Projets de recherche

Code: ISF17B03BS 

Recombinant erythropoietins (EPOs) have long been one of the most frequently abused drugs for increasing blood oxygen capacity. However, numerous compounds with EPO-like activity but completely different chemical structures (either short peptides or even nonpeptidic synthetic molecules) have been developed so far and due to such chemical diversity, detection of their potential abuse is notoriously difficult. To our knowledge, no universal and reliable analytical method is available to confirm the presence of all EPO mimetics in biological sample regardless of their chemical structure. Here, we describe development of a rapid and universal assay for detection of any ligand binding to the erythropoietin receptor (EpoR) which could be used as a low-cost screening test in doping control to identify individuals for further detailed examination. Our innovative platform comprises combination of immunoprecipitation steps and two modified ELISA tests where recombinant filamentous phage particles displaying EpoR are used instead of primary antibodies. As evaluated with spiked artificial urine, such assay can detect the presence of EpoR ligands chemically unrelated to EPO in specimens. Site-specific proteolytic cleavage and isolation of EpoR:ligand complex from phage particles enables analysis of the complex by mass spectrometry and potential identification of previously unknown EpoR ligand. The described platform may also be further developed into the lateral flow immunoassay format similar to common over-the-counter pregnancy test which would greatly increase its applicability out of the laboratory. To emphasize, such concept can also be applied to detection and identification of other growth factor or hormone mimetics by implementation of appropriate combination of receptor, ligands and antibodies. Moreover, based on our platform a multiplex assay capable of simultaneous detection of substances with distinct activity can be developed by utilizing Luminex® technology and appropriate combination of recombinant phage particles displaying different hormone and growth factor receptors. 

Code: 17A28PA 

Detection of the use of the anabolic steroid nandrolone is based primarily upon the identification of the main urinary metabolite, 19-norandrosterone. Laboratories use the sophisticated technique of gas chromatography mass spectrometry to detect 19-norandrosterone in athletes' urine. WADA have recently taken steps to harmonize the analysis of nandrolone, issuing WADA Technical Document TD2016NA in 2016.

Reference materials are a vital tool for validating analytical testing methods and for ongoing laboratory quality assurance. International best practice specifies that CRMs beused as part of routine laboratory quality control. However CRMs for the measurement of steroid metabolites in human urine are not readily available. The project involves certification of the stable carbon isotope delta value of 19-norandrosterone (19-NA) in CRM MX017, currently being prepared for WADA project 15J04JM. Laboratories will reconstitute this material with water, and then analyse it alongside samples of urine taken from athletes for doping control. The testing laboratory can the compare the measured values for the CRM to those certified, providing assurance of the validity of the testing procedure.

Main Findings:

A new freeze-dried human urine NMIA MX017 certified reference material (CRM) has been prepared by NMIA, under the funding rom WADA, to replace the depleted MX005 CRM. Approximately 2400 units had been produced and made available to WADA laboratories since April 2019. This material will assist WADA laboratories to demonstrate comparability of measurements for the Athlete Biological Passport introduced in 2014. The replacement material will continue to support the longitudinal profiling measurements for detection of testosterone abuse and to provide SI-traceable values for the mass fraction and mass concentration of the glucuronides of six steroids specified in the WADA technical document TD2018EAAS: testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5a-androstane-3a,17b-diol (5a-Adiol) and 5b-androstane-3a,17b-diol (5b-Adiol).

In February 2020, the carbon isotope delta value of (δ13CVPDB) 19-norandrosterone (19-NA) has been added to the MX017 certificate. The new property value (δ13CVPDB of 19-NA) is traceable to the VPDB reference via the NMIA MX018 steroid carbon isotopic reference materials normalised by two secondary isotopic reference materials IAEA-CH-6 (sucrose) and IAEA-CH-7 (polyethylene). The reference value, δ13CVPDB -29.82 ± 0.41 , was determined using 36 bottles of MX017 analysed in five batches of experiments. The combined standard uncertainties of the reference value was expanded with a coverage factor of 2.0 to provide a 95% level of confidence.

Analysis was performed on 20 mL of reconstituted urine CRM (MX017) after hydrolysis with β-glucuronidase (E.colie). Free steroids were extracted into hexane and purified by two-dimensional HPLC. In-house quality control samples including water spikes, matrix spikes and solvent standards were included to monitor fractionation due to sample transformation procedures. The purified fractions were dried and reconstituted in cyclohexan/2-propanol (4:1) for anaylis by GC-C-IRMS. The identical treatment priniciples was followed to ensure samples and calibration soltions were combusted and transferred the same way into the IRMS. Each samples was injected twice in a randomised order bracketed by calibration standards. Frequent bracketing of the test samples with calibration solutions allowed any drift in the instrument to be fully captured. An approximate δ-value of the internal working gas was used to calculate all raw δ-values for samples and standards. A multi-point isotopic bracketing calibration approach was adopted to normalise all the measured δ-values of samples using steroid isotopic CRM mixtures NMIA MX018-1 and MX018-3. A linear regression line (R2> 0.99) fitted through the measured and the reference delta values from the eight steroid compounds, in the two calibration mixtures, allowed the measured δ-values of 19-norandrosterone in the MX017 to be normalised to δ13CVPDB the VPDB scale (Figure 1). The multi-point steroid isotopic calibration approach has allowed the property value to be assigned with ower uncertainty than the measurement approach employing calibrated reference gas.

Code: ISF17D05FM & ISF17E06RG

In a funded WADA study recently completed (“Novel molecular biomarkers for detection of autologous blood transfusion in sport: fetal hemoglobin and microRNAs”) we tested the hypothesis that novel biomarkers as fetal hemoglobin (HbF) and related microRNAs might show changes of interest in a group of trained healthy volunteers exposed to ABT respect to controls. The first result of this study is the production of a validated WADA-UNIFE-Biobank constituted of around 2000 plasma samples from control and ABT trained subjects. It is established that hypoxic and hyperoxic stimuli and blood manipulation during procedures of withdrawal and reinfusion (distress, ageing, apoptosis/degradation of circulating blood cells, effects of preservative substances, etc.) might induce a predictable and an unpredictable series of changes of miRNAs expression. Therefore, we hypothesized that after significant blood collection and autologous reinfusion, the miRNA network in the athlete’s plasma is changed, allowing to generate integrated molecular profiles permitting to predict ABT. Therefore, the hypothesis is that a miRNA pathway might be much more informative than a single miRNA, even if demonstrated to be associated with ABT in a sub-set of athletes. To this end, a small sample of subjects enrolled for the cited study was recently spontaneously tested in our laboratory for a full genome analysis. Therefore, the aim of this study is the validation of the first release of ABT-miRNA list and the identification of novel miRNAs of putative interest in predicting ABT following global miRNA analyses.

Main Findings

This project was aimed at identifying novel molecular markers useful in the anti-doping field to detect autologous blood transfusion (ABT). The hypothesis was that both the phases of ABT, blood withdrawal and reinfusion, might be accompanied by changes in different parameters to be identified by OMICS approaches in the level of microRNAs (miRNAs), small non-coding RNAs that regulate gene expression. In a previous funded study “Novel molecular biomarkers for detection of autologous blood transfusion in sport: fetal hemoglobin and microRNAs” twenty-four healthy trained male subjects were enrolled and randomized into Transfusion (T) and a Control (C) groups. The T subjects underwent nine seriated blood samples before and after the procedures of withdrawal and reinfusion. Among erythroid-related miRNAs tested, following ABT a pool of 7 miRNAs associated with fetal hemoglobin and regulating transcriptional repressors of gamma-globin gene was found stable in C and differently expressed in three out of six T subjects at 12 days time-point after re-infusion.

In the present study, using samples stored in the biobank, we aimed to validate the first release of ABT-miRNA list at other time-points (i.e. D-25 and D+3) to confirm the interest of this miRNA list in predicting ABT. ABT prediction was also demonstrated after global miRNA analyses, thus identifying novel ABT-informative miRNAs. The levels of selected informative microRNAs in relation to sex was determined and protocols/assays for ABT detection were developed. of specific interest. To this aim, microarray analysis and droplet digital RT-ddPCR were also performed for subjects of the T group and C groups for selected time-points.

The data obtained studying the global miRNome pattern demonstrated clustering of ABT-treated plasma samples at D+3 and D+15. These results support the concept that identification of ABT can be performed in one-step using the developed microarrays procedure for miRNA global analysis. The blood withdrawal was not found to be a “confounding factor”. Secondarily, the data obtained by RT-ddPCR performed in plasma samples from ABT trained subjects at D-25, D+3 and D+15 time points were first aimed at validation the “erythroid-associated miRNA list” (let-7a-5p, miR-126-3p, miR-144-3p, miR-191-3p, miR-197-3p, miR-486-3p, miR-486-5p and miR-92a-3p) representing ABT associated miRNAs involved in hypoxia, erythroid differentiation and fetal hemoglobin production. This set of 8 erythroid associated miRNAs were informative in detecting ABT in most (5/6) of the ABT-treated subjects at all the time-points considered. We then considered miRNA found specifically dysregulated at D-25, D+3 and D+15 time points according with the miRNA microarray analyses. This additional list was proposed in consideration of the miRNA fold changes, the availability of the assays for miRNA amplification and the necessity to have both up- and down-regulated miRNAs to test as possible ABT-associated biomarkers. A final list of six miRNAs was identified and selected for further experimental activity. While the data obtained for down-regulated miRNAs were not informative (no major differences with the control set), differential expression of miR-766-3p (D-25), miR-636 (D-25), miR-425-3p (D+3), miR-4284 (D+3), miR-3151-3p (D+15) and miR-5787 (D+15) was found. When the two miRNA lists (erythroid and global) were used, only one ABT subject was not identified as abnormal or potentially positive for ABT (score 11/12 = 92%). Despite a high heterogeneity within the male and female groups, minimal sex-biased miRNAs differences were observed. Interestingly several miRNAs found up-regulated at the D-25 time point, which is 10 days after the blood withdrawal, may represent “follow-me” markers to identify athletes at risk of ABT to be tested in the proximity of major agonistic events. The results of the present project allow to validate a list of 14 miRNA useful, by RT-ddPCR based analysis, to detect ABT in the majority (over 90%) of the ABT-treated subjects.

Code: 17D15XD 

The detection of pseudoendogenous steroids is based on the alterations of the parameters of the steroid profile included in the steroid module of the Athletes’s Biological Passport (ABP). A Bayesian statistical inference approach allows fixing the boundaries for the ABP parameters of a given individual based on the previous data collected. In the case of a suspicious sequence of data or data outside the population based ranges when previous values are not available, a specific confirmatory method based on isotope ratio mass spectrometry (GC-C-IRMS) is mandatorily applied to the suspicious sample to disclose the synthetic from endogenous origin of the steroids detected. The current approach, although scientifically valid, has some limitations: (1) delay in taking appropriate decisions by the results management authorities (RMA) if the first value of the sequence is abnormal since the model needs the collection of additional samples to detect it; (2) ineffective for those athletes not tested with some frequency; (3) unable to detect low dosages with physiological effect.

In addition, new pharmaceutical formulations of synthetic steroids are not easily detectable by GC-C-IRMS if isotopic analyses of additional samples of the athlete are not performed. The aim of this study is to expand the use of IRMS at a screening level for those disciplines at higher risk of pseudoendogenous steroids abuse, simplifying the current confirmatory procedure. This would allow reducing the time of evaluating an abuse of pseudoendogenous steroids including those athletes less frequently tested, detecting lower dosages of administrations and for a longer period of time and finally being able to detect those preparations not currently detectable by the longitudinal evaluation of the IRMS data since these data would be now available.

Code: ISF17R02NN

This project is aimed at identifying markers of rHuEPO misuse and separate these from markers of natural hypoxic exposure by metabolomics. Male (n=20) and female (n=20) non-competing athletes are exposed to 4 weeks sea-level investigation and after a full washout period, participants are exposed to hypobaric hypoxia at 2.320 m for four weeks. Four groups are formed in a blinded randomized and gender balanced fashion: One group (n=20) receives EPO during the sea-level period and placebo during the stay
at altitude. Another group (n=8) receives EPO both at sea-level and altitude. A third group (n=8) receives placebo both at sea-level and altitude and the fourth group (n=4) receives placebo at sea-level and EPO at altitude. The design facilitates identification of markers of EPO misuse at sea-level (n=28) as well as markers of natural altitude exposure (n=28). Each participant’s treatment response is analyzed in relation to a 4 week baseline period. Moreover, the design includes an intra-individual possibility to verify identified targets expected to be unique to rHuEPO misuse, since 8 participants are treated with rHuEPO both at sea-level and altitude. Runners are enrolled due to the good possibility of recruitment and conduction of training at altitude. The project results in a highly valuable biobank for identification of markers sensitive to initiation and termination of rHuEPO injections. Metabolomics discovery is carried out in collaboration with a world-leading research group as well as the experts at University of Cologne, Germany. Importantly, the project is a joint effort between world-leading University research, Anti-doping organizations and WADA laboratories.

Main Findings

With the present study we were able to demonstrate that recombinant human erythropoietin (rhEpo) treatment combined with altitude exposure provide an additive erythropoietic response compared to rhEpo treatment or altitude exposure alone. Accordingly, analysing the results in the Athlete

Biological Passport revealed that the passport is more sensitive to rhEpo treatment at altitude than at sea level, but a concurrent compromised specificity exist with altitude exposure.

We also investigated the potential of immature reticulocyte fraction (IRF) and the ratio between immature reticulocyte and red blood cells (IR/RBC) as novel biomarkers for rhEpo treatment. With this study we demonstrate that a low-dose rhEpo treatment alters IRF and IR/RBC compared with placebo, which also exceeds the fluctuations induced by altitude exposure in the post-treatment period. Similarly, during rhEpo treatment at altitude, both biomarkers exceeded the fluctuations induced by placebo injections at altitude. When individual thresholds were calculated, we were able to identify ~90% of the rhEpo treated participants at sea level with each variable, whereas we were able to identify ~30% and ~65% of the rhEpo treated participants at altitude for IRF and IR/RBC, respectively. Importantly, these sensitivities were obtained with a specificity >99%.

Plasma samples have been subjected to untagerted metabolomics followed and bioinformatic analyses. No biomarkers with sufficient sensitivity and specificity to discriminate between sea-level or altitude exposure could be identified. Likewise, no biomarkers able to discriminate rhEpo and placebo treatment could be identified.

Finally, the present study demonstrated that the novel markers of iron homeostasis, hepcidin and erythroferrone, are more sensitive markers of altered iron homeostasis within one week of changes in iron demands than routine iron markers. When individual thresholds were calculated, 30-40% of the rhEpo treated participants exceeded the thresholds. Our results demonstrate that erythroferrone, and to some extent hepcidin, may hold promise as novel biomarkers for detecting rhEpo misuse at altitude, but intra-individual variability is of concern and additional studies are required

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.