Projets de recherche

Code: 18A28PA 

The aim of this project is to produce certified reference materials (CRMs) to ensure the accuracy and traceability of measurements of the stable carbon isotope ratios of steroids used to confirm Adverse Analytical Findings (AAF) in sports doping analysis. These CRMs will be used for validation of GC-C-IRMS methods and confirmation of Adverse Analytical Findings in accordance with WADA Technical Document TD2016IRMS. The ability of WADA-accredited laboratories to comply with this document is reliant on the availability of reference materials of appropriate steroids certified with traceable values for 13C isotope ratios. The availability of such materials is currently limited.

The proposed substances are boldenone, boldenone M1 and formestane.  Certified values for the δ13C values of each steroid and their associated measurement uncertainties will be determined by a combination of reference measurements with metrological traceability to VPDB made by NMIA using Elemental Analysis (EA-IRMS) and Gas Chromatography (GC-C-IRMS) Carbon Isotope Ratio Mass Spectrometry.

Main Findings: 

Two new CRMs have been prepared providing three steroids certified for stable carbon isotope delta values (δ13CVPDB). These materials have been designed to assist anti-doping laboratories to validate their calibration method for stable carbon isotope measurements of boldenone, boldenone metabolite and formestane to ensure accuracy and traceability in compliance with WADA Technical Document TD2019IRMS. The CRMs are packaged as dried steroids sealed in ampoules. MX020 consists of a mixture of boldenone and boldenone metabolite 1 and MX021 contains a single analyte, formestane.

Elemental Analyser Isotope Ratio Mass Spectrometry (EA-IRMS) was employed as the priamry reference method to assign δ-values for pure steroid starting materials due to the low uncertainty associated with this technique. Calibration was performed using a two-point normalization approach [2] which employed USGS-40 and IAEA-CH-7 as calibration standards, permitting traceability to the internationally recognized Vienna Pee Dee Belemnite (VPDB) carbon isotope reference standard. Potential bias in the assigned EA-IRMS values was investigated both through theoretical modelling using the steroid starting material’s purity data and through data obtained using the gas chromatography coupled to combustion isotope ratio mass spectrometry (GC-C-IRMS). Calibration and normalization of the GC-IRMS results were performed using the MX018 certified steroid standards with traceability to the VPDB reference. Homogeneity assessments of MX020 and MX021 were carried out using fifteen randomly selected ampoules. Stability of the CRMs were verified by analysis of randomly selected ampoules after storage at 4 °C for a period of upt to 5 months and at 40 °C for a period up to 30 days.

Code: ISF18R01AB 

In this project, we propose to perform additional validation of the EPO gene doping test with blood samples on the conditions for samples storage.  This includes factors affecting centrifugation and thawing, assay performance, improve separation of Tm peaks, storage temperature.  The assay will be validated with the optimal conditions.

The further validation of the EPO gene doping test will facilitate completion of the Technical Document for gene doping detection in blood and implementation of the test in the near future.

Main Findings: 

We performed additional validation of the polymerase chain reaction (PCR)-based erythropoietin (EPO) gene doping test using blood samples. We tested various conditions for prolonged sample storage and determined storage temperatures and duration, as well as sample processing prior to freezing, which do not compromise reliability of detecting the EPO doping gene in blood. We also improved test reliability by using a more suitable form of Uracil-DNA Glycosylase in PCR mix and tested other modifications to the test protocol with the intention to make the test easier and more legally defensible.
The results and outcomes from this project were incorporated in the Test Protocol. We conclude that the PCR-based EPO gene doping test using whole blood samples was fit to be implemented in doping control.

Code: 18A17MP 

The identification of anabolic androgenic steroids (AAS) is a vital issue in doping control. Due to the performance enhancing properties of AAS, the World Anti-Doping Association (WADA) banned their use but according to the annual report of WADA, steroids are still very popular amongst athletes and are responsible for 40% of all adverse analytical findings. The search for metabolites with longer detection times remains an important task and the introduction of new long-term metabolites for exogenous AAS such as for example stanozolol, methanedione and dehydrochloromethyltestosterone, led
to a 4 - 80-fold increase of adverse analytical findings due to the prolonged detection time.

Recently, our laboratory developed a new strategy for finding new long term metabolites. The approach uses low energy EI GC-QTOF and it is applicable to a wide range of different anabolic steroids. The technique has been tested in a proof-of-concept setting and the combination with a robust polar column allows the analysis of both derivatized and non-derivatized steroids, further expanding the application window and increasing the chances of finding new long term metabolites.

Main Findings: 

A search for new metabolites was performed by applying the low energy EI GC-QTOF product ion scan strategy. To increase the chances of finding new long-term metabolites this strategy was applied using different (complementary) approaches: Anaylses of both the derivatized and non-derivatized steroid form in the glucuronidated sulfated and free steroid fraction.

The main conclusion is that the strategy suffers from a lack of sensitivity that cannot be sufficiently compensated by the increased selectivity. Unfortunately, this meant that the procedue proved to be inferior to the MRM CI GC-MS/MS approach for detection of new metabolites (WADA project 16A01MP). Consequently, ne new metabolites could be found within this study and the approach failed to find many of the metabolites that were detected with the MRM CI GC-MS/MS detection protocol.

However, during this study we did acquired more knowledge on the analysis of sulfated steroids on GC-MS and this provided us with some new insides on their GC-MS behavious, prompting us to pursue a more in depth study of directly injecting non-hydrolyzed sulfated steroid on GC-MS. We are convinced that this direct injection strategy has great potential to, in the future, lead to the discovery of new long term metabolites and/or will allow the inclusion of long term sulfated metabolites in a general GC-MS initial testing procedure.

Code: 18C05CR

Chapter S2 of WADA’s Prohibited List 2018 (“Peptide hormones, growth factors, related substances, and mimetics”) lists EPO-Fc under sub-chapter 1.1 (“Erythropoietin-Receptor Agonists”). The current version of WADA’s technical document on ESA-analytics (TD2014EPO) describes general criteria of positivity for ESAs (e.g. rEPO, NESP, CERA). Since EPO-Fc cannot be directly detected by IEF-PAGE, SDS- or SAR-PAGE has to be used. However, chapter 2.1.2.4 "EPO-Fc" of the technical document does not define detailed criteria. The reason is that no data from human administration studies of EPO-Fc exist. Hence, it is unclear if both bands, which are typically observed for the standard (the strong band of the monomer AND the weak band of the dimer), have to be present for an adverse analytical finding.

So far, no approved EPO-Fc pharmaceuticals are available. Hence, administration of EPO-Fc to human test persons will be ethically not justifiable. For that reason we plan a study with rats. The test animals will receive EPO-Fc at a dosage, which can be still clearly detected after 48 hours in serum (160 μg/kg) according to literature.  Subsequently, serum and urine will be collected and tested for EPO-Fc by SAR- or SDS-PAGE. The study will help to clarify if (1) both bands of EPO-Fc are still observable after 48 hours of circulation in blood, and (2) EPO-Fc can also be detected in urine. Based on these results, more precise criteria for EPO-Fc might be specified in TD2014EPO.

Main Findings:

An administration study of EPO-Fc was performed with rats. After a sungle subcutaneous injection of 160 μg/kg EPO-Fc, serum and urine samples were collected and analyzed by SAR-PAGE after immunoaffinity purification. EPO-Fc could be clearly detected in just 10 μl of serum. No degradation products or interferences due to non-specific binding were observed. Both, the bands of the monomeric and dimeric EPO-Fc were detectable in all samples. EPO-Fc was also found in the urine samples, but at a muc lower concentration. However, additional bands were also observed, which might be degradation products of EPO-Fc. In conclusion, blood should be the preferred matrix for detecting EPO-Fc in doping control samples. The simultaneous detection of both bands (monomer and dimer) on SAR- or SDS-PAGE should pose the main criteria for the presence of EPO-Fc, when these methods are applied.

Code: 18B09CR

Chapter S4 of WADA’s Prohibited List 2018 (“Hormone and metabolic modulators”) lists myostatin inhibitors under sub-chapter 4 (“Agents modifying myostatin function(s)”). Follistatin (FST) suppresses signaling of myostatin and subsequently leads to an increase in muscle mass and loss of body fat. FST is a secreted glycoprotein, which can be found in many tissues and organs (e.g. pituitary, bone marrow, ovary, kidney, liver, blood vessels). Due to alternative splicing, three FST-isoforms exist (FS-288, FS-300, and FS-315). The isoform with 315 amino acids (FS-315) is the dominant one. FS-315 can also be detected in blood. Typical concentrations in serum and plasma are in the range of 2-3 ng/mL.  

So far, no approved follistatin pharmaceuticals are available.  Nevertheless, follistatins can be bought from many internet providers for “research purposes”. Their products are labelled either “Follistatin”, “Follistatin 344”, or “Follistatin 315”. Most of these proteins are expressed in E. coli and hence lack the characteristic glycosylation of human endogenous follistatins. This fact will be exploited in order to detect doping with follistatins. After immunoaffinity purification (serum/plasma, urine), FST will be separated by electrophoresis (SDS-, SAR-, or IEF-PAGE) and detected by Western blotting. Due to the missing glycosylation, “black market” FSTs will not only differ in molecular mass but also isoelectric point (pI) from the endogenous FSTs.

Main Findings:

Follistatin (FS), a myostatin-inhibiting protein, is prohibited according to chapter S4 of the "WADA Prohibited List 2022". While currently no approved pharmaceutical formulations of Follistatin are available, Follistatin can be bought on the black market. Most of the products are labelled "Follistatin 344" (FS344), few "Follistatin 315". A study on FS344 black market products was performed and an electrophoretic detection method for serum and urine developed. While only 9 of the 17 tested products actually contained Follistatin, in some of the others growth promoting peptides were found (e.g. MGF, GHRP-2). Suprisingly, all nine products contained His-tagged FS344 and a high degree of its oligomoers. The detection method is based on immunomagnetic purification followed by SDS-PAGE and Western bloting with a monoclonal anti-His antibody. Alternatively, a monoclonal anti-FS antibody can be used. For immunoprecipitation (IP), a polyclonal anti-Follistatin antibody is applied. An evaluation of suitable antibodies for IP and immunoblotting is also presented. Furthermore, practicall all currently available Follistatin standards were investigated. The detection limit of the method for back market FS344 in urine is ca 0.1 ng/mL for 10 mL. For a sample volume of 100 μL, an LOD of 5 ng/mL could be achieved for serum. Due to the presence of His-tags an unambiguous differentiation from endogenous Follistatin is possible.

Code: 18C02MT 

Testosterone misuse still remains a challenging task for doping control laboratories as this steroid is produced naturally by each and everybody. As it is detectable in all urine samples, concentration-based thresholds have been established to uncover testosterone administration. As soon as these thresholds are exceeded, samples are forwarded to isotope ratio mass spectrometry determinations (IRMS) to elucidate the steroid´s source and to unambiguously differentiate between naturally elevated testosterone concentrations and doping.

Recently a promising new target analyte for IRMS determinations was reported, epiandrosterone (EPIA). This steroid enables to prolong the detection of a single testosterone or testosterone prohormone administration from 24 h to more than 100 h using IRMS. Unfortunately, EPIA is only excreted into urine in it sulfoconjugated form while all other steroids routinely employed in IRMS are excreted glucuronidated. To investigate EPIA an additional time consuming step in sample preparation is inevitable.

The aim of this study is to investigate the potential of two other possible long term markers in IRMS excreted glucuronidated and therefore avoiding the additional step. The so called “Epidiols” are known long term metabolites of epitestosterone and might also work for other steroid administrations. In a preliminary study employing dehydroepiandrosterone both Epidiols were remarkably influenced by the steroid administration supporting the hypothesis that these new markers will improve steroid detection by IRMS. To further substantiate this finding we are going to re-analyze other excretion studies after improving and validating the already existing method for IRMS determinations of both Epidiols.

Main Findings: 

A novel IRMS method for determination of carbon isotope ratios (CIR) of 5a- and 5bEpiD was developed and fully validated in line with WADA requirements encompassing limits of detection, linear range and absence of isotopic fractionation. By means of investigations on a reference population encompassing n=72 individuals it was possible to derive referece-based decision limits, which demonstrated that the novel method is fit-for-purpose. Several administration trials were investigated to elucidate the potential of both markers. For a single oral T administration, the effect on both 5a- and 5bEpiB was less pronounced compared to traditional markers and offreed merely short detection windows. After consecutive transdermal T-Gel administrations, especially 5bEpiD was significantly influenced while 5aEpiD shows similar CIR,s as 5a- and 5bDIOL. Unfortunately, the first sample after cessation of T-Gel administration was collected after 5 days, and here all CIR values had returnedd to natural abundance. The administration of a single oral dose of androstenedione resulted in a prolonged depletion of CIRs for 5a- and 5bEpiD compared to other target compunds, but the influence was less pronounced, i.e. the CIR values were not found to be influenced beyond the established reference-based decision limits.

While a direct application of 5a- and 5bEpiD as long-term markers for steroid administrations appears to offer limited added value, these additional target compounds may be beneficial in detecting multiple transdermal T-Gel administration even if so called low-doses are applied. Moreover, they could support the differentiation between single administrations (including inadvertent exposure) and multiple and therefore intended administrations. Further research will be necessary to elucidate this potential of 5a- and 5bEpiD.

Code: 18A03MT 

Ghrelin and Ghrelin mimetics belong to the prohibited substances according to the current WADA Prohibited List. After administration, they induce the secretion of growth hormone into the circulation and are considered as performance enhancing accordingly. While Ghrelin itself is an endogenous peptide hormone, Ghrelin mimetics (such as Capromorelin, Macimorelin and Tabimorelin) are low molecular mass synthetic peptides (or peptide similars) which act as agonists at the ghrelin receptor. Although prohibited, efficient detection methods for these drugs are scarce. Thus, it is planned to develop analytical methods to determine the potential misuse of the new hGhrelin mimetics (Capromorelin, Macimorelin and Tabimorelin) in doping control samples. This will include the investigation of efficient extraction procedures, metabolism studies (in-vitro/ in-vivo) and identification of reliable target analytes in the respective matrix. Results will be validated and published.

Main Findings: 

Analytical methods to determine the potential misuse of the ghrelin mimetics capromorelin (CP-424,391), macimorelin (macrilen, EP-01572), and tabimorelin (NN703) in sports were developed. Therefore, different extraction strategies, i.e. solid-phase extraction, protein precipitation, as well as a ‘dilute-and-inject’ approach, from urine and EDTA-plasma were assessed and comprehensive in vitro/vivo experiments were conducted, enabling the identification of reliable target analytes by means of high resolution mass spectrometry. The drugs’ biotransformation led to the preliminary identification of 51 metabolites of capromorelin, 12 metabolites of macimorelin, and 13 metabolites of tabimorelin. Seven major metabolites detected in rat urine samples collected post-administration of 0.5-1.0 mg of a single oral dose underwent in-depth characterization, facilitating their implementation into future confirmatory test methods. In particular, two macimorelin metabolites exhibiting considerable abundances in post-administration rat urine samples were detected, which might contribute to an improved sensitivity, specificity, and detection window in case of human sports drug testing programs. Further, the intact drugs were implemented into World Anti-Doping Agency (WADA)-compliant initial testing (LOD: 0.02-0.60 ng/mL) and confirmation procedures (LOI: 0.18-0.89 ng/mL) for human urine and blood matrices. The obtained results allow extending the test spectrum of doping agents in multi-target screening assays for growth hormone-releasing factors from human urine.

For ghrelin and its desacylated analog, a quantitative test method with an LOD of 20 pg/mL and an LOQ of 100 pg/mL was developed and subsequently, a representative population of athletes’ blood samples was analyzed. The intact (non-digested) ghrelin exhibited poor chromatographic properties due to the presence of 7 basic amino acids in the sequence and, therefore, the approach was designed to include an enzymatic hydrolysis step which yielded the tryptic peptide t1 with 11 amino acids and only one Arg residue at the C-terminus. In accordance with literature data, the analyzed plasma samples from professional athletes yielded concentrations between 26 and 177 pg/mL, which is significantly lower than concentrations reported for ghrelin post-administration plasma samples that reached peak levels of 17500 pg/mL in clinical studies.

Code: 18A16AK 

In anti-doping control, the detection of steroid conjugates is of increasing relevance. The wide detection window of certain conjugates in combination with efficient and highly sensitive LC-MS technology enables a prolonged traceability of hormone misuse. The reliable quantitation of steroid conjugates requires isotope-labelled reference materials, which are only available for a minority of doping relevant conjugates. An approach to generate steroid sulfates is the in vitro incubation with human liver S9 fraction.

The objective of the present study is to produce isotope-labelled steroid sulfates in vitro. This will be accomplished by the incubation of relevant steroids or their metabolites with liver S9 fraction and 34S-sodium sulfate. The in vitro generated reference material can be directly used for dilute-and-shoot quantification of the respective steroid sulfates in human urine samples. Moreover, the established approach could be rapidly expanded to comparable steroids and methods or materials may be shared with other WADA accredited laboratories.

Main Findings: 

The project aimed at the generation of 34S-labelled epiandrosterone sulfate as internal standard for doping routine analysis. This in vitro-synthesis should be accomplished by incubation of epiandrosterone with liver S9 fraction and 34S-labelled sodium sulfate as co-factor. Results: We introduced a modification of a previously described protocol which replaces phosphoadenosin-5’-phosphosulfate by sodium sulfate and adenosine-5’-triphosphate in the S9 fraction incubation. By using isotope-labelled sodium sulfate (Na234SO4), we generated 34S-labelled epiandrosterone-sulfate with a purity of ≥99.9 %. With this purity, the in vitro generated steroid sulfate fulfills the requirement for an internal standard for quantitation purposes. The sulfonation rate was found to be very low (13 %) and attempts to increase the reaction yield were not successful. 
Conclusions: In conclusion, we successfully introduced a protocol to generate isotope-labelled epiandrosterone sulfate with purity. Due to the low percentage of the epiandrosterone sulfonated by the S9 fraction, we were not able to provide the intented amount of min. 5 mg epiandrosterone-34SO4. Future plans: As the modified protocol was proven to generate 34S-isotope-labelled epiandrosterone, it is possible to expand investigations on comparable steroids. Future investigations should focus on the optimization of the sulonation rate of the subsrate.

Code: 18B04AM 

The increasing number of biosimilars of the first generation EPOs (epoetin alfa and beta) produced all over the world raise the question of their detection. Due to small structural changes, for some EPO Biosimilars the identification criteria edicted by WADA for rEPO may not always be reached, which would render their use by athletes undetectable. Preliminary results have identified Hemax and Epotin authorized in Algeria and Jimaixin authorized in China as rEPOs with apparent molecular weights lower than the original epoetin alfa (Eprex) and closer to endogenous EPO. Thus identification using SDS/SAR-PAGE method could be problematic while their IEF profiles always remain very basic and distinct from endogenous EPO. To go further, evaluating detection from samples resulting of a real administration in healthy subjects is needed.

The objectives of this project are:
- to analyze blood and urine samples using IEF and SAR-PAGE methods according to the TD2014EPO and to compare identification capacities and establish the window of detection for each rEPO tested.
- to test complementary strategies to improve detection of EPO biosimilars: neuraminidase treatment that has been shown to improve the migration distance between rEPO and endogenous EPO as well as a 2-D separation gel approach (mixing IEF and SDS separation) will be tested by AFLD on samples that show a problematic rEPO identification.

In addition specific glycosylations of each Biosimilar will be characterized by LC-MS coupled to fluorescence.

Main Findings: 

Recombinant erythropoietin (rEPO) biosimilars are generic epoetin drugs developed following an expired patent. All licensed EPO biosimilars shall demonstrate the same saferty and efficacy for therapeutic use as the original drug but small structural differences compared to the reference product due to some variations in the production process can be accepted. After analyzing various EPO-biosimilars, three different kinds were selected for further characterization due to their slightly lower apparent molecular weight (MW) compared to the original epoetin alpha drug Eprex®.  Jimaixin™ authorized in China, and Hemax® and Epotin™ authorized in Algeria.

The aims of this research were:
i) to study the electrophorectic profiles obtained by IEF and SDS-PAGE of the three Biosimilars spiked in urine and plasma and to evaluate their identification following WADA's criterai (TD2014EPO),
ii) to test complementary strategies to improve detection of EPO biosimilars using a two-dimensional electrophoresis approach (SDS separation following IEF) and a neuraminidase treatment of the sample shown to increase the separation between recombinant and endogenous EPO by SDS-PAGE,
iii) to evaluate by mass spectrometry the specific N-glycosylation pattern of each biosimilar and compare with the original rEPO Eprex®. 

Experiments with spiked urine and plasma samples showed that samples spiked witht he Biosimilars were more challenging to identify for rEPO compared to Eprex in particular at low amount. Differences were seen according to the method of identification used and the biosimilar spiked. Epotin and Jimaixin were more difficult to identify by IEF compared to SDS-PAGE while it was the opposite for Hemax. The SDS-PAGE method applied to urine samples had the higher identificaiton rate considering the three biosimilars. Two-dimensional electrophoresis experiments did not improve the detection. This analysis proved complex to perform and no clear criteria could be used to identify rEPO.

Samples pre-treated with Neuraminidase gave promising results. Using the SDS-PAGE, the EPO bands were slightly broader compard to untreated samples. Neuraminidase-treated Dynepo could be used as a sparation marker between endogoenous and exogenous signals and this could improve the identification of low doses of biosimilars. By IEF, an interesting pattern for the biosimilars treated with neuraminidase was found. Using a 2-10 pH gradient gel was necessary to detect three thin additional bands inserted between the main EPO isoforms. This characteristic was observed for the three biosimilars while these bands were totally absent in non-spiked samples.

N-glycosylations of Eprex® and the biosimilars were identified by MALDI-TOF and adundance of the various glycan forms were compared.
All three biosimilars were enriched in bi and tri antennae forms while a decrease was observed in particular for the main glycan forms of Eprex® (tetraantennae tri- and tetra-sialylated forms). Hemax had the glycan profile the closest to Eprex® while Epotin™ and Jimaixin™ presented more loss of sialic acids. These results on N-glycan were in good agreement with the presence of additional basic isoforms in their IEF-PAGE profile.

In conclusion, even if these biosimilars may present some challenges at low concentration, current methods used in the anti-doping laboratories and if necessary an easy-to-implement neuraminidase pretreatment of the samples can assure detection of these compounds

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