Projets de recherche

Code: R20M01MS

Primarily, the objective of this project is to conduct an exploratory study to monitor the blood steroid profile of women subjects over two menstrual cycles to compare the intra- and inter-individual variability of the blood steroid profile of women subjects during a menstrual cycle. Secondly, transdermal testosterone gel (Androgel 1%, AbbVie, North Chicago, IL) will be administered to all volunteers once. The study will enable the pharmacokinetic study of transdermal testosterone gel in women in addition to its detection and will allow to determine putative differences of steroid metabolism between men and women. Simultaneously, the urinary steroid profile will be monitored to allow comparison of the steroid profile between both matrices. This study should help to develop further the blood steroidal of the ABP and to refine the identification of doping behaviours in females with adequate investigations allowing to define reference values for specific female athletic populations. This study will also allow to lay the foundations for a further untargeted metabolomics project for the discovery of new steroidomic biomarkers for steroid detection in female athletes. The design of the study was modified and extended to three menstrual cycles and the treatment regimen was modified. Instead of a single application, testosterone gel (Tostran 20mg/g, 0.5g applied corresponding to 10mg of testosterone) is applied daily for a menstrual cycle (corresponding to 28 days).

Main Findings

In women, hormonal fluctuations related to menstrual cycle may impose a great source of variability for some urinary biomarkers of testosterone (T) administration, which can ultimately disrupt the sensitivity of their longitudinal monitoring. Additional biomarkers and alternative matrices need therefore to be investigated to improve the detection capability for doping practices with T, especially in female athletes. The aim of this study was therefore to investigate the impact of menstrual cycle combined with T gel administration on the biomarkers of the ABP (steroidal and haematological module) and on serum steroid biomarkers in females. It allowed to directly compare the sensitivity of T gel detection between urinary and blood steroid profiling either for targeting samples for IRMS or for longitudinal evaluation. To achieve this, a clinical trial involving fourteen healthy women subjects was conducted over three consecutive menstrual cycles with the second cycle combined with a daily administration of T gel for 28 days. The sensitivity of the current urinary and haematological markers of the Athlete Biological Passport (ABP), as well as serum steroid biomarkers was investigated for the monitoring of the T gel treatment. Additionally, endogenous fluctuation of these parameters were monitored within the menstrual cycle.

Code: 20D09OP

In previous WADA funded projects (11A9RV, 12A13OP, 14A29OP) our research group discovered two endogenous steroid (EAAS) metabolites, namely, 6β-hydroxyandrosterone-3-glucuronide (6bOHA-3G), and 6β-hydroxyetiocholanolone-3-glucuronide (6bOHEtio-3G) particularly selective markers of the administration of testosterone. Small quantities of the compounds were synthesized and fully characterized. They showed to be resistant to enzymatic hydrolysis, a reason for its late discovery.

An analytical method has already been developed, fully validated and published for the direct analysis of those substances together with other regular steroid profile glucuronide markers. One way to improve the the application of the method would be to have access to labelled internal standards which will decrease the variability of the basal values.

These two new markers have proven to be sensitive to testosterone administration (oral, intramuscular and even transdermal, improving its detection window, as compared to the current T/E ratio. We are  currently testing their specificity to differentiate testosterone administration from ethanol consumption or the combination (WADA project ISF18D13OP). Preliminary results of this on-going project show that while TG/EG increases both with T and EtOH administration, the new markers increase specifically after T administration while they do not increase after EtOH administration. This differential behavior adds specificity and improves the steroid profile. 

The current project (inter-ALMA) aims at finally incorporating these new markers into a routine screening by performing an interlaboratory validation incorporating 5 different laboratories. For that purpose, analytical methodology, standards (already synthesize in the frame of the 2019 PCC project entitled “Essential reference materials to advance the long term detection of testosterone abuse”) and their deuterated analogues (synthesized in the frame of iner-ALMA) will be provided to the laboratories. The outcome after using common or comparable methodologies to analyze selected samples will be compared.

Code: 20D03JH

The use of information technology within sport has significantly increased over recent years. The availability of data for longitudinal tracking of athletes over the course of their careers is critical to identify an individual’s performance progress. As such, a key component of this longitudinal monitoring is to be able to differentiate between “normal” increases in performance caused by maturation and training, from an “unnatural” improvement caused by doping. Our initial work demonstrates that it is possible to identify specific performance profiles characteristics for athletes with previous ADRVs, which appear to be consistent across the track and field disciplines we have investigated (100-800m, jumps & throws). However, there is a need to extend the current model and translate it into a useful tool for anti-doping authorities. Therefore, the purpose of this study is to refine and further develop our existing Bayesian modelling approach. Specifically, this project will enable us Page 2/8 to explore confounding factors for modeling performance data, and at the same time explore the effects of these confounders on the probability level needed to flag a suspicious individual, balancing false positives (clean athletes identified as doping) and false negatives (doping athletes which are not flagged). We will therefore develop a “risk” score for suspicious performance profiles. A key aspect of the method will be its impact on decisions made via the traditional Athlete Biological Passport (ABP). Working in conjunction with the AIU, we will retrospectively explore whether performance data adds value to decisions made by expert witnesses in historical passport cases. Using this approach, we will evaluate whether adding performance data to existing ABP data is more effective than current methods for identifying athletes who demonstrate may present adverse, or uncertain passport profiles findings.

Main findings

As the aim of any doping regime is to improve sporting performance, it has been suggested that analysis of athlete competitive results might be informative in identifying those at greater risk of doping. The aim of this research project was to investigate the utility of a statistical performance model to discriminate between athletes who have a previous anti-doping rule violation (ADRV) and those who do not.

We analysed performances of male and female 100 – 10,000m runners obtained from the World Athletics results database using a Bayesian spline model. Measures of unusual improvement in performance were quantified by comparing the yearly change athlete's performance (delta excess performance) to quantiles of performance (50%, 75% and 90%) in their age matched peers from the database population. Sudden or unexpected changes in an athlete's level of excess performance the exceed these quantiles might therefore be indicative of doping. The discriminative ability of these risk measures was investigated using the area under the ROC curve (AUC) with the highest values being observed using the 75% quantile (AUC = 0.78-0.80). To assess the specificity of the model using the 75% quantile at different age points we assessed the False Positive rate across different probability levels for delta excess performance. The true positive rate ranges between 0.20 and 0.67 across the ages due to the changes in the number of observed true positives (i.e. ADRVs) recorded at each age, and athletes within the database.

Further, we investigated the ability of delta excess performance to discriminate between athletes with and without adverse analytical findings (AAFs), adverse passport findings (APFs) and Anti-Doping Rule Violations (ADRVs). The 75% quantile for delta excess performance demonstrated AUC values of ~0.60 at age points with the highest numbers of APFs. However, by comparison, the model showed a better ability to discriminate the ADRV status of athletes by AUC values of ~0.65 to 0.75 at the same corresponding age points.

The findings of this project demonstrate the utility of performance monitoring to discriminate between athletes on the basis of their doping status. However, it is important to recognise that high levels of delta excess performance are not sufficient to prove an athlete is doping, and that information obtained from this type of analysis should be integrated with other data as part of a wider intelligence gathering approach to anti-doping.

Publication:

Hopker JG, Griffin, JE. Hinoveanu, LC. Saugy, J. Faiss, R. (2023) Competitive performance as a discriminator of doping status in elite athletes. Drug Testing and Analysis. 16:473-81. doi: 10.1002/dta.3563.

Code: 20D02FP

Volumetric Absorptive Microsampling (VAMS) is a recent microsampling technique used to obtain dried specimens of biological fluids that could represent in the near future a valid alternative to urine, whole blood and serum sampling for anti-doping purposes. Although similar to Dried Blood Spots (DBS), VAMS promise to bring a number of significant advantages over them: i) improving quantitative performance thanks to higher sample volume acccuracy; ii) obtaining the extraction yield and reproducibility without and impact of hematocrit (HCT) value; iii) enhancing the overall analytical performance, resulting in better correlatio with plasma values; iv) simplifying collection procedures by homogeneous samples with a lower reject rate; v) availability of 96-well formats, opening the access to automation. In this study, the suitability of VAMS, in 30µL format, as an innovative matrix for the measurement of novel highlighted blood markers of Endogenous Anabolic Androgenic Steroids (EAAS) doping will be evaluated. A UHPLC-MS/MS method for the quantification of a significant panel of circulating steroid hormones and their phase II metabolites will be developed. Mass spectrometric parameters and transitions will be fine-tuned to achieve the maximal sensitivity needed to detect ad quantify as many markers as possible. Chromatographic conditions will also be thoroughly optimized in terms of separation of isomers with the aim of finding the best compromise between resolution and analysis time. Validation procedure will be carried out following the WADA requirements. Various parameters, such as limits of detection and quantification, precision and accuracy, matrix effects, selectivity, repeatability and robustness will be investigated for each measured analyte. The developed analytical method will be transferred to a WADA-accredited laboratory and an inter-laboratory comparison will be performed. Finally, results will be discussed putting particular emphasis on the systematic comparison with more conventional hematological matrices, such as serum and plasma.

Main findings

A novel LC-MS/MS method for the measurement of 18 steroids panel, including main endogenous steroid hormones and androgen phase II metabolites, was developed and validated in compliance with WADA requirements for quantitative methods. A simple sample preparation procedure was optimized and resulted in extraction recoveries ranging from 69.8% to 92.5%, while showing ion suppression-related matrix effect around 30% for most of target analytes. The validation protocol allowed demonstrating the satisfactory performance of developed method in terms of selectivity (separation of isomers and exclusion of interferences), trueness, repeatability, precision, combined uncertainty, linearity range, LLOQ, carry-over and overall robustness. Finally, the method was successfully transferred to the Lausanne WADA-accredited laboratory. A stability study involving 20 healthy subjects (10 males and 10 females) demonstrated that steroid concentrations measured in VAMS samples stored at room temperature, 4°C, -20°C and -80°C, do not deviate from the values measured in baseline samples, with calculated mean percentage differences being always lower than Reference Change Value threshold which estimated method’s analytical variability. Moreover, the influence of up to three consecutive freeze and thaw cycles was evaluated as not significant. VAMS proved to be a valid collection strategy for measuring steroid hormones in blood that could be employed in doping control analysis with the goal of increasing sampling frequency dedicated to the newly implemented Blood Steroid Profile (BSP). The stability of steroid hormones in blood microsampling collected on VAMS support opens the way of interesting advantages for blood samples’ transportation and storage for anti-doping purposes. Nevertheless, prior to the introduction of VAMS sampling for BSP analysis, further studies aiming at evaluating the correlation between steroid concentration levels measured in VAMS (capillary whole blood) and in currently employed serum samples should be performed in the future to gather an exhaustive overview of VAMS potential in anti-doping context.

Publications/Presentations related to the project

Publications:

- F. Ponzetto, M. Parasiliti Caprino, L. Leoni, L. Marinelli, A. Nonnato, R. Nicoli, T. Kuuranne, E. Ghigo, G. Mengozzi, F. Settanni, LC-MS/MS measurement of endogenous steroid hormones and phase II metabolites in blood volumetric absorptive microsampling (VAMS) for doping control purposes, Clin. Chim. Acta (2024), under review.

Code: 20C20MH

The updated 2019 Global Initiative for Asthma (GINA) guidelines, now recommending inhaled corticosteroid (ICS) in combination with the long-acting beta2-agonist (LABA) formoterol as preferred controller step 1 & 2 for asthma-related symptoms in adults and adolescences. Therefore, the use of formoterol in combination with an ICS will surely increase among athletes, given the high prevalence of asthma and exercise-induced bronchoconstriction (EIB) in the athletic population. Given the potential performance enhancing effects of beta2-agonists, WADA has imposed a urine threshold for formoterol of 40 ng/ml with a maximum dose of 54 µg over 24 hours. However, inhalation of 54 µg formoterol has been shown to enhance muscle strength and sprint performance. Thus, it is necessary to re-evaluated the current maximum doses especially considering that more athletes will receive formoterol for treating asthma and EIB. Nonetheless, no studies have examined whether daily inhalation of formoterol in therapeutic doses exert anabolic actions or can cause potentil adverse in trained males and females. The project consists of two studies. Study I will investigate the chronic effect of inhaled formoterol on exercise performance and body composition in therapeutic doses in males and females. Study II will examine the pharmacokinetic profile of inhaled formoterol with or without budesonide and oral ingestion of formoterol in therapeutic doses in males and females.

Main findings

Use of the inhaled beta2-agonist formoterol is increasing among athletes due to updated asthma management guidelines. However, the acute and long-term effects of formoterol on exercise performance and muscle function in trained individuals remain inadequately explored. In a series of randomized placebo-controlled trials, we explored these effects in endurance-trained individuals. The first study investigated the effect of therapeutic doses of inhaled formoterol (24 μg twice daily for 6 weeks) versus placebo in 51 endurance-trained men and women. Participants were assessed for aerobic capacity, body composition, muscle mitochondrial function, and cardiac parameters. Results showed that formoterol increased lean body mass but significantly impaired maximal oxygen uptake (V̇O2max), incremental exercise performance, and muscle mitochondrial oxidative capacity. No changes were observed in cardiac function or blood volume.

The second study examined the acute effects of a high, one-off inhaled dose of formoterol (54 μg) versus placebo in 21 elite male cyclists. After administration, cyclists performed sprints and a 4-minute all-out cycling test. Formoterol led to a slight enhancement of sprint and short intense exercise performance. The third study compared the acute effect of inhaled formoterol (with and without budesonide) and oral formoterol versus placebo in 20 well-trained males and females. Only the combination of inhaled formoterol and budesonide produced a small improvement in sprint performance and time to peak twitch force, with no effect on isometric muscle strength or 4-minute time trial performance. Collectively, these studies indicate that while inhaled formoterol may acutely enhance sprint performance, chronic use at therapeutic doses can impair aerobic capacity in endurance athletes, likely through negative effects on muscle mitochondrial function. The combination with budesonide may offer minor sprint benefits, but further research is needed on long-term use in athletes.

Code: 20C17CR

Chapter S4 of WADA’s Prohibited List 2020 (“Hormone and Metabolic Modulators”) lists myostatin propeptide under sub-chapter 4 (“Agents preventing activing receptor IIB activation, Myostatin inhibitors”) as prohibited substance. So far, no approved myostatin propeptide pharmaceuticals are available. On the other hand, myostatin propeptide is sold on the black market (labelled “MyoPro”, “HMP”, “Myostatin-Propeptide (HMP)”, or erroneously “GDF-8” and “Myostatin”). But the administration of black market myostatin propeptide to human test persons will be ethically not justifiable. For that reason we plan a study with rats. The test animals will receive black market myostatin propeptide at a dosage, which can be clearly detected in serum (10 mg/kg BW). After 24, 48 and 168 hours, serum and urine will be collected and tested for myostatin propeptide by electrophoresis and Western blotting. The study will help to clarify (1) how long black market myostatin propeptide is detectable in blood, and (2) if it can also be observed in urine. We have already shown that black market myostatin propeptide can be differentiated from endogenous myostatin propeptide by electrophoresis (SDS-PAGE) and Western blotting.

Main findings

Chapter S4 of WADA’s Prohibited List 2024 (“Hormone and Metabolic Modulators”) lists myostatin propeptide under sub-chapter S4.3. (“Agents preventing activin receptor IIB activation, Myostatin inhibitors such as myostatin-binding proteins (e.g. follistatin, myostatin propeptide)”) as prohibited substance. Currently, myostatin propeptide is only available on the black market. Since administration of black market products to humans is ethically not justifiable, a study with rats was performed.

Aims of the project were:

- an animal study (administration of black market myostatin propeptide (“GDF-8”) and recombinant myostation propeptide standard to rats followed by collection of serum and urine)

- an investigation of the electrophoretic detectability of black market GDF-8 in rat serum and urine after circulation in blood for 24, 48, and 168 hours. Additionally, the detectability of recombinant myostation propeptide standard after 24 hours was also investigated

- an in vitro metabolism study of myostation propeptide using human and rat liver microsomes

Results:

After a single dose administration of black market GDF-8 (10 mg/kg body weight) to rats, the protein was detectable in all serum samples after 24 hours. However, it was no longer traceable in the samples taken after 48 h and 168 h. No GDF-8 was found in the urine samples at all three time-points. Recombinant myostation propeptide was neither detectable in serum nor urine 24 hours post administration.

In order to reveal possible differences in the metabolism of myostation propeptide between humans and rats, it was incubated with human and rat liver microsomes (5, 60, 120, 300 min, 24 hours). The protein proved quite stable - only after 24 hours degradation of the main band was observed.

Conclusions:

Black market myostation propeptide can be detected in rat serum samples by SDS-PAGE and immunoblotting with a monoclonal myostation propeptide-specific antibody. For the extraction from rat serum and urine, polyclonal myostation propeptide-antibodies captured by magnetic beads or coated on ELISA-wells were used. After single dose administration, the protein remained detectable for only 24 hours in rat serum samples. No signals were obtained on Western blots in serum after 48 and 168 hours and at all three time-points in urine samples.

Code: 20C15XD

Clostebol (4-chloro-testosterone; 4-chloro-4-androsten-17-ol-3-one) is an anabolic androgenic steroid (AAS) derivative from testosterone. In humans, among the legitimate therapeutic indications of anabolic steroids, clostebol acetate is approved for topical use in dermatological and ophthalmological preparations. The esterification on the 17 position also permits its oral use, protecting the compound from an extensive firstpass metabolism. Clostebol has also been used in cattle to improve the animal growth. Due to its anabolic properties the International Olympic Committee (IOC) in the past and today the World Antidoping Agency (WADA) have included clostebol and the other AAS in the yearly renewed list of prohibited substances in sports[1]. The human [2,3] and animal[4] metabolism of clostebol allowed to establish adequate methods to 
detect its illicit use. The detection of clostebol intake is traditionally based on the detection of its main metabolite (4-chloro-4-androsten-3a-ol-17-one) excreted into urine glucuronoconjugated (Figure 1.). In order to improve the detection capabilities, antidoping laboratories have developed in the last decade methods based on gas chromatography (GC) coupled to tandem mass spectrometry (MS/MS) lowering the limits of detections or have implemented methods for the direct investigation of phase II metabolites (sulphates) using liquid chromatography coupled to mass spectrometry (LC-MS/MS) [5,6]. The presence of clostebol metabolite in an athlete’s urine sample may be due to its illicit use as anabolic, the ingestion of contaminated meat[7] and finally some case reports describe the accidental contact with cream preparations containing clostebol[8]
. 
In the last years, the improvement of the detection capabilities of the antidoping laboratories has led to a moderate increase of clostebol detection worldwide, and especially in Italy where the use of a cream containing clostebol acetate and neomicine is quite extended. Trofodermin® is a pharmaceutical preparation containing 5% clostebol acetate and 5% neomicine sulphate that can be applied by cream or spray, that can be used for the following treatments: abrasions and erosions of the skin, injuries and wounds, such as varicose ulcers, due to poor blood circulation, bedsores (due to immobility in bed) sores or trauma, fissures (cuts) on the nipple (which can occur during breastfeeding), anal fissures (small cuts around the anus), burn wounds, infected wounds, wounds that delay to form the scar, irritation, redness, and sensitization of the skin that appears after radiotherapy (radiodermatitis), dryness, cracking or peeling of the skin with ulceration. According to the Italian law, a visible symbol on the packaging indicating the presence of a substance included in the WADA list of prohibited substances must be present. Although clostebol is prohibited by all administration routes, the aim of this study is to investigate the presence of clostebol metabolite in urine after an accidental contact with the substance due to the therapeutic application of clostebol acetate in a different individual.

Main findings

The accidental contamination of an individual after getting into close contact with another individual using transdermal clostebol acetate has been demonstrated after different degrees of exposure.This project aimed to search for specific clostebol metabolic markers or concentrations thresholds that may help to distinguish an illicit use of clostebol as AAS after an oral administration from an accidental contamination after a transdermal application, or to distinguish between a transdermal or oral administration, helping to establish adequate criteria to be adopted by the antidoping community. This kind of thresholds have been already applied for other groups of substances

The metabolism of clostebol after oral and transdermal applications has been extensively described using the more common tools used by WADA accredited Laboratories, gas chromatography coupled to mass spectrometry. Ten metabolites were detected after oral administration of which 5 could not be detected after transdermal application under the assay conditions here applied. A suitable criterion to disclose an oral (late excretion) from a transdermal application was achieve. The use of concentrations of any of the metabolite is quite difficult because the variability in the individual absorption, metabolism and/or excretion. Instead, the ratios between specific metabolites M4, M3 and M2 to M1 showed plausible results to discriminated between both administrations.

The proposed metabolic ratios have a fundamental limitation for being applied worldwide by all WADA accredited laboratories. The response ratios (even among isomers) depend on the signals (transitions) selected by every laboratory. To overcome this point the metabolites considered more diagnostic need to be synthetized to allow an estimation of the concentration that would be then independent from the analytical method applied. This would allow in addition to confirm definitively the configuration of the proposed metabolites. Some metabolites are excreted in urine only after oral administrations but their response is much lower and there are more potential structure fitting with the candidates proposed here. The sulfate fraction that was in previous communications considered as a potential solution to the problem presented, were not considered here since this is a fraction not used in ITP conditions. Based on the previous considerations, we propose M2, M3 and M4 as the best targets worth to be synthetized and characterized for this purpose.

Code: 20C13GJ

Objective: To determine urine thresholds consistent with maximum therapeutic dose of: salmeterol.

Study A: Salmeterol
A salmeterol threshold study will be conducted using a similar protocol to that previously used by our team, consisting of two acute urine PK trials 25 over one week; one under normal conditions at baseline, followed by salmeterol dosing for one week and then a second acute PK trial performed under stressful conditions with heat and dehydration22. During the acute phase, we will also assess the effect of salmeterol on muscle strength, peak power and time trial performance utilising a placebo-controlled crossover design. After the second PK trial, subjects are randomly allocated 5 weeks of treatment with placebo or salmeterol (1:1) to assess the effect of salmeterol on muscle mass, fat mass, muscle strength, time trial performance and peak power during cycling measured at six weeks (completion).

Subjects: Thirty six healthy trained men and women (equal male:female) will be recruited for the study, non-smokers, no history of chronic disease, and recreationally active (>5 h/wk). 

Clinical Study Procedure: Pre-intervention will consist of general health screen, lung function test, and VO2 max will be determined by incremental bike ergometer to exhaustion using standard laboratory protocols. A baseline Wingate test protocol will be administered to participants during this period.5 Pharmacokinetic (PK) intervention in all subjects (n=36) will consist of salmeterol delivered by pMDI (200 µg dose) at time 0, followed by urine collection at 0.5, 1, 2, 4, 8 and 24 hours under resting conditions. Salmeterol will be continued to be delivered daily (100 µg twice daily – home administration with supervision monitored by Skype/Facetime) with spot urine collection until the 7th day at which point a 200 µg dose will be administered and a second acute phase PK study will be conducted under identical time (urine collection at 0.5, 1, 2, 4, 8 and 24 hours). The second acute PK study, however, will encompass stressful conditions, namely accumulation of dose (as noted previously using a similar protocol for terbutaline 25) but also with exercise (time trial) under heat (30-35°C) as per our previous work with terbutaline22. Subjects will then be randomised to salmeterol:placebo 1:1 and continue treatment for a further 5 weeks (6 weeks total duration) and undertake a second Wingate test to assess effects of the intervention on exercise performance using our previously applied protocols 5 – further spot urine sampling will not occur during this time. Subjects will be asked to avoid any CYP3A4 inhibitors such as grapefruit juice during the chronic phase. 

Analytical: Urine samples will be analysed by racemic assay (a simple modification based on our previous racemic work 26). We will investigate the alpha-hydroxysalmeterol metabolite and salmeterol in urine, with a total of around 612 samples for the acute and the intermediate chronic spot sampling phase treatment arm.

Code: 20C09GG

Today, the steroid profiling mainly relies on the analysis of glucuronide metabolites. After the selective hydrolysis of the glucuronide moiety, the corresponding free steroid is analyzed by gas chromatography – mass spectrometry (GC-MS) subsequent to silylation of hydroxyl - and keto-groups. The inclusion of sulfate metabolites has previously been difficult due to a non-efficient hydrolysis to the corresponding phase I metabolites. Consequently, an important piece of information about alterations in the steroid metabolism after testosterone doping is not monitored so far. During the last years, the direct analysis of phase II steroid metabolites by liquid chromatography – mass spectrometry (LC-MS) has been described.

 In a previous study, conducted by the current research team, the implementation of sulfate androgen metabolites in the steroid profile was investigated after the intramuscular administration of a single dose of testosterone esters to six healthy volunteers. For this purpose, a LC-MS method was developed and validated which allowed for the quantification of eight conjugated steroids within the same run. As an outcome of this study, a promising potential marker for the intake of exogenous testosterone, the ratio of [testosterone glucuronide/testosterone sulfate]/[epitestosterone glucuronide /epitestosterone sulfate] = (TG/TS/EG/ES). This ratio is further called “combined ratio”. 

The hereby proposed study represents a follow up project aiming at the investigation of the impact of endogenous sulfate steroid metabolites on the steroid profile after additional routes of administration – oral and transdermal, in addition to the already examined one - intramuscular. Also, as an additional element, the current study will be expanded to include male and female subjects. Furthermore, the already existing analytical data for the Phase II metabolites of Testosterone will be enriched. The proposed study is expected to increase knowledge of the usefulness of the combined ratio as a complementary biomarker for testosterone abuse

Code: 20C07MP

Based on earlier results of our group ecdysterone is included in the 2020 Monitoring Program. As ecdysterone containing plants may be part of common human diet, discrimination between common dietary levels, excessive dietary intake of ecdysterone and supplementation for misuse is highly desired.

It is intended to perceive markers for classification of the type of ingestion. This may help to confirm or contradict athletes' claims in future anti-doping investigations.

Within the project high amounts of regular dietary compounds, i.e. spinach or quinoa, will be ingested by volunteers and urinary excretion compared to post administration urines after pure ecdysterone. Concentrations of ecdysterone will be monitored and potential metabolites or additional ecdysteroids from the plants sources will be uncovered. An investigation of the influence of the human microbiome on ecdysteroid metabolism will be included in the project.

Main Findings

In this study, ecdysterone was quantified in spinach and quinoa. Subsequently, quantitative analysis of ecdysterone and its metabolites in urine samples of human volunteers was conducted, following four different administration studies of sautéed spinach, smoothie from sautéed spinach leaves, quinoa and a combination of sautéed spinach and quinoa.

The purpose of the study was to investigate the human excretion of ecdysterone and metabolites following the consumption of ecdysterone-containing food sources, spinach and quinoa.

After all four administrations to humans (n=8, same participants for each) ecdysterone was excreted and quantified in all volunteers. The metabolite 14-deoxy-ecdysterone was also excreted by all volunteers although in certain cases in quantities below the lowest calibration point of the present study. The second metabolite, 14-deoxy-poststerone, was only detected and quantified in the urine of three volunteers (the same for all study arms). The maximum concentration of ecdysterone ranged between 0.08 and 5.5 μg/mL while for 14-deoxy-ecdysterone from 0.02 to 1.45 μg/mL, and for metabolite 14-deoxy-poststerone from 0.03 to 1.9 μg/mL.

Ecdysterone was not always the most abundant analyte found in the urine, since in certain volunteers 14-deoxy-ecdysterone or 14-deoxy-poststerone was excreted in higher amounts. Another aim of the current study was the comparison of ecdysterone and its metabolites excretion after the ingestion of spinach in different preparations and after the ingestion of sole quinoa and combined with spinach. For both sautéed and smoothie administrations the mean (SD) of the total excreted amount (%) of ecdysterone as unchanged plus the metabolites in urine was 1.4 (1.0) %, consequently no significant difference was observed. On the other hand, significant difference was found in the total excreted amount (%) between the consumption of sole quinoa (mean (SD) 2.6 (1.1) %) and combined with spinach (mean (SD) 1.7 (0.9) %).