Projets de recherche

Code: ISF17A12MT

In sports drug testing, the use of alternative matrices such as dried blood/plasma spots (DBS/DPS), oral fluid (OF), hair, and exhaled breath (EB) can be favorable in terms of the duration, intrusiveness and invasiveness of the sampling procedure as well as analyte stability and overall costs for transportation and storage. While DBS already have become an emerging complementary matrix for a broad range of clinical and forensic applications, the utility for doping control purposes was just recently demonstrated. For a variety of low molecular weight analytes such as anabolic agents and stimulants, detection strategies were successfully developed, however, only a few protein-and peptide-based drugs (e.g. Synacthen and IGF-I) were considered so far. Therapeutic proteins have emerged to an important class of new pharmaceuticals and comprise many drug candidates with potential performance-enhancing properties. The misuse of specific protein-/peptide-based drugs in sports is prohibited and both immunological as well as mass spectrometry-based proteomic approaches are currently used for their detection from urine and/or serum samples. Within this study, analytical strategies for the analysis of peptide hormones from DBS specimens will be developed by using different proteomic approaches such as (immuno-)affinity purification, proteolytic digestion, and LC-MS/MS. As extraction, processing and analysis of DBS are automatable, a novel workstation specially designed for a standalone-preparation of DBS samples will be tested and, if successful, employed for routine doping controls. Both “classical” and novel protein and peptide therapeutics will be used as model compounds for method development and characterization: Growth hormone releasing peptides (GHRPs), a myostatin inhibiting therapeutic antibody, and erythropoiesis-stimulating Fc fusion proteins (Sotatercept, ActRIIA-Fc, ACE-011). This study will contribute to the expansion and improvement of available test methods for performance-enhancing proteins and peptides from DBS.

Main Findings

Dried blood spots (DBS) are a relatively new alternative matrix in sports drug testing, which is advantageous with regards to the invasiveness and intrusiveness of the sample collection procedure, the effort and costs for sample transportation and storage, and the analyte stability. Most of the existing doping control DBS assays include low molecular mass analytes (e.g. anabolic agents and stimulants) and only a few methods for peptidic drugs such as Synacthen and IGF-I have been developed so far.

Within this research project, a DBS detection method facilitating the analysis of insulin and its synthetic or animal analogues was established and comprehensively characterized. The successful analysis of these substances at physiologically relevant concentrations was realized after ultrasonication-assisted extraction, immunoaffinity purification, and liquid chromatographic separation followed by high resolution mass spectrometric detection (with or without ion mobility). During method development, major challenges were an efficient purification of the target peptides from the DBS matrix in combination with the low sample volume of 20 µL. Therefore, DBS analysis of insulins at the present stage cannot reach the sensitivity and simplicity of established serum or plasma analysis. Thus, classical plasma/serum or urine analysis is still superior and recommended in case of quantitative analysis. But for the analysis of non-fasting / non-basal insulin levels, this method can provide reliable qualitative results and opens the possibility to simplify the sample collection, transfer, and storage procedures.

Additionally, two complementary LC-HRMS detection methods for the emerging erythropoiesis-stimulating agent Sotatercept (ActRIIA-Fc) from DBS were developed and validated: An initial testing and a confirmation procedure. Both methods comprise an ultrasonication-assisted extraction, affinity enrichment, proteolytic digestion, and HRMS detection by Orbitrap MS. Due to the generic extraction, the multi-analyte initial testing procedure enables the collection of retrospective data and therefore a simultaneous detection of different IgG-based drug analytes. As proof-of-concept, artificial samples fortified with the emerging protein drugs Luspatercept and Bimagrumab as well as authentic post-administration samples containing Bimagrumab were successfully analyzed.

Finally, an automated extraction method for GHRPs from DBS was set up by using the Gerstel MultiPurposeSampler (MPS). Different parameters such as the composition of the extraction buffer, the cartridge used for subsequent online solid-phase extraction (SPE), and the SPE elution buffer were optimized, enabling detection limits at ng/mL levels.

Publications:

Lange T, Walpurgis K, Thomas A, Geyer H, Thevis M. Development of two complementary LC–HRMS methods for analyzing sotatercept in dried blood spots for doping controls. Bioanalysis 2019;11:923–40

Code: 17A21CG 

It has been found that a number of supplements available internationally via the internet contain banned substances which are not specifically listed on the World Anti-Doping Agency (WADA) Prohibited List. These new compounds are generally termed ‘designer durgs’. Even though they are not specifically named in the WADA Prohibited List, the List allows for inclusion of new substances with similar structure or biological actions to drugs already on the
List. However this presumes that the substance can be detected by the normal drug screening procedures. To achieve the sensitivity required in screening methods they need to target specific compounds and this information is gained by studying the metabolism of the substances in the body. Every time a new substance is introduced a detection ‘gap’ is present until the substance’s metabolism has been studied extensively.

Currently there are many such compounds to be investigated. The aim of this project is to obtain supplements currently sold on the internet, establish the identity of the main ingredients and study the metabolism of a select number of these substances and determine procedures to allow them to be introduced into current testing schemes across all WADA-accredited laboratories.

Main Findings: 

The use of performance and image enhancing drugs (PIEDs) in the community is increasing. The types of drugs are moving from the traditional anabolic-androgenic steroids of the past, increasingly toward selective androgen receptor modulators (SARMs) and metabolic modulators. Most PIEDs have yet to complete clinical trials, so their toxicity and drug-drug interactions are unknown. Due to limited regulatory interest, these PIEDs are easily accessible online and often sold alongside sports supplements. Studies on products containing these PIEDs are also limited, and there is a lack of knowledge about their quality and availability. 
A survey of 107 oral liquid products purporting to contain PIEDs were purchased from Australian online retailers. A further 4 PIEDs were obtained from a seizure. The websites of the suppliers and the products themselves were appraised to gather intelligence on the marketing and presentation of these products. The PIEDs products themselves were analysed by liquid chromatography-high resolution quadrupole mass spectrometry (LC-qHRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) to assess the contamination, concentration, and homogeneity of the PIED products. 
It was found that most PIED products contained drugs, were highly contaminated, and were of poor quality. The SARMs were generally better quality than the other PIEDs classes assessed, and quality issues were generally consistent across suppliers. Additionally, the compound AC-262,536 was found to be in two of the products, meaning it is accessible in the market. In vitro metabolism studies using human liver microsomes (HLM) and S9 liver fraction were conducted, revealing that hydroxylation and glucuronidation of the parent and hydroxylated compound were the main metabolites.

Code: 17A27SD 

In 2002 7a-methyl-19-nortestosterone was shown to demonstrate advantageous hormone replacement therapeutic properties compared to testosterone and its 17 esters. It was also demonstrated to be a potent inhibitor of spermogenesis and received considerable interest as a male contraceptive. Despite the commercial potential of this androgenic anabolic steroid it was never officially approved for clinical use. A recent seizure of 1 by law enforcement agencies in Germany has alerted the doping control community to its availability on the black market and raised concerns of potential abuse by athletes wishing to take advantage of the performance enhancing properties. To ensure the WADA accredited anti-doping laboratories can readily identify the abuse of 7a-methyl-19-nortestosterone scientists at the National Measurement Institute Australia will synthesise three major metabolites detected in human urine. Each compound will be certified to the highest level ensuring unequivocal confirmation of structure using a range of techniques, including mass and NMR spectroscopy. The purity of each calibration standard will be assigned as a mass fraction and a suitable quantity (e.g. 1-5 mg) of each certified reference material will be provided to each WADA accredited laboratory accompanied with a certificate of analysis.

Main Findings: 

Scientists at the National Measurement Institute Australia have synthesized a sample of 7α-methyl-19-nortestosterone and two metabolites observed in human urine post administration. Each compund underwent comprehensive certification confirming the identification and mass racation of the main component. Upon certification all three calibration standards were made available to the WADA accredited sports doping laboratories, accompanied with a certificated of analysis.

Calibration standards of 7α-methylnandrolone (NMI collection number 2048), 7α-methyl-4-estren-3α-ol-17-one (S050) and 7α-methyl-5β-estran-3α-ol-17-one (2047) and associated certficates of analysis have been made available to the WADA accredited laboratories. The certification of each candidate material was performed in accordance with ISO 17034 accreditation as a reference material producer. Each material been certified for identity and pruity as a mass fraction and can be considered to be a primary calibrator. The certification, identification and purity assessment, will be reviewed by a panel of experts comprising staff within NMI and academecs from local university chemistry departments. NMI's external review committee was established to ensure the quality of NMI certified reference materials is maintained.

Code: 17A11CG 

An increasing number of peptidic analytes are being added to the WADA prohibited list, multi-residue testing procedures have become routine for the detection of many of these compounds and their metabolites by LCHRMS or LCMSMS in anti-doping laboratories. We propose to develop a multi-analyte mixed standard, covering the majority of lutenising hormone releasing hormones (LHRHs), growth hormone releasing peptides (GHRPs), growth hormone secretagogues (GHSs) and their metabolites. The multi-analyte standard would be available to all WADA accredited laboratories as a standardised mix across all laboratories world-wide.

Main Findings: 

The aim of this study was to prepare a small peptide (< 2 kDa) mixed standard and determine the best storage conditions to maintain the stability of all peptides within the standard. In this study, ASDTL commissioned Auspep Pty Ltd (Tullamarine, Australia) to synthesise and prepare two small peptide (<2 kDa) mixed standards. The first (SPMIX1) contains 41 small peptides and metabolites, while the second (SPMIX2) contains the same 41 small peptides but also utilises bovine insulin to act as a carrier peptide to minimise any binding of the peptides to the glass vials. The peptides were selected based on published metabolism studies to include the parent peptides and the main metabolites excreted. Minor metabolites and parent peptides that were found to be completely metabolised were not included so that the cost of the mixed standard could remain as low as possible to ensure all WADA accredited laboratories would have access.
Initial homogeneity testing was performed on both SP Mixes. The results obtained for the three peptides for which isotopically labelled internal standards were available - Leuprolide, GHRP-2 and GHRP-2 (1-3)-OH, were used to evaluate the homogeneity of SPMIX1 and SPMIX2 as they provided the most precise data set. The repeatability precision of the measurements was sufficiently small as to show a statistically significant inhomogeneity between vials. Analysis of variance (ANOVA) fo the homogeneity results obtained for each analyte was used to indicate, if at a level of confidence of 95%, whether the variability observed between-bottles was significantly different to the variability observed within bottles (duplicate analysis). The uncertainty in the mass of peptide in each vial due to inhomogeneity was conservatively estimated to be ≤2.52 % for SPMIX1 and ≤ 2.16 % for SPMIX2.
Stability assessment was carried out over a 12 month period under the following 4 different conditions for both SPMIX1 and SPMIX2: 1) in solution at -20°C; 2) in solution at -80°C; 3) dried prior to storage at -20°C; and 4) dried prior to storage at -80°C. The majority of peptides were best stored at  -80°C either in solution or dried, except for Hexarelin, its metabolites and AOD-9604 (7-16). At  -80°C, there was very little difference over the 12 month period between being stored in solution or dried for either SMPIX1 or SPMIX2, with dried peptides only marginally better. However for Hexarelin and its metabolites, drying the solutions prior to storage resulted in a decrease in analyte concentration, especially when stored at  -20°C with losses of 10-30% observed in both SP MIXees. Losses of around 15% were observed for AOD-9604 (7-16), when stored in solution at  -20°C but were not observed to the same extent under the other conditions tested.
The mixed peptide standards were distributed to all WADA accredited laboratories during the 38th Manfred Donike Workshop 2020. Each laboratory was asked to assess both SP MIXes against in-house mix standards with respect to the peptides included in the mix, the addition of a cattier peptide and whether it would be cost effective to purchase SP MIXes instead of buying individual standards. Eleven laboratories reported results and found the quality of the SP MIXes compared well to what they currently used and indicated they would be prepared to purchase SP MIXes in the future, provided additional peptides are added so that they are similar in composition to their current in-house peptide standard mixes. Only 2 laboratories noted a preference for bovine insulin to be added as a peptide carrier.

Code: 17A16CG 

The implementation project will review the capability of the proposed new technology (ultra-high performance supercritical fluid chromatography) within an anti-doping environment. The project will replicate the work done by Desfontaine, Novakova et al (2015/2016) and apply it into screening analysis for three months (circa 1500 samples). This will allow the methodology to be assessed with real samples which can be quitetdirty and more difficult to analyse. The method will also be considered as an orthogonal technology which could be ideally suited for use in confirmation methodology by anti-doping laboratories.

Main Findings: 

An Agilent 1260 Infinity II Hybrid SFC/UHPLC-MS system provided on loan by Agilent Technologies (Sydney, Australia) was used to review the capability of ultra-high performance supercritical fluid chromatography (SFC-MSMS) within a high throughput anti-doping environment by applying it to the screening of routine samples. The SFC-MSMS methodology was provided by Dr Bernhard Wüst, Agilent Technologies, Waldbronn, Germany and was developed by Parr et al (1) at the Institute of Pharmacy, Freie Universität Berlin. As part of this project the method was modified to include an additional 159 compounds with 10 compounds removed. The final modified method monitored 906 MRM transitions to screen for 400 compounds. This did not constitute the full suite of compounds currently monitored by the ASDTL UHPLC-HRMS (Orbitrap) screen however with further optimisation and rationalisation it may be possible to include additional compounds. The MS was operated in dynamic MRM mode with up to 281 concurrent transitions.
The current ASDTL automated sample preparation procedure with a minor modification was used to prepare extracts for SFC-MSMS analysis. Validation data was acquired for the 365 compounds in the laboratory's current multi-analyte standard mixes. A total of 306 compounds met the required validation performance criteria of detection at 50% MRPL (TD2019MRPL) in all ten samples with 310 compounds detected at 100% MRPL in all samples. Carry over was evident with 101 compounds visible in the third blank injected after injection of a sample spiked at 400% MRPL, with peak areas less than 1% of the 400% MRPL injection for most compounds. The SFC-MSMS system proved capable of screening for a large number of substances from multiple classes in a single analysis.
The scope of this project wasimpacted from on-going instrumnt issues that ultimately limited the size of the data set acquired during the routine sample analysis component of the project. Only 336 of the planned 1500 samples were analysed. This individual UHPSFC-MSMS system supplied for this project did not prove to be sufficiently robust during the period over which it was tested. SFC-MSMS however does offer an alternate selectivity and orthogonality to reverse phase chromatography which is complementary to existing methogologies and is therefore a potentialy useful orthogonal confirmatory technique. The volume of organic solvent consumed by the SFC-MSMS was significantly greater than that used by the UHPLC-HRMS making it less 'green' than anticipated.

Code: ISF17A17OJ 

In this project, we want to detect recombinant EPO in plasma samples by liquid chromatography - mass spectrometry through the analysis of intact glycosylated peptides.

We hypothesize that with the new fragmentation techniques such as EThcD, high energy HCD and laser photodissociation, present in the latest orbitrap MS analyzers, it is feasible to detect EPO in body fluids after thorough purification protocols. We aim thus to develop a purification protocol that after a specific EPO immunocapture from plasma samples, it extracts and fractionates glycopeptides based on their disctinct glycan structures through different LC resins. After comprehensive offline fractionation, then we will quantify these intact glycopeptides through parallel reaction monitoring (PRM) acquisition. We will independently monitor each different type of recombinant EPO through trypsin or AspN proteotypic peptides, characterized by different glycan structures that will elute and fragment distinctively
 

Main Findings:

Electrophoretic methods provide te sensitivity for distinguising recombinant and endogenous EPO in human body fluid samples. However, the detection of EPO in biological samples containing both endogenous and recombinant EPO remains challenging. Mass spectrometry (MS) has emerged as a powerful tool to efficiently provide a direct view of glycoprotein profiles and it is useful for assessing differences in natural and recombinant EPO variants.

In this research project, we demonstrated the potential of native MS analysis at the intact protein level for the determination fo complex glycosylation profiles of EPO. We demonstrated the feasibility of using intact glycopeptide profiles for chracerizing EPO produced in CHO cells and HEK293 cells, respectively. We generated a large list of potential targets for distinguising recombinant and endogenous EPO in human body fluid samples. The selection criteria will include the feasibility of lowering the limit of detection compatible with human sample analysis, and selection of non- overlapping glucoforms for specific and sensitive EPO detection.

We propose an integrative approach combining native MS analysis at the intact EPO protein level with intact EPO-glycopeptide MS analysis to facilitate comprehensive characterization of EPO produced in different cell systems. Integration of data from these two approaches will confirm the feasibility of EPO-glycopeptide characterization, and ultimately facilitate the selection of potential mass spectrometry-based biomarkers for doping control.

Code: 17A07GR 

The World Anti-Doping Agency (WADA) establishes two procedures to detect recombinant human growth hormone abuse (rhGH) in sports. The first, the “hGH Isoforms Test”, is based on the separate measurement of growth hormone (GH) isoforms using immunoassays. The second, the “hGH Biomarkers Test”, involves the analysis of two GH-responsive proteins, IGF-1 and P-III-NP, using either immunoassays and/or mass spectrometry (MS)-based techniques. Although being currently applied in routine analysis in anti-doping laboratories, both present some limitations. Main drawback of the “hGH isoforms Test” is the short window of opportunity whereas for the “hGH biomarkers Test” is their dependence of age and sex parameters and the more rapid elimination of IGF-1 compared to P-III-NP reducing retrospectivity.

Recent studies have described fibronectin (FN) as a long-term biomarker of rhGH abuse. These results have been obtained applying enzyme-linked immunoassays (ELISA) to serum and dried blood spots (DBS). Thus, the inclusion of FN in the evaluation of potential rhGH misuse would improve detection of cheating athletes. However, immunological tests suffer from some limitations that MS-based methodologies could overpass. This project aims to develop and validate a liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) method to measure FN concentrations in serum and DBS. The potential advantages of the MS-based
methodology will be assessed and special focus will be done on pre-analytical effects of storage conditions and the introduction of freeze-thaw cycles. Preliminary results from our laboratory show that FN determination is affected by these pre-analytical effects. The new method will be used to quantify FN in samples from an administration study with subjects treated with rhGH and in a control group of healthy subjects to obtain preliminary reference values and preliminary potential decision limits.

Main Findings:

Detection of recombinant human growth hormone (rhGH) abuse in sports is one of the major concerns of anti-doping agencies. The discovery of new biomarkers targeting rhGH misuse would help to improve the current methods carried out routinely in anti-doping laboratories. Recently, immunoreactive fibronectin (FN) protein has been described as a potential biomarker of rhGH abuse that could increase the window of opportunity of the already approved biomarkers of rhGH misuse, IGF-1 and P-III-NP.  Traditionally, immunoassays have been the method of choice to measure protein levels in biological samples. However, these antibody-based approaches could present analytical issues, as cross-reactivity, dependence on tertiary/quaternary protein structures or interferences with endogenous antibodies, which could impact final determination. To overcome immunoassay drawbacks, this project aimed to develop a mass spectrometry-based method to quantify FN in blood samples. A panel of four tryptic peptides (out of six initially proposed) located in different domains of FN sequence was selected to be used as surrogates of FN protein for quantification. The digestion process with trypsin was improved in view of the matrix characteristics and protein expected levels (in the range of µg/mL). Once optimized, an LC-MRM-MS method was developed and validated in accordance to the World Anti-Doping Agency in the Code International Standard for Laboratories (ISL) in terms of specificity, carryover, LoD (1.2 - 2.2 fmol/µL), LoQ (range 3 - 6 fmol/μL), linearity (mean r2 > 0.99), precision (within-run 3.0 - 3.9 %; between-run 9.5 - 44.3 %), accuracy (-16.7 - 5.7 %), recovery (mean 87.7 %), matrix effect (mean 14.6 %) and stability (-15.4 - 18.3 %). This procedure was applied to a set of samples from two different studies. The first was an administration study in which 10 subjects were treated for three consecutive days with a moderate dose of rhGH (0.027 mg/kg) and compared with two untreated subjects. The analysis of FN concentration using the mass spectrometry validated method indicated that no differences were present due to the drug treatment, in disagreement with previous results by immunoassay. For the second study, and because FN has been described as a potential biomarker of Dystrophic Muscular Duchenne (DMD), a population of DMD (mostly teenagers) patients was compared with a group of healthy control children. Similarly, no differences in FN levels were detected between both groups. However, when these samples were tested by immunoassay, significant differences were observed between both groups.  

These apparent divergences between results obtained with mass spectrometry and those using immunoassay could be potentially explained by the high structural and functional complexity of FN, which could impact differently depending on the antibodies’ selectivity used in immunoanalytical methods. In summary, our results spotlight the importance of method selection in peptide/protein quantification when intended to be used for bioanalytical discriminating purposes.

Code: 17A31MT 

Trenbolone is a veterinary product marketed as an effective growth promoter in animal husbandry and has never obtained medical approval for humans. Nevertheless, it is presumed to be regularly misused by athletes due to its performance enhancing properties and resulted in various adverse analytical findings in the recent years. By the end of 2016, the issue of trenbolone in sports was particularly emphasized by the McLaren report as one of the anabolic steroids extensively misused in Russia. The cocktail administered to athletes consisted of oxandrolone, methenolone and trenbolone. While the first 2 steroids have been carefully investigated in recent years and long-term metabolites were identified, this research is still pending for trenbolone. Since more than 25 years doping control laboratories search for trenbolone itself and its major urinary metabolite epitrenbolone. Detectability of trenbolone was only improved by using liquid chromatography-mass spectrometry instead of gas chromatography-mass spectrometry.

Aim of this study will be the in-depth investigation of the trenbolone metabolism in humans using a recently established methodology developed especially for metabolite detection in sports drug testing.  After administration of deuterium-labeled trenbolone, all metabolites carrying this label will be identified unambiguously by hydrogen isotope ratio mass spectrometry. Then all analytes of interest will be quantified and identified by means of high-resolution and high-accuracy mass spectrometry. Within this study we will focus on the detection of long-term metabolites prolonging the retrospective detection of trenbolone misuse.

Main Findings: 

Trenbolone is a synthetic anabolic-androgenic steroid, which has been misused for performance enhancement in sports. The detection of trenbolone doping in routine sports drug testing programs is complex as methods utilizing gas chromatography/ mass spectrometry are complicated by unspecific derivatization products and artefacts, and liquid chomatography/ mass spectrometry-based assyas have shown to allow for comparably high limits-of-detection only. The number of previously reported metabolites in human urine is limited, and most analytical methods rely on targeting epitrenbolone, trenbolone glucuronide, and epitrenbolone glucuronide.

In order to probe for the presence of additional trenbolone metabolites and to re-investigate the metabolism, an elimination study was conducted. One single dose of 10 mg of 5-fold deuterated trenbolone was administered to a healthy male volunteer and urine samples were collected for 30 days. For sample processing, published protocols were combined considering unconjugated, glucuronic acid-, sulfo- and alkaline-labile conjugated steroid metabolites. The sample preparation strategy consisted of solid-phase extractions, liquid-liquid extractions, metabolite de-conjugation, HPLC fractionation, and derivatization. Analytical methods included gas chromatography/ thermal conversion/ hydrogen isotope ratio mass spectrometry combined with single quadrupole mass spectrometry as well as liquid chromatography/ high accuracy/ high resolution mass spectrometry of the hydrolyzed and non-hydrolyzed samples.

Twenty deuterium-labelled metabolites were identified including glucuronic acid-, sulfo- and potential cysteine-conjugates, and characterized by parallel reaction monitoring experiments yielding corresponding produc ion mass spectra. Main metabolites were attributed to trenbolone-diol and potential trenbolone-diketone derivatives excreted as glucuronic acid and sulfo-conjugated analytes with detection windows of 5 respectively 6 days. Further characterization was conducted with pseudo MS3 experiments of the intact conjugates and by comparison of resulting product ion mass spectra with reference material.

Code: 17A14MT 

Compared to the metabolism of low molecular mass drugs (such as anabolic agents, stimulants etc.), the biotransformation of peptide-based drugs after (subcutaneous) administration is largely unknown. Especially for larger peptides and proteins, dedicated in-vitro models simulating the parenteral administration have been missing. Since mass spectrometric methods commonly rely on detailed information about the active drug and particular its metabolites existing in the circulation, studies providing these data are critical. In the present project it is planned to apply a sophisticated in-vitro model using skin tissue microsomes for prohibited peptides such as synacthen, insulin like growth factor, growth hormone releasing hormone and others. Selected amino acids of the utilized reference peptides will be isotopically labeled, which facilitates and accelerates the identification of formed metabolites by means of diagnostic reporter ions but does not affect the metabolic reactions of the substance of interest. High resolution mass spectrometry enables finally to identify the molecular mass and the amino acid sequence of the formed metabolites, which eventually can serve as target peptides for efficient doping controls using this Isotope-labeled Reporter Ion Detection strategy.

Main Findings: 

Investigations into the metabolism of peptidic drugs stil represents a subtantial challenge in doping controls, and the detection of the intact non-metabolized drug (condidate) is the most commonly employed strategy for most of the established detection assays. In contrast to drugs of low molecular mass (e.g. anabolic androgenic steroids, stimulants, etc.), in vitro experimental apporaches baseed on e.g. liver microsomes do not simulate appropriately the conditions for peptide and protein metabolism, as these compunds are almost exclusively applied by parenteral routes and alternative approaches have been required.

In the present project, the strategy to use skin tissue microsomes for in vitro metabolism studies of prohibited peptide hormones (insulin, synacthen and corticotropin) was shown to be a particularly informative option. Combined with the use of stable isotope-labelled peptides, the identification of resulting metabolites was significantly facilitated due to the formation of diagnostic reporter ions (derived from amino acid-generated immonium ions) still bearing the 13C of 2H isotope label(s) by means of high-resolution/high accuracy mass spectrometry. Applying this stable Isotope-labeled Reporter Ion Screening (IRIS) approach to selected prohibited peptides (insulin, synacthen and cortocotropin) yielded nearly 20 metabolites for these peptides with truncated amino acid chains. Especially for metabolically less stable peptides such as synacthen, the identification of these new metabolites will support prolonging the detection window in doping control samples. In addition, also for metabolically more stable peptides such as insulin and corticotropin, identified metabolites showed diagnostic potential concerning the differentiation between endogenous secretion and subcutaneious administration. Finally, a generically applicable approach with simplified sample preparation protocol was developed by means of mixed-mode cation exchange solif-phase extraction, which facilitates the preparation of blood samples for doping control analysis without the need for immunoaffinity purification.

Code: R16SC02SM

To analyze biological samples using SomaLogic's proprietary SOMAscan™ proteomic assay with the objective of potential discovery of new biomarkers of hGH abuse. The services will be provided in accordance with the terms and conditions set forth in the Standard SOMAscan Services Agreement attached hereto as Exhibit A.

Main findings

As shown in this study, hGH treatment results in detectable changes of SOMAmer-targeted protein analytes. Across the 10-day study period, some analytes exhibit a quadratic, or squared-type change, which generally track the hGH treatment period. Such parabolic responses that have a maximum or minimum at Day 4, and then return toward baseline levels by day 10, represent analytes that change during the administration of hGH, such as those theorized or observed in the previous WADA/SomaLogic pilot study. However, analytes discovered via a quadratic model in which measurement levels return to baseline within 6 days do not represent detectable differences > 6 days after treatment has stopped. The linear models in Section 4.2 show targets that change across the entire 10 day period, remaining elevated (or depressed) several days post-treatment. Finally, the actual magnitude of change at different timepoints may be important. For this reason, this report includes log2 of fold-change compared to baseline at several key timepoints, as well as test statistics from paired Student’s t-tests. These “key” timepoints include baseline vs. 8 hours (the initial post-treatment follow-up), baseline vs. Day 4 (the final treatment day), and baseline vs. Day 10 (the final study day). The 8-hour analysis shows the highest number of significant analyte changes, but the metabolic targets (e.g. insulin and pancreatic hormone) are suggestive of a potential post-prandial effect. The Day 10 analysis shows the least number of significant changes. The paired analysis results should be combined with the linear mixed-model (quadratic or linear) results to suggest protein targets that have plausible magnitude and directional changes as well as longitudinal trends. To this end, FDR and Bonferroni-adjusted p-values are provided, as well as unadjusted p-values are provided in the external spreadsheet “WADA_SomaLogic_HGH.xlsx”). Appropriate usage of statistically-significant results, combined with specific treatment hypotheses and biological knowledge should be combined holistically and purposefully. This report contains annotated visualizations, as well as the combined, interactive spreadsheet mentioned above, to aid in hypothesis testing so that trade-offs can be made between model type, false discovery, and particular protein targets of interest.