Projets de recherche

Code: 19A11MM 

The 5a-reductase enzyme system is involved in the metabolism of endogenous and exogenous steroids. The substrates are the C19/C21 steroids with a keto group at the carbon 3 and a double bond between carbons 4 and 5. The reaction mechanism is complex and involves the binding of a reducted pyridine nucleotide cofactor to the enzyme followed by the substrate.

The modulation of the 5α-reductase activity by 4-azasteroids is used in therapy for the treatment of benign prostatic hyperplasia or androgenic alopecia. In the anti-doping field, the possibility to use the 4-aza-steroids (finasteride and dutasteride) to manipulate the steroid excretion profiles and, consequently, to mask the abuse of both pseudoendogenous and exogenous steroids was demonstrated. From 2005 to 2009, this class of compounds was included in the WADA list in the section S5 “Diuretics and other Masking agents”; whereas since 2014 the 5α-reductase inhibitors are included in the Technical Document TDEAAS “Endogenous Anabolic Androgenic Steroids Measurement and Reporting” as confounding factor. Indeed, the administration of these agents leads to a decrease of the 5α-steroids with a consequent alteration of the ratios between androsterone and etiocholanolone; 5α-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol; and androsterone and testosterone.

Different analytical procedures are reported in literature to determine finasteride in biological fluids, whereas for dutasteride only determinations in blood samples are reported due to its pharmacokinetics properties.

The aim of this project is to select the most appropriate analytical strategy and matrix to detect dutasteride during doping control test in order to avoid the risk to give uncorrected results.

Code: 19C03AM 

Tramadol is potent narcotic analgesic that acts on the opioid system. Data from the WADA Monitoring Programme and from athlete testimonies suggest that this drug is used across multiple sports in order to reduce exertional pain and allow the athlete to work even harder. In doing so, it is likely that tramadol is being used to provide the athlete with a performance advantage. However, there is currently no convincing research evidence to support or reject whether tramadol is performance enhancing in highly trained, healthy athletes. This project will employ an experimental design that focuses purely on the question of whether tramadol allows athletes to work harder by reducing pain, and thus allow for a better performance. Therefore, the main outcome of this project will be to provide robust experimental evidence to inform whether the use of tramadol in competition should be regulated. Thirty highly trained road racing cyclists will be recruited for this study, as the efficacy of performance enhancing interventions vary according to athletic ability. In a randomized, controlled, double-blinded crossover design, these cyclists will complete a laboratory cycling task that replicates the time/intensity demands of professional road cycling following the ingestion of tramadol or a placebo. The cycling task will involve fixed intensity and self-paced time trial cycling, amounting to approximately 1.5 hours of hard cycling. The intensity/time of the task is critical, as the ergogenic effects of tramadol are likely to be reduced in shorter duration exercise that induces less fatigue, and is therefore not representative of the context in which it is purportedly taken. Exercise performance (completion time) and perceptual responses (perceived pain and effort) will be compared between conditions, with results used to inform consultation with WADA regarding the S7 Narcotics category of the Prohibited List.

Code: 19B06MM 

Recently a wide variety of peptide hormones were included in the World Anti-Doping Agency List of prohibited substances and methods. The analytical approach of choice to detect these substances in biological fluids is most of the times linked to (i) the matrix selected, (ii) the concentration of both the analyte of interest and the interferences, (iii) the natural occurrence in human body and finally (iv) the molecular weight of the compound under investigation. Concerning, specifically, those peptide hormones with a molecular weight in the interval of 2000-10000 Da (i.e. insulins, GHRHs, synacthen or IGF-1 and its analogues), they are excreted in urine, either as parent compounds and/or as metabolites/degradation products, in very low levels.

Different analytical approaches have already been developed and published for the detection of these agents in doping control specimens (plasma/serum or urine samples) employing immunoafinity purification and liquid chromatographic-mass spectrometric-based techniques. The sample pre-treatment protocols proposed in literature are mainly based on ultrafiltration followed by the use of immunoaffinity purification: magnetic beads, immune-affinity chromatography or antibody pre-coated ELISA plates. Although better recoveries were achieved by using immune-affinity chromatography (>70%), the strategies currently adopted by the anti-doping laboratories are based on the use of magnetic beads (<30%) or of antibody pre-coated ELISA plate, mainly due to their simpler protocols and to the possibility to fully automate.

This project is focused on the evaluation of alternative off-line immunocapture technqiues, such as for example, pre-coated pipette tips or spin trap column with the aim to improve the efficacy, repeatability and robustness of the protocols currently used by the anti-doping laboratories to extract large peptides from biological fluids.

Code: 19A10MP

In the fight against doping the laboratories are confronted with an increasing number of substanced to screen on. Thus, a comprehensive screening for different classes of substanced using dilute-and-inject methods in anti-doping screening is desirable. As lots of xenobiotics are excreted as conjugates a detection of the intact conjugates is performed by this approach. While chemical synthesis of sulfoconjugates works efficiently for compounds having only one potential conjugation site, several analogous compounds could not be chemically synthesized effectively, due to their more complex chemical structure. For the synthesis of the phase-II metabolites (glucuronides and sulfates) of these compounds a biotechnological production will be implemented.

In part 1 of the project fission yeast strains, that enable the biotechnological production of glucuronies and sulfates that cannot be synthesized efficiently via classical chemical synthesis were generated. In part 2 they will used to produce the relevant human conjugates of prohibited substances. It is planned to address the sulfoconjugation of some challenging compounds such as salbutamol-sulfate, salbutamol-glucoronide, fenoterol-sulfate and 4-hydroxy-DHEA-sulfate within the project.

The produced reference material can be used for method set-up for direct detection. If laboratories still rely on hydrolysis of the conjugates, these reference compounds may serve as control for hydrolysis efficiency and quality assurance. Furthermore, artifact generation during conjugate cleavage can be evaluated by the help of this newly generated reference compounds. The relevance of the generated reference substances in doping control analysis will be demonstrated by comparison with authentic samples from doping control analysis.

Code: ISF19E03AM

Blood doping and in particular transfusions were used by athletes for decades, as an easy way to increase red blood cells, oxygen transportation to muscles and endurance. Homologous transfusion (blood from a compatible donor) was the simplest method used.

However, in 2004 a method based on flow cytometry was implemented in anti-doping laboratory to identify homologous transfusion. While being efficient to identify around 2% stranger blood in a sample, it requires the use of mnay antibodies and still could lead to false negative results. Nowadays, only few anti-doping laboratories are still running flow cytometry and athletes could feel free to come back to homologous transfusion.

With the development of forensic science, DNA has proven to be a reliable source of identification of the presence of two different DNA in a single blood sample. The power of amplification of DNA based techniques is sufficient to start from very small volumes and Dried Blood Spots (DBS) is an interesting matrix for the future of doping controls. The aim of the project is to evaluate the interest of implementing a new test to detect homologous transfusion by using a forensic DNA-based protocol on DBS for anti-doping purpose.

The objectives of the project are:

1. To validate the conditions to propose a robust protocol for detection of DNA mixture from Dried Blood Spots.

2. To estimate the window of detection after transfusion of healthy volunteers by analyzing DBS spotted from venous blood and to compare these results with those of the current flow cytometry method for detection of homologous blood transfusion.

Main findings

The protocol was successfully developed and validated. With 100μL of dried blood collected on a card treated to protect nucleic acids. A mix of two DNA was robustly identified when the minor blood proportion was 2% or higher in vitro when two whole bloods were mixed. Following one month of storage of the cards, at room temperature, the sensitivity of detection was not altered.

A blinded in vitro study with samples containing mixed compatible, bloods in different proportions as well as negative samples (one DNA) was conducted. For DNA analysis of DBS the limit of detection was confirmed at 2% donor blood. while the flow cytometry method performed on fresh blood was slightly more sensitive and could sometimes detect 1% donor blood in a sample.

However, the in vivo evaluation of this method with transfusion of 150 mL of a red blood cells concentrate (RBCC) using the forensic DNA approach gave disappointing results as no analyzed DBS showed any presence of a second DNA even one day following transfusion.

This result is most likely due to a lack of a sufficient number of white cells (the only blood cells containing DNA) from the donor in the RBCC and consequently in the DBS. On the contrary the flow cytometry method was very efficient and the transfusion could still be detected for more than 40 days. The classic flow cytometry method was performed with a tube by tube analysis of 11 antigens (C, c, E, e, Jka, Jkb, S, K, Fya, Fyb and s). A new simplified protocol for sample analysis by flow cytometry was also developed and evaluated using 96 well plates with 9 antigens detected (C, c,E, Jka, Jkb, S, Fya, Fyb and s). In each well two antibodies against two different RBC antigens can be mixed if one is an Immunoglobulin subtype G (IgG) and the other an immunoglobulin subtype M (IgM). Two secondary antibodies with two different fluorophores are added in each well to identify presence/absence or mixed expression of each antigen. Both flow cytometry methods detected well the double populations for several antigens after transfusion and were sufficiently sensitive to confirm at least two antigens with a double population at day 42 and day 50 post-transfusion.

In conclusion, analysis of DBS with the validated forensic DNA method to detect DNA mixes did not demonstrate enough performance to be considered as an alternative to the flow cytometry method for detection of HBT. In particular, it completely failed to detect the transfusion of the RBCC while the flow cytometry method was very efficient with a window of detection of 1.5 months. To simplify the laboratory work needed to analyse blood by flow cytometry, a technical evolution of the method was evaluated with success and the new protocol will soon be fully validated for a future application to doping control samples.

Code: 19A08LM 

Microsampling provides a wide range of applications that may offer advantages over traditional fluid samples on logistics and bioanalytical workflow. Among microsampling methods, dried matrix spots represent a feasible method for the microsampling of biological matrices to obtain for example dried urine spots (DUS). Moreover, volumetric absorptive microsampling (VAMS) has been recently introduced for the sampling of small, accurate biological fluid volumes. Dried microsamples can usually be stored under ambient conditions, although comprehensive analyte stability assessments are still under research for many compounds. Following the promising results previously obtained from the definition of mid-term stability of some doping-relevant peptides (e.g. GnRH analogues) in urine collected as DUS and VAMS, aim of this research is to carry out a systematic study on the stability in a wider time frame of such compounds in urine sampled as DMS and VAMS and to expand the study to additional peptides and to relevant compounds included into the Athlete Biological Passport (ABP) steroidal module. All variables involved in the sampling process will be assessed: humidity, temperature, light exposure will be evaluated to determine the optimal sampling, storage and transport conditions, and to evaluate results obtained from microsamples. In addition, possible scenarios will be simulated, representing the life cycle of an anti-doping sample: from collection to shipment, storage and handling before being subjected to pretreatment procedures and LC-MS/MS and LC-HRMS analysis. The project goal is to establish feasible and reliable workflows for microsample collection, stably storable and shippable with minimum precautions. These procedures could then be proposed as effective anti-doping strategies to be compared to conventional fluids. 

Main Findings: 

Two different dried microsampling and analysis workflows, based on dried urine spots (DUS)-LC-MS/MS and volumetric absorptive microsampling (VAMS)-LC-MS/MS respectively, were developed and validated for the testing of S2 peptides and of steroids included in the ABP. Validation included sampling time and drying time assays. Moreover, different microsampling volumes (10, 20, 30 μL) were tested to provide better versatility; 30 μL always granted the best sensitivity/selectivity compromise. Eleven-months stability assays showed that both microsampling matrices, stored at 25°C, provided good stability for all analytes at all time points, with end-point stability in the 52-66% range for peptides and in the 79-85% range for steroids. Similarly, both DUS and VAMS at 25°C produced better stability than fluid urine frozen at either -18°C or -80°C, with large differences (28-32.5%) for peptides and marginal ones (2-12%) for peptides. VAMS produced slightly better results than DUS: Stability in the former was 1-8% higher than in in the latter for peptides and 1-3% higher for steroids. Urine microsample storage for 11 months in sub-optimal conditions (up to 35°C, up to 40% HR, 12 h light exposure) caused larger analyte degradation, but again with better performance for VAMS over DUS (-9% vs. -18%, respectively, in comparison to optimal conditions). 11-months stability studies were completed on doping-relevant peptides and ABP-listed steroids, providing positive, interesting results. In particular, innovative microsampling and pretreatment procedures based on dried matrices (DUS and urine VAMS) were optimised for application to urine specimens for anti-doping purposes. Analytical platforms based on LC coupled to different MS detectors were exploited to develop two sets of validated analytical methods, one for the analysis of peptides and another for the analysis of steroids, in dried urine microsamples. Solid, satisfactory performance and good throughput were obtained for each analyte group. After 11 months, peptide stability in dried micro-matrices was above 51% and steroid stability was above 78%. Regarding peptides, stability in the micromatrices at RT was vastly superior to that of frozen or ultra-frozen urine. Results for steroids were less starkly defined, with both microsampling matrices providing marginal advantages in comparison to urine. In all cases, there was no need for any refrigerated or frozen microsample storage. Among the two microsampling strategies, VAMS granted significantly superior performance over DUS for peptides, with much smaller differences for steroids. However, VAMS generally gave better precision, extraction yield and matrix effect in the analytical workflow than DUS. In conclusion, dried urine micromatrices are a very promising tool for enhancing the stability of peptides and steroids in an anti-doping testing perspective. They may also provide reduced storage and shipping requirements and expenses, and simpler pretreatment procedures.

Code: 19A04MP 

In 2018, the current GC-QQQMS routine screening method (i.e., initial testing procedure) for human doping control was successfully converted into an equivalent and complete GC high resolution acquisition screening method for routine purposes by using low energy electron ionization (EI) GCQTOF. This GC-QTOF screening method is compliant with the WADA requirements and allows the detection of 294 target compounds (and 14 internal standards), including diuretics, stimulants, narcotics, beta-2-agonists, beta-blockers, hormone modulators, anabolic agents and the quantification of 14 endogenous steroids in a single fast run (14.1 min). Because of the full scan high resolution data acquisition ability of TOF technology (and the corresponding retrospective capabilities), this proved to be a big step forward in comparison with the current GC-QQQMS routine screening methods. Taking into account that anti-doping samples can be stored and reanalyzed for up to ten years, the retrospectivity and sensitivity offered by GC-QTOF opened the door to a cleaner sport. Sensitivity is obviously compound depended, but in general the sensitivity of the low energy EI GCQTOF is situated between EI GC-QQQMS and CI GC-QQQMS.

Chemical ionization (CI) is a softer ionization than low energy EI and has the potential to further increase the sensitivity, in parallel with our previous EI/CI work on GC-QQQMS. Combining GC-QTOF with CI is the next logical step and this project aims at exploring, testing and exploiting the potential of CI GC-QTOF in all its aspects. This will result in the development of a high-resolution acquisition screening method with higher sensitivities. Higher sensitivities lead to more flexibility, longer detection times and a more extended list of compounds that can be monitored.

Main Findings

The main objective of this project was to improve the sensitivity of the GC-QTOF by using CI instead of EI, in order to maximize the capabilities of the GC-QTOF. Our experiments show that at high concentrations, CI is indeed more sensitive than EI, as we expected. Unfortunately, at lower concentrations, in general, the signal generated by CI ionization drops substantially faster than with EI ionization, making CI less sensitive than EI at those crucial low concentrations. To make It worthwhile to shift from EI to CI ionization on the GC-QTOF, the sensitivity at the low levels should be at least 5 times higher. Only then, it becomes worthwhile shifting from EI to CI ionization as CI also has some important inherent disadvantages such as the requirement for laborious and frequent instrument maintenance. The performance of CI GC-QTOF is insufficient and was found unsuitable for ITP purposes. At high concentrations, CI indeed outperforms EI, as we expected. Unfortunately, at lower concentrations, in general, the signal generated by CI ionization drops substantially faster than with EI ionization, making CI less suitable than EI at those low concentrations. The reason for this phenomena is up to this day unclear to us and we are at this stage cooperating with the R&D department of Agilent the share our experiences and to examine possible causes and solutions. From a theoretically point of view CI should outperform EI. However, in practice, this is only the case at high concentrations. That is the main cause and issue, preventing the use of CI for an ITP. Due to the gain/loss of sensitivity/specificity when comparing CI GC-QTOF versus EI GC-QTOF, CI might be useful to use as a CP. However, this needs to be checked on a substance to substance basis and depends on sample prep, GC parameters, etc. For example, for some AAS, CI will be a better option, for others it will not.

Code: 19D06RF

The goal of our first study is to investigate the correlation between the Athlete Biological Passport (ABP) variables and performance data from competitive elite athletes analyzed from training and racing power outputs (elite cyclists), apneic times, depth or distance (trained apneic divers) and endurance capacity (sport students). In this way, our study would challenge the complexity of confounding factors affecting the interpretation of the ABP. Testing the hypothesis that variations in performances are related to variations in the ABP in different sport disciplines would allow the ABP model to be strengthened. Finally, our project will evaluate the range of variability of identified confounding factors altering the blood formula in a population of trained apnea divers. It is hypothesized that breath-holding methods and specific apnea training techniques may significanlty alter blood parameters from the ABP. Indeed, it has already been shown that 3 repeated breath-holdings increase hemoglobin concentration ([Hb]) in divers, skiers and untrained humans acutely while an extension of such findings in the context of the ABP is still needed. Overall, out study aims at improving the ABP by challenging the individual whithin-subject variance in light of known confounding factors. The objectives of this study are threefold: firstly, this study will allow to further investigate the relationship between Hbmass and aerobic performances in three distinct populations (elite cyclists, apnea divers, sport students). Second, blood variables data collected monthly will allow us to discriminate between the influence of specific confounding factors thanks to the multi-sourcing data and heterogeneous groups (cyclists, apnea divers, sport students). Overall, this study will enable us to strengthen the ABP after looking at the within-subject variations to propose a robust evaluation of known underlying confoundng factors.

Second, we will conduct a study investigating the influence of the menstrual cycle on hematological varaibles to identify potential variations factors specific to blood losses and hormonal variations.

Thirdly, we will conduct a study on elite race walkers to describe hematological variations observed before, during and after a prolonged exposure to i) altitude and ii) heat in their final preparation block before a major competition.

Main Findings

In the supported project, we first conducted a study to investigate the relationship between Hbmass and aerobic performances in three distinct populations (elite cyclists, apnea divers, sport students) with blood variables data collected monthly to discriminate between the influence of specific confounding factors thanks to the multi-sourcing data and heterogeneous groups (cyclists, apnea divers, sport students). This resulted in a peer-reviewed publication (1). Although individual hematological variations were observed, all ABP variables remained within the individually calculated limits. We showed that acute training load variations in elite cyclists significantly affect (Hb), likely due to short-term PV fluctuations, underlining the importance of considering training load when interpreting individual ABP variations for antidoping purposes.

Second, we conducted a study investigating the influence of the menstrual cycle on hematological variables (total Hbmass, ABP variables & indicators of plasma volume changes) to identify potential variations factors specific to blood losses and hormonal variations. This resulted in another peer-reviewed publication (2). A multi-parametric model previously validated in elite cyclists was applied to compare inferred and actual PV variations. Some significant intra-individual PV variations were observed, in good agreement with the estimated PV changes; and it can be concluded that estimating PV variations may help interpret individual ABP haematological profiles in women.

Thirdly, we had planned to conduct a study on elite race walkers to describe hematological variations observed before, during and after a prolonged exposure to i) altitude and ii) heat in their final preparation block before a major competition. However, this could not be completed yet, due to 2020/2021 travel restrictions with the context of the pandemic. We worked however instead on the monitoring data from the apnea divers cohort. This resulted in a peer reviewed publication (3). Whilst we hypothesized that regular breath-hold training could affect or alter ABP variables, the results of our study do not outline any particular significant effect of regular breath-hold training on ABP variables. Overall, this project will enable us to strengthen the ABP after looking at the within-subject variations to propose a robust evaluation of known underlying confounding factors.

PUBLICATIONS

(1) Astolfi T, Crettaz von Roten F, Kayser B, Saugy M, Faiss R. The Influence of Training Load on Hematological Athlete Biological Passport Variables in Elite Cyclists. Front Sports Act Living. 2021 Mar 18;3:618285. doi:10.3389/fspor.2021.618285. PMID: 33817634

(2) Basile Moreillon, Tristan Equey, Tiffany Astolfi, Olivier Salamin 1 3, Raphael Faiss. Removal of the influence of plasma volume fluctuations for the athlete biological passport and stability of haematological variables in active women taking oral contraception. Drug Test Anal 2022 Jun;14(6):1004-1016.doi: 10.1002/dta.3218.

(3) Astolfi, T., Crettaz Von Roten, F., Kayser, B., Saugy, M., & Faiss, R. Hematological variables in recreational breath-hold divers: a longitudinal study. J Sports Med Phys Fitness. 2021 Sep 9. doi: 10.23736/S00224707.21.12918-4.

Code: 19C02MT

The anti-estrogen clomiphene is prohibited at all times in sport and since 2011 a continuous trend in increasing numbers of adverse analytical findings is noted. Recent studies have outlines a particularly long detection window for clomiphene in human urine; further, few studies have demonstrated a significantly enhanced egg production if layig hens are treated with clomiphene. Hence, concerns have ben raised whether trace amounts of clomiphene are present in eggs or poultry due to a potential use of clomiphene in the farming industry, and whether such trace amounts could lead to adverse analytical findings in doping controls. In order to protect the athletic community, a controlled administration study is planeed, where clomiphene is administered to laying hens, and both the produced aggs as well as the edible tissue will be tested for residues of clomiphene. Fruther, eggs and edible tissue will be consumed by study volunteers, and urine sample will be subjected to routine doping control analytical assays to prove for th presence of the prohibited substance. The information gained from this study is vital to fair result managment and decision-making processes in case of clomiphene findings in sports drug testing programs. If the results prove the possibility of clomiphene contamination in dietary products such as eggs of poultry, athletes and anti-doping organizations must be warned and informed.

Main Findings: 

Athletes are permanently at risk of inadvertent ingestion of prohibited substances, as shown so prominently across countries by the clenbuterol scandal. In consideration of the growing number of AAFs related to clomiphene, it cannot be excluded that food contamination is one reason of this phenomenon. In this study, the possible contamination of eggs and chicken meat with clomiphene was investigated since it had been shown that clomiphene administration can lead to an increased egg production rate in hens.

When a drug is administed to a chicken, it can accumulate in certain parts of the body or even in the eggs. This distribution depends on physical and chemical properties such as the diffusivity of trhe substances, the molecular mass, lipophilicity and on other properties such as the ability to bind to plasma proteins. All this has an influence on if clomiphene accumulates in the meat and eggs of hens fed with the drug and if yes, how fast, how long and how much of it can be found.

The results of this study reinforce the concern that clomiphene given orally to laying hens migrates to and is stored in eggs. The determined clomiphene levels result in absolute amounts of 10 to 20 µg per egg. These amounts are more than 1000-fold below the daily therapeutic dose, so no effect would be expected with the consumption of a contaminated egg. However, many athletes have a high-protein diet and may consume clomiphene-contaminated eggs in larger quantities. This could subsequently lead to clomiphene residues in urine, leading to adverse analytical findings. This was simulated in the second part of the study where healthy male volunteers consumed the eggs produced in the animal administration study. A single consumption of two clomiphene-containing eggs led to maximum hydroxy-clomiphene concentrations between 82 and 266 pg/mL in the tested urine. Discrimination of the isomers already took place in eggs. Therefore, it was not surprising that almost exclusively (Z)-clomiphene and its metabolites were detected in urine. The (Z)-form of clomiphene has a longer half-life and is mostly found as the only isomer in urine long after clomiphene therapy. Hence, the findings indicated that it is possible to generate an adverse analytical finding by consuming clomiphene-containing eggs. Consequently, a more detailed study of the metabolites was conducted.

The intake of the substance differed between the both studies: While the microdose study volunteers took a preparation containing 42 % (Z)- and 58 % (E)-clomiphene, the egg consumers took almost only (Z)-clomiphene. Additionally, the egg consumers took 10-20 % of the clomiphene amount as hydroxy clomiphene. As (Z) 4 HC was already present in eggs, it was suggested that this had an influence on the formation of the metabolites. By analyzing the phase-II metabolites, it was found that the preferably formed hydroxy metabolite must differ between egg consumers and the comparator group, since the glucuronide peaks differed in retention time and product ion mass spectra. This was confirmed when analyzing the samples with the method for differentiation of the hydroxy metabolites of clomiphene by derivatization with dansyl chloride to achieve chromatographic separation. After consumption of clomiphene-containing eggs, over 90 % of the hydroxy metabolites was (Z)-4-HC, which was found neither in samples from the microdose study nor in the re-analyzed samples with clomiphene AAFs. 

In conclusion, a method was developed to assist in distinguishing between the consumption of eggs containing clomiphene and ingestion of the drug.

Code: ISF19C05MT

Due to the erythropoiesis-stimulating effects, the misuse of cobalt and cobalt salts in sports is prohibited both in- and out-of-competition. While total urinary cobalt levels can be determined by means of inductively coupled plasma-mass spectrometry (ICP-MS), there are currently no assays for the detection of inorganic cobalt which exclude cobalt-containing molecules such as Vitamin-B12. But especially in cases of atypical findings with elevated cobalt concentrations, the analysis of Vitamin-B12-depleted urine is required to provide accurate information on the ionic cobalt content of the sample. Therefore, a quantitative test method for inorganic urinary cobalt will be developed within this study by using different depletion approaches such as solid phase extraction (SPE) or liquid chromatography (LC) in combination with ICP-MS. In particular during prolonged exposure to high concentrations, cobalt was found to be irreversibly incorporated into red blood cells. As the determination of the cobalt content in erythrocytes could be highly relevant to uncover long-term cobalt exposure in a doping control context, an assay for the quantitative determination of cobalt from a defined amount of erythrocytes will be additionally set up. Both assays will eventually be used to analyze blood and urine samples collected within two administration studies with cobalt chloride and Vitamin-B12 (dose: 1 mg/day over a period of 14 days). The Vitamin B12 administration study will provide important insights into the influence of Vitamin-B12 supplementation – which is legitimately used by many athletes – on urinary cobalt levels.

Main Findings

The manipulation of blood and blood components, commonly referred to as “blood doping” is one of the continuing challenges in the field of sports drug testing. For that purpose, specific and sensitive detection methods enabling the detection of prohibited substances and methods of doping are required. As a cheap an easy available alternative to illicit blood transfusions, erythropoiesis stimulating agents have been shown to be misused in sport. To illegally improve the athlete's aerobic capacity and endurance performance, the administration of ionic cobalt (Co2+, e.g. CoCl2) can be used to stimulate the endogenous erythropoietin (EPO) biosynthesis. By contrast, several organic Co-containing compounds such as cyanocobalamin (vitamin B12) are not prohibited in sports, and thus, the need of analytical differentiation of urinary Co-concentrations is desirable. To this end, an excretion study with daily applications of either 1 mg of CoCl2 or 1 mg of cyanocobalamin was conducted with 20 volunteers over a period of 14 consecutive days where urine, plasma, and concentrated red blood cells were analyzed. The samples were collected starting 7 days before the administration until 7 days after. For total cobalt analyses, inductively coupled plasma mass spectrometry (ICP-MS), which yielded significantly elevated levels exclusively after inorganic cobalt intake, was utilized. Moreover, a liquid chromatography (LC)-ICP-MS approach was established and employed for the simultaneous determination of organically bound and inorganic cobalt by chromatographic separation within one single run. Especially for illegal Co2+ supplementation in sports this approach can be complemented to a prospective detection method.

Finally, for adequate method characterization and quantitative analyses, one or more internal standards need to be implemented and the chromatographic separation of additional cobalt-containing organic species as well as the stability of different variants of cyanocobalamin, especially with regard to photolytic degradation and possible conversions, need to be clarified. Nevertheless, despite the fast and preparative chromatographic run, inorganic cobalt is clearly separated and Co2+ concentrations attributed to unbound cobalt and exceeding future threshold levels will be regarded as antidoping rule violations. With regard to routine doping controls the presented approach offers an initial testing tool in order to identify those doping control samples that justify subsequent accurate cobalt quantification.