Projets de recherche

Code: ISF19C05MT

Due to the erythropoiesis-stimulating effects, the misuse of cobalt and cobalt salts in sports is prohibited both in- and out-of-competition. While total urinary cobalt levels can be determined by means of inductively coupled plasma-mass spectrometry (ICP-MS), there are currently no assays for the detection of inorganic cobalt which exclude cobalt-containing molecules such as Vitamin-B12. But especially in cases of atypical findings with elevated cobalt concentrations, the analysis of Vitamin-B12-depleted urine is required to provide accurate information on the ionic cobalt content of the sample. Therefore, a quantitative test method for inorganic urinary cobalt will be developed within this study by using different depletion approaches such as solid phase extraction (SPE) or liquid chromatography (LC) in combination with ICP-MS. In particular during prolonged exposure to high concentrations, cobalt was found to be irreversibly incorporated into red blood cells. As the determination of the cobalt content in erythrocytes could be highly relevant to uncover long-term cobalt exposure in a doping control context, an assay for the quantitative determination of cobalt from a defined amount of erythrocytes will be additionally set up. Both assays will eventually be used to analyze blood and urine samples collected within two administration studies with cobalt chloride and Vitamin-B12 (dose: 1 mg/day over a period of 14 days). The Vitamin B12 administration study will provide important insights into the influence of Vitamin-B12 supplementation – which is legitimately used by many athletes – on urinary cobalt levels.

Main Findings

The manipulation of blood and blood components, commonly referred to as “blood doping” is one of the continuing challenges in the field of sports drug testing. For that purpose, specific and sensitive detection methods enabling the detection of prohibited substances and methods of doping are required. As a cheap an easy available alternative to illicit blood transfusions, erythropoiesis stimulating agents have been shown to be misused in sport. To illegally improve the athlete's aerobic capacity and endurance performance, the administration of ionic cobalt (Co2+, e.g. CoCl2) can be used to stimulate the endogenous erythropoietin (EPO) biosynthesis. By contrast, several organic Co-containing compounds such as cyanocobalamin (vitamin B12) are not prohibited in sports, and thus, the need of analytical differentiation of urinary Co-concentrations is desirable. To this end, an excretion study with daily applications of either 1 mg of CoCl2 or 1 mg of cyanocobalamin was conducted with 20 volunteers over a period of 14 consecutive days where urine, plasma, and concentrated red blood cells were analyzed. The samples were collected starting 7 days before the administration until 7 days after. For total cobalt analyses, inductively coupled plasma mass spectrometry (ICP-MS), which yielded significantly elevated levels exclusively after inorganic cobalt intake, was utilized. Moreover, a liquid chromatography (LC)-ICP-MS approach was established and employed for the simultaneous determination of organically bound and inorganic cobalt by chromatographic separation within one single run. Especially for illegal Co2+ supplementation in sports this approach can be complemented to a prospective detection method.

Finally, for adequate method characterization and quantitative analyses, one or more internal standards need to be implemented and the chromatographic separation of additional cobalt-containing organic species as well as the stability of different variants of cyanocobalamin, especially with regard to photolytic degradation and possible conversions, need to be clarified. Nevertheless, despite the fast and preparative chromatographic run, inorganic cobalt is clearly separated and Co2+ concentrations attributed to unbound cobalt and exceeding future threshold levels will be regarded as antidoping rule violations. With regard to routine doping controls the presented approach offers an initial testing tool in order to identify those doping control samples that justify subsequent accurate cobalt quantification.

Code: 19B05CR

Class S4 of WADA’s Prohibited List 2019 (“Hormone and metabolic modulators”) lists myostatin inhibitors under sub-chapter 4 (“Agents preventing activin receptor IIB activation”). Like follistatin, myostatin-propeptide suppresses signaling of myostatin and subsequently leads to an increase in muscle mass and loss of body fat. In serum, >70% of myostatin is bound to myostatin-propeptide and thus myostatin-propeptide regulates skeletal muscle mass, i.e. if myostatin-propeptide is administered, more myostatin will be inhibited and then more muscle mass will be developed. Myostatin-propeptide is a glycoprotein containing one N-glycosylation site and 243 amino acids. Typical concentrations in serum and plasma are in the range of ng/mL.

So far, no approved myostatin-propeptide pharmaceuticals are available. Nevertheless, myostatin-propeptides can be bought on the black market for “research purposes”. They are labelled either “MyoPro”, “HMP”, “Myostatin-Propeptide (HMP)”, or erroneously “GDF-8” and “Myostatin”. All of these proteins are expressed in E. coli and hence lack the characteristic glycosylation of human endogenous myostatin-propeptide. This fact will be exploited in order to detect doping with myostatin-propeptide. After immunoaffinity purification (serum, urine), myostatin-propeptide will be separated by electrophoresis (SDS- or IEF-PAGE) and detected by Western blotting. Due to the missing glycosylation, “black market” products will not only differ in molecular mass but also isoelectric point (pI) from endogenous myostatin-propeptide.

Main Findings

Myostatin propeptide is prohibited according to chapter S4 of the “WADA 2022 List of Prohibited Substances and Methods.” So far, no approved myostatin-propeptide pharmaceuticals are available. Nevertheless, myostatin-propeptides can be bought on the black market for “research purposes.” A study on black market myostatin propeptide products was performed and electrophoretic detection methods for serum and urine were develeoped. Out of the 12 tested products, only nine actually contained the protein. Separation by SDS-PAGE revealed that the nine products were relatively impure and that the main compound had a much higher mass (approximately 54–55 kDa) than expected (approximately 33 kDa). Further analyses by mass spectrometry showed that the elevated molecular mass was due to the presence of a full length GST-tag on the propeptide. The developed detection method for serum is based on immunoprecipitation (IP) followed by SDS-PAGE and Western blotting. In total, three antimyostatin propeptide antibodies were tested. All of them were well suited for either IP or immunoblotting. The final protocol applies a biotinylated polyclonal antibody, streptavidin-coated magnetic beads, and a monoclonal detection antibody. For a sample volume of 500 μL serum, the detection limit of the method is approximately 2.5 ng/mL. The urine method applies a commercial ELISA for IP and performs with a limit of detection (LOD) of approximately 0.4 ng/mL. Furthermore, practically all currently available myostatin propeptide standards were also investigated. Due to the significant molecular mass difference of the black market products, an unambiguous differentiation from endogenous myostatin propeptide is possible. Publication: Reichel C, Gmeiner G, Thevis M. Electrophoretic detection of black market myostatin propeptide. Drug Test Anal. 2022;14(11-12):1812-1824.

Code: 19A14MT 

Several new performance enhancing peptides have necessitated particular attention of doping control laboratories. These peptides own significant energy modulating properties by acting as insulin receptor modulators (S507, S519). Athletes will benefit from these modulations and availability is given via internet-based sources for the non-approved candidates or via the pharmacy for approved compounds. For this study, peptides will be purchased, in-vitro metabolized and characterized by mass spectrometric methods. Afterwards, simple and fast detection methods will be developed by means of solid phase extraction and mass spectrometry. Ideally, it is aimed that these simplified methods will be combined with the already established assays for large peptides (insulin, GRFs etc.) to obtain one single multiplexed initial testing procedure for a large number of different prohibited peptides.

Main Findings: 

Due to their insulin mimetic properties, the two bioactive peptide-based drugs S519 and S597 represent prohibited compounds in sports. They act as selective insulin receptor modulators and can potentially trigger performance enhancing effects comparable to insulin or its analogs. So far, no analytical method exists to uncover the misuse of these peptides in sports. Within this study, a detection assay was developed to determine S519 and S597 in human plasma by means of liquid chromatography – mass spectrometry (LC-MS) after solid-phase extraction (SPE). The peptides together with their stable isotope labelled internal standards were custom synthesized and characterized by mass spectrometry. Moreover, the method was comprehensively characterized and found to show excellent specificity and sufficient limits of detection (< 0.5 ng/mL). In addition, different in-vitro experiments were conducted with both peptides and 15 different metabolic products were identified by means of high-resolution mass spectrometry (HRMS).

Code: 19D08OP

Screening for EAAS misuse remains one of the main challenges in doping control. Currently, relevant EAAS are quantified by enzymatic hydrolysis, TMS-derivatization and GC-MS determination. However, several markers might be either lost or underestimated by this approach.

In a previous WADA funded project (15A29OP, BICEPS), we obtained promising results with the detection of two steroid bis-sulfates, which substantially improved the retrospectivity of the T/E marker for oral testosterone misuse. Based on their MS behaviour, we hypothesize that these markers are two isomeric forms of the compund 3,16-dihydroxy-5-androstane-17-one bis-sulfate (16PHAnd_EtioSS1 and 16OHAnd_EtioSS2). Synthess of reference materials is required to confirm these results. In BICEPS, we also evaluated the occurence of steroid glucoronide-sulfates in human urine. We found that one of them (5a-androstane-3β,17β-diol 3-sulfate 17-glucoronide) clearly increased after oral administration of testosterone supporting its potential usefulness for doping control.

This follow-up project (BICEPS II) aims to continue with the evaluation of the potential of steroid bis-conjugates for the detection of EAAS misuse. The project will be divided in three parts: Part I will be focused on the elucidation of the exact structure of 16OHAnd_EtioSS1 and 16OHAnd_EtioSS2. Reference materials for a range of isomers will be sythesized and the confirmation of the marker identity will be performed by comparison with excretion urines already available at IMIM. Part II will be focused on the evaluation of the potential of mixed steroid glucuronide-sulfate conjugates for the detection of testosterone misuse by developing an untareted screening method. Part III will evaluate the actual potential of these conjugates for the detection of testosterone misuse. For that purpose, a quantative analytical methodology will be validated and applied to samples from excretion studies already available at IMIM.

Code: ISF19D02JP 

Ethanol affects the steroid profile in a way that may mask testosterone administration. Our group has shown that urinary ratios 6OHAndrosterone3G/Epitestosterone17G and 6OHEtiocholanolone3G/Epitestosterone17G increase after testosterone administration while preliminary results show they decrease after ethanol consumption. This behavious suggests that those two glucoronides 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.

A project to investogate those markers was approved in 2018 (ISF18D13OP). The clinical trial includes the administration of placebo, testosterone, alcohol and the combination of testosterone plus alcohol. Samples of urine, blood and saliva are collected. However, a budget reduction in the approved grant prevented investigating the new biomarkers not just in saliva, but even in blood.

The steroid profile in blood is very relevant. Previous attempts to develop a blood steroid profile lost the focus including a mixture of a few androgens, plus estrogens and corticoids. However, the key analytes in blood will also be steroid conjugates. The ratio of free to conjugated testosterone is known to change greatly after oral testosterone administration. Our primary results show how the new glucurono-conjugated biomarkers 6OH-A3G and 6OH-Etio-3G and others can be monitored in blood. The administration of ethanol affects phase II metabolism and therefore this specific blood steroid profile needs to be studied.

This project aims at studying the blood samples collected in project ISF18D13OP to study the behaviour of the new biomarkers 6OH-A-3G and 6OH-Etio-3G in blood as part of a more selective steroid profile, and the usefulness of the combination of phase I plus phase II metabolites in blood to differentiate between the consumption of alcohol alone and its consumption during testosterone administration.

Main Findings

The steroid profile is subject to several confounding factors. The use of alcohol is probably the most prevalent one. This projects aimed at finding new markers to tell the difference between the administration of testosterone and the administration of alcohol or the combination of both substances.

In the present study, urine and whlole blood samples (as for ABP hematological module) were collected from four volunteers after the administration of placebo, alcohol, testosterone transdermal or the combination alcohol plus testosterone.

The analysis of the plasma from blood samples collected as per ABP hematological module purposes, showed that from the many markers studied, androsterone glucuronide was the one showing a clearly different behaviour when administering alcohol (decrease) or testosterone (increase). This has been the key factor in the development of a new marker calculated as the product of the concentrations of testosterone glucuronide and androsterone glucuronide (T-G x AN-G) in plasma samples. This new marker has shown to be:

· sensitive to the administration of testosterone (transdermal)

· insensitive to the administration of alcohol

· still sensitive to testosterone when administered jointly with alcohol

Other markers, i.e. the recently proposed ratio testosterone over androstenedione (T/A4) have shown unable to discriminate between both substances.

The new marker (T-G x AN-G) has a great potential for the detection of testosterone use even when co-administered with alcohol. Furthermore, it can be readily implemented by accredited laboratories as these analytes are commercially available and laboratories have experience in the analysis of those substances. In order to confirm and validate this new marker, further studies wil be necessary increasing the cohort of subjects and to test its behaviour for other dosages and routes administration of testosterone, the impact of genetic/ethnic differences and other potential confounding factors.

Considering the latest publication of the WADA guideline Quantification of Endogenous Steroids in Blood for the Athlete Biological Passport (July 2023), analysis of these new markers in serum instead of plasma would be advisable for consistency with the approach followed by WADA.

Code: 19C09CR 

Class S4 of WADA's Prohibited List 2019 ("Hormone and Metabolic Modulators") lists follistatin under sub-class 4 ("Agents preventing activin receptor IIB activation, Myostatin inhibitors") as prohibited substances.

So far, no approved follistatin pharmaceutricals are available. On the other hand, there are two groups of follistatins sold on the black market (FST-344 and FST-315). But the administratio of black market follistatins to human test persons will be ethically not justifiable. For that reason, we plan a study with rats. The test animals will receive black maket FST-344 (group 1) and FST-315 (group 2) at a dosage, which can be clearly detected after 48 hours in serum (1 mg/rat, weight-adjusted). Subsequently, serum and urine will be collected and tested for follistatin with eletrophoresis and Western blotting.

The study will help to clarify (1) if both FSTs (FST-344, FST-315) are still observable after 48 hours of circulation in blood, and (2) if these FSTs can also be detected in urine. We have already shown that black market FSTs can be clearly differentiated from endogenous FSTs by electrophoresis (SDS-PAGE) and Western blotting.

Main findings

The main findings are not available due to the sensitivity of the information and results developed in this project.

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.

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.