Projets de recherche

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.

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.

Code: 21A05XT

The design and synthesis of new anabolic androgenic steroids, characterized by reduced side effects and better oral bioavailability, begun shortly after the structure of testosterone was identified in 1935. Since then, the discovery of new steroids has been accompanied by studies on their metabolism, to understand how they could be metabolized by phase I and II enzyme isoforms that alter their toxicity, action, and elimination. The presence of pharmacologically active metabolites, their variable formation, and their potential interaction with other drugs and/or nutritional supplements, may indeed interfere with the analytical results of doping controls. Furthermore, although metabolite detection allows identification of the metabolic pathways involved in drug absorption, molecules with related structures and similar physicochemical properties may follow generally common metabolic pathways, but different biotransformation steps, making more challenging the selective and unambiguous identification of the original compound. The process of detecting, characterizing, and confirming metabolites is time-consuming and resource-intensive. The analytical strategy currently used to identify drug metabolites involves several steps, some of which are more influenced by knowledge bias. The combination of knowledge-based and machine-learning approaches represents an innovative and effective way to predict, identify and characterize drug metabolites, not only in anti-doping but also in clinical or forensic fields. 

To this end, the proposed systematic analytical protocol, capable of combining targeted and untargeted analyses, full scan, MS/MS experiments, and previous knowledge, represents a strategic tool for the rapid detection of drug metabolites. The use of accurate mass measurements by high-resolution mass spectrometry (HRMS) and the complementarity of liquid and gas chromatography ensures a high degree of completeness to the results. The processing of the raw data by multivariate analysis allows to focus on the specific features of the condition, without performing any a priori bias.

Code: 21A03MT

Phenethylamine (PEA) is listed on WADA´s Prohibited List as a stimulant and therefore its administration is forbidden in competition. As the human body naturally produces PEA, its presence in urine samples alone is not an indication for its misuse. Already in 2015, a potential metabolite indicative for the administration of PEA ((2-(3-hydroxyphenyl)acetamide sulfate) has been reported, based on pilot study results conducted with two individuals. During a follow up study encompassing more volunteers and different administration protocols, other potential metabolites were identified. Both PEA and its metabolites show are large inter-individual variance, i.e. the urinary concentrations of these compounds can be substantially elevated even without any administration simply due to the individual´s metabolism. This phenomenon is well described for urinary steroids and their concentrations as demonstrated for testosterone. Here, the carbon isotope ratios (CIR) are used to confirm the exogenous origin of elevated concentrations if found in urine samples of athletes. Similarly, within this research project a method suitable to determine Page 2/8 the CIR of PEA and its metabolites will be developed and validated according to WADA guidelines. Besides the development of a sophisticated sample clean-up procedure possible derivatization techniques will be investigated to ensure valid results. Finally, a reference population will be investigated to figure out the expected range of isotopic ratios for PEA and its metabolites. This will enable a clear differentiation between endogenous (produced naturally inside the human body) and exogenous (administered) PEA found in urine specimens.

Main Findings

Phenethylamine (PEA) is itemized as a stimulant on the Prohibited List of the World Anti-Doping Agency since 2015. It represents a naturally occurring trace amine and has been investigated in the 1970s and 80s regarding its medical potential as a modulator in the central nervous system. Today it is sold as a dietary supplement, marketed for its mood enhancing effects and as a potential treatment for weight loss.

Its detection in sports drug testing remains complicated as PEA is an endogenous substance, i.e. it is present in urine, at least in trace amounts. Investigations into a reference population demonstrated that the found urinary concentrations show a large inter-individual variation. Interestingly, after the oral administration of PEA, its urinary concentrations were found only slightly elevated. Establishing a urinary concentration threshold for PEA was therefore not successful and further studies focused on potential metabolites of PEA identifying one sulphated minor metabolite and phenylacetylglutamine (PAG) as the most abundant metabolite of PEA in urine. Again, the biological variability of these urinary metabolites complicated establishing a threshold and only the combination of both parameters in a binary logistic regression enabled to identify post-administration samples.

Aim of this research was the development and validation of an isotope ratio mass spectrometry-based method for PEA and PAG to differentiate between the endogenous or exogenous source of these metabolites considering their carbon isotope ratio (CIR). Especially for PAG, the developed method met all expectations and reference population-based thresholds were established. Unfortunately, both endogenous PEA and PAG showed unexpectedly depleted CIR and the found difference between exogenous and endogenous PEA was only around 3 ‰. According to these relatively similar CIR, the opportunities for detecting an oral administration of PEA were very short, and after a single oral dose none of the volunteers was found with CIR outside the population-based thresholds.

In athletes with relatively enriched CIR the developed method will most probably not result in an adverse finding as here the impact of the exogenous PEA will not influence the determined CIR of urinary PAG beyond the established threshold.

Further investigations will be necessary to elucidate the potential of IRMS to detect the misuse of PEA, e.g. by means of stable isotopes other than carbon.

Code: 21A02MP

In human sports the application of glucocorticoids is limited by anti-doping regulations, however, recent statements of professional cyclists underline the high potential of misuse. Currently, for glucocorticoids a reporting level of 30 ng/mL in urine is established in doping control analysis. Etamivan is classified as respiratory stimulant, that is also prohibited in sports. Due to its high metabolic sulfonation in humans, in recent external quality assessment schemes (EQUAS) etamivan-sulfate was explicitly integrated as target analyte besides the parent compound etamivan. At present a reporting limit of 50 ng/mL for stimulants is specified. Both classes are highly prone to matrix interferences in LC-ESI-MS/MS that may confound quantitation and thus influence decision of reporting. As orthogonal technique, SFC-MS/MS may help to overcome these issues. Thus, the project aims to investigate its potential for determination of glucocorticosteroids and etamivan and its sulfoconjugate. Furthermore, the LC- and SFC-based methods will be compared in terms of matrix interferences and accuracy of determination.

Code: 21A04RM

In the glucuronated fraction, the differences among Δδ13C pairs are known to reflect the metabolic isotopic fractionation and potential method bias. This is known as well, to be magnified in the sulfate fraction. After the publication of some results concerning the sulfate fraction analysis advantages, WADA TD2021IRMS states that Epiandrosterone could be used as an additional TC for GC-C-IRMS
analysis to determine the administration of testosterone or its precursors and the decision rule for IRMS positive test is based on the absolute Δδ13C value of ERC-EpiA pair higher than 4‰. No
specification is explicitly done about which ERC should be used to evaluate an analyte from the sulfate fraction and in which fraction the ERC should be present.

The project aims to study the differences between the δ13C and Δδ13C values obtained taking into consideration the ERC in glucuronated and sulfate fraction of urine, during the evaluation of
sulfated target compounds. It will be studied the classic ERCs: Pregnanediol, Pregnanetriol, 11β-hydroxy-androsterone,11β-hydroxy-etiocholanolone and Androstenol. Sulfoconjugation of these compounds is not extensively published but we do not expect more than 10% based on the structure. In this case, a preliminary and alternative study of estrogens will be carrying out as well as androst-5-ene-3β,17α-diol proposed previously.

If the goals of the proposed project are reached, we should be able to align the results based on ERC and TC both excreted as sulfo-conjugates. Besides, the bias should be minimized because the
already described difference between CIR of glucuronate and sulfate fractions could be avoided. Finally, we expected to increase the knowledge regarding the δ13C of ERCs and Δδ13C pairs, all in
sulfate fraction from the Reference Population Data (at least for males) which can contribute to the selection of a sensitive and/or affordable ERC for IRMS confirmation in sulfate fraction of the urine.

Dr. Nikola Jojic, University Business Academy Novi Sad, Faculty of Pharmacy Novi Sad

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