Projets de recherche

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.

Main findings

Since the detection of the latest long-term metabolite of metandienone a lot of effort is put in the discovery of similar metabolites with 17β-hydroxymethyl-17α-methyl-13-ene structure for long-term detection of other 17-methyl steroids. Thereby 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, the elimination of metandienone with special respect to A-ring reduced metabolites was investigated as well. Various diastereomers of 17-hydroxymethyl-17-methyl-18-norandrost-13-en-3-ol (20OHNorTHMT) were chemically synthesized as well as diastereomers of 17-methylandrostanediol (THMT) for analytical method optimization, and characterized by high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Their excretion in human urine after a single dose administration of metandienone was monitored using GC-MS. Both THMT and 20OHNorTHMT were detected in the post administration urines. Final excretion data and excretion kinetics was evaluated for the various metabolites in six volunteers. As diastereomers 3α,5β-20OHNorTHMT, 3α,5β-THMT and 3α,5β-epiTHMT as well as 3α,5β-NorTHMT were unambiguously confirmed in the post-administration urines, besides the classical metandienone metabolites. Stereochemical assignments were achieved by comparison of retention times and mass spectral data with authentic reference material. While the commonly considered metandienone long-term metabolite, 20OHNorMD was detectable for up to 34 days in the GC-QQQ-MS analysis, the above-mentioned metabolites with fully reduced A-ring were detectable for much shorter post-administration windows (approximately 5-9 days post-administration). Thus, the reduced metabolites are obviously not amenable for long-term detection of a metandienone administration, which is in contrast to its closely related chlorinated analog DHCMT. However, a promising new candidate long-term metabolite was assigned to the 1-ene analogue of 3α,5β-20OHNorTHMT (17-hydroxymethyl-17-methyl-18-nor-5β-androst-1,13-dien-3α-ol). While its quantifier transition allowed for detection for up to 38 days after administration using GC-QQQ-MS in one volunteer its confirmation will most likely require improved clean-up procedures or optimized chromatographic separation to eliminate interferences from the urinary matrix.

In addition to further assembling the picture of human metabolism of AAS with newly discovered metabolites, NorTHMT turned out to be a promising candidate for a new long term marker in anti doping analyses. However, the current method did not allow for proper confirmation in the late excretion urines due to matrix interferences. Thus, method adaptations have to be evaluated in the future to provide a reasonable method for later routine use as confirmatory procedure.

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.

Code: DBS20CT34JM

The minimally invasive dried blood spot (DBS) technique has the potential to improve the time-and-cost efficiency compared to traditional matrices in doping control. Understanding the athletes’ preferred sampling site will support the drafting of WADA Collection Guidelines/ISTI in the implementation process. Furthermore, the potential impact of the sampling site e.g. finger vs. arm on the concentration of target analytes needs to be established. Doping Control Officers (DCOs) will collect capillary blood from the finger (finger-prick) and from the upper arm (specific collection device) from 108 athletes (males and females) of various sport disciplines. The DCOs will record the time needed to collect the samples, register the number of unsuccessful attempts and evaluate the usability of the collection devices and whether they prefer the collection of DBS from the fingertip or the upper arm. The athletes will fill out a questionnaire regarding the perception and painfulness of the two DBS collections and whether the collections processes have had any impact on their sport activities afterwards. The lab staff will to fill out a questionnaire once they have received the samples to understand the suitability of the samples for analysis. The DBS samples will be analysed for the concentration of endogenous testosterone.

Main findings

Currently several DBS collection devices exists allowing collection of capillary blood from different anatomical sites. Nevertheless, the suitability for collecting DBS samples in an anti-doping context depends on the sample collection experience by the athletes, doping control officers (DCO) and the handling of the sample by the laboratory staff. Furthermore, agreement between quantitative measurements is important if more than one collection method (device and/or sampling site) is approved. In this project, a total of 108 matched DBS samples from the fingertip (HemaSpot HF; lancet device) and the upper arm (Tasso-M20; microneedle device) were collected from 49 female and 59 male national level athletes from various sports (handball, weightlifting, football, running and wheelchair rugby). Following sampling, the collection process was evaluated by athletes and DCOs. Furthermore, upon reception of the samples, the laboratory staff compared the quality and usability of the samples from the two devices. The testosterone concentration was measured in all samples and the correlation between concentrations determined.

Overall, the DBS sampling was associated with minimal sensation of pain and a high general experience by the athletes, but the perceived pain was rated lower (-0.4 ± 1.6, p < 0.05) and the general experience rated higher (+0.6 ± 2.3, p < 0.001) during upper-arm DBS collection than during DBS collection from the fingertip. Likewise, the DCOs rated the general experience with the upper-arm DBS collection higher (+1.6 ± 1.1, p < 0.01) than the fingerprick DBS collection, partly due to problems occurring more frequently during the DBS collection from the fingertip (28% of collections) than from the upper arm (6% of collections). Both procedures were equally fast, lasting only around two minutes on average, which is a great advantage compared to urine. When choosing, the great majority of DCOs and athletes, independent of gender and discipline, preferred the automated DBS collection from the upper arm over the manual collection from the fingertip, and both DBS collections over conventional sample collection methods (urine and venous blood collection).

Both devices provided easy access to the DBS sample and the overall analysis time was not affected by the choice of DBS material, however, the Tasso-M20 device was the preferred device amongst the four analysts preparing the samples due to less sample handling prior to sample preparation. Endogenous testosterone was quantified in DBS samples from both devices with good repeatability (RSD < 5%) and reproducibility (RSD < 10%). The quantitative analyses showed good correlation between samples collected from the fingertip and the upper arm from athletes (r = 0.921, p<0.0001), as well as non-significant difference of the median testosterone concentrations (1.70 ng/mL for Tasso and 1.67 ng/mL for HemaSpot, p = 0.503). A small, measured bias between Tasso and HemaSpot (-7.45%) was observed, likely due to unsatisfactory volume control in the HemaSpot compared to the Tasso device. Collectively, the results suggest that there is no physiological difference in the basal testosterone concentrations in capillary blood samples collected from the fingertip and the upper arm.

In conclusion, both an automated upper-arm DBS collection device [Tasso-M20] and a manual fingerprick DBS system [HemaSpot HF] could be used for DBS collection in an anti-doping setting. However, for subsequent quantitative analyses, the volumetric control of the Tasso-M20 spots seemed more robust than for HemaSpot HF. Both DCOs and laboratory personnel seem to prefer the upper-arm DBS collection with the Tasso-M20 device. If more than one collection method (device and/or sampling site) is approved, it appears crucial that the analytical assays are validated and calibrated on the respective DBS-devices/materials.

Code: DBS20AS7JM

The minimally invasive dried blood spot (DBS) technique has the potential to improve the time-and-cost efficiency compared to traditional matrices in doping control. The potential impact of the sampling site e.g. finger vs. arm on the concentration of target analytes needs to be established, especially when analyzing for threshold substances prohibited in-competition only. Eight healthy male volunteers will receive a single oral administration of ~20 mg (‘low dosage’) and 60 mg (‘high dosage’) of ephedrine in a randomized crossover design with one week between the interventions. Parallel DBS samples from the fingertip and upper arm will be collected at 0 (pre-administration control sample), 1, 2, 4, 6 and 8 hours post-administration. From the DBS samples the ephedrine concentration will be determined. Additionally, venous blood samples will be collected through a peripheral venous catheter on the same time points to compare the DBS concentrations of ephedrine with those in plasma.

Main Findings

Dried blood spot (DBS) testing allows for fast, easy, and minimally invasive collection of microvolumes of blood. In an anti-doping context, DBS testing has particularly relevance for substances prohibited in-competition only, as it can determine the presence of pharmacologically relevant blood concentrations during the in-competition period. A wide range of collection methods and devices exist for DBS collection allowing collection of capillary blood from different anatomical sites, but the possibility to use different devices in an anti-doping setting would rely on agreement in substance concentrations between sampling sites and between devices. Furthermore, it is of interest to evaluate the agreement between concentrations of target analytes in DBS and conventional venous plasma samples. Herein, we collected matched upper-arm DBS, finger prick DBS and venous plasma samples from 8 healthy, male subjects in an 8-hour period following oral administrations of 20 mg (‘low dose’) and 60 mg (‘high dose’) of ephedrine. We observed no consistent trend in the dependence of ephedrine concentration on blood sampling site or sampling device, and the correlations between ephedrine concentrations in finger prick DBS and upper-arm DBS were very high (Pearson’s r > 0.80) after both low and high dose administration. These results indicate that DBS originating from finger prick and automated upper-arm collection, along with conventional venous blood samples, can be used for quantification of ephedrine in doping control.

Code: DBS20AS7AT

This project will assess the introduction of cut-off limits for substances that are only prohibited in competition (stimulants, corticoids, cannabinoids, and narcotics). Data about the pharmacologically relevant levels of each substance are required to interpret the measured results correctly.

Main Findings

In competition sport drug testing represents an important aspect in doping controls. Here the analysis of blood samples owns a considerable benefit compared to urine samples due to the determination of actually relevant blood concentrations during the competition. DBS sampling represents a simple, reliable, cost-efficient and robust approach to ideally support the classical urine analysis, especially for in competition testing. In the present study potential analytical cut-off limits for stimulants, corticoids, cannabinoids, and narcotics are proposed. The values are based on literature review for pharmacokinetic data as well as already existing levels valid for driving under the influence of drugs.

Code: DBS20AS2AT

This project will investigate the possibility to measure the hematocrit value of the dried blood spot (DBS) sample by near-infrared spectroscopy (NIR). This can have an influence on the result, especially when the data are evaluated quantitatively. For this purpose, the results of the NIR measurements will be compared with reference data (Sysmex etc.) obtained simultaneously.

Main Findings

In contrast to established blood sampling strategies (yielding serum or EDTA plasma), DBS consist of whole blood. Therefore, the knowledge about the hematocrit (as percentage of red blood cells) of the DBS sample represents an important parameter especially for quantitative results interpretation. Here, the hematocrit measurement with near-infrared spectroscopy (NIR) from cellulose-based DBS paper represents a valid and reliable approach. After complete drying of the cards, the non-destructive NIR-analysis enables robust hematocrit (Hct) measurements over weeks and presumably months. The correction for the actual hematocrit of the finger prick DBS samples facilitates the accurate correlation to the resulting and comparable plasma levels of the respective drugs. With regard to in-competition DBS sampling, this possibility will enhance the result interpretation significantly. In the present project, different whole blood sampling strategies (venous, finger prick, TAP, Tasso, capillary) and the subsequent hematocrit measurement (Sysmex, NIR, centrifugation) were compared. All measurements for venous EDTA-blood and capillary finger blood (finger prick) were < 10% of relative deviation compared to the ‘true’ Sysmex value. This is also true regardless of the method of measurement. However, collection of capillary blood on heparin (here using the TAP device) and subsequent analysis by NIR led to greater Hct values and a mean relative deviation ˃10%. Transfer to other laboratory is not hindered, because NIR analysis is based on calibration-based technology, which can be adapted and transferred to any other instrument using the same technology. It was additionally shown that the repeated exposure to NIR does not have a measurable impact on the subsequent chemical analysis of the prohibited drugs.