800. The normalized collision energy (NCE) and
precursor ions are listed in Table2. For product ion scan
experiments, the isolation window of the quadrupole was
set to 1m/z. Nitrogen was generated by the CMC nitrogen
generator (Eschborn, Germany) and used as collision gas.
Table 1 Characteristics of peptides of semenogelin I and the internal standard (hemoglobin) used in the LC–MS/MS assay [13]
Analyte Peptide Amino-acid sequence Sum formula Precursor ion (m/z) Qualifier ion (m/z) Qualifier ion (m/z) NCE (%)
Semenogelin I (human) T21 GTQNPSQDQGNSPSGK C62H10N22O28 801.36 1201.54 388.22 25
T46 QITIPSQEQEHSQK C69H113N21O26 826.92 1197.55 214.16 30
T57 EQDLLSHEQK C51H83N15O20 613.8 628.31 741.39 25
Hemoglobin (bovine) T6 FFESFGDLSTA-
1DAVMNNPK
C93H136N22O31S 1045.48 1147.55 1432.7 30
Table 2 Characteristics of GW1516, GW1516-Sulfoxide, GW1516-Sulfone and the internal standard used in the LC–HRMS-confirmation assay
RT retention time
Analyte Sum formula Precursorion (m/z) Production (m/z) Production (m/z) NCE (%) RT (min)
GW1516 C21H19O3NF3S2454.0765 257.0477 188.0525 50 10.23
GW1516-Sulfoxide C21H19O4NF3S2470.0709 257.0477 188.0525 50 8.01
GW1516-Sulfone C21H19O5NF3S2486.0658 257.0477 188.0525 50 8.70
Stanozolol-d3 (ISTD) C21H30N2OD3332.2775 81.0447 65 6.87
Forensic Toxicology
The Thermo Scientific Orbitrap Exploris™ 480 was cali-
brated regularly with the manufacturer’s calibration solution.
Results
After the publication of a method for the detection of seme-
nogelin [13], a protein specific for seminal fluid and thus a
marker for unprotected sexual intercourse, an aliquot of the
urine sample from the cases described above was requested
for retrospective analysis. The initial-testing procedure
by a laminar flow immunologic test strip method yielded
a positive result of semenogelin for one sample (GW1516
metabolites/LGD-4033 metabolite), but not the other sam-
ple (GW1516 metabolites/letrozole metabolite) (data not
shown). The presence of semenogelin in the urine sample
was confirmed by the detection of three specific peptides
in MS/MS experiments (see Table1 and Fig. 1). Both
analytical results thus showed that seminal fluid was present
in the urine sample and, therefore, confirmed the possibil-
ity that GW1516-containing seminal fluid was a potential
contaminant of the athlete’s doping control urine sample. In
addition, the results confirm the athlete’s statement that she
had intimate contact within hours prior to the sample col-
lection. However, the absence of semenogelin in the sample
of case 2, despite the clear evidence of intimate contact on
the day of sample collection suggest an important limitation
of relying solely on a semen-exposure marker as definitive
evidence of intimate contact, as due to female anatomy, it is
reasonable to not always expect semen-contamination of a
female urine doping control sample post-coitus.
To probe for the potential and extent of a transfer of drugs
into seminal fluid, a total of 361 seminal fluid samples was
obtained from the University Hospital in Münster (Ger-
many). The specimens were analyzed to determine the con-
centration of therapeutics and illicit substances classified as
Fig. 1 Extracted-ion chromatograms of a blank urine (a), a urine
specimen containing 100nL of semen (b), the authentic doping con-
trol urine sample containing GW1516 and LGD-4033 metabolites (c)
and the authentic doping control urine sample containing GW1516
and letrozole metabolites (d)
Forensic Toxicology
doping agents in the general population (publication pend-
ing). Therefore, a screening method was developed using a
TQ-MS to test the samples for 56 different target analytes,
and samples suspected to contain one or more doping agents
were further subjected to an LC–HRMS/MS confirmation
procedure to probe for the presence and quantity of the tar-
get analyte and potential (example for GW1516 shown in
Table2).
One of these investigated seminal fluid samples was
found to contain GW1516. Using the confirmatory method,
the concentration of GW1516 was determined to be approxi-
mately 48ng/mL. In addition, the metabolites GW1516
sulfoxide and GW1516sulfone (Fig.2) were detected.
Although the administered amount of GW1516 is unknown,
these results show that high concentrations of GW1516 can
occur in seminal fluid and could lead to an AAF, even when
considering the dilution factor in urine.
Discussion
These case reports provide a dataset that plausibly supports
the scenario of seminal fluid as a possible source of contami-
nation leading to an AAF. The estimated concentration of
GW1516 sulfoxide and sulfone were 4pg/mL and 5pg/mL,
respectively; the athlete’s doping control urine sample was
proven to contain semenogelin I (a seminal fluid-specific
protein), and GW1516 (and its metabolites) was shown to
transfer into seminal fluid as demonstrated by means of a
clinical sample.
Yet, several aspects of this case remain unknown (e.g., the
presumed concentration of GW1516 in the athlete’s partner’s
ejaculate, the volume of ejaculate potentially contained in
the doping control urine sample) and, hence, the AAF can-
not exclusively be attributed to a contamination scenario
[13]. Hence, additional aspects assisting in assessing the
Fig. 2 Extracted-ion chromatograms of the authentic seminal fluid sample containing GW1516 and GW1516 metabolites (left) and a blank sem-
inal fluid sample (right)
Forensic Toxicology
likelihood of the scenario should be factored-in, such as
the average volume of 3.7mL of seminal fluid [14] trans-
ferred during sexual intercourse and the minimum volume of
90mL of urine collected in a routine doping control setting
according to WADA regulations.
When applying the GW1516 concentration observed in
the clinical sample to the athlete’s doping control scenario,
a 1/100 fraction (i.e., 37 µL) of seminal fluid transmitted
during sexual intercourse and introduced into the doping
control urine sample would theoretically suffice to produce
the reported AAF.
The interindividual transfer of drugs has also been
described in other situations. For instance, in a recent case
report, the absorption of drugs into the cervical mucosa via
seminal fluid was described [15]. There, severe symptoms
corresponding to acute opioid withdrawal occurred after
sexual intercourse. This adds another layer of complexity,
namely that not only are substances transmitted during sex-
ual intercourse and can lead to possible contamination by
introduction of (drug-containing) seminal fluid into the dop-
ing control urine specimen, but they can also be absorbed,
metabolized, and eliminated.
Conclusions
The herein presented data support the assumption that dop-
ing control urine samples can be contaminated with doping
agents caused by sexual intercourse. The detection of seme-
nogelin in a doping control sample that returned an AAF
for GW1516 confirmed the intimate contact claimed by the
athlete. Semenogelin presence can be used as strong sup-
porting evidence for prohibited-substance exposure via the
introduction of male ejaculate contaminating a female ath-
lete’s doping control urine sample, however there are some
important limitations, therefore the presence (or absence)
cannot be used as unequivocal evidence of prohibited-sub-
stance exposure via sexual transmission. It has also been
shown that doping agents such as GW1516 can be present
in seminal fluid in concentrations sufficient to cause an AAF
when introduced into a female athlete’s urine at volumes of
37 µL, and that commonly observed GW1516 metabolites
are also present in seminal fluid when GW1516 is used. Fur-
ther studies on the concentrations and metabolite profiles
of prohibited substances, which also have a relevance for
therapeutic use in the general population, are important to be
able to provide the data required for assessing probabilities
of debated scenarios.
Complementary matrices have also been suggested to
contribute information for case management, e.g., hair test-
ing of the athlete and/or the partner (allegedly) using the
prohibited substance [11]. However, also here analytical as
well as legal issues might apply, as the person who is not
in the doping control system and asked to provide the hair
sample might refuse to do so or no hair segments covering
the relevant time period are available.
Acknowledgements The authors wish to acknowledge support from
the Manfred-Donike Society for Doping Analysis (Cologne, Germany)
and the Federal Ministry of the Interior, Building and Community (Ber-
lin, Germany). Open access funding enabled and organized by Projekt
DEAL.
Authors´ contributions J.Breuer conducted sample preparation and
analysis work, performed results interpretation, and wrote the origi-
nal draft. AM. Garzinsky conducted research processes and reviewed
the manuscript. S.Kliesch and E. Nieschlag provided study materials.
M.Fedoruk provided information on the study materials, reviewed
and edited the final manuscript. J.Breuer, A.Thomas, H.Geyer, and
M.Thevis planned and designed the project, and reviewed and edited
the final manuscript.
Funding Open Access funding enabled and organized by Projekt
DEAL. The study was funded by the Federal Ministry of the Interior,
Building and Community of the Federal Republic of Germany (Ber-
lin, Germany) and the Manfred-Donike Institute for Doping Analysis
(Cologne, Germany).
Data availability The datasets generated and analyzed as part of this
study are available upon request from the corresponding author.
Declarations
Conflicts of interest The authors have no conflict of interest to declare.
Ethics approval The seminal fluid samples were collected as part of
routine andrological examinations at Muenster University Hospital
(Germany) and consent was obtained for further research purposes.
No animal experiments were conducted within this study.
Open Access This article is licensed under a Creative Commons Attri-
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need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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Publisher's Note Springer Nature remains neutral with regard to
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... The peroxisome proliferator-activated receptor delta (PPARδ) agonist GW1516 was detected in one of the seminal fluid samples, which was already described in detail in an earlier case report (Breuer et al., 2024). Using the confirmatory method, the concentration of GW1516 was determined to be approximately 48 ng/mL. ...
... The analysis of clinical ejaculate samples presented here provided preliminary data on an authentic concentration of GW1516 and its metabolites in human seminal fluid. The combined facts support the possibility of an AAF caused by unprotected sexual intercourse and the plausibility of the case-related arguments, as detailed in a case report (Breuer et al., 2024). ...
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Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called Adverse Analytical Finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offences and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from non-athletes were analysed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e. samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the non-approved PPARδ agonist GW1516) in a total of 81 samples, with 91 % of these suspected cases being verified by the confirmation method. Besides the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. Significance Statement This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing.
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The 17th edition of the annual banned‐substance review on analytical approaches in human sports drug testing is dedicated to literature published between October 2023 and September 2024. As in previous years, focus is put particularly on new or enhanced analytical options in human doping controls as well as investigations into the metabolism and elimination of compounds of interest, which represent central (while not exclusive) cornerstones of the global anti‐doping mission. New information published within the past 12 months on established doping agents as well as new potentially relevant substances are reviewed and discussed in the context of the World Anti‐Doping Agency's 2024 Prohibited List. Thereby, analytical challenges, especially with regard to the continuously growing number of target compounds and potentially relevant drug classes as well as the exigency (and consequences) of utmost analytical retrospectivity, are thematized and contextualized. Investigations especially into anabolic agents, peptide hormones, and strategies for the detection of gene doping were identified as core areas of anti‐doping research in the reviewed period.
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Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes’ urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033—substances listed under anabolic substances (S1) on the World Anti-Doping Agency’s Prohibited List—in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02–0.40 ng/mL) and in sf (0.01–0.25 ng/mL) as well as of LGD-4033 in bp (0.21–2.00 ng/mL) and in sf (0.03–0.68 ng/mL) post-administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine. Graphical Abstract
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The high sensitivity of antidoping detection tests creates the possibility of inadvertent doping due to an athlete’s unknowing ingestion of contaminated environmental sources such as dietary supplements, food, or drinks. Recently athletes denying use of a prohibited substance have claimed that the positive antidoping tests was due to exchange of bodily fluids with a non‐athlete partner using a prohibited substance. Measurement of drugs in semen is largely limited to one or very few samples due to the inaccessibility of sufficiently frequent semen samples for detailed pharmacokinetics. An emerging issue in semen drug measurements is that semen samples may contain residual urine from ejaculation left in the urethra; however, the urine content in semen samples has not been studied. In the present study we employed concurrent creatinine measurements in urine and seminal plasma to determine the urine content of semen samples.
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Rationale An increasing number of adverse analytical findings (AAFs) in routine doping controls has been suspected and debated to presumably result from intimate contact with bodily fluids (including ejaculate), potentially facilitating the transfer of prohibited substances. More precisely, the possibility of prohibited drugs being present in ejaculate and introduced by sexual intercourse into the vagina of an athlete and, subsequently, into doping control urine samples, was discussed. Methods Two testing strategies to determine trace amounts of semenogelin I, a major and specific constituent of semen, were assessed as to their applicability to urine samples. First, the testing protocol of a lateral flow immunochromatographic test directed against semenogelin was adapted. Second, a liquid chromatography/tandem mass spectrometry (LC‐MS/MS)‐based method was established, employing solid‐phase extraction of urine, trypsinization of the retained protein content, and subsequent detection of semenogelin I‐specific peptides. Sensitivity, specificity, and reproducibility, but also recovery, linearity, precision, and identification capability of the approaches were assessed. Both assays were used to determine the analyte stability in urine (at 3 µL/mL) at room temperature, +4°C, and ‐20°C, and authentic urine samples collected either after (self‐reported) celibacy or sexual intercourse were subjected to the established assays for proof‐of‐concept. Results No signals for semenogelin were observed in either assay when analyzing blank urine specimens, demonstrating the methods’ specificity. Limits of detection were estimated with 1 µL and 10 nL of ejaculate per mL of urine for the immunochromatographic and the mass spectrometric approach, respectively, and figures of merit for the latter assay further included intra‐ and interday imprecision (4.5‐10.7% and 3.8‐21.6%), recovery (44%), and linearity within the working range of 0‐100 nL/mL. Spiked urine tested positive for semenogelin under all storage conditions up to 12 weeks, and specimens collected after sexual intercourse were found to contain trace amounts of semenogelin up to 55‐72 h.
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Semen quality is taken as a surrogate measure of male fecundity in clinical andrology, male fertility, reproductive toxicology, epidemiology and pregnancy risk assessments. Reference intervals for values of semen parameters from a fertile population could provide data from which prognosis of fertility or diagnosis of infertility can be extrapolated. Semen samples from over 4500 men in 14 countries on four continents were obtained from retrospective and prospective analyses on fertile men, men of unknown fertility status and men selected as normozoospermic. Men whose partners had a time-to-pregnancy (TTP) of < or =12 months were chosen as individuals to provide reference distributions for semen parameters. Distributions were also generated for a population assumed to represent the general population. The following one-sided lower reference limits, the fifth centiles (with 95th percent confidence intervals), were generated from men whose partners had TTP < or = 12 months: semen volume, 1.5 ml (1.4-1.7); total sperm number, 39 million per ejaculate (33-46); sperm concentration, 15 million per ml (12-16); vitality, 58% live (55-63); progressive motility, 32% (31-34); total (progressive + non-progressive) motility, 40% (38-42); morphologically normal forms, 4.0% (3.0-4.0). Semen quality of the reference population was superior to that of the men from the general population and normozoospermic men. The data represent sound reference distributions of semen characteristics of fertile men in a number of countries. They provide an appropriate tool in conjunction with clinical data to evaluate a patient's semen quality and prospects for fertility.
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The benefits of endurance exercise on general health make it desirable to identify orally active agents that would mimic or potentiate the effects of exercise to treat metabolic diseases. Although certain natural compounds, such as reseveratrol, have endurance-enhancing activities, their exact metabolic targets remain elusive. We therefore tested the effect of pathway-specific drugs on endurance capacities of mice in a treadmill running test. We found that PPARbeta/delta agonist and exercise training synergistically increase oxidative myofibers and running endurance in adult mice. Because training activates AMPK and PGC1alpha, we then tested whether the orally active AMPK agonist AICAR might be sufficient to overcome the exercise requirement. Unexpectedly, even in sedentary mice, 4 weeks of AICAR treatment alone induced metabolic genes and enhanced running endurance by 44%. These results demonstrate that AMPK-PPARdelta pathway can be targeted by orally active drugs to enhance training adaptation or even to increase endurance without exercise.
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Buprenorphine, a partial mu-opioid receptor agonist, is a commonly prescribed medication for opioid use disorder (OUD). There is evidence that drugs may enter the male genitourinary tract by an ion-trapping process, based on the lipid solubility and degree of ionization (1). While little is known about the pharmacokinetics of drugs in seminal fluid, pH is thought to play an integral role. Limited evidence exists surrounding cervical absorption of drugs via seminal fluid transmission. This also prompts survey of the frequency of this event and the influence on treatment within this population.
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The presence of a prohibited substance or its metabolites or its markers in an athlete's sample constitutes the more frequent anti-doping rules violation. In the world anti-doping code, it is indicated (point 10.5) that if someone establishes in an individual case that the athlete bears no fault or negligence, then the otherwise applicable period of ineligibility shall be eliminated. The conditions that have to be met to fix the no fault or negligence evidence are described in several other points of the code. The following two points are of paramount importance: 1. the athlete or his/her legal representative must present verified circumstances of contamination and the source of contamination must be identified; and 2. there must be verified claims by the athlete about the fact that he/she did not knowingly take the prohibited substance, i.e., that the violation was not intentional.In recent years, several cases of contamination involving drug transfer during intimate moments have been reported. This later situation was first reported in 2009 with the Richard Gasquet case. Since that time, several athletes have been allowed to return to competition with no charge based on strong evidence that the source of contamination was drug transfer during intimate moments. As some of these cases are public and because the author performed hair tests for the majority of the international athletes involved in such procedures, the strategy of the defence and the scientific bases of discussion are reviewed in this article.
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Similar to the general population, elite athletes are exposed to a complex set of environmental factors including chemicals, radiation, but also biological and physical stressors, which constitute an exposome that is, unlike for the general population, subjected to specific scrutiny for athletes due to applicable anti‐doping regulations and associated (frequent) routine doping controls. Hence, investigations into the athlete’s exposome and how to distinguish between deliberate drug use and different contamination scenarios has become a central topic of anti‐doping research, as a delicate balance is to be managed between the vital and continually evolving developments of sensitive analytical techniques on the one hand, and the risk of the athletes’ exposome potentially causing adverse analytical findings on the other.
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The number of compounds and doping methods in sports is in a state of constant flux. In addition to 'traditional' doping agents, such as anabolic androgenic steroids or erythropoietin, new therapeutics and emerging drugs have considerable potential for misuse in elite sport. Such compounds are commonly based on new chemical structures, and the mechanisms underlying their modes of action represent new therapeutic approaches arising from recent advances in medical research; therefore, sports drug testing procedures need to be continuously modified and complementary methods developed, preferably based on mass spectrometry, to enable comprehensive doping controls. This tutorial not only discusses emerging drugs that can be categorized as anabolic agents (selective androgen receptor modulators, SARMs), gene doping [hypoxia-inducible factor stabilizers, peroxisome-proliferator-activated receptor (PPAR)delta-agonists] and erythropoietin-mimetics (Hematide) but also compounds with potentially performance-enhancing properties that are not classified in the current list of the World Anti-Doping Agency. Compounds such as ryanodine-calstabin-complex modulators (benzothiazepines) are included, their mass spectrometric properties discussed, and current approaches in sports drug testing outlined.