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Description forthcoming.

Code: R18M01OM

The study was conducted based on the WADA's interest in conducting an excretion study with trimetoquinol in order to determine the possibility of establishing a reporting threshold for this substance in the anti-doping context.

Trimetoquinol (tretoquinol, 1-(3', 4', 5'-trimetoxybenzyl)-6,7-dihydroxy-1,2,3-tetrahydroisoquinoline), non-selective β-agonist, [1] is explicitly listed as a banned substance on the WADA Prohibited List 2019.[2] The corresponding substance is therapeutically used for treating asthma, and the pharmaceutical product Inolin® is available in Japan, Indonesia and Chinese Taipei.[3] The over-the-counter pharmaceuticals are also available (max. dosage 6 mg/day) on Japanese market, and the fact means that it is easy to purchase for Japanese athletes.

In the previous metabolism studies,[4,5] free form, glucoronide conjugate, and sulphate conjugate of trimetoquinol and the O-methylated trimetoquinol were reported (Scheme 1).Trimetoquinol is metabolized by Catechol-O-methyl transferase (COMT), and the β-stimulating effect of the methoxymetabolites is lower than that of trimetoquinol.[6] Moreover, the β-blocking action of 6-methoxytrimetoquinol is a tenth that of propranolo. Similarly, these findings have been reported in other β-stimulants, popssessing a catechol moiety, e.g. 3-methoxyisoproterenol (isoproterenol metabolite) and coclaurine (higenamine metabolite).[6,7] Therefore, the concentration of the O-methylated metabolites and their conjugated metabolites was not considered in this study, such as our previous study in higenamine.[8]

This minimum required performance level (MRPL) of β2-agonist is set to 20 ng/mL.[9] The reporting threshold for higenamine and salmeterol is defined as 10 ng/mL (50% MRPL of each parent compound).[9] Moreover, the WADA threshold concentration is based on the sum of free form and the glucuronide conjugate of parent β2-agonists (i.e., salbutamol and formoterol).[10] However, little information is available concerning the urinary concentration in which trimetoquinol can be identified in human urine after administration.

Based on these backgrounds, in this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of trimetoquinol (free pluc glucuronid conjugate) in human urine was developed. Moreover, trimetoquinol hydrochloride hydrate was orally administered to six Japanese volunteers (3 males and 3 females). Urine samples were analysed using the developed method and the excretion profile was examined.

Main findings

Trimetoquinol (tretoquinol) is a non-selective beta-2 agonist, which has been explicitly listed as an example of prohibited beta-2 agonists under class S3 of the 2019 Prohibited List. Trimetoquinol is used therapeutically for the treatment of asthma (sold as Inolin® in several Asian countries) and is also used as an ingredient of over-the-counter (OTC) cold and flu medications.

Studies on trimetoquinol metabolism have shown that it is excreted in urine either as free form (minor) of phase-II conjugates (glucuronide and sulfate). However, there is no information available about the urinary concentrations after oral administration.

In this study, volunteers were administered 6 mg of oral trimetoquinol hydrochloride hydrate (as per manufacturer's recommendations) to determine the excretion kinetics of trimetoquinol. The results indicate that a conservative reporting limit of 20 ng/m of trimetoquinol (free plus glucuronde conjugate) should avoid the reporting of an Adverse Finding which may have resulted from the inadvertent use of the trimetoquinol-containing OTC medications before the 2019 Prohibited List comes into effect on 1 January 2019.

Code: 18E08CB

Glucocorticoids are prohibited in sport in competition and only when administered by oral, intravenous, intramuscular or rectal routes. The World Anti Doping Agency (WADA) proposed a criteria of 30 ng/mL above which a result should be reported for this class of substances. However such criteria cannot be applied to cortisone and hydrocortisone which are naturally present in urine samples. Regarding these two substances confirmatory method by GC-C-IRMS is applicable for the determination of endogenous or exogenous origin. However, no efficient screening criterion is available and applicable in Antidoping laboratories to trigger GC-C-IRMS analysis. Our project aims to use omics strategies to highlight biomarkers of effect and pattern changes in urine and blood samples able to give evidence of hydrocortisone administration. For this project, a clinical study will be conducted with two routes of administration: one prohibited such as oral route in order to find biomarkers of effects of hydrocortisone abuse and one authorized route such as topic application with a cream in order to confront the potential biomarkers found with prohibited administration. The impact of topical application on carbon isotopic ratio of cortisol and its metabolites will also be explored in a second step.

Main findings

In the present project, two administration studies with hydrocortisone were performed, one with a prohibited route (oral) and one with an authorized route (topic). The effects of such treatments on urinary concentrations and 13C-delta values of a set of endogenous corticosteroids were studied. The clinical trial was conducted with 20 healthy recreational athletes. For the prohibited route, 50 mg hydrocortisone was taken orally by 10 subjects every day for 5 days. For the authorized route study, 10 other subjects used a cream with 1% hydrocortisone at the therapeutic dose of 25 mg twice a day also for 5 days. The overall goal of this study was twofold, to find biomarkers of the hydrocortisone treatment but also to investigate if the two routes tested could be differentiated.

In the first part of this project, blood and saliva samples collected in this study were analyzed using ELISA kits. It was found that therapeutic oral administration of hydrocortisone once daily for 5 days modified adrenal DHEA secretion by inhibiting pituitary ACTH. However, this effect was only transient, with no significant alteration in basal adrenal or pituitary function 24 hours after administration. Given the high correlations between plasma and saliva, noninvasive saliva sampling may therefore be a sensitive alternative to invasive venous blood sampling for estimating pituitary and adrenal function after hydrocortisone treatment.

Urine samples were analyzed by LC-MS/MS to determine the concentration and by GC-C-IRMS to determine carbon isotopic ratios focusing on hydrocortisone and its metabolites: cortisone, tetrahydrocortisol (THF), allo-THF, tetrahydro-11-desoxycortisol (THS), 11-keto-Etiocholanolone, 11β- OH-Androsterone, 11β-OH-Etiocholanolone, β-cortol and 6β-hydrocortisol. As a main result, the topical application of 1% hydrocortisone cream on safe skin (25 mg twice daily for 5 days) had no impact on cortisone, hydrocortisone or metabolite concentrations. On the contrary, as expected, a significant increase in concentrations could be observed after oral treatment for all compounds studied except 11bOH-etiocholanolone. This compound being a minor metabolite of cortisone, a significant impact on this substance was expected. On the basis of the results obtained with oral treatment, we were also able to observe that the detection windows in the context of anti-doping controls were dependent on the metabolite considered and on the subject. A clear impact on concentrations could be observed on all the target compounds during less than 24 hours except for b-cortol which seems to be impacted for a longer period of time (return to basal values in 48 hours). The multivariate statistical analysis carried out on all the data obtained made it possible to highlight new markers of detection of unauthorized hydrocortisone intake. Sixteen markers based on specific target metabolites or ratios of these metabolites were selected to create a statistical model for screening samples from doping controls. The steroid profile was also measured in all samples collected. No clear trend could be confirmed regarding the impact of hydrocortisone intake on these steroids.

The GC-C-IRMS analysis was then performed on the urine samples from this study. These analyses concluded that 1% hydrocortisone cream does not exhibit transdermal activity when used under therapeutic conditions and apply on safe skin (25 mg twice daily for 5 days): no changes in 13C/12C isotope ratios were observed for all subjects. The results of the IRMS analysis showed that in addition to the metabolites already studied in the literature such as allo-THF or allo-THE or 11keto-Etiocholanolone. The compound 11bOH-etiocholanolone could also be a good target for this application but with a detection window observed up to 36h, the interpretation of results in the context of out-of-competition treatment could be more complex.

Code: 18A22CR

Chapter S2 of WADA’s Prohibited List 2017 (“Peptide hormones, growth factors, related substances, and mimetics”) lists carbamylated EPO under sub-chapter 1.2 (“Non-erythropoietic EPO-Receptor agonists”). Carbamylated EPO (also called “CEPO”) is not an erythropoiesis stimulating agent (ESA) but acts tissue protective and can be used for therapeutic or prophylactic treatment of human diseases. In this function, it may also be misused by cheating athletes. However, CEPO is still under clinical investigation and not available as a pharmaceutical or analytical standard. Aim of the project is the synthesis of CEPO by controlled reaction of rEPO with cyanate, followed by purification, mass spectrometric characterization and electrophoresis (IEF-, SAR-, SDS-PAGE according to WADA TD2014EPO). Carbamylation will lead to a loss of positive charges and an increase in molecular mass (43 Da for each carbamylated amino group). Consequently, it is expected that the isoform cluster of CEPO is shifted towards the anode on IEF-PAGE, i.e. towards the “endogenous area” – thus making CEPO undetectable. Contrary to that, the relatively small increase in molecular mass (in total 387 Da, if the N-terminus and all 8 lysines are carbamylated) will lead to hardly any changes on SDS- or SAR-PAGE, i.e. CEPO will be detected within the same mass range as recombinant EPO and hence misusers of CEPO will also be tested positive. Additionally, the project will answer the question if CEPO remains detectable by the highly sensitive anti EPO antibody used for Western blotting in doping control (clone AE7A5). The antibody is directed against the first 26 amino acids of the N-terminus of EPO, which contains two modified (carbamylated) amino acids in CEPO. The synthesized compound will be made available to all WADA-accredited labs for use as standard in routine ESA-testing.

Main findings

CEPO was successfully synthesized and characterized by mass spectrometry. It is detectable by SAR-, SDS-, and IEF-PAGE and can be differentiated from other ESAs by these methods. Differentiation between CEPO and rEPO is possible by digestion with endopeptidase Lys-C. Since Lys-C cannot cleave at carbamylated lysines only rEPO is degraded and CEPO remains intact. Conclusions: CEPO is detectable by the currently applied electrophoretic methods for ESA-doping control. Although there is a band-overlap with rEPO and endogenous EPO on SAR- and SDS-PAGE, it can be relatively easy differentiated from them by Lys-C digestion. However, since usage of CEPO is prohibited, this differentiation may be not necessary.

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Code: 18C18MP

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Part 1 of the project demonstrated the performance enhancement of an ecdysterone supplementation in combination with resistance training in humans. To allow for detection of an ecdysterone administration in doping control, the metabolism of ecdysterone and its urinary excretion will be investigated. For method development additional in vitro experiments will be conducted to help targeting the right potential metabolites. Using the prospected metabolites a targeted method will be used to analyse post-administration urines. This approach will be complemented by non-targeted analyses using high-resolution mass spectrometry. Finally the project aims in providing a method useful for screening of ecdysterone and its metabolites in compliance with existing screening procedures in anti-doping laboratories.

Using these results the prevalence of ecdysterone in samples from human sports anti-doping control from different regions will be monitored.

Main Findings: 

Ecdysterone is a phytosteroid widely discussed for its various pharmacological, growth-promoting, and anabolic effects, mediated by the activation of estrogen receptor beta (ERbeta). Performanceenhancement in sports was demonstrated recently, and, in 2020, ecdysterone was consequently included in the Monitoring Program of the World Anti-Doping Agency to detect potential patterns of misuse in sport. Dietary supplements containing ecdysterone were analyzed for their quality. Assay revealed to be poor for the majority of the products. Only a few studies on the pharmacokinetics of ecdysterone in humans have been reported so far. In this study, a single oral dose of 50 mg of ecdysterone was administered to ten volunteers (five males, five females). After a washout period, two out of the female volunteers were tested a second time. Analysis of serum samples was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after solid-phase extraction. Kinetic parameters were determined based on this data. Additionally, post-administration urine samples were analyzed using dilute-and-inject LC-MS/MS. Identification and quantitation of ecdysterone and of two metabolites, 14-deoxy-ecdysterone and 14- deoxy-poststerone, were achieved. Ecdysterone was the most abundant analyte present in postadministration urine samples, detected for more than two days, with a maximum concentration (Cmax) in the 2.8–8.5 h urine (Cmax = 4.4–30.0 µg/mL). The metabolites 14-deoxy-ecdysterone and 14- deoxy-poststerone were detected later, reaching the maximum concentrations at 8.5–39.5 h (Cmax = 0.1–6.0 µg/mL) and 23.3–41.3 h (Cmax = 0.1–1.5 µg/mL), respectively. Sex-specific differences were not observed. Cumulative urinary excretion yielded average values of 18%, 2.3%, and 1.5% for ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone, respectively. Ecdysterone and 14- deoxy-ecdysterone were excreted following first-order kinetics with half-lives calculated with three hours, while pharmacokinetics of 14-deoxy-poststerone needs further evaluation. Due to the potential generation of metabolites by gut bacteria that may cause significant variations in the metabolic profile, an integration of further isomers and analogues may also be appropriate. Analytical properties of ecdysterone and its metabolites were evaluated to assess the possibility of integrating them into existing procedures currently used for screening in anti-doping laboratories. Ecdysterone and its metabolites may be easily integrated into current initial testing procedures (ITP) for monitoring the prevalence in elite sports. LC-MS/MS showed excellent eligibility for these analytes. Alternatively, GC-MS analysis is possible after TMS derivatization. If extraction is required or desired, SPE was found to be by far superior to LLE. Enzymatic hydrolysis did not provide advantages over the analysis of the unconjugated fraction only. Out of 1100 doping control samples 2% revealed positive findings for ecdysterone. The samples were collected from athletes performing a broad variety of sports. Up to now, no classification of “high prevalence” type of sports is seen. Compared with the concentrations found after oral administration of 50 mg of ecdysterone, their concentrations were rather low.

Code: ISF18D10LL

Fluctuations in plasma volume present a major confounding factor to the analysis of an Athlete's Biological Passport (ABP) profile.  A recently developed model for plasma volume estimation via a simple blood test will be applied to samples collected during a cycling stage race. The model will be used to correct the concentration based hematological markers of the ABP, in order to account for the natural progressive plasma volume expansion induced by the multi day stage racing. The proposed research serves to validate the model in an anti doping context, which if successful presents an exciting step forward for the ABP and anti doping authorities.

Main findings: 

Fluctuations in plasma volume (PV) present potential confounders within the concentration-based markers of the hematological Athlete Biological Passport (ABP). Here, a multi-parametric approach involving a simple blood test is applied to the current ABP adaptive model in an attempt to remove the influence of PV expansion, induced by a cycling stage race. Blood samples were obtained from 29 professional cyclists (15 female, 14 male) before, during, and after consecutive days of cycle racing (Tour Down Under, Adelaide, South Australia). Whole blood was analyzed in accordance with the World Anti-Doping Agency (WADA) ABP guidelines at the WADA Accredited Laboratory in Sydney (ASDTL). Serum and plasma were analyzed for hemoglobin concentration ([Hb]), platelets, transferring, albumin, calcium, creatinine, total protein, and low-density lipoprotein. Significant reductions in [Hb] and the OFF-score were observed in female cyclists after 3 and 4 days of racing, with accompanying increases in PV, which returned to baseline values 4 days post competition. Similarly, a significant increase in PV was observed in male cyclists after 3 and 5 days of racing. PV was calculated using the CO-rebreathing method. When individual calculations of PV were applied to the adaptive model, the upper and lower reference predictions of [Hb] and the OFF-score were refined such that all outliers consistent with racing induced PV changes were removed. The multi-parametric PV model appears capable of reducing the influence of PV on volumetric markers during competition. This is an important step towards the inclusion of the PV correction in the hematological module of the ABP. Future work should consider intra laboratory and intra analyzer variation as well as adquirig more longitudinal data on elite athletes from different disciplines.  

Code: ISF18D13OP 

The screening of the misuse of endogenous androgenic anabolic steroids (EAAS) is currently performed by the quantification of the urinary steroid profile. One of the main problems related with this approach is the great impact of alcohol consumption on the profile. The fact that ethanol affects and invalidates the steroid profile opens the door to the use of ethanol as masking agent for testosterone administration. 

Our group has shown that urinary ratios 6OH-Etio3G/EG and 6OH-Andros3G /EG increase after EAAS administration. Conversely, preliminary results show that those ratios decrease after ethanol consumption. This behavior suggests that those two glucuronides may be useful to distinguish between changes in T/E due to ethanol consumption and those due to the combined administration of testosterone and ethanol (used as masking agent). Additionally, the determination of phase II metabolites in alternative matrices is a promising tool for the screening of EAAS misuse and might provide new insights in differentiating testosterone administration from (simultaneous) ethanol consumption. 

This project aims to (i) evaluate the usefulness of phase II metabolites to differentiate between the consumption of alcohol alone and its consumption during testosterone administration, (ii) explore the potential of the determination of phase II metabolites in alternative matrices for the screening of testosterone misuse and (iii) look for the differences in a comprehensive steroid profile (in urine, plasma and saliva) between samples collected after testosterone administration and after the combination of testosterone and ethanol. For this purpose, we plan to perform a pilot study with 4 male volunteers. Each volunteer will be treated with placebo, testosterone gel, ethanol and the combination of ethanol and testosterone. Urine, plasma and saliva will be analyzed to look for differences between the different conditions.

Main findings:

Considering that the most common situation is the consumption of moderate amounts of alcohol and that the transdermal application of testosterone is the most challenging to detect, we aimed at testing those new markers for those difficult conditions. Following our clinical trial scheme, 4 volunteers received (using a randomized protocol) either placebo, 100 mg/day for 3 days transdermal testosterone, 30 g/day for 3 days ethanol, or the combination of the same dosages of testosterone plus ethanol.

Code: T18M04NB 

The International Standard for Testing and Investigations requires that blood samples for the Athlete Biological Passport (ABP) are collected at least two hours after training or competition, in order to harmonize the passport data by reducing the impact of variation in volume of blood plasma that is induced by intensive exercise. Removal of this requirement would facilitate more frequent, convenient, cost-effective sample collection for athletes and testing authorities right after competition. A set of common clinical chemistry markers have recently been demonstrated to correct for fluctuations in plasma volume (PV) in experiments with healthy volunteers, indicating that they have potential to refine the ability of the ABP to detect blood manipulations by improving the sensitivity of the primary ABP markers. This project will evaluate the feasibility and validity of these markers in routine anti-doping settings by analyzing the markers in four WADA-accredited anti-doping laboratories. Successful validation of the PV correction method in this study will support the future implementation of this approach as a pertinent component of the ABP hematological module and enable the collection of ABP blood samples immediately after training or competition.