Projets de recherche

Code: 20B06DE

A single administration of subcutaneous luspatercept (0.25 mg/kg) is proposed for four healthy participants, two males and two females. While detection of luspatercept has been studied before by spiking standard into samples, the excretion profile of the drug has not yet been determined in an anti-doping setting. These excretion samples are vital for proper validation of the detection method(s) to be applied to athlete samples. Additionally, the detection window of the drug has yet to be determined in serum/plasma and DBS (and, if applicable, urine), which provides important information for results management authorities.

Finally, Phase I clinical data from luspatercept in healthy individuals suggests that there will be an effect on the hematological module of the ABP, if abused by athletes. As such, it is important to provide a well-characterized timeline of these changes.

Main Findings

The main objective of this study was to perform an administration of Reblozyl® (luspatercept-aamt), a newly approved drug to treat anemia, in healthy volunteers and evaluate the detection of luspatercept in serum, dried capillary blood spots (collected with the Tasso M20), and urine for antidoping purpose. Four volunteers, two males and two females, received one subtherapeutic dose of luspatercept (0.25mg/kg) followed 3 weeks after by a second dose. Samples were collected from before administration to 7 weeks after the second dose. Evaluation of Luspatercept detection in the samples was performed after an immunoextraction step with magnetic beads coated with anti-ActRIIB antibodies, followed by electrophoretic separation by SDS-PAGE and a single-blot and immunodetection using a biotinylated anti-ActRIIB. To propose a confirmation analysis, direct detection was also assessed by SDS/SAR-PAGE followed by double-blotting using a second ActRIIB detection antibody or by IEF-PAGE and single-blot. Indirect effects were examined by measuring endogenous EPO concentrations and by evaluating hematological parameters variations using the ABP model.

Despite the supraphysiological dose administered, intense signals were identified for luspatercept in serum from the day after the administration until the end of the study, 7 weeks after the second dose, and detection is likely possible for even longer time. This administration study also confirmed that the drug is excreted unchanged in urine and regularly eliminated, allowing detection in this matrix. 20μL-DBS also showed sufficient sensitivity to detect the drug until the end of the study. The three electrophoretic methods used in Anti-Doping laboratories: SDS-PAGE, SAR-PAGE and IEF-PAGE were all appropriate for both screening and confirmation and could be used for one or the other. They showed very similar sensitivity.

The impact of the luspatercept on indirect markers was evaluated: ABP approach could flag luspatercept administration in some subjects, especially when ABPS and HGB were both atypically increased, but the effects on RET% were limited and the time/amplitude of the effects varied between subject. Endogenous EPO expression was also not strongly affected and was not indicative of the use of an erythropoietic agent.

In conclusion, this study demonstrated that luspatercept can be detected for a long time using electrophoretic methods in all the matrices relevant for doping. Its survey can be easily implemented in antidoping laboratories.

Publication: Marchand A, Miller G, Martin L, Gobbo C, Crouch AK, Eichner D, Ericsson M. Detection of erythropoiesis stimulating agent Luspatercept after administration to healthy volunteers for antidoping purposes. Drug Test Anal. 2022 Jul 5. doi: 10.1002/dta.3341.

Code: 20B05MT

Every year, approximately 2-5% of the urine samples routinely tested for the presence of erythropoiesis-stimulating agents by the Cologne Doping Control Laboratory are "no-shows" with undetectable endogenous (and recombinant) EPO. Possible origins for this phenomenon can be certain urine properties such as extreme specific gravity or low EPO concentration as well as the addition of adulterants such as proteases.

The aim of this research project is to investigate whether EPO detection can also be manipulated by adding oral fluid to doping control urine sample. Saliva contains many different enzymes including carbohydrases and proteases/peptidases, which can potentially interfere with the detection of a highly glycosylated protein such as EPO. In order to elucidate the effects of urinary oral fluid contaminations on EPO analysis, urine samples fortified with different erythropoietins will be mixed with varying amounts of oral fluid collected from healthy volunteers, stored for different periods of time at different temperatures, and subjected to EPO routine analysis.

Moreover, urine samples will be fortified with different amounts of oral fluid and analyzed by means of ultrafiltration, SDS-PAGE separation, tryptic digestion, and LC-HRMS/MS in order to identify saliva-specific proteins suitable as markers to reveal possible oral fluid contaminations in doping control urine samples.

Main Findings

The aim of this study was to investigate this assumption and to develop a detection assay in order to identify present OF in urine doping control samples.

For this purpose, a total of 1080 urine samples were subjected to EPO analysis and evaluated with regard to variations due to the subject, sex, volume of OF, time point of OF-sampling, and storage conditions. The results showed, that OF can indeed lead to masking of ESA abuse. In particular, interindividual differences as well as the sex and the timing of OF-sampling (pre- and post-prandial) were observed to have an impact on the analysis. In addition, the volume of OF in urine is of major relevance, but realistic amounts, which can be achieved e.g. by spitting once or twice, were found to impair the EPO analysis in a significant number of cases.

In order to identify contaminations or urine samples with OF, detection methods targeting human salivary α-amylase (sAA) were developed, as it was found to be a specific and most abundant protein in OF. For this purpose, both a lateral flow strip test (rapid test) and a bottom-up proteomic assay involving tryptic digestion followed by LC-HRMS/MS analysis were evaluated in terms of selectivity, sensitivity, and stability. Carry-over effect as well as linearity were additionally assessed for the bottom-up proteomic approach. Both approaches successfully identified sAA in urine, and the negative controls and OF-enriched samples could be clearly distinguished from each other. However, the naturally excreted level of sAA in urine presented a major challenge. A proof-of-concept study revealed an intersection between individuals with naturally occurring high levels of sAA in urine and those with low levels despite contaminations with OF, e.g. due to degradation processes caused by high concentrations of proteases in OF. First follow-up experiments demonstrated that peptides of the protein “salivary acidic proline-rich phosphoprotein 1/2” could be used as complementary biomarkers, but further research is required to confirm and subsequently optimize this approach in terms of its applicability.

Results of the project were presented at the 41st Cologne Workshop on Doping Analysis 2023, 26.2.-03.03.2023.

Code: 20A16DE

Peptides, IGF-1 and insulin analogues have significant ergogenic properties, however, are very difficult to test for in all samples currently tested for in doping control. To date, only a limited number of specific and targeted samples are tested for these substance. We have developed a high-throughput mass-spectrometry method that will enable anti-doping organisations to screen for large peptides, e.g., GHRHs, insulin analogues and IGF-1 on all samples. This method will enable all WADA-accredited laboratories to effectively screen for these substance.

 1.    To validate a high throughput, sensitive, and inexpensive method to screen several classes of large peptides in urine.
2.    To validate a similar method in serum and plasma.
3.    To test the method for detection of insulin analogues from urine and blood samples from diabetic patients.

Code: 20A13MT

The detection of novel doping-related substances is challenging for doping control laboratories, especially if these compounds are also produced endogenously by the common human metabolism. In these cases not only the detection of these compounds or related metabolites is sufficient but (semi)quantification becomes necessary followed by confirmation of the exogenous source of the urinary metabolites by isotope ratio mass spectrometry. This procedure is well known for testosterone and its metabolites.

The novel doping agent under investigation in this study will be the 11-keto derivative of testosterone and its urinary metabolites. 11-Ketotestosterone (11KT) is described as a potent anabolic doping agent that is of special interest as it additionally showed the ability to lower cortisol levels. It is easily available via internet-based providers and at the moment no analytical strategy exists to detect its misuse. 11KT is closely related to a prohormone called adrenosterone (androste-4-ene-3,11,17-trione) but the proposed method for detection of adrenosterone will not be suitable for 11KT as it is only focusing on the major metabolites also produced in large amounts in the adrenal glands strongly limiting the sensitivity of this approach.

Within this research project, all currently known metabolites of 11KT will be taken into consideration to identify the most promising candidates for doping controls. First, the initial testing procedure will be developed to semi-quantitate the urinary amounts of all compounds of interest. Secondly, a confirmation procedure based on IRMS will be developed and validated. Both methods will be used to investigate an excretion study performed with 11KT in order to identify those metabolites most suitable for sports drug testing. This research will be completed by the investigation of routine doping control samples enabling to identify urinary baseline concentrations and carbon isotope ratios at natural abundance.

Main Findings

In this study, the human metabolism of KT was investigated in order to provide additional means for the detection of KT and its prohormone OHA4. Two volunteers (one female and one male) orally administered 20 mg of KT each, and urine samples were collected for 5 days. Urinary concentrations of KT and its metabolites were investigated, and a reference population encompassing 220 male and female athletes was investigated in order to elucidate preliminary thresholds. As confirmation procedure, an isotope ratio mass spectrometry-based method was developed in order to determine the CIR of KT and relevant metabolites. The developed methods enabled the detection of exogenous KT for more than 20 h after a single oral administration, which is comparable to a single oral testosterone administration.

Code: 20A12LM

Current illicit doping practices include the use of peptide hormones and their peptide release factors, with different kinds of activity (gonadotrophic, corticotrophic, growth factor, etc.), with the common purpose of increasing sport performance. These substances are included in the WADA Prohibited List, section S2. One of the main analytical hurdles for the detection of peptides is their low stability in blood (and other biological fluids). This makes sample storage and shipping critical steps, possibly causing the decrease of peptide levels to amounts that are no longer detectable due to degradation.

Dried microsampling provides logistic and analytical advantages over fluid samples: water loss can effectively stop most degradation and biotransformation reactions, leading to higher peptide stability and to more favorable, cheaper storage and transportation conditions. Among microsampling methods, dried blood spots (DBS) is the most well-known and widely applied, but more innovative alternatives are also available (“smart DBS”), such as special cards and devices that obviate some drawbacks of “classical DBS”, like dependency of sampling volume on haematocrit and lack of sampling accuracy. Other dried microsampling approaches, such as volumetric absorption microsampling (VAMS), exist as well. Following the promising results obtained from stability studies on doping-relevant peptides in urine microsamples (Project funded within the 2017 Scientific Research Grants, successfully developed and concluded by this research team), aim of the present project is to carry out a systematic study on the stability of prohibited peptides in dried blood spots. The most important variables involved in the sampling process will be studied, such as humidity, temperature and light exposure, to determine optimal sampling, storage and shipping conditions, and to evaluate the results obtained from microsamples.

The project goal is to establish feasible and reliable workflows for dried blood microsample collection, which could be proposed as effective strategies for anti-doping testing.

Main Findings

Background – With the introduction of dried blood spots (DBS) into official anti-doping testing workflows, scientific studies on their performance have been increasing. DBS-based analytical platforms can be advantageous over conventional methods of blood drawing and handling. Increased analyte stability due to lack of water (and consequent increased reliability of delayed analyses) is one of the most attractive features of dried microsampling, however it has to be verified for each analyte or at least for each chemical class of analytes. Project aims – Prompted by the results obtained from previous WADA-funded projects regarding doping-relevant peptide stability in urine-derived dried micromatrices, we have undertaken the task of assessing the mid-term (3-month) stability of 19 peptides in DBS. Both classical and “smart” DBS platforms were tested; the latter are based on microfluidics to obtain fixed-volume DBS from blood drops. Peptide stability in DBS stored under subpar and worst-case conditions was also evaluated. Results were compared with stability data obtained from plasma samples stored at -20 or -80°C (Figure 1). Results – Validated, original LC-MS/MS methods were developed for the simultaneous determination of the 19 analytes. Using them, peptide stability was reliably evaluated. Stability in DBS stored at room temperature was always good (78-84% recovery) and significantly (5-35%) higher than stability in plasma stored at freezing temperatures. It was also observed that subpar and worst-case handling and storage conditions can have a noticeable impact on analyte stability in DBS (up to 15% lower after 90 days), without compromising overall reliability and storage, handling, and cost advantages. Conclusions – The one-year project was successfully completed according to the planned timeline. Innovative microsampling, preparation and analysis platforms were developed, based on classical and microfluidic DBS exploitation. The results of mid-term stability assays confirmed that the dried microsampling approach is a viable alternative to classical venipuncture and plasma analysis, providing better stability and sampling feasibility coupled to considerably lower shipping and storage costs. Future plans – A systematic study on the long-term (1+ years) stability of peptides in DBS would be the natural prosecution of the present project. Other forms of microsampling could also be tested, from volumetric adsorptive (VAMS) devices to microfluidic or membrane-based devices for the automatic creation of dried plasma spots (DPS) from blood drops to water-soluble supports, and others. Finally, more peptides could be added to the current analytical panel, to increase the validity and significance of the produced results for anti-doping testing advancement.

Code: 20A07MT

All doping agents that may also be produced endogenously, i.e. as part of the human metabolism, necessitate further investigations in order to differentiate between a doping rule violation and endogenous production. Testosterone is the prime examples for this procedue and in many cases, only the determination of carbon isotope ratios (CIR) allows for an unambiguous discrimination between the endogenous production of elevated amounts of testosterone and the administration of this doping agent. A similar approach has been established for boldenone, a so called pseudo-endogenous compound, which is not produced by everyone but can occasionally be found in urine specimens. In such cases, the determination of CIR is the best solution to identify the source of these steroids.

Recently, the World Anti-Doping Agency (WADA) has identified several new pseudo-endogenous steroids that should be addressed with caution by doping control laboratories and strongly recommended the use of CIR to identify the origin of these agents. For the majority of compounds (1-androstenediol, 1-testosterone, 1-androstenedione, 1-androsterone and androstatrienedione), no isotope ratio mass spectrometry-based methods exist, so the aim of this research project is the development and validation of these approaches. For the steroid 66α-hydroxy-androstenedione, a method has already been published, but is currently not fully validated according to the recently established WADA guidelines. Therefore, this compound will also be included in this study.

Following method optimization and validation, EQAS and/or excretion study samples already present in our laboratory will be analyzed as proof-of-concept together with samples derived from the routine screening showing low concentration of the steroids under investigation.

Main findings

Our study confirms that the ratio 6OH-Etio-3G/EG can complement the results obtained by T/E for testosterone administration. The ratio 6OH-Etio-3G/EG was found to be subject to a high intraindividual vatiation, therefore making the selection of a threshold based on its variance very difficult. On one hand, a proper isotopically labeleled internal standard Is still needed to reduce the analytical variability. On the other, there must be a circadian variabllity that would require adding other markers to compensate for it. Finally, both T/E and 6OH-Etio-3G/EG were not able to differentiate between alcohol and testosterone administrations under the mild conditions used (transdermal testosterone and low ethanol dosage).

Code: 20A06MP

Since the detection of the latest long-term metabolite of metandienone a lot of effort is put in the uncoverage of similar metabolites with 17β-hydroxymethyl-17α-methyl-13-ene structure for long-term detection of other 17-methyl steroids. The closely related 4-chlorometandienone (Oral Turinabol, DHCMT) was found to be excreted as analogous metabolites, with A-ring reduced metabolites being even longer detectable.

Thus, in the current project we aim to investigate the excretion of metandienone with special respect to A-ring reduced metabolites as well. Their integration in doping control analysis may further extend the detection window of a misuse of metandienone and open new possibilities to catch cheaters that adjusted their habits to experiences after the integration of 17β-hydroxymethyl-17α-methylandrosta-1,4,13-triene-3-one into routine screening. Deduced from DHCMT metabolism, it is expected that also after metandienone administration a potentially further increased detection window may be achieved from screening for the analogue metabolite 17β-hydroxymethyl-17α-methyl-18-norandrost-13-en-3-ol.

Code: T20M04JB

Salbutamol is a fast- and short-acting β2-adrenergic receptor agonist, indicated for the treatment of asthma attacks and the prevention of exercise-induced bronchospasm. It is one of the medications most frequently used by athletes, generally for its bronchodilating effect, but also sometimes for its ergogenic properties. Some evidence indicates a positive effect of systemic salbutamol on physical performance while no significant effect is demonstrated for inhaled salbutamol [1-3]. After oral administration, salbutamol undergoes a significant first-pass metabolism. It is predominantly metabolized by SULT1A3, expressed primarily in the intestine [4], into an inactive sulfoconjugated metabolite [5]. After oral intake, a roughly one third of the dose is excreted in the urine unchanged (free), and one half as sulfoconjugate [5]. Another small fraction (<3%) is found as glucuroconjugated metabolite [6]. Conversely, salbutamol is not extensively metabolized in the lungs [7] and after inhalation, the fraction of the dose actually absorbed in the circulation through this route is mainly eliminated in the urine as the free form, while a low proportion of sulfoconjugate and a negligible fraction of glucuroconjugated metabolite are excreted [6]. However, the remaining fraction administered by inhalation settles along the oral cavity and the throat or is carried back from the tracheobronchial by mucociliary clearance, thus resulting into ingestion and gastrointestinal absorption.

Main findings

The objective of this work was to evaluate the capacity of the current WADA approach to differentiate salbutamol therapeutic use from violation. A population PK analysis including both individual and 
aggregate data from the literature was developed using up-to-date MBMAtechniquesto characterize salbutamol plasma and urine PK profiles after inhalation or oral administration, under various dosing 
regimens and conditions.

In conclusion, although not entirely satisfactory, the current WADA rules regarding the definition of AAFs related to salbutamol could be globally maintained, and possibly improved by a slight and 
reasonable modification of inhalation dosages allowed in therapeutic exemption. Moreover, the use of such a model might help WADA experts in their evaluation of individual AAFs through the 
confrontation of the athlete’s allegation about treatment intake.

Code: T20M01MT

Higenamine, also known as norcolaurine, is a non-selective β2-agonist naturally occuring in different plants such as Nandida domestica, Tinospora crispa, and Annona quamosa [1-5]. Due to its bronchodilatative and stimulating effects, the misuse of higenamine in sports is prohibited at all times [6]. According to the relevant WADA technical document, a reporting level of 10 ng/mL (50% of the MInimum Required Performance Level (MRPL)) applies for the detection fo β2-agonists in doping control urine samples [7]. From 2016 till 2019, 201 samples werer reported as an adverse analytical finding (AAF) for higenamine [8-11].

Although higenamine has never been approved as a drug by the US Food and Drug Administration (FDA) [11], it plays an important role in traditional Chinese herbal medicine [1, 4]. Moreover, it was found tto be an (un)labeled ingredient of different weight loss and sports supplements [2-4, 12], which have caused several cases of unintentional doping during the last years [13-18].

As also some tropical fruit plants of the Annonaceae family were founhd to contain higenamine [2, 5], the aim of this study was to investigate whether the ingestion of such fruits can lead to AAFs in sports. The results of this reserach project can be of great value for anti-doping routine work, as they will help to ensure fair result management and decision-making processes in case of higenamine findings in sports drug testing programs.

Therefore, elimination studies were conducted in order to characterize the time-dependent urinary excretion of higenamine. Previous unpublished reports for L. C. Cameron's laboratory showed that the consumption of A. muricata (n=3) or A. squamosa (n=4) produced detectable higenamine in 100% of the subjects' urine (n=7). Otherwise, higenamine was not detectable in the C. papaya (control) group (n=3). In total, two administration studies with single dose of a fruit puree from Anonna muricata (330 g) and Anonna cherimola (330 g) were conducted, and the collected urine samples wre analyzed by means of liquid chromatography - tandem mass spectrometry (LC-MS/MS). HIgenamine detection times in general and, specifically, urinary concentrations of higenamine were desirable in support of an improved interpretation of AAFs, especially when scenarios of proven supplement contamination are debated and supplement administration protocols exist.

Main findings

The β2-agonist higenamine is prohibited in sports at all times and available, amongst others, as ingreditnets in various dietary supplements.
Further, a variety of plants including tropical fruits of the Annonaceae family have been reported to contain higenamine, and the aim of this study is to investigate whether the ingestion of such fruits can lead to AAFs in sports. For that purpose, single-dose elimination studies were conducted with A. muricata and a second higenamine-containing fruit (A. cherimola). Post-administration urine samples were analyzed concerning their higenamine content and assessed with regards to the currently enforced reporting level of 10 ng/mL.
All study volunteers produced urine samples containing higenamine after ingestion of the fruit preparations; however, under the chosen conditions, all observed urinary concentrations ranged exclusively below 5 ng/mL and, thus, the established MRL.

The herein obtained data can be considered in result management and decision-making processes in case of higenamine findings in sports drug testing programs. The outcome supports the position that single-dose administrations of these fruit species are rather unlikely to lead to AAFs in sports. Yet, substantial variability of the natural higenamine content in fruits exists, potentially influenced by seasonal/spatiotemporal factors, and also fruit processing and storage might affect the overall dietary higenamine availability. Hence, whilst less likely, it cannot be excluded that under specific circumstances the current MRL is exceeded by nutrition-derived higenamine uptake.

Code: DBS20AS1AT

Due to the aimed implementation of dried blood spot (DBS) sampling in doping controls for the upcoming Olympic events (Summer Olympic / Paralympic Games 2020 in Tokyo and Winter Olympic / Paralympic Games 2022 in Bejing), several targeted research projects were initiated by WADA’s DBS steering committee early 2020. To support realization of the ambitious timeline, the following projects are being applied for as targeted research. The first subproject deals with the stability of DBS samples. Of particular interest is how the stability of the substances in the dried state appears on the paper of the card. Factors such as temperature, light, and humidity should also be considered. Several model compounds from various classes of prohibited substances will be included in this study.

Main Findings

The stability of prohibited substances on dried blood spots (DBS) is generally assumed as superior to the storage under liquid conditions. Nevertheless, stability is not warranted per se for all target analytes and all conditions. Under consideration of the reanalysis of samples that were fortified with model compounds (originally stored for 365 days at room temperature, 4 °C or -20°C with/without desiccant) from various classes (anabolics, peptide hormones, β2-agonists, metabolic modulators, diuretics, stimulants, narcotics, glucocorticoids and β –blockers) and subsequently stored for another two years at -20°C showed excellent stability. All model compounds included were still detectable. Additionally, the potential impact of an intercontinental flight was found to be negligible for the selected model drugs. Another newly performed stability study over five months (at room temperature, 4 °C or -20°C with/without desiccant) with new model compounds (incl. HIF-stabilizers etc.) showed that storing at room temperature without desiccant and exposure to light will cause considerable losses for some of the highly volatile compounds. Results for storing at 4°C and -20°C showed equivalent results to the formerly performed study. Thus, storage at 4° C (or -20°C) with desiccant in the dark is recommended for long-term storage of DBS doping control samples.