Projets de recherche

Code: 21D06RV

The steroid profile is used to detect administration of testosterone and other endogenous anabolic androgenic steroids. Metabolites included in the steroid profile have both gonadal and adrenal origin. Glucocorticosteroids (GC) inhibit the hypothalamic-pituitary-adrenal axis, and reduce the adrenal steroid production. For that reason, an effect on the urinary steroid profile might be expected. 

In a previous project, we evaluated the effect of single oral or intramuscular administration of GC on the steroid profile. A significant decrease in the excretion rates of steroid profile metabolites was observed after GC administration that was found to be associated with the dose. However, the ratios between metabolites evaluated in the athlete’s biological passport were not significantly altered, although all volunteers receiving the highest intramuscular dose showed a clear decrease in some of the ratios. Given the significant decrease in the excretion rates of steroid profile metabolites after a single dose and the alterations of some of the ratios, the question of what would happen after repeated administration of GC arose.

 The aim of the present project is to evaluate the effect of repeated oral doses of dexamethasone (DEX) or methylprednisolone (MP) on the steroid profile. Repeated oral doses of DEX or MP will be administered to eight healthy volunteers. The steroid profile will be measured by GC-MS/MS in urines collected before and after administration. The excretion rates of the metabolites will be calculated. In addition the adaptive module will be applied: samples collected before administration will be used to establish the reference ranges for each volunteer, and post-administration samples will be individually compared with these reference ranges.

Main findings

The steroid profile (SP) is a powerful tool to detect the misuse of endogenous anabolic androgenic steroids in sports. The SP consists of the longitudinal monitoring of concentrations of testosterone (T), its related metabolites (epitestosterone, E; androsterone, A; etiocholanolone, Etio; 5α-androstane-3α,17β-diol, 5aAdiol; and 5β-androstane-3α,17β-diol, 5bAdiol), and the ratios between them (T/E, 5aAdiol/E, A/T, A/Etio and 5aAdiol/5bAdiol). Alterations on the SP cannot only be associated with doping practices, but also with the presence of confounding factors. Glucocorticoids (GC) could be a confounding factor to the SP since they inhibit the HPA axis, and the SP metabolites have partial adrenal origin. In fact, in a study conducted by our group, GC treatments involving single-dose systemic administration produced a reduction of the excretion rates of the SP metabolites, as well as a decrease of the A/T and 5aAdiol/E ratios. However, the reduction in the SP ratios did not result in atypical profiles. Considering these results, the question regarding the potential effect of repeated GC administrations on the SP arose.

In this study, the impact of multiple oral administrations of GCs on the SP was evaluated in two studies, using two of the GCs most detected in sports drug testing: methylprednisolone (MP) and dexamethasone (DEX) (n=8 volunteers each study). In MP study, 12 mg of MP were administered per day during 3 consecutive days. In the DEX study, 2 mg of DEX were administered every 12 h for 5 consecutive days. Urine samples were collected before, during and after the GC treatments, and the SP was measured in all samples using gas chromatography-tandem mass spectrometry. The multiple dose oral administration of GCs resulted in a treatment-dependent reduction of the excretion rates of some SP metabolites (5aAdiol, A and Etio) and the SP ratios A/T and 5aAdiol/E. The T/E ratio was not significantly affected.

Overall, although the consumption of GC could result in atypical profiles for A/T and 5aAdiol/E, according to the cost/benefit assessment, GC should not be considered a confounding factor to the SP since misunderstandings in the evaluation of the SP would only take place in very specific situations and, in those cases, the analysis by GC/C/IRMS of the sample triggering the atypical profile would demonstrate the endogenous origin of the SP metabolites.

Code: 21C19EI

Our research hypothesis is that combined intake of diosgenin and ecdysterone is an effective strategy to induce anabolic responses resulting in the enhancement of performance, in particular strength and speed. Therefore, we want to test the dose-dependent anabolic activity of different combinations of ecdysterone and diosgenin in our well-established in vitro test system for anabolic activity. Diosgenin and ecdysterone will be tested dose-dependently as single compounds and in combinations in comparison to other anabolic substances (testosterone, estradiol and IGF1). 

Mechanistic studies will provide more evidence which molecular pathways are involved in its anabolic activity. Comparison with the recently characterised plant-derived anabolic substance ecdysterone in our in vitro test system will provide first information regarding its potential anabolic potency if abused orally in athletes. Ecdysterone was first identified to be anabolic in this test system, which was later confirmed in a human training study [10]. Taken together, the results of our investigations will be part in the pharmacological characterisation of these substances. This knowledge will be helpful to verify and justify the already existing limits of the combinations of ecdysterone and diosgenin for positive testing, to get additional information about side effects and to improve already existing doping tests for these substances. 

Study 1: Binding affinity of ecdysterone and diosgenin combination to the androgen and estrogen receptor.
In our yeast transactivation system, we will examine the binding affinity of ecdysterone and diosgenin combination to the androgen and estrogen receptor in comparison to testosterone, ecdysterone and diosgenin separately. We will determine the respective Michaelis constant (MC) of the combined substances.

Study 2: Anabolic potency of ecdysterone and diosgenin combination
In our C2C12 cell culture system, we will examine dose-dependent anabolic effects of ecdysterone and diosgenin combination in comparison to testosterone, IGF1 and ecdysterone and diosgenin. This could allow to estimate the anabolic potency of a combination of phytosteroids in comparison to the other anabolic substances.

Study 3: Mechanisms of action
In coincubation experiments with anti-estrogens, antiandrogens, glucocorticoids and antagonists to the IGF-1 receptor (like Losartan), we will determine signal transduction pathways mainly involved in the anabolic activity of this combinations. 

Main Findings

For decades, plant-derived phytosteroids like ecdysterone (Ecdy) have been used to enhance physical performance, especially in terms of strength, due to their anabolic properties. Recent findings indicate that Ecdy is increasingly being used in combination with other phytosteroids such as diosgenin (Dio). However, there is limited data available on the combinatorial effects of phytosteroids. Our study aimed to investigate the anabolic effects and underlying molecular mechanisms of various combinations of Dio and Ecdy in C2C12 myotubes in a dose-dependent manner.

In differentiated C2C12 cells, dose-dependent effects of Dio and Ecdy and combinations (ranging from 2 µM to 0.1 nM) on myotube size were investigated. In addition, the mRNA expression of genes associated with muscle hypertrophy, such as IGF-1 and PIK3R1, was analyzed by RT PCR. To delve into the molecular mechanisms, we co-incubated Dio with inhibitors of the estrogen receptor (ER) and androgen receptor (AR), along with dexamethasone (DEX), and evaluated myotube diameter. Moreover, the interaction of Dio with the AR and the ER was examined in a yeast transactivation assay.

Different combinations of Dio and Ecdy had positive effects on the myotube diameter. Combinations of Dio and Ecdy also affected mRNA expression of IGF-1 and PIK3R1. Contrary to the ER inhibitor ZK 191703, the AR inhibitor Flutamide, and DEX treatment effectively counteracted Dio induced myotube diameter growth. Dio did not exhibit agonistic activity in the AR and ER yeast transactivation assays, but it displayed antagonistic effects on the AR rather than the ER. The full mechanism affecting myotube diameter needs to be further elucidated.

Our in vitro findings provide evidence that combinations of Dio and Ecdy display anabolic activity in C2C12 cells in an additive manner. Dio has anti-androgenic activity in the yeast cell model, while Ecdy has anti-oestrogenic activity. Moreover, our data suggest that activation of the PI3K/Akt/mTOR pathway is essential for Dio effects on myotubule diameter. Considering that these results were obtained in vitro in myoblast cells (C2C12) from rodents, future studies are needed to investigate these effects in human training interventions to conclude on any anabolic effect in human.

Code: 21C11CR

Chapter S4 of WADA’s Prohibited List 2021 (“Hormone and Metabolic Modulators”) lists ACE-031 under sub-chapter 3 (“Agents preventing activating receptor IIB activation, Activin receptor IIB competitors – Decoy activating receptors”) as prohibited substance. ACE-031 (Ramatercept) is a soluble fusion protein, which blocks activin receptor type II B (ActRIIB) ligands including myostatin. So far, no approved ACE-031 pharmaceuticals are available. On the other hand, ACE-031 is sold on the black market. However, the administration of black market ACE-031 to human test persons will be ethically not justifiable. For that reason we plan a study with rats. Page 2/7 The test animals will receive black market ACE-031 at a dosage, which can be clearly detected in serum (10 mg/kg BW). After 24, 48 and 168 hours, serum and urine will be collected and tested for ACE-031 by electrophoresis and Western blotting. In addition, the metabolism of black market ACE-031 will be investigated in human microsomes as the metabolites in rat may be different from those in humans. The study will help to clarify (1) how long black market ACE-031 is detectable in blood, (2) if it can also be observed in urine, and (3) if metabolites are different in rats and humans. We have already developed a method for the detection of black market ACE-031 after electrophoretic separation and Western blotting (SDS-, SAR-PAGE).

Main Findings

Chapter S4 of WADA’s Prohibited List 2024 (“Hormone and Metabolic Modulators”) lists ACE-031 under sub-chapter S4.3 (“Agents preventing activating receptor IIB activation, Activin receptor IIB competitors such as decoy activin receptors (e.g. ACE-031)”) as prohibited substance. Currently, ACE-031 is only available on the black market. Since administration of black market ACE-031 to humans is ethically not justifiable, a study with rats was performed.

Aims of the project were:

- the characterization of 14 black market ACE-031 products by SDS-PAGE followed by Coomassie staining or Western blotting using ACVR2B-, follistatin- and His-tag-specific antibodies

- an investigation of the electrophoretic detectability of black market ACE-031 in rat serum and urine after circulation in blood for 24, 48, and 168 hours

- an in vitro metabolism study of ACE-031 using human and rat liver microsomes

Results:

Of the 14 tested ACE-031 products, 13 contained Coomassie-stainable proteins. Western blotting revealed that 12 products were indeed ACVR2B-related proteins. One product was mislabelled and contained black market follistatin instead. It could be further shown that on all 12 ACE-031 products His-tags were present as already demonstrated for black market follistatin.

After a single dose administration of black market ACE-031 (10 mg/kg body weight) to rats, the protein was detectable in all serum samples after 24 hours. While it remained traceable in the majority of the 48 h samples, it was undetectable after 7 days (168 h). No ACE-031 was found in the urine samples at all three time-points.

In order to reveal possible differences in the metabolism of ACE-031 between humans and rats, ACE-031 was incubated with human and rat liver microsomes (5, 60, 120, 300 min, 24 hours). The protein proved quite stable - only after 24 hours degradation of the main band was observed.

Conclusions:

Black market ACE-031 can be detected by SDS-PAGE and immunoblotting with a monoclonal ACVR2B-specific antibody. Additionally, it contains immunoreactive His-tags. For extraction of ACE-031 from rat serum and urine samples, a polyclonal ACVR2B-antibody linked to magnetic beads was used. After single dose administration, the protein remained detectable for at least 48 hours in most rat serum samples. No signals were obtained on Western blots after 168 hours in serum and in all urine samples.

Code: 21C09MP

The administration of anabolic androgenic steroids (AAS) is prohibited as doping in sports. Anti-doping analysis tries to target long-term metabolites of AAS to extend the detection windows. For several 17-methylsteroids the detection of 17β-hydroxymethyl-17α-methyl-13-enes demonstrated superior detection times over other metabolites monitored so far. In dehydrochloromethyltestosterone (DHCMT), active pharmaceutical ingredient of Oral Turinabol, four metabolites with this structure have been reported so far, (Sobolevsky “I”, “M2”, “M3”, and “M4”). The integration of these metabolites into initial testing procedures resulted in a strong increase of adverse analytical findings in doping control, that are currently considered to trace back an administration of DHCMT. Compared to DHCMT methylclostebol (chloromethyltestosterone, ClMT) lacks the 1(2) double bond in the A-ring. Based on common knowledge on steroid metabolism it is expected that "M3" may also be excreted after methylclostebol administration, while the other metabolites might be better suited for discrimination of the administered parent drug. The project therefore aims to investigate the excretion of the above mentioned metabolites after methylclostebol administration and to monitor their excretion kinetics. Integration of the “classical” methylclostebol metabolites will complement the investigation. A comparison with the data obtained for DHCMT is also planned.

Code: 21C06MT

The possible performance-enhancing effects and medical benefits of ecdysterone (ECD) have been discussed several times throughout the last decades.[1-4] In 2020, WADA decided to include ECD in their monitoring program and continued this prevalence study in 2021.[5] Only little is known about the metabolism of ECD. In calf urine, the intact compound was eliminated rapidly, and three hydroxylated and de-hydroxylated metabolites were identified.[6] In mice, mainly de-hydroxylation and side-chain cleavage at C-20 were detected.[7] The only study performed on human subjects in the field of sports drug testing was already conducted 2001.[8] Besides the parent compound, 2-deoxyecdysterone (2DE)and deoxyecdysone (DE) (Figure 1) were identified in post-administration urine samples.

i. Sample collection from the human participant – details of the interventions. In order to detect and to comprehensively identify urinary human metabolites of ECD, two consecutive administration trials will be performed. During the first investigation, which serves the purpose of qualitatively detect the occurrence of metabolites, 20 mg of deuterium labelled ECD will be administered orally to one person and samples will be collected before the administration (n = 3) and afterwards. During the first two days after administration every urine specimen will be sampled, followed by morning and evening urines throughout days 3 to 7. Then, for the next two weeks, only morning urine specimens will be collected. The collection scheme for the second administration study conducted with 20 mg of unlabelled ECD will be conducted in accordance with the first trial and may be adopted to gather more data at those time points that were found to be important during the first investigation. Here, three study participants will be enrolled. Within both studies, a washout period of at least one month will take place. The volunteer will be recruited from the male population of employees of the German Sports 3University, Cologne. All samples will be collected in 250 ml PET flasks and stored at -20°C without adding any preservative.

ii. Analytical methods
A) Hydrogen isotope ratio mass spectrometry
Pre and post administration samples will be subjected to established sample preparation protocols enabling to separate unconjugated, glucuronidated and sulfoconjugated phase-II-metabolites. The different urinary fractions will be de-conjugated and further purified by high performance liquid chromatography prior to trimethylsilyl-derivatization. During the injection on the GC/TC/IRMS system the hydrogen isotope ratios (HIR) will enable to identify those steroids still showing a deuterium label and therefore attributable to the administered ECD. Simultaneously, low-resolution mass spectra will be acquired using the hyphenated triple quadrupole MS. These low-resolution mass spectra facilitate relocation of metabolites during HR-MS measurements. 

B) Structural elucidation employing HR-MS All samples containing deuterium labelled compounds will be re-injected on a gas chromatography/high resolution mass spectrometry based system in order to gain structural information of possible ECD metabolites. The full scan high-resolution mass spectra obtained will enable to calculate the elemental composition of metabolites and tandem mass spectrometry based experiments will help to identify the chemical structure of metabolites. Those metabolites most 
promising for sports drug testing may be further characterized employing e.g. reverence materials (if available) or partial chemical synthesis (if possible).

iii. Targets/analyses/variablesThe urine samples collected during the administration trial encompassing deuterated ECD will be processes in order to identify possible metabolites of ECD. Known metabolites will of course be considered in a targeted manner to demonstrate the validity of the approach. Samples collected within the non-deuterated study will be investigated regarding the potential to implement found metabolites into current doping control methods. Both samples (deuterated and non-deuterated) will enable structural elucidation of metabolites taking into account the known site of deuteration.

Main Findings

In a first trial, one male volunteer was administered with deuterated ECDY to enable the detection and potential identification of all urinary metabolites still comprising the deuterium label by employing hydrogen isotope ratio mass spectrometry and high resolution/high accuracy mass spectrometry. Samples were collected for up to 14 days and metabolites excreted unconjugated, glucuronidated and sulphated were investigated separately.  The detected deuterated metabolites were confirmed in a second administration trial encompassing two male and one female volunteers. After the administration of 50 mg native (i.e. non-deuterated) ECDY, urine samples were collected for up to 7 days. Besides the already described urinary metabolites of ECDY, more than 20 new metabolites were detected encompassing all expected metabolic conversions including the described side chain cleavage at C-21. A significant inter-individual variation in the amounts of excreted ECDY and its metabolites was noted. Defining a urinary threshold for ECDY will become extremely complicated as either acceptable sensitivity or specificity will most probably not be achievable. Considering other detected metabolites will also be challenging as a potential solution for this issue as, also here, a large inter-individual variation was visible and considerable differences in abundances of early- and late-excretion phase metabolites were observed.

Code: 21C05GH

Objectives: 

The use of anabolic androgenic steroids (AAS) can increase body dimensions, muscular strength, and lean body mass in athletes. 6β-chloro-4-androsten-17β-ol-3-one (6β-chlorotestosterone), a new designer steroid substance, with an added 6-chloro group in the B-ring of a testosterone derivative, was reported existing in many dietary supplements [1]. This steroid substance is usually undetectable due to the lack of detection window in routine analysis [2]. Cheating athletes have a strong motive to take this designer steroid in order to both achieve performance enhancement and to escape from testing positive in anti-doping tests [3]. 

Methodology and experimental design:

1. Excretion studies will be conducted after 6β-chlorotestosterone took by 4 volunteers (two male, two female) at least. Urine samples will be collected and detected for 2 months or longer.
2. In order to evaluate the effect of 6β-chlorotestosterone to steroid profile, 6β-chlorotestosterone excretion urine samples will be analyzed by GC-MSn.
3. Steroid-like drugs are always extensive metabolized in human. Phase I metabolite and phase II metabolites will be extracted by different methods such as SPE or LLE. Enrichment procedure such as preparative performance liquid chromatography may also be employed for low concentration metabolites. 
4. Metabolites especially long term metabolites will be identified and characterized by LC-Q Extractive MS. Currently, liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) represents the most powerful metabolomics platform. Metabolites will be analyzed and identified in targeted mode and untargeted mode using accurate mass measurements.

Code: 21A17EP

LGD-4033 (Ligandrol), is a Selective Androgen Receptor Modulator (SARM) with a pyrrolidinyl-benzonitrile core structure. Although it displays promising muscle-and bone-anabolic properties, it is not yet
a drug approved for clinical use and it is included in the World Anti-Doping Agency’s (WADA’s) Prohibited List. Nonetheless, lately numerous related adverse analytical findings have been reported by
doping control laboratories worldwide.

Prior studies related to the detection of LGD-4033’s illicit use by LC–MS analysis of human urine have indicated two isomeric dihydroxylated metabolites as the preferred target analytes. Their monitoring could extend the detection for at least twenty-one days post-administration of the parent compound. For doping control laboratories to check, improve and properly validate their methods of detection of these metabolites it is crucial that relevant reference materials are available for comparison of chromatographic retention times and mass spectrometric properties. However, these compounds are not readily available and their structure has only partially been elucidated. Furthermore, their bis-hemiaminal nature questions their stability for long-term handling and storage. To address the abovementioned challenges, proposed herein is the chemical synthesis of all possible diastereoisomers of the main dihydroxylated LGD-4033 metabolite. Two alternative synthetic approaches that employ as starting material either the parent compound or commercially available building blocks will be investigated at small scale. NMR and LC-MS analysis of the synthesized diastereoisomers in comparison with the ones reported for the targeted metabolite is anticipated to clarify its stereochemistry and stability. To secure material to be distributed among WADA accredited laboratories, the synthesis of the diastereoisomer(s) that match the chromatographic and mass spectrometric properties of the in vivo observed metabolite(s) and exhibit stability that permits their use a reference material will be pursued at a larger scale.

Main findings

The main objective of this project was to secure through chemical synthesis reference material for the main dihydroxylated metabolite of LGD-4033 (M5b), a long-term marker for the detection of the illicit use of this WADA-banned SARM employing LC-HRMS/MS methods.

In the course of the project, the originally proposed structure for this metabolite was revised to (4R,5R)-4-{[4-cyano-3-(trifluoromethyl)phenyl]amino}-6,6,6trifluoro-5-hydroxyhexanoic acid. This material was synthesized in multi-mg scale, and shown, by LC-HRMS/MS and NMR, to be identical to the metabolite detected in post-administration urine samples. Thus, it can serve as reference material for this important metabolic marker. Details can be found in the research article Organic & Biomolecular Chemistry 2022, 20, 9112–9116. https://doi.org/10.1039/D2OB01907H and in the international patent application PCT/EP2023/063907, which has been published in WIPO under publication number WO2024046605 (publication date 07.04.2024) (https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2024046605).

Furthermore, two diastereoisomeric pyrrolidinone-type derivatives—the 4{(5R)-2-oxo-5-[(1R)-2,2,2-trifluoro-1-hydroxyethyl]pyrrolidin-1-yl}-2-(trifluoromethyl)benzonitrile and the 4-{(5R)-2-oxo-5-[(1S)-2,2,2-trifluoro-1-hydroxyethyl]pyrrolidin-1-yl}-2-(trifluoromethyl)benzonitrile)—as well as the (4R,5S)diastereoisomer of M5b were synthesized and fully characterized (specific optical rotation, IR, 1H and 13C NMR, HRMS).

LC-HRMS/MS comparison of the above synthetic materials with a postadministration urine sample, obtained in the frame of a previous related excretion study, revealed that:

1. the (4R,5S)-diastereoisomer of M5b corresponds to the minor dihydroxylated long-term metabolite of LGD-4033 (M5a), which elutes in the same chromatographic window with M5b and is monitored simultaneously by WADA-accredited laboratories during their ITP;

2. the two synthesized pyrrolidinones correspond to two of the pyrrolidinonetype metabolites (M2) detected in LGD-4033 post-administration urine samples;

3. they are true metabolites and not, as previously suggested, analytical artifacts.

Some of the above results have been published in ChemPlusChem 2024, e202300634. https://doi.org/10.1002/cplu.202300634

The efficient synthetic sequence developed for M5b was adapted for the preparation of molecules that could correspond to the (R,R)-diastereoisomer of the ring-opened hydroxylated metabolite M4, and two of the potential isomers of the trishydroxylated metabolite M6. Their comparison (LC-HRMS/MS) with the corresponding in vivo-observed metabolites allowed structural elucidation of these metabolites.

Finally, a targeted metabolic investigation of a post-administration human urine sample, employing as reference material a pyrrole derivative that was obtained in the course of synthetic studies performed in the frame of the project, suggested that it corresponds to a new, not previously reported, short-term metabolite of LGD-4033.

Code: 21A08HK

Administration of some peptide hormones for the purpose to improve physical abilities is prohibited. We have investigated antibody-free analytical methods using PAC-LC-MS to quantify their endogenous concentrations of peptide hormones, particularly growth hormones in the plasma. The latest method enables quantification of endogenous growth hormone (major variant) in human plasma and detection of 11 peptide hormones. Our objectives are to improve the sensitivity of this method to quantify both major and minor variants of endogenous growth hormones in human plasma by improving selectivity for the peptides, and to apply our analytical methods to quantify other peptide hormones in the plasma. We will optimize ion mobility to find the best parameters to separate the target peptides from contaminants. We will also expand our PAC-LC-MS/MS methods to quantify various prohibited peptide drugs, including GHRH and CG, by optimizing the pretreatment process, chromatographic conditions, and MS/MS detection. Human plasma specimens will be collected from healthy subjects who receive growth hormone to monitor the ratio of major variant of GH to minor variant, and the plasma concentration-time profiles of the other peptide hormones with or without GH administration.

Code: 21A07RN

The detection of testosterone misuse in elite sports is still one of the mayor challenges for anti-doping laboratories. Since the human body also produces testosterone and its metabolites, the presence of abnormal concentrations of endogenous androgens in urine sample(s) of an athlete is not sufficient to declare an Adverse Analytical Finding (AAF). Their concentrations and ratios are monitored longitudinally in the urinary steroidal module of the athlete biological passport (ABP). If a sample is outside of the individual reference limits for the athlete, it is considered as atypical, and the origin of testosterone has to be confirmed. Currently, the methodology to routinely distinguish between endogenous and exogenous testosterone is the gas chromatography–combustion-isotopic ratio mass spectrometry (GC/C/IRMS) analysis. This type of investigation is accompanied by labour-intensive sample preparation that is prone to deficiencies. The results can sometimes be inconclusive, most often due to limited sensitivity, which is the main problem associated with GC-C-IRMS method, and which requires also a significant volume of urine. In this context, in a recent study carried out at LAD, the use of blood matrix was proposed as a complementary tool for improving the urinary steroidal module of the ABP. Through the quantification of testosterone (T), androstenedione (AND) and 5-dihydrotestosterone (DHT) in serum samples, the development and consideration of a future longitudinal “blood steroidal profile (BSP)” was hypothesized. This approach already showed a particularly good detection windows for patch and oral T administrations [1] . Also WADA has recently established a working group to further investigate this BSP approach and the possibility of setting up future regulations for the concept. In addition to the BSP approach, the serum matrix will also be very useful to detect the testosterone esters. In fact, oral and/or intramuscular applications are usually administered not in the form of free testosterone, but its esters. This is to prevent a fast diurnal excretion and provide the body with a prolonged supply of free testosterone through the esters hydrolysis in blood. Those esters cannot be synthesised by the human body, therefore their presence in the blood matrix is a direct sign of illicit testosterone intake and an unambiguous proof of doping. In the case of testosterone ester positive findings, this would even negate the need for an IRMS analysis. With this in mind, and in compliance with the prevailing WADA TD2018EAAS, different gas or liquid chromatography-mass spectrometry methods have been developed by few anti-doping laboratories in the last years (see below). The combination of results obtained with the analysis of endogenous steroids and testosterone esters in serum could provide additional and complementary information to the urinary steroid profile and IRMS results. With this approach, we would also emphasize the usefulness of simultaneous collection of urine and blood samples within a sample collection session [2] .

The project is divided into four different sub-projects, which focus each on different aspects of the detection of testosterone via the analysis of the esters. The first part focuses on the method development and validation of an initial testing procedure (ITP, “screening”) for the majority of ester derivatives (e.g. propionate, cypionate, decanoate, undecanoate) at LAD, in the second part this method will be utilised to analyse serum samples from a previous clinical study performed at LAD which included an oral TU administration to 19 healthy men volunteers. In the third part of the project, GC/C/IRMS analyses will be performed on 50 urine samples of the same clinical study and results, in terms of detection window, will be compared to that of testosterone esters. Finally, a comparison between the different methods for the testosterone ester analysis will be performed through an inter-laboratory study, which is arranged and managed by the LAD.

Main findings

An LC-MS/MS method for the quantification of three endogenous steroids (T, A4 and DHT) as well as qualitative analysis of a selected menu of exogenous steroids and steroid esters with a single aliquot of human serum was developed and validated. With this approach it was possible to combine the direct detection of synthetic steroids and steroid esters with the indirect detection of steroid doping in the context of the Athlete Biological Passport (ABP). The method was then applied to the analysis of approximately 200 serum samples coming from a previously described testosterone undecanoate (TU) oral administration study. Results demonstrated that elevated TU serum concentrations had a profound impact on the endogenous steroids, allowing distinction between the control phase (no TU administration) and samples after oral TU administration. The two markers, which were found to be most influenced by the oral TU administration, are TU itself and DHT, which was elevated even at low TU serum concentrations.

In the second part of this study, urine and serum samples from the same administration study were analysed to compare the TU detection window with the urinary GC/C/IRMS analysis used in the routine environment. Intact TU was detected in serum samples for at least 4 h up to 24 h after the administration. While urinary IRMS analysis led to slightly longer overall detection windows (especially for 5α/5β-diols) from 12 to 48 hours, this analysis was associated with increased efforts needed in comparison with the serum analysis.

Finally, an inter-laboratory study on the detection of testosterone esters in serum samples was performed showing good results in terms of detection capabilities by the different participating laboratories. All substances spiked in the serum samples were detected correctly by different ITP and estimations of concentrations were judged satisfactory for qualitative methods in the low ng/mL range of concentrations.

Code: 21A06AM

The detection of endogenous anabolic agents still represents one of the major challenges in doping control. Gas chromatographic separation in combination with combustion and isotopic ratio mass spectrometry (GC/C/IRMS) is needed to discriminate between intake and natural production of the endogenous steroids. However, the technique requires a very tedious and lengthy sample preparation that makes it inapplicable to screening analysis and limits its use to those samples that have been flagged suspicious in steroidal module of the Athlete Biological Passport (ABP) after GC-MSn analysis. Historically based on gas-chromatography, the analytical approach for anabolic agents has later expanded to include liquid chromatography, and more recently investigations have considered supercritical fluid chromatography (SFC) as well. The introduction in 2012 of the ACQUITY Ultra Performance Convergence Chromatography (UPC²) system, with improved robustness and performance, suggested that a widespread implementation in routine analysis would follow. However, laboratories have been slow in introducing the change, assumedly at least partly because of the limited research being performed and published so far. We intend to perform an in-depth investigation of the potential of UHPSFC combined with mass spectrometry for the analysis of endogenous anabolic agents in urine samples. In particular, this project will investigate the separation and quantification of endogenous steroids both in the free and conjugated form by UHPSFC-MS and evaluate the various factors influencing sensitivity. The optimal conditions for chromatography and MS will be established, such as the use of co-solvents, the effect of the MS parameters, in combination with an appropriate sample preparation procedure to maximise selectivity and sensitivity as well as achieving the required chromatographic separation. This project will provide an alternative or complementary analytical method to current GC/MSn methods traditionally used in anti-doping laboratories. Additionally, we aim to explore the potential use of UHPSFC combined to fraction collection to simplify and automate sample preparation for GC/C/IRMS analysis.

Main findings

We have developed a method based on UHPSFC-MS/MS to measure the urinary steroid profile for the athlete biological passport (ABP). The goal of the project was to understand whether UHPSFC-MS/MS can be considered a valid alternative to GC-MS for the analysis of endogenous steroids. A thorough investigation was performed on the main parameters influencing method performance and after method optimisation a complete validation was carried out. Depending on the steroid, the method is capable of quantification from 0.5 ng/mL up to 10 μg/mL by employing a weighted quadratic regression model. This is in line with current WADA requirements.

To demonstrate the applicability of the method, a comparison with our routine GC-MS method was performed. Authentic urine samples (128) were analysed using both method and the results compared using Passing-Bablok regression analysis. The results showed an excellent correlation and comparable results between the two methods. In our opinion this demonstrate the good performance of the method proposed. Further studies with a much larger sample population would be needed to confirm our results and eventually propose the approach as a valid alternative to the conventional GC-MS.

In a second part of the project, we have investigated the potential of UHPSFC combined with fraction collection for the sample purification prior to GC/C/IRMS analysis. Thanks to the presence of a splitter between the chromatographic column and the mass spectrometer, it is possible to collect fraction while analysing the remaining portion by MS on the same injection. As a proof of principle, we have manually collected the fraction corresponding to the different steroids, then analysed them by GC/C/IRMS. The same positive and negative control samples were also analysed with our routine method, that involves a sample preparation by a double HPLC purification prior to IRMS. Results obtained in terms of peak purity are encouraging and comparable between the routine method and the newly proposed approach. This suggests that further investigations should be pursued to automate the collection (using a fraction collector) and increase the sensitivity, which for the moment is limited by the maximum volume that can be injected on the system without compromising the chromatography.