Studies of glucocorticoids after oral administration: evaluation of reporting levels and washout periods
By
Investigateur principal
R.
Ventura
Institut Municipal d’Investigacio Medica (IMIM)
Espagne
―
2020
―
En vigueur
Sommaire
Code: T20M03RV
Recent research has demonstrated that the criterion of discrimination between allowed and prohibited administrations of GCs needs to be compound specific. For some GCs, largely detected in doping controls, no sufficient data is available, and the objective of this project is to perform excretion studies with these GCs to generate the data needed to define the reporting levels and the washout periods.
The project will be focused on dexamethasone (DEX), methylprednisolone (MP) and deflazacort (DEF).
The research will be focused on the following specific objectives:
To perform excretion studies of DEX, MP and DEF with administration of one single oral dose. For DEX, multiple oral doses will be also studied.
To measure concentrations in urine of the parent compounds and the main metabolite in case of DEF, to evaluate the reporting lebels and washout periods.
To measure concentrations of the compounds and cortisol in plasma, to evalulate the concordance of the urinary reporting levels and washout periods proposed with plasma concentrations of the active drug and the systemic effect
Establishing and optimizing hGH capture antibody-coated assay microplates as solid phases for the CMZ Differential Immunoassays for hGH Isoforms (CMZ hGH LIA)
By
Investigateur principal
D.
Müller
CMZ-ASSAY GMBH
Allemagne
―
2020
―
En vigueur
Sommaire
Code: T20M02DM
The differential immunoassays to detect doping with recombinant human growth hormone (hGH) have been in use at WADA accredited laboratories for more than 10 years now. From the beginning, the assays were produced in the coated tube (CT) format. In the past, this format, where the specific antibodies are coated to plastic tubes, and the final assay signal is determined by a tube luminometer, was very popular for immunoassays used in laboratory medicine and research applications. However, it meanwhile has been largely abandoned, and the respective assays were switched to the so-called microtiter plate (MTP) format. Here, the antibodies are coated to 96 wells of a plastic plate, and the final assay signal is read by a plate luminometer. The microplate format has advantages in terms of production, but also in terms of workflow in the laboratory. As a consequence of the grossly reduced use of CT assays in laboratories worldwide, production capacities for such CT assays become rare, and might become unavailable in the near future. To ensure continued production and availability of the differential immunoassays it is suggested to convert these assays to the MTP format now. While assay ingredients (and particularly the monoclonal antibodies involved) remain unaffected by the conversion to the MTP format, and it has been demonstrated for many assays used in clinical routine that key parameters such as assay specificity, linearity, reproducibility etc. remain unaffected by the change, the MTP format nevertheless has to be optimized and validated for each individual assay. The current project aims to establish the MTP format for the differential immunoassays.
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.
Evaluation of a novel gene doping detection approach via high multiplex MALDI-TOF MassArray analysis
By
Investigateur principal
M.
Thevis
German Sport University
Allemagne
―
2021
―
En vigueur
Sommaire
Code: 22E05MT
Gene doping has been classified as a prohibited method in the WADA prohibited list for many years. As gene therapeutic approaches improve and concerns regarding their safe clinical application decline, concerns about their illicit use in elite sports are rising sharply. The outlined project aims at the evaluation of a novel gene doping detection approach emplying high multiplex MALD-TOF mass spectrometry (MS). As preliminary work, a prototype panel able of the multiplex detection of transgenic expression of seven relevant gene doping targets (EPO, FST, GH-1, IGF-1 MSTN(-Propeptide), VEGF-A, VEGF-D) has been designed and pre-tested for its general specificity and applicability. Central aim of the proposed project is the in-depth validation of the prototype panel with regard to its specificity, sensitivity, and reproducibility. Futhermore, suitable reference material will be designed and tested taking advantage of the single-base extension and detection technique of the proposed chemistry and detection method. In a final step, the optimized and validated panel and reference material will be tested for its applicability to routine testine procedures with a first set of 1000 (doping control) specimens consented for research purposes. If successful, the described high multiplex gene doping detection panel and MALDI-TOF MS detection approach would allow a time- and cost-effective detection of multiple gene doping relevant targets at once and could thus expand the range of available tests for gene doping detection.
Development of a screening tool to detect identical urine samples within the athlete biological passport based on artificial intelligence
By
Investigateur principal
M.
Thevis
German Sport University
Allemagne
―
2022
―
En vigueur
Sommaire
Code: 22D06MT
Several cases of so-called sample swapping have been detected in recent years. During sample swapping an athlete substitutes the presumable positive doping control sample by another negative urine sample in order to circumvent an adverse analytical finding. For the exchange of urine, a sample from the same individual or from a different individual may be used. While the latter can result in a significantly different steroid profile, employing a urine sample from the same individual collected prior to administration of a doping agent will not show any differences in the steroid profile. Here it can be expected that the stored clean sample will be used several times and that this may result in steroid profiles with an atypical similarity. Two cases of this type of sample swapping have already been detected, but only by chance as the similar samples were analyzed in the same laboratory in a timely manner.
The aim of this research project is the development of a screening tool based on machine learning and artificial intelligence which will enable to detect steroid profiles with an atypical similarity in all steroid profiles determined world-wide in different accredited doping control laboratories. The developed software will be able to search within the existing profiles for similar ones and will be trained to compared new entries to the dataset to existing ones in order to indentify highly similar patterns. Employing artificial intelligence will enable to train the software to become highly sensitive, selective and robust. In order to confirm potentially similar samples to belong to the same athlete, DNA analysis of these urine samples will be applied where applicable.
Further investigations into urinary higenamine – metabolite patterns and stability
By
Investigateur principal
M.
Thevis
German Sport University
Allemagne
―
2022
―
En vigueur
Sommaire
Code: 22C05MT
Higenamine is a non-selective β2-agonist, which has been described as a key component of a great variety of natural plants such as Nandina domestica, Tinospora crispa, and Annona squamosa. Although higenamine has never been approved as a drug by the US Food and Drug Administration (FDA), it plays an important role in traditional Chinese herbal medicine (it has been approved for clinical research by the China Food and Drug Administration - CFDA), and has been found to be an ingredient - often unlabeled - of different weight loss and sports supplements, which has been suspected or proven to have caused cases of unintentional anti-doping rule violations.
Within the scope of a previous elimination study aiming at the urinary quantification of higenamine after administration of tropical fruit plants from the Annonaceae family, it could be shown that single-dose administrations of these fruit species are rather unlikely to lead to AAFs in sports. The aim of this project is to conduct further investigation which complement the previous datasey. For that purpose, stability evaluation of componuds of interest, and specific contribution of the different higenamine conjugates will be performed. Besides, two different administrations studies with a total of 6 healthy volunteers (3 x ♂, 3 x ♀) are planned. Each participant will eat 3 servings on Annona fruit puree with 24 h (8:00 am, 6:00 pm, and next day 8:00 am) and, at after washout, a single dose of two different higenamine-containing nutritional supplements. Urine samples will be collected up to three days following administration in both study phases, and comprehensive analyses are planned to support result management and decision-making processes in case of higenamine findings in sports drug testing programs, preferably by means of new potential biomarkers of plant-derived higenamine administrations.
Development on Confirmation Method for recombinant EPO Analysis in Individuals with c.577del in EPO Gene
By
Investigateur principal
X.
Zhou
National Anti-Doping Laboratory
Chine
―
2022
―
En vigueur
Sommaire
Code: 22B06XZ
Upon our previous research, a variant in the EPO gene can encode a variant EPO (VAR-EPO) which has a similar molecule weight with recombinant EPO (rEPO), thus cannot be distinguished using the current method of rEPO analysis. Though WADA has established a detailed procedure for investigation and further analysis, it is a burden for both laboratories, Testing Authoity and WADA, nevertheless, rEPO abused by this variant carriers can be hardly detected. For this situation, we are going to develop an effective confirmation method that can discriminate the wild-type EPO, VAR-EPO and rEPO directly, by removing or eliminating the detection of VAR-EPO using the VAR-specific antibody. Two methods will be developed and validated in this project:: reverse-normal immunopurification and step-recognition western blotting. The rEPO administration study on individuals who carry this varient will also be conducted. The collected positive urine and blood samples will be used to evaluate the method. With out goals, the new method can not only protect the "clean" athlete with that variant but also effectively penalize the rEPO abused athelete whether he or she carries that variant. Another significant advantage, this method would be easily adopted by other WADA accredited anti-doping laboratories.
A novel photoclick hydrogel electrophoretic and in situ immunoblotting method for EPO-receptor agonists screening
By
Investigateur principal
P.
Chen
Shanghai Anti-Doping Laboratory
Chine
―
2022
―
En vigueur
Sommaire
Code: 22B05PC
Erythropoietin receptor agonists (ERAs), e.g. darbepoetins (dEPO), erythropoietins (EPO), EPO-Fc were listed in WADA's Prohibited List under Class S2.1. Traditional polyacrylamide gel-electrophoretic analytic methods were applied by most of the WADA certified laboratories for ERAs detection for the last 20 years. In this project, a novel photoclick hydrogel electrophoretic and in situ immunoblotting method for the screening of ERAs in urine and blood samples of athletes will be developed with high sensitivity and high throughput. A tetrazole-functionalized photoclick hydrogel based chip will be designed first which can satisfy the processing of 15 samples simultaneously. Optimization of the parameters in SAR-PAGE and intensity immunoblotting assay will be carried out on this chip to realize the separation of ERAs, especially the distinguishment of endogenous EPO and recombinant human EPO )rhEPO). Along with the confocal microscope for image acquisition, the LOD of this method can be lower than 1 IU/L (plasma) and 0.1 IU/L (urine) with limited sample volumes. Meanwhile, the whole analysis time can be reduced within 6 hours for plasma samples. Finally, an intravenous administration of rhEPO drugs on healthy subjects will be carried out to verify the practicability of this new method.
Direct Coupling of SPME to Mass Spectrometry and Ion Mobility Spectrometry for the Analysis of Prohibited Substances in Biofluids
By
Investigateur principal
J.
Pawliszyn
University of Waterloo
Canada
―
2022
―
En vigueur
Sommaire
Code: 22A10JP
There is a high demand for rapid screening methods using mass spectrometry (MS) that can decrease the turnaround time, cost, and limits of quantitation of existing methodologies. It is important to emphasize that appropriate sample preparation is required to perform proper sample cleanup and analyte enrichment. We are focused on using matrix-compatible SPME-based devices for direct immersion extraction of small molecules from biofluids and direct coupling to MS. In this context, matrix-compatible SPME-based devices have extraction phases comprised of high-capacity sorbent embedded in a polyacrylonitrile binder that ensures small molecules can be extracted while minimizing the co-extraction of macromolecules. This provides better performance and more sensitive analysis of a wide range of small molecules including many of those found on the 2022 Prohibited List. Futhermore, we are focused on couple SPME-based devices to MS via the microfluidic open interface (MOI) and coated blade spray (CBS) technology, as well as developing more efficient ways to separate isobaric/isomeric analytes in the gas phase before MS detection using ion mobility spectrometry (IMS). These developments are critical to decrease the detection limits and discern between permitted and prohibited substances while increasing the speed of analysis and the range of screened and quantified molecules. In this proposal, we build on this success and propose applying SPME technologies directly to MS which have been recently developed in our laboratory to further improve the speed of screening directly during sports events. In the long term, the goal of the team's research is to take advantage of ongoing advances to develop powerful new analytical technologies to assist in the ongoing fight against doping in sport, with an emphasis on lowering the detection limits to better distinguish between permitted from prohibited use, as well as improving the detection window of prohibited substances.
LC-IRMS in antidoping analysis: confirming the exogenous administration of AICAR, endogenous bioamines, and IGF-1
By
Investigateur principal
X.
de la Torre
Federazione Medico Sportiva Italiana
Italie
―
2022
―
En vigueur
Sommaire
Code: 22A08XT
For substances produced endogenously and included in the WADA Prohibited List, its abuse is based on: 10 identification of the best biomarker of abuse to be applied at the ITP level and 2) confirmation by a procedure able to demonstrate the application of the drug or its origin. Usually, this is performed by establishing the population distribution of the biomarker in ITP and the application isotope ratio mass spectrometry (IRMS) as a confirmatory procedure. This has been applied successfully using the steroidal module of the ABP and GC-IRMS.
In addition to steroidal hormones, several other prohibited compounds may benefit from a similar approach. We intend to investigate the application of IRMS coupled to liquid chromatography to the confirmation of compounds that because of their physicochemical properties cannot be analyzed by GC and for which a definiticive confirmation procedure does not exist.
The analysis of AICAR by GC-C-IRMS has been described after silylation and its comparison with a steroid (pregnanediol) as an endogenous reference compound (ERC). The current limitations of this approach can be overcome by the use of LC-IRMS, allowing the underivatized compound analysis and the use of an ERC closer to its metabolic path.
The main 2-phenethylamine and octopamine target analytes are excreted as sulfates, whose hydrolysis is not obvious. The confirmation by GC-C-IRMS is impaired by the high interaction with GC systems. The direct analysis of their sulfated conjugates by LC-IRMS will unambiguously confirm their origin. The main objective is to prove the applicability for antidoping purposes of LC-IRMS in the analyses of intact sulfates.
For IGF-1 the main objective is the use of LC-IRMS for the analysis of recombinant preparations and preparations of human origin, to prove its applicability for anti-doping purposes. The protein will be analyzed intact or after trypsin digestion.