Funded Projects
The PCC has supported world-class research since 2008, investing more than $35.6 M to drive novel science. Research and grant-making are the foundation of the PCC and are the focus of everyday business activity. PCC-supported research contributes to a movement in addressing doping’s root causes and ultimately decreasing the use of performance-enhancing drugs by all participants in all sports at all levels of play.
Funded PCC projects represent proposals from 23 different countries and three continents. More than 100 principle investigators have been awarded grants to advance anti-doping knowledge through more than 250 unique research projects. Projects which showcase alignment with the PCC research priorities are given funding precedence.
Please contact us if you have questions regarding individual projects listed here.
Fellowships
Funded: 2024
Fellow:
Sophia Krombholz, German Sport University Cologne
Mentor:
Dr. Mario Thevis, German Sport University Cologne
Research Title:
Effect of alternative routes of drug administration on metabolic profiles in doping controls – exposure vs. intake
Funded: 2022
Fellow:
Dr. Jenna Goodrum, Sports Medicine Research and Testing Laboratory (SMRTL)
Mentor:
Dr. Goeff Miller, SMRTL
Research Title:
Biotin as a masking agent for hCG
Funded: 2022
Fellow:
Dr. Sara Amalie Solheim, Oslo University Hospital and the Norwegian School of Sport Sciences
Mentor:
Dr. Yvette Dehnes, Laboratory and APMU Director, Norwegian Doping Control Laboratory
Research Title:
Further development of blood testing: ABP, deterrence and virtual testing
Funded: 2022
Fellow:
Dr. Huu Hien Huynh, University of Washington
Mentor:
Dr. Andrew Hoofnagle; University of Washington
Research Title:
Quantification of type III procollagen fragments in capillary blood
Funded: 2019
Fellow:
Dr. Federico Ponzetto, University of Turin
Mentor:
Professor Ezio Ghigo; University of Turin
Research Title:
Implementation of a blood steroidal module in the framework of the athlete
Research Summary:
The research project entitled “Implementation of a blood steroidal module in the framework of the Athlete Biological Passport” and carried out at the Department of Medical Sciences of the University of Turin, aimed at providing a robust analytical tool for investigating the variations of Blood Steroid Profile. During the 2 years of the project a novel LC-MS/MS method for the quantification of 27endogenous steroids, including major circulating hormones as well as a wide panel of androgens’ phase II metabolites, was developed and validated according to WADA guidelines. This method was then used for calculating preliminary reference intervals of target analytes, some of which were never reported to date, analyzing serum samples collected from healthy volunteers accessing to the Blood Bank of Turin University Hospital. Furthermore, the potential impact of circadian rhythm and physical exercise on Blood Steroid Profile markers was investigated thanks to the analysis of serum/plasma samples collected during two dedicated clinical studies involving 19 healthy male volunteers and 31professional football players. The outcomes of these studies , beside proving the usefulness of the developed analytical platform for gathering additional information on steroid metabolism, useful for the introduction of blood steroid profiling in anti-doping context, highlighted that serum concentrations of testosterone(T) and dihydrotestosterone (DHT)as well as of androgens phase II metabolites are not significantly perturbated by diurnal variations linked to circadian rhythm and by a moderate physical exercise such as a football training session. These results strongly support the introduction of such markers in the future implementation of Blood Steroid Profile for doping control purposes, remarking the utility of monitoring not only T and DHT but also more concentrated phase II metabolites such as etiocholanolone glucuronide, androsterone glucuronide, epiandrosterone sulphate and androsterone sulphate. During the performed studies it was also observed that the administration of 5α-reductase inhibitors (e.g., Finasteride) could have a strong impact on target markers, stressing the need of dedicated study for better characterizing steroid response to such intake. Finally, with the aim of suggesting an easier sample collection procedure for Blood Steroid Profile, a further analytical method for the measurement of most concentrated steroidal compounds from Volumetric Absorptive Micro-Sampling (VAMS) was developed, validated and applied to assess the medium-term stability of target analytes in blood micro-matrices. The promising results obtained in this proof-of-concept study, underlining the stability of VAMS samples up to 100 days at-80°C,opened the way to the use of such technology for simplifying blood sample collection, storage and transportation in the future studies that are needed to fully characterize the perturbations of endogenous and exogenous potential confounding factors on Blood Steroid Profile. Although the advances obtained thank to the present projects, there is still lack of population studies for the definition of reference intervals in elite athletes’ populations as well as for the assessment of long-term intra-individual variation of most promising serum markers of EAAS doping. These topics could represent the challenge for future research project in the field of steroid analysis in anti-doping context.
Funded: 2018
Fellow:
Dr. Scott Lacombe, Dell Pediatric Research Institute, University of Texas at Austin.
Mentor:
Dr. Herbert Tobias; Dell Pediatric Institute, University of Texas at Austin
Dr. Tom Brenna; Dell Pediatric Institute, University of Texas at Austin
Research Title:
Validation of ARC Catalytic Combustion Reactors for Fast GCC-IRMS and
GC×GCC-IRMS
Funded: 2018
Fellow:
Dr. Jacob Bejder, University of Copenhagen
Mentor:
Dr. Nikolai Nordsborg, University of Copenhagen
Research Title:
Improving detection of erythropoiesis stimulating agents and glucocorticoids
Funded: 2018
Fellow:
Dr. Danielle Moncrieffe, King’s College London Drug Control Centre
Mentor:
Prof. David Cowan; King’s College London Drug Control Centre
Research Title:
Improving sample preparation for the quantification of low level proteins in blood
Research Summary:
Accurate protein quantification is relevant to anti-doping. With the advances in mass spectrometry, LC-MS now offers sufficient sensitivity and selectivity to facilitate the development of analytical methodologies for proteins. Analysed in blood, the effect of interferents originating from this biological matrix, which may be whole blood, serum, plasma or dried blood, is unknown for many analytes of interest. It is important to consider the analytical challenges- associated with sample preparations of proteins for LC-MS. For this research the growth hormone biomarker peptides insulin growth factor 1 (IGF-I, 7.5k Da, typically present in concentrations of>100 ng/mL) and procollagen III amino terminal pro-peptide (P-III-NP, 42 k Da, typically present in concentrations of~5 ng/mL) were targeted for analysis, as they represent two different groups of protein based on size and concentration.
IGF-I, being the smaller and more abundant protein, is suitable for analysis by LC-MS using both intact and digest approaches. Generating much interest in clinical and anti-doping science, several validated methods exist using both approaches, with few showing investigating inter-laboratory imprecision. In collaboration with the PCC funded IGF-I working group, intact IGF-I methods were developed and tested, achieving good inter-laboratory agreement of serum sample measurements. This complements the previous methodology of the group, where IGF-I measurements were based on two trypsin-digestion peptides, T1 and T2.
P-III-NP, being the more challenging of the two analytes, has no validated approach for LC-MS analysis. Some of my previous work has shown a possible LC-MS measurement approach from serum after immunocapture and trypsin digestion, where the liberated tryptic peptides T1 and T5 are targeted for analysis. Of significant importance to the progress of method development is access to reference standards (both normal and heavy labeled). No international reference material exists for human P-III-NP and previous research has been based on other species, such as bovine P-III-NP that shares 97 % sequence homology. Using solid-phase protein synthesis, peptides for T1 and T5 have been synthesized in-house. Accurate quantification using amino acid analysis has been initiated. It is anticipated that, with the successful completion of this work, better accuracy will be achieved for the developed methods for P-III-NP.
For P-III-NP and IGF-I, serum is the target method for analysis. Other blood matrices- (e.g. whole blood and dried whole blood) should be considered due to their increased use within the field. Method transfer between the different blood matrices is not always straight-forward. Hence investigation of analytical variance and possible interferents originating from the different blood matrix should be investigated.
It is important to note that as a result of the research constraints imposed by the COVID-19pandemic,the project deliverables have been affected.
Funded: 2018
Fellow:
Dr. Laura Lewis; Australian Catholic University
Mentor:
Dr. Daniel Eichner, SMRTL
Research Title:
Influence of Relative Energy Deficiency in Sport (RED-S) on the Athlete Biological Passport
Funded: 2016
Fellow:
Dr. Liying Jiang; King’s College London Drug Control Centre
Mentor:
Dr. David Cowan; King’s College London Drug Control Centre
Research Title:
Strategy for cost reduction in doping test using UPLC-ESPI-TOFMS for urine and DESI-QTOFMS for DBS
Funded: 2014
Fellow:
Dr. Geoff Miller; Sports Medicine Research and Testing Laboratory (SMRTL)
Mentor:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Research Title:
Assessing hydration status through evaluation of albumin osmolality and lactate for the ABP
Funded: 2012
Fellow:
Dr. Bryan Kelly; Sports Medicine Research and Testing Laboratory (SMRTL)
Mentor:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Micro-Grants
Funded: 2022
Principal Investigator:
Dr. Nicholas Leuenberger, Swiss Laboratory for Doping
Funded: 2021
Principal Investigator:
Dr. Mario Thevis, German Sport University Cologne / Institute of Biochemistry
Funded: 2021
Principal Investigator:
Dr. Guenter Gmeiner, Seibersdorf Labor GmbH
Funded: 2021
Principal Investigator:
Dr. David Cowan, King’s College London
Funded: 2021
Principal Investigator:
Nasser Al Ansari, Anti-Doping Lab Qatar
Funded: 2021
Principal Investigator:
Dr. Holly Cox, SMRTL
Funded: 2021
Principal Investigator:
Dr. Mario Thevis, German Sport University Cologne / Institute of Biochemistry
Research Summary:
In order to detect the misuse of testosterone (T), urinary steroid concentrations and concentration ratios are quantified and monitored in a longitudinal manner to enable the identification of samples exhibiting atypical test results. These suspicious samples are then forwarded to isotope ratio mass spectrometry(IRMS)-based methods for confirmation. Especially concentration ratios like T over epitestosterone (E)or 5α-androstanediol over E proved to be valuable markers. Unfortunately, depending on the UGT2B17genotypeand/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively on urine samples.
In recent years, the potential of plasma steroids has been investigated and were found to be suitable to detect T administrations especially in female volunteers. A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations are solely derived from an administration of T and cannot be attributed to confounding factors. Therefore, an IRMS method for plasma steroids was developed and validated considering the comparably limited sample volume. As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone-sulfate were found suitable, while androsterone and epiandrosterone (both sulfo-conjugated) were chosen as target analytes.
The developed method is based on multi-dimensional gas chromatography coupled to IRMS in order to optimize the overall assay sensitivity. The approach was validated, and a reference population encompassing n = 65 males and females was investigated to calculate population-based thresholds. As proof-of-concept, samples from volunteers receiving T-replacement therapies and excretion study samples were investigated.
Funded: 2021
Principal Investigator:
Dr. Mario Thevis, German Sport University Cologne / Institute of Biochemistry
Research Summary:
A fundamental challenge in preventive doping research is the study of metabolic pathways of substances banned in sport. However, the pharmacological predictions obtained by conventional in vitro or in vivo animal studies are occasionally of limited transferability to humans according to an inability of in vitro models to mimic higher-order system physiology or due to various species-specific differences using animal models. A more recently established technology for simulating human physiology is the “organ-on-a-chip” principle. In a multi-channel microfluidic cell culture chip, 3-dimensional tissue spheroids, which can constitute artificial and interconnected microscale organs, imitate principles of the human physiology. The objective of this study was to determine if the technology is suitable to adequately predict metabolic profiles of prohibited substances in sport. As model compounds, the frequently misused anabolic steroids, stanozolol and dehydrochloromethyltestosterone (DHCMT) were subjected to human liver spheroids in microfluidic cell culture chips. The metabolite patterns produced and circulating in the chip media were then assessed by LC-HRMS/(MS) at different time-points of up to 14days of incubation at 37°C. The overall profile of observed glucurono-conjugated stanozolol metabolites excellently matched the commonly found urinary pattern of metabolites, including 3’OH-stanozolol-glucuronde and stanozolol-N-glucuronides. Similarly, but to a lower extent, the DHCMT metabolic profile was in agreement with phase-I and phase-II biotransformation products regularly seen in post-administration urine specimens.
Funded: 2021
Principal Investigator:
Dr. Nicolas Leuenberger, Swiss Laboratory for Doping Analyses
Research Summary:
Aim: We assessed the feasibility of using hematological parameters (such as hemoglobin and reticulocyte mRNA) in dried blood spot (DBS) samples to test athletes for doping and to improve patient care.
Methods: Hemoglobin and erythropoiesis-related mRNAs were measured in venous blood and DBSs from both healthy athletes and hemodialysis patients.
Results: We accurately measured hemoglobin changes overtime in both venous blood and DBS samples. Combining hemoglobin and mRNA analyses, we detected erythropoietin injection in DBSs more sensitively and with higher efficiency by using the DBS OFF-score than by using the athlete biological passport OFF-score.
Conclusion: DBS-based measurements are practical for calculating hemoglobin levels and athlete biological passport OFF-scores. This approach may help detect blood doping and help predict patient response to EPO.
Funded: 2021
Principal Investigator:
Dr. Andy Hoofnagle, University of Washington
Research Summary:
In collaboration with the Collagen Turnover Working Group, the Hoofnagle Laboratory finished development and validation of a novel assay to quantify P-III-CP and type III collagen breakdown products in human plasma, which we hope will be useful for the sensitive detection of growth hormone doping by athletes. The assay uses the approach of trypsin digestion of proteins in serum and peptide immunoaffinity enrichment of surrogate peptides prior to the LC-MS/MS simultaneous quantification of unlabeled, endogenous peptide along with an isotope-labeled analog internal standard peptide. The ratio of the chromatographic peak area of the unlabeled peptide to the labeled peptide, also known as the peak area ratio, is used to quantify the peptides, which is referred back to calculate the protein concentration in the original sample. Our laboratory has developed two monoclonal antibodies to peptides in type III collagen, which we used to develop the assay. The approach to the validation of the assay borrowed from guidelines published by the Clinical and Laboratory Standards Institute (C62 and C64). Experiments included precision, linearity, interferences, limits of quantification, and stability. This work culminated in a paper being accepted to the journal Clinical Chemistry, the most prominent journal in its field.
Funded: 2021
Principal Investigator:
Dr. David Handelsman, ANZAC Research Institute
Funded: 2020
Principal Investigator:
Dr. Oscar Pozo, IMIM Hospital del Mar Research Institute – Bioanalysis Research Group
Research Summary:
The detection of exogenous recombinant hGH (rhGH) abuse in doping control analysis is challenging. Currently, rhGH misuse is detected by two approaches: the isoforms test and the hGH biomarkers test. Due to several limitations, the development of alternative biomarkers of rhGH misuse is of great interest for doping control purposes. Based on decades of research suggest that relevant alterations in circulating lipids are likely to take place after rhGH administration. GhAELIC project aimed to evaluate the potential of lipid determination as screening tool for hGH administration by determining the lipidomics fingerprint alterations produced after recombinant human Growth Hormone (rhGH) administration. For that purpose, we determined 104 lipidic biomarkers by applying the targeted lipidomics LC-MS/MS methodology developed by our group for the analysis of 6 families of lipids (monoacyl glycerols, diacylglycerols, ceramides, hexosylceramides, lysophosphatidylcholines, and free fatty acids) to a set of plasma samples collected after rhGH administration to 10 volunteers from a previous clinical trial performed in our institution. Our results showed that rhGH administration promotes moderate changes in the lipidome. These changes are mainly produced in the balance between monoacylglycerols (MAG) and diacylglycerols (DAG). In particular, we selected two potential biomarkers related with the ratio between mono-and di-acyl glycerols. Based on the results obtained for the two volunteers with did not received rhGH, we could estimate the maximum intra-individual variation of the two biomarkers. We tested the suitability of the two selected biomarkers for the screening of rhGH abuse using individual thresholds in the 10 volunteers receiving rhGH. Although we found that these specific alterations in the lipidomeare statistically significant and biologically relevant, they are not robust enough to be used as screening tool in doping control analysis. Therefore, results from GhAELIC project advise against using the accurate measurement of the lipidome for screening purposes.
Funded: 2020
Principal Investigator:
Dr. Dustin Nabhan, United States Olympic and Paralympic Committee
Funded: 2020
Principal Investigator:
Dr. Gustavo Cavalcanti, Laboratorio Brasileiro de Controle de Dopagem (LBCD), Federal University of Rio de Janeiro, Brazil
Research Summary:
Collision Cross Section (CCS) is expected to be a valuable analytical parameter to provide additional structural information for the analytes of interest. Unlike the retention time and MS/MS spectra which can depend on experimental conditions, the CCS value is highly reproducible across various instruments and laboratories, making it easily standardized.
Moreover, in addition to the retention time and accurate mass measurement, CCS measurements will supply an additional molecular descriptor to ascertain the metabolite identity and thus increase the confidence level of metabolite identification. Unfortunately, very few reference materials for phase II steroid metabolites are commercially available. For this reason, there are a very limited number of experimental CCS values in existence for steroid metabolites commonly monitored in doping control analysis. To increase the coverage of steroid metabolites in CCS databases, reference urine samples can be used for the determination of CCS. Implementation of reference urine will yield more accurate CCS values than theoretical modeling methods which provide large relative error in terms of the in-silico CCS predictions.
Funded: 2020
Principal Investigator:
Dr. James Hopker, University of Kent
Research Summary:
This report provides an overview of the work conducted as part of a Micro Grant aimed at investigating the utility of performance monitoring to be used for anti-doping. For the project we have been working with USADA to explore performance data from male 100m sprinters who are affiliated to USATF. We implemented a Bayesian hierarchical model to analyze athlete career trajectories accounting for the effects of ageing and confounders of 100m sprinting performance (i.e. wind velocity). After building a model of population-wide model we were able to explore how individual performance trajectories differed from the overall average, with those exceeding this level being deemed to display ‘excess performance’. However, as good, or exceptional performance is not necessarily linked to doping, we decided to explore changes in ‘excess performance’ over fixed period of time (1, 2 or 3 years)–termed ‘yearly excess performance’. We identified that shorter periods of time resulted in better model predictions than those of longer duration. To enable us to account for the top performing athletes showing consistent levels of excess performance when compared against the population average, we used an Empirical Distribution Function to explore how performance trajectory is affected by athlete performance level. By dividing our sprinters in to 4 quartiles we were able to adjust our measure of excess performance based upon the performance level of the athlete. Using the probability for an athlete to demonstrate a ‘yearly excess performance’ we subsequently demonstrated that our performance model was able to discriminate between sprinters who have a historical anti-doping rule violation and those who do not (AUC = 0.83).The report also highlights the challenges associated with data quantity and quality which impact on the robustness of its predictive capacity and reduce chances of bias and false positives arising from the analytical process. Finally, we highlight the opportunities for this work to be transferred into anti-doping practice via our collaboration with USADA. However, we also acknowledge that further research is required to explore the transference of our generalized performance model to other sports and disciplines in advance of it becoming incorporated into anti-doping practice within ADOs.
Funded: 2020
Principal Investigator:
Dr. Geoff Miller, Sports Medicine Research & Testing Laboratory (SMRTL)
Funded: 2020
Principal Investigator:
Dr. Danielle Moncrieffe, King’s College London Drug Control Centre
Funded: 2020
Principal Investigator:
Dr. Ole N. Jensen, University of Southern Denmark
Funded: 2020
Principal Investigator:
Dr. Janusz Pawliszyn, University of Waterloo
Research Summary:
In this project, we successfully applied solid phase microextraction (SPME) combined with direct/ambient mass spectrometry (MS) methods to analyze more than twenty drugs of abuse in the oral fluid matrix. This technique combines sample preparation and extraction in one step, eliminates LC or GC separation procedure, which allows to decrease the total analysis time and the amount of solvent used in the experiment. Our results have clearly demonstrated that SPME technique combined with direct/ambient MS was capable of simultaneous detection of 20 targeted compounds, at low concentration level (LODs between 0.005-10 ppb) with short analysis time (less than 90 s for each sample) in saliva samples without any other sample preparation.
Three state-of-the-art SPME-direct/ambient MS techniques including direct analysis in real-time (DART), microfluidic open interface (MOI) and coated blade spray (CBS) were developed, optimized and applied for achieving the above objective. All the experiment parameters including the physical shape of SPME device, coating materials, extraction time, desorption conditions, etc. has been optimized regarding the method sensitivity, reproducibility and usability. In addition, the most interesting part of the project is that we re-designed the devices of SPME-MOI-MS and CBS-MS interfaces, to make it specifically for the application in this project, and has the potential to be fully automated in the near future. Finally, the above three methods were applied for the rapid analysis of more than twenty drugs of abuse in human saliva samples and excellent performance regarding the analytical speed, sensitivity, selectivity and accuracy were observed.
Funded: 2020
Principal Investigator:
Dr. Francesco Botre, Laboratorio Antidoping FMSI
Funded: 2020
Principal Investigator:
Dr. Guenter Gmeiner, Seibersdorf Labor GmbH
Funded: 2020
Principal Investigator:
Dr. Dilshadbek Usmanov, Institute of Ion Plasma and Laser Technologies, Uzbekistan Academy of Sciences
Funded: 2020
Principal Investigator:
Dr. Andy Hoofnagle, University of Washington
Funded: 2020
Principal Investigator:
Dr. Siri Dorum, Norwegian Doping Control Laboratory
Funded: 2019
Principal Investigator:
Dr. Yvette Dehnes, Norwegian Doping Control Laboratory
Funded: 2019
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Funded: 2019
Principal Investigator:
Dr. Mario Thevis; German Sport University Cologne
Research Summary:
Ostarine, also known as enobosarm/S-22, is an arylpropionamide-based selective androgen receptor modulator (SARM) with tissue-selective anabolic effects and its misuse in sports is therefore prohibited since 2008 [1-3]. Although no SARM drug candidates have obtained clinical approval yet, they are distributed online for research purposes as well as on the black-market. As especially the number of ostarine findings in routine doping controls has continuously increased within the last years [1-10], the possibility of nutritional supplement/dietary product contamination with minute amounts of the anabolic agent has become a major concern for both athletes and result managing authorities [11]. The analytical sensitivity of anti-doping laboratories has been optimized to allow for utmost retrospectivity in sports drug testing; the problem arising from this performance however is that also trace amounts introduced into an athlete’s organism by contaminated food or supplements are detected, and according to the World Anti-Doping Code (WADC) and its rule of strict liability [12], the athlete needs to provide conclusive information demonstrating that the AAF results from contamination and not from doping. This is naturally a complex task, but anti-doping laboratories could support the process of fair and comprehensive case management by providing additional analytical data, more precisely ostarine elimination (and potentially metabolite) profiles, that contribute to determining whether it is more likely that a minute amount of the drug was recently ingested or if a larger amount (pharmacologically relevant) of the doping agent was administered several days/weeks ago.
Therefore, the aim of this research project was to investigate the elimination profiles of microdosed ostarine in order to characterize the time-dependent urinary excretion of the compound as well as its phase-I and-II metabolites. In total, six administration studies with single and multi-doses of 1, 10, and 50 μg were conducted and the collected urine samples analyzed by solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) as well as enzymatic hydrolysis, liquid-liquid-extraction (LLE), and LC-MS/MS. Both in the single and multi-dose studies, ostarine and its metabolites M1a (OH-ostarine), M1b (OH-ostarine), M3 (glucuronidated ostarine), and M4 (glucuronidated OH-ostarine) were detected in post-administration urine samples. The maximum urine concentrations (cmax) of ostarine as well as the detection times of the drug and its metabolites were found to correlate with the administered drug dose. However, also ahigh inter-individual variation with regards to absolute values as well as time points of the peak urine concentrations was observed. The high variability of the cmax-values noticed in the multi-dose studies indicates that drug accumulation under the chosen conditions is unlikely.
In a microdose pilot study, a distinct metabolic pattern predominantly characterized by the ostarine/OH-ostarine ratio was observed. Unfortunately, these results could not be confirmed within this study, where the ostarine/OH-ostarine ratio did not change systematically within the study group over time. However, interpreting the elimination profiles of individual target analytes, i.e. phase-I and phase-II metabolites, suggest a utility of short-lived metabolites as indicators for a recent ingestion of ostarine, also if amounts representing contamination scenarios are repeatedly administered. Further, ostarine (metabolite) detection times in general and, specifically, urinary concentrations of ostarine following different administration schemes are now available that support an improved interpretation of AAFs, especially when scenarios of proven supplement contamination are debated and supplement administration protocols exist. The herein obtained data particularly assist the assessment of plausibility and hence support an objective result management of adverse analytical findings.
Funded: 2019
Principal Investigators:
Dr. Owen Davies, Loughborough University
Funded: 2018
Principal Investigator:
Dr. Ole Jensen, University of Southern Denmark
Research Summary:
A current challenge is to develop straightforward and reliable bioanalytical methods to detect the origin of EPO in micro-dose doping practice. Biological samples often contain both endogenous and recombinant EPO, giving rise to mixed EPO profiles. Such mixed EPO profiles are mainly due to heterogenous N-glycosylation of EPO that are difficult to resolve by current laboratory protocols, giving rise to inaccurate results and ambiguous conclusions.
We here report our objectives, results and new key findings based on our research catalyzed by a generous PCC Microgrant.
We aimed to improve the electrophoretic separation of EPO to resolve mixtures of differentially glycosylated EPO species. Tricine-SDS PAGE gel analysis and high accuracy tandem mass spectrometry are proposed as alternative methods to characterize trypsin and Asp-N derived EPO glycopeptide profiles.
Highlights of research program:
-Asp-N generates EPO glycopeptides suitable for glycosylation profiling by Tricine SDS PAGE.
-Intact EPO-glycopeptide analysis by mass spectrometry enables detailed characterization of N- and O-glycopeptides.
-Feasibility of intact glycopeptide profiling by Tricine-SDS PAGE and High-Resolution Mass Spectrometry for characterizing EPO produced in CHO cells, HEK293 cells and new EPO-glycoengineered variants according to differences in glycosylation profiles.
-Discovery and novel use of Flavastacin protease for specific cleavage of EPO to generate distinct EPO-glycopeptides with potential for novel detection methods (follow-up by recently submitted PCC Micro Grant proposal, 2. Dec. 2019).
Our methods offer detailed analytical assessment of EPO-glycopeptide heterogeneity subsequent to trypsin or Asp-N cleavage. Further investigation of flavastacin efficiency, specificity, and mechanism for generation of EPO N- and O-linked glycopeptides is required (follow-up proposal, PCC Micro Grant). We believe that this new approach has great potential to investigate the O-glycan composition and profiles of new EPO variants, since the specific targets for O-glycan engineering are less obvious than for N-glycan engineering.
Funded: 2018
Principal Investigator:
Dr. Malcolm McLeod, Australian National University
Research Summary:
Direct detection of steroid phase II metabolites is rapidly emerging as a useful tool in the screening of steroid misuse. Whereas the addition of phase II metabolites into screening methods for exogenous androgenic anabolic steroids has been largely accepted, the usefulness of these conjugates for the screening of endogenous androgenic anabolic steroids (EAAS) is less well established. In order to check the suitability of these conjugates for EAAS screening, the development of quantitative methods is required in order to select the most appropriate markers. One of the main limitations of developing such quantitative methods is the absence of commercially available reference materials for some potentially useful metabolites.
This PCC Micro-Grant targeted the synthesis of two conjugated reference materials 6β-hydroxyandrosterone 3-glucuronide (6OH-And-3G 1) and 6β-hydroxyetiocholanolone 3-glucuronide (6OH-Etio-3G 2) that have been reported as long-term markers EAAS abuse. The synthesis of conjugated reference materials is essential for the study of these metabolites by LC-MS as they are resistant to Escherichia coli (E. coli) β-glucuronidase hydrolysis. The reference materials 6OH-And-3G 1 and 6OH-Etio-3G 2 were synthesised in 12 and 8 steps respectively in moderate yield and >10mg scale, with all compounds fully characterised by spectroscopic and spectrometric methods. The reference material identities were also confirmed against urinary metabolites by UHPLC-MS/MS analysis of two diagnostic transition in accordance with WADA Technical Document–TD2015IDCRby Dr. Oscar Pozo, IMIM, Barcelona, Spain.
The provision of these reference materials will enable a range of future research including: (i) an inter-laboratory comparison of the determination of 6OH-And-3G 1 and 6OH-Etio-3G 2 as markers of testosterone misuse, (ii) work evaluating the determination of 6OH-And-3G 1 and 6OH-Etio-3G 2 as markers for the misuse of other EAAS, and (iii) investigating the potential of 6OH-And-3G 1 and 6OH-Etio-3G 2 in discerning ethanol consumption as masking agent for testosterone administration. Negotiations are currently underway to establish an inter-laboratory comparison of 6OH-And-3G 1 and 6OH-Etio-3G 2 as markers of testosterone misuse for the forthcoming WADA 2020 Scientific Research Grants round.
Funded: 2018
Principal Investigator:
Dr. Nikolai Nordsborg, University of Copenhagen
Research Summary:
On April 12th2018 the PCC granted 48.000 USD to a pilot study of flowcytometric based determination of red blood cell (RBC) surface protein abundance related to the RBC life cycle. Experiments were initiated on June 20th2018 when the Grant was released at UCPH.
The primary objective was to evaluate a panel of nine fluorophore anti-bodies in a flow-cytometry based identification of red blood cell characteristics. Successful positive signals were obtained from eight antibodies (CD35, CD47, CD55, CD59, CD71,CD147, CD235, CD4) and one DNA/RNA binding reagent (Thiazole Orange). Staining and analyzing for AE1, AQP1, MCT1 was not successful due to the need of intracellular staining, which were not possible because the used permeabilization protocol lead to lysis of all erythroid cells. Furthermore, Annexin V staining was successful, but due to use of a specific binding buffer the protocol required flow cytometric analysis of the samples within one hour, which was not possible when analyzing several samples in the same run. Of the included anti-bodies, CD71 seems as a promising marker of altered erythropoiesis (unpublished pilot data).
Funded: 2018
Principal Investigator:
Dr. Usmanov D.T., Academy of Science Uzbekistan
Funded: 2018
Principal Investigator:
Dr. G. Reverter Fundació, IMIM
Research Summary:
The use of dried blood spots (DBS) for sample collection is increasingly attracting the interest of the antidoping analytical community because of their multiple advantages [1]. DBS consists on the deposition onto a filter card paper of a few drops of blood obtained from a finger-prick [2]. DBS eliminate the need of specialized personnel (phlebotomist) at the collection site, reduce sample collection invasiveness, and facilitate transport and storage by decreasing the associated logistics [3,4]. Moreover, stability has been shown to be maintained in almost all the studied substances [5,6]. Hence, DBS constitute a cost-effective procedure with potential to effectively permit an increase in blood sampling, an ancient ambition in the fight against doping [7]. However, DBS also present some drawbacks that need to be addressed when considering their use for analytical tests. These difficulties include the small volume of sample collected, the hematocrit effects, or the need of additional validation steps.
In sport, recombinant human growth hormone (rhGH) abuse is one of the main concerns of antidoping authorities [8]. The World Anti-doping Agency (WADA) includes growth hormone (GH) and their releasing factors in its yearly reviewed Prohibited List [9]. Among GH releasing factors, GH-releasing peptides (GHRPs) are acquiring relevance because of the extension of their suspected misuse. GHRPs are small synthetic peptides capable of interacting with the GHS receptor 1a (GHS-R1a) to stimulate GH release by the pituitary gland [10]. Initially developed for therapeutic purposes, the list includes, among others, GHRP-1, 2, 4, 5, 6, hexarelin, alexamorelin, and ipamorelin peptides [10]. So far, only GHRP-2 has been approved for clinical use to detect GH deficiency in Japan [10]. Even prohibited, GHRPs are easily accessible on internet websites and present in different formulations. Thus, they have been found on police seized vials and nutritional supplements [11,12]. Adding concern, the list of GHRPs is in continuous expansion as new analogues are being continuously discovered on black market products [13,14]. Thereby, the confirmation of GHRPs abuse has been described in urine samples from athletes using mass spectrometry-based techniques [15].
In this study, GHRP-2 has been used as a proof-of-concept model to evaluate capabilities of DBS for GH-releasing peptides detection in anti-doping tests. Thus, a method to detect GHRP-2 after DBS extraction and analysis by mass spectrometry (LC-MS/MS) has been developed. The method has been validated in terms of specificity, limit of detection (LoD), identification capability, robustness and carry-over. Specific consideration to factors having a potential effect on DBS analysis has been also studied, namely, capillary vs. venous blood comparison and long-term storage stability. The method has been applied to samples collected during an already executed clinical trial in our institution which involved the administration of a single intravenous dose of GHRP-2 (100 μg). Finally, an evaluation of the analytical performance for reference anti-doping laboratories has also been elaborated.
The developed methodology shows that it is possible to detect GHRP-2 down to a concentration about 50 pg/mL in DBS. No interferences were observed at the analyte retention time after analysis of blank samples indicating good identification capabilities. The robustness was assessed in terms of hematocrit effect in the 30-60% range and variationsbetween-15.26 to 1.68%were obtained. No carry-over was observed after samples injected following the highest calibrator. Blood site collection (capillary vs. venous) showed to influence GHRP-2 concentrations. Preliminary data comparing long-term storage (> 2 years) conditions exhibited similar outcomes independently of DBS having been stored either at 4ºC or RT.
The analysis of the DBS samples collected in the administration study (eight GHRP-2-treated subjects and two non-treated subjects) showed that it was possible to detect the drug on the DBS in all subjects at 1.5 h post-administration. Additionally, in 7 out of 8 of the subjects, the window of opportunity reached the 2 h and in 4 out of 8 of the subjects the 3 h. Exceptionally, it was possible to detect the drug at 4 h in 1 out of 8 of the volunteers. The GHRP-2 concentrations estimated at the analyzed times were (mean ± SD) 530.5 ± 202.0; 210.3 ± 93.6; 145.6 ± 76.7; 143.4 ± 74.85; 109.8 ± 16.5 and 51.2 ± ND pg/mL at 0.5 h, 1 h, 2 h, 3 h and 4 h after drug administration, respectively. The detection times after administration are in line with the known pharmacokinetic characteristics of GHRP-2. In addition, the comparison of these data with the corresponding serum samples measured in the anti-doping laboratory of Moscow (Dr. Krotow, personal communication) and in-house by a radioreceptor displacing technique 16 permitted to assess some analytical considerations. Serum concentrations resembled more those observed in venous DBS than in capillary DBS levels, indicating that the type of blood (venous vs. capillary) influence GHRP-2 levels. Nevertheless, these observed differences by the type of blood appear not affecting the window of detection. On the other hand, the long term storage of the samples has not avoided GHRP-2 detection, supporting the use of already collected DBS samples for analysis. Additionally, based on the reported GHRP-2 concentrations here it is possible to propose a required minimal analytical performance by antidoping laboratories for the detection of GHRP-2 in DBS at 50 pg/mL.
In summary, data provided here add new support to the existing literature proposing DBS sampling as an alternative matrix of analysis for anti-doping tests. This study using GHRP-2 as a proof-of-concept for the GHRPs family reveals that this synthetic peptide can be effectively recovered from the dried blood spotted onto a filter card. And not only, extracts from the administration study (long-term stored) permit to confirm that GHRP-2 could be detectable up to a maximum of 4 h post-administration with the sensitivity achieved with current instrumentation.
Funded: 2018
Principal Investigator:
Dr. Roger Giese, Northeastern University
Research Summary:
Impact. We believe that our project has achieved an impact. The impact is a new way to collect saliva for drug analysis with many advantages: (1) inexpensive (40 cents); (2) off-the-shelf components (commercially available, wide mouth, small plastic jar containing 2 mL of isopropanol (rubbing alcohol); (3) rapid (one spit); (4) the alcohol overcomes the viscosity of the saliva as well as stabilizes and preserves the saliva at room temperature; (5) no proprietary ingredients; (6) safe to use (is made of tough plastic); (8) avoids adsorption losses of drugs (the plastic is polypropylene); (7) comes commercially in a sterile form ready for loading with the alcohol; (8) the spitting step is discreet rather than disgusting since itis masked by the jar; (9) because the jar is leak proof against isopropanol, the jar can be mailed as such, ready for instant use; and (10) the isopropanol facilitates subsequent nontargeted analysis by mass spectrometry, which, we believe, should be the future of testing athletes for performance-enhancing drugs. Because subsequent detection by mass spectrometry is very sensitive, a small amount of saliva can be collected, enabling testing of a subject with a dry mouth, in principle.
Demonstration of Impact. We have applied our technology to a subject who smoked marijuana (8 puffs). Saliva samples were collected by our new technique before the smoking, after 1 hour, and after 16 hours. The samples intentionally were stored at room temperature for 6 days prior to mass spectral analysis. No cannabinoids were found in the pre-smoking sample. Six cannabinoids (including one unknown) were discovered in the 1 hour sample. The 16 hour sample only showed a low level of one residual cannabinoid. The equivalent of 2 uL of saliva was injected into the mass spectrometer.
New Collaboration. A collaboration is about to begin with the Bridge Clinic of Substance Abuse Disorders at Massachusetts General Hospital, Harvard Medical School. The goal is to increase our list of detected drugs (so far we have detected 9), and bring on nontargeted drug analysis.
Intellectual Property. In regard to this technology, a US patent has issued; a utility patent application has been submitted; a second utility patent application is about to be submitted; a provisional patent application has been submitted; and a second provisional patent application is about to be submitted.
Funded: 2018
Principal Investigator:
Dr. James Hopker, University of Kent
Research Summary:
This PCC Micro-grant was used to fund a Research Assistant to work on a 6-month project in order to establish the efficacy of performance monitoring in the identification of doping in sports. We used a retrospective analysis of existing performance results to determine how performance evolves over an athlete’s career by applying various statistical and machine learning methods. We initially aimed to extend our previous work involving the use of a Bayesian Latent Factor Regression model to construct performance trajectories of male shot put athlete’s careers. We hypothesized that these modelled trajectories would enable us to identify differences between the careers of doped and presumed clean athletes. Unfortunately, our model proved not to be sensitive enough to detect the variability in athlete performance in order to detect small instances of performance enhancement from doping, especially across different seasons. We therefore considered an alternative Bayesian methodology called change-point analysis. The change-point approach sought to standardize an athlete’s performance across the course of their career taking into consideration a predicted career trajectory derived from presumed clean athletes in the same dataset. This change-point analysis was able to identify patterns of performance trajectory changes common across doped athletes in the dataset that are not evident in the majority of presumed clean athletes. However, we were also able to identify “clean” athletes within the dataset who had similar performance trajectories to previously sanctioned athletes. The performance trajectories we observed in the male shot put athletes were also persistent across other disciplines of track and field athletes we studied (women’s 800m,female shot put, and men’s 100m). These results suggest there is a need to conduct further work on the use of the Bayesian change-point analysis in order to refine modelling of rate and change in rate of performance. Moreover, there is a need to test the model’s ability to predict doping status in athletes, given their performance trajectory. Alongside the statistical approaches, we also investigated whether unsupervised machine learning was able to identify patterns of performance evolution indicative of doped and clean athletes. Unfortunately, this proved to be unsuccessful and was not able identify common patterns of performance trajectories for doped and presumed clean athletes. Further work is therefore required in order to refine and optimize our machine learning approaches involving clustering and neural network algorithms.
Funded: 2017
Principal Investigator:
Dr. Robert Chapman, Indiana University
Research Summary:
The newest class of erythropoietic stimulating agents (ESAs) are substances that stabilize Hypoxia Inducible Factor (HIF-1a) and result in an upregulation of transcription and production of erythropoietin (EPO). Efforts by pharmaceutical companies to identify various HIF-1a stabilizers represents one of the greatest potential threats to anti-doping efforts, due to the ever increasing number of identified compounds that stabilize HIF-1a, including many common over the counter medications and legal supplements. One such legal supplement is n-acetyl cysteine (NAC), which has been shown to increase both resting EPO levels and the EPO response to acute hypoxia in non-athletes. Whether well-trained athletes show the same EPO response at rest and after acute hypoxic exposure with chronic NAC supplementation is not known.
The objective of this pilot project was to obtain controlled erythropoietic data from a cohort of highly trained endurance athletes of the effects of chronic NAC supplementation: a) after 7 days of supplementation (Deliverable #1), and b) with acute exposure to hypoxia (Deliverable #2).
A total of 10 highly trained men completed measures of serum EPO concentration, total hemoglobin mass (via carbon monoxide rebreathing), hemoglobin concentration, and reticulocytes before and after 7 days of supplementation with 1800 mg/day of NAC or placebo in a repeated measures, cross-over design with washout. Additionally, the same cohort was exposed after the supplementation periods (NAC and placebo) to 6 hours of normobaric hypoxia (16.1% O2, simulating 2500m / 8000ft), with serial measures of serum EPO and arterial oxyhemoglobin saturation (via pulse oximetry).
No significant changes were noted in hemoglobin concentration, total hemoglobin mass, or resting EPO concentration after 7 days of NAC supplementation (or 7d of placebo supplementation). As expected, EPO was significantly elevated in both NAC and placebo conditions with acute hypoxic exposure. However, no significant differences were found in EPO at 2h, 4h, and 6h of hypoxic exposure between NAC and placebo conditions. In examining individual responses, it does not appear that there are cases where individuals could be classified as “responders” or “non-responders” to NAC supplementation.
Although other studies in non-athlete and clinical cohorts suggests that NAC may be a beneficial treatment for increasing erythropoiesis, our data in an athletic cohort suggests NAC does not affect erythropoietic variables. This may be due to athletes having different baseline redox / glutathione states than non-athletes, resulting in no hematological effect of NAC supplementation in athletes. As such, our data would suggest that the PCC, USADA, WADA does not have reason to pursue studies of NAC for inclusion on the list of prohibited substances.
Heme Triggered Immune Responses and Redox Pathways as Targets to Detect Autologous Blood Transfusion
Funded: 2017
Principal Investigator:
Dr. Mariana Renovato Martins; Instituto Federal do Rio de Janeiro
Research Summary:
The oxygen carrying capacity of the body is a limiting factor of performance in most sports of long duration and endurance. It is known that oxygen absorption can be naturally increased through intense and frequent resistance training, because the cardiac cavities are enlarged, triggering an increase in the red blood cells. But these adaptations are slowly acquired over many years. Thus, the athletes created the need for artificial induction of erythropoiesis as an ergogenic resource. The greater oxygen supply can also be achieved by homologous or autologous transfusion. In homologous transfusion a red blood cell concentrate obtained from a compatible donor is reinfused into the athlete’s circulation. While in the autologous transfusion, red blood cells are removed from the athlete, being preserved (stored) for a minimum period of 21 days, and then reinfused in it. However, since these procedures confer an advantage over resistance competitions, they are considered doping. The use of blood doping has been banned by the World Anti-Doping Agency since 1985 because it is a method capable of promoting changes that artificially improve the athlete’s performance. However, autologous blood infusions cannot yet be detected directly by any current “anti-doping” method. Therefore, our objective was to evaluate in monocytes/macrophages as key proteins of the metabolism of heme andiron are altered by red blood cells by the cooling process. Thus, monocytes/macrophage, previously co-cultivated with red blood cells, we evaluated the protein expression of Heme Oxigenase 1 (HO-1), Ferritin and the Spi-C transcription factor. By Western Blotting technique we observed that the storage of red blood cells induced increased heme release, triggering an increase in the expression of ferritin and Spi-C in monocytes/macrophages compared to non-stored blood. The latter was presented as a sensor promising in the detection of free heme probably due to its ability to selectively control the development of red pulp macrophages (RPM), which phagocytose senescent red blood cells. Significant changes in the dosage of heme and hemoglobin and release of extracellular vesicles by red blood cells were also observed in stored blood. Therefore, through the identification of new targets, our study opens the way for the development of bioanalytical assays for the detection of autologous transfusion in athletes.
Funded: 2017
Principal Investigator:
Dr. Pozo Mendoza; IMIM: Institut Hospital del Mar d’Investigacions Mèdiques
Research Summary:
The occurrence of new psychoactive substances (NPS) is growing steadily every year. Many NPS display stimulant properties and, therefore, they belong to the class S6 of the WADA’s Prohibited List. Doping control laboratories must constantly adapt their methodologies to include their urinary detection complying with the MRPL requirements for stimulants.
The typical experimental strategy is the detection of the parent compound excreted unconjugated in urine. However, NPS, their pharmacological effects, metabolic clearance, fraction excreted of the parent compound and stability in biological matrices are unknown due to the difficulties of obtaining the substance and performing controlled excretion studies. Some NPS can be extensively metabolized and the urinary recovery of the unchanged- parent drug might correspond to a minor percentage of the total dose. Therefore, the analytical strategies to comply with MRPLs for the NPS need to be properly studied.
Our group has already obtained urine samples collected in naturalistic studies after the administration of mephedrone, 3-MMC, methylone, 6-APB and 2-CE. The aim of the proposed project is to use those excretion studies to develop the analytical methodology to incorporate those substances and/or their metabolites into routine screenings in doping control while evaluating which particular analyte is shall be determined to properly comply with the MRPL.
For that purpose, we have developed and validated an analytical methodology for the quantification of 5 NPS in urine. The methodology has been applied for the quantification of the unconjugated parent drug in urine samples collected from naturalistic studies. Our results show that the detection of the unconjugated drug using 50% MRPL as reporting level is able to detect the misuse of methylone, 3-MMC and 6-APB in all samples collected. However, this strategy was doubtful for some mephedrone samples and failed in most of the 2C-E samples.
For these two NPS we performed metabolic studies in order to evaluate alternative markers for their detection. In the case of mephedrone, we performed metabolic studies by a targeted method already available in the laboratory. We found that one of its metabolites (carboxy-mephedrone) is excreted at concentrations 10 fold higher than the parent drug. Therefore, its selection allowed to clearly determining the misuse in all samples by using the current MRPL. However, after determining the metabolite in samples from a controlled study we showed that the MRPL was also exceeded in several samples collected 2 days after the administration. This result opens the door to the detection in in-competition samples of administrations performed some days before the competitions. Based on our results we can not discard that this situation is also happening in other NPS such as methylone.
On the other hand, we evaluated the metabolism of 2C-E by untargeted approaches. We found a hydroxylated metabolite with responses between 3-6 fold higher than the parent compound. Thus, the determination of this metabolite might be useful for the determination of 2C-E administration. However, the ultimate identification of the metabolite structure, its synthesis and the development, validation and application of a method for its determination are required before confirming the suitability of this metabolite. Alternatively, a reduction of the MRPL for some compounds especially for those active at low doses could be envisaged.
Funded: 2017
Principal Investigator:
Dr. Robert Chapman; Indiana University
Funded: 2016
Principal Investigator:
Dr. Herb Tobias; Dell Pediatric Research Institute
Funded: 2016
Principal Investigator:
Dr. Jack Henion; Q2 Solutions
Research Summary:
A prototype 3D printed dried plasmas pot (DPS) card equipped with a tamper-proof security cover has been developed which is shown in Figure 1. This 3D printed design resembles the construction and design of the earlier prototype paper card; however, it offers additional benefits beyond those of the paper card design such as the tamper-proof security cover and the option to subject the card directly for on-line LC/MS/MS analysis. Previously, to attain the automated on-line analysis, the cellulose substrate of the paper card design was removed and then adhered to an on-line amenable cardstock. This 3D prototype eliminates that step. Details of the entire work flow from sample collection to the fully automated on-line analysis can be found in the experimental section.
The functionality of this prototype was evaluated by 1) visual evaluation of the generated plasma spots ensuring that the 3D card is capable of producing hemolysis-free plasma spots and 2) automated on-line analysis using the previously validated DPS-SPE-LC-ESI-MS/MS (dried plasma spot-solid phase extraction–liquid chromatography-electrospray ionization–tandem Mass Spectrometry) method for quantitative determination of hydrocodone using deuterium-labeled internal standard. The analysis was performed on incurred samples which were collected using the 3D printed cards at 11 time points in three replicates from 0 to 24 hours after an intake of 5 mg Hydrocodone orally by a heathy volunteer male. Results showed a fully functional card through a successful generation of hemolysis-free plasma spots from whole blood. The LC-MS/MS analysis results showed good linearity (R2≥0.9963) with a linear range from 5 to 1000 ng/mL. Incurred sample analysis showed a typical pharmacokinetic (PK) curve for Hydrocodone where half-life of the drug was observed at around 4 hours after intake.
Additionally, a side project of developing an analytical method for DPS analysis of cannabinoids using the fully automated on-line platform has also been conducted. In this platform, analysis of cannabinoids was challenging. We have proposed solutions to address the challenges which can be found in this report.
In summary, the 3D-printed DPS card improves the quality of the paper DPS card by providing additional benefits of on-line amenable capability, tamper proof design, and a better manufacturing process. However, it is recommended that the future mass production of this DPS card device will require automation of the current hand-made procedure.
Funded: 2016
Principal Investigator:
Prof. Dr. Maria Kristina Parr; Freie Universitat Berlin
Research Summary:
For long-term detection of a misuse of Oral-Turinabol (DHCMT) anti-doping laboratories analysis mainly rely on the detection of 4-chloro-17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol (20ξOH-NorTHCMT) as TMS derivative when utilizing GC-MS(/MS). This metabolite was proposed by Sobolevsky et al (J Steroid Biochem Mol Biol 128 (2012) 121-127). However their proposal was only based on mass spectrometric data without assignment of the stereochemical orientation. At present, the use of post administration urines instead of purified reference material has been accepted in confirmatory analyses. Nevertheless, scientific proof of the structures and well defined reference material would be helpful. It is planned to generate reference material for- structure confirmation and future use in anti-doping analyses by single-step hydroxylation from 17, 17-dimethyl-18-nor-13-ene precursor in a whole-cell biotransformation assay (part of WADA15A21MP). The educt 4ξ-chloro-17α-methyl-5ξ-androstane-3ξ,17β-diol is also not commercially available. Generation by hydrogenation of DHCMT of 4-chloromethyltestosterone failed due to a concomitant loss of chlorine. Thus, alternative routes for synthesis will be performed in the project. Starting from methyltestosterone desoxygenation of the A-ring and subsequent hydroxychlorination of the double bond was successfully performed and different isomers of 3/4ξ-chloro-17α-methyl-5ξ-androstane-3/4ξ,17β-diol were obtained. The isomers and side products from intermediates and final products were separated by HPLC prior to MS and NMR confirmation of the structures.
Funded: 2016
Principal Investigator:
Dr. Lena Ekstrom; Karolinska Instituet
Research Summary:
The main results are:
Three days treatment with codeine increase the serum total testosterone levels, whereas LH and FSH are unaffected
In one of the participant a 3 days codeine use increased the T/E ratio above the individual threshold in ABP
The administration of 500 and 125 mg testosterone enanthate were detected in all study subjects when three baseline T/E values were added to the ABP and the co-administration of codeine did not conceal the results
One of the participants showed a high morphine/codeine ratio post codeine treatment, indicating that discriminating between morphine and codeine use could be difficult
There were no association between UGT2B7*2 polymorphism and urinary concentrations of morphine and codeine glucuronides
Funded: 2016
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Funded: 2016
Principal Investigator:
Professor David Handelsman; ANZAC Research Institute
Research Summary:
The present study indicates that the sex steroid and gonadotropin profiles of testosterone-treated transgender men are very similar to those of testosterone-treated hypogonadal men while also confirming that both testosterone treated groups have distinctive differences from untreated healthy eugonadal control men. Differences between the testosterone-treated groups in the urine or serum sex steroid profiles were limited to higher serum estradiol, estrone, DHEA and LH in transgender men compared with hypogonadal men. These differences were mostly attributable to transgender men who had undergone ovariectomy.
Time since injection was an important predictor of serum and urine testosterone, serum DHT and urine T/E ratio but not of other serum or urine sex steroids. From an anti-doping perspective, these differences indicate both the potential advantages of greater sensitivity for serum steroid profile as in the steroidal module of the Athletes Biological Passport (ABP) to detect exogenous testosterone use. Conversely, however, the time since last testosterone dose is essentially an uncontrollable variable in the anti-doping context which limits the better overall sensitivity of serum analytes for anti-doping testing for exogenous testosterone use. Hence monitoring serum steroids may enhance the sensitivity of the ABP steroid module but its specificity is subject to time since last testosterone dose. Sensitivity of steroid module of ABP to exogenous T is low and blood testing which may augment sensitivity especially soon after the last done but less reliably later.
Limitations of this study include that we did not examine transdermal testosterone gel where the different route-dependent pharmacokinetics from injectable testosterone esters may alter the steroid profiles and consequently the utility of anti-doping detection tests. The injectable testosterone ester used in this study was 1000 mg testosterone undecanoate which is marketed world-wide except in the US where it is marketed as a 750 mg formulation so that the findings may differ according to injection dose. However, for anti-doping purposes the lack of substantial difference between the profiles of transgender and hypogonadal men in this study suggests that the urine T/E ratio is most likely not to differ systematically between these groups of testosterone treated men for any testosterone products including transdermal gel, cream or solutions although this warrants confirmation.
We conclude that urine anti-doping detection tests in urine of T-treated transgender men can be interpreted like those of T-treated hypogonadal men and are largely unaffected by time since last T dose, unlike serum steroids. Urine steroids may be considered a more time integrated measure of steroid exposure, production and excretion which may buffer out fluctuations evident in serum steroid concentrations.
Funded: 2016
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Funded: 2016
Principal Investigator:
Dr. Mario Thevis; German Sport University Cologne
Research Summary:
Meldonium (trade name: Mildronate) is an anti-ischemic drug which was originally developed in the late 1970s for veterinary applications due to proposed growth-promoting effects in animals [1]. Meanwhile, it has become an approved drug in selected Eastern European countries and is the subject of ongoing clinical trials focusing on the compound’s anti-ischemic and cardio protective properties as well as potential applications regarding diabetes, neurodegenerative disorders, and bronchopulmonary diseases [2, 3]. As meldonium was also reported to increase the endurance performance of athletes, to improve the rehabilitation after exercise, to protect against stress, and to enhance the activation of central nervous system (CNS) functions, it has also potential for being misused as doping agent in sports [4-6]. In 2015, a one-year monitoring program initiated by the World Anti-Doping Agency (WADA) demonstrated a considerable extent of meldonium use by athletes [7, 8], which was further corroborated by a significant number of declarations of use and analytical findings at the Baku 2015 European Games [9]. Therefore, it was added to the section S4 (Hormone and Metabolic Modulators) of the WADA prohibited list in 2016 [10].
So far, the pharmacokinetic properties of meldonium were investigated in the course of single-and multiple-dose administration studies with healthy volunteers [11, 12]. The drug’s elimination behavior was monitored in plasma over 24 h post-administration and characterized by non-linear pharmacokinetics. To date, doping controls are based on urine and blood as test matrices, but a variety of alternative matrices including amongst others dried blood spots (DBS) and dried plasma spots (DPS) have been considered lately [13]. In comparison to the classical matrices, DBS 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. Within this research project, the first mass spectrometric detection method for meldonium in DBS was developed and successfully validated. As proof-of-concept, a pilot excretion study was conducted and both DBS and urine samples were collected from a healthy male volunteer who orally administered a single meldonium dose of 500 mg. For the analysis of the urine samples, hydrophilic interaction liquid chromatography-high resolution high accuracy mass spectrometry (HILIC-HR-MS) was employed [7]. The analysis of both the DBS and urine samples resulted in an atypical elimination behavior of meldonium: An initial phase with a rapid clearance of the active substance within the first 12-24 h was followed by a second substantially slower elimination phase, where concentrations of ca. 80 ng/mL were observed up to 16 days post-application in DBS and ca. 10-200 ng/mL for up to 49 days in urine.
Factors potentially influencing the elimination of meldonium were not investigated yet, but are of particular importance to sports drug testing. In order to clarify the atypical excretion profile and investigate the role of erythrocytes and their ability to affect the detection windows of meldonium in doping controls (e.g. by incorporation), an additional multi-dose administration study (3 x 500mg/day over a period of 6 consecutive days) was conducted. For that purpose, Na2-EDTA-stabilized whole blood samples were collected on days 4 and 28 post-administration, and used to prepare DBS, DPS, and spots consisting of washed erythrocytes. Additionally, pre-, inter-, and post-administration urine samples were collected until study day 33. Samples were analyzed by using the novel automated isotope-dilution MS approach and HILIC-HR-MS. The analysis of the urine samples confirmed the biphasic elimination behavior observed in the previous study: Within 72 hours, urinary meldonium concentrations decreased from 960 μg/mL to ca. 9 μg/mL and until day 33, concentrations ranged from 1 to 9 μg/mL. As these post-administration concentration profiles of meldonium suggested an incorporation of the substance into the cellular fraction of blood, DPS and spots consisting of washed erythrocytes were additionally analyzed. Despite intense washing of the intact erythrocytes, meldonium was still abundantly present in the RBC fraction with a concentration of approximately 1800 ng/ml on day 28. In the corresponding plasma spot, a ca. 30-fold lower concentration was found, which corroborated the hypothesis that meldonium can be incorporated into RBCs. The sustained liberation of meldonium during eryptosis would plausibly explain the observed and beforehand unexpected detection window for meldonium. As a long-term administration of high but yet therapeutic amounts would result in detection windows of several weeks or months, these results are of great importance for sports drug testing–especially with regard to the result management concerning meldonium findings in doping controls. Therefore, further studies investigating the underlying mechanisms should be conducted in the near future.
Funded: 2016
Principal Investigator:
Dr. Mario Thevis; German Sport University Cologne
Funded: 2014
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Funded: 2014
Principal Investigator:
Dr. Mario Thevis; German Sport University Cologne
Funded: 2014
Principal Investigator:
Dr. Pauline Rudd; National Institute for Bioprocessing Research and Training (NIBRT)
Research Summary:
In 2011, funding was initially received from the PCC to evaluate “Structural differences as a biomarker of EPO use”, and this enabled N-glycan profiling of 15 biosimilar recombinant α erythropoietin’s using a 2-AB label. Limited amounts of sample coupled with the low sensitivity of LC-MS analysis of 2-AB labelled glycans unfortunately meant we were unable to perform orthogonal structural analysis. We proposed to utilise procainamide (PA), a label that has higher fluorescence intensity and ionisation efficiency to analyse 9 biosimilars from the original project and also the human urinary (hu) EPO standard by Liquid Chromatography Mass Spectrometry (LC-MS). Preliminary experiments using PA labelling of Eprex and analysis by HILIC UPLC and LC-MS demonstrated that 100 μg quantities was needed to identify glycan variants of interest such as acetylated variants or N-glycolyneuraminic acid containing variants. Due to the low remaining quantity (5 μg) of biosimilar and the inability to source more, we were unable to characterise them using LC-MS. This was also the case for huEPO. The huEPO standard is a crude urinary preparation containing many other glycoproteins. We purified EPO from this preparation using MAIIA diagnostics anti-EPO affinity purification kit. The standard is estimated to contain 80 ng of EPO and due to the sensitivity of PA it was not possible to characterise this. SDS-PAGE analysis of the biosimilars revealed the presence of a number of contaminant proteins. Extraction of these gel bands followed by trypsin digestion and analysis by LC-MS identified a number of human serum proteins that are present in the final formulations. It is proposed that these contaminants are introduced by the use of human serum albumin in the formulation. We collated all of our findings from both projects and created a manuscript entitled “Challenges and potential of using glycan analysis for the detection of doping with recombinant erythropoietin biosimilars” to Drug Testing and Analysis in September 2016. Unfortunately this was found to be not suitable for publication in its current form in November 2016.
Working Groups
Principal Investigator:
Dr. John Higgins; Massachusetts General Hospital
Dr. Nikolai Nordsborg; University of Copenhagen
Dr. Jakob Bejder; University of Copenhagen
Dr. Daniel Eichner, SMRTL
Dr. James Cox, University of Utah
Dr. Merav Socolovsky, U Mass Medical School
Dr. Maziyar Baran Pouyan, University of Pittsburgh Medical School
Dr. John Phillips, University of Utah
Dr. Steve Elliott, PCC Scientific Advisory Board
Dr. Mike Sawka, PCC Scientific Advisory Board
Principal Investigator:
Dr. Marilyn Huestis; National Institute on Drug Abuse (NIH)
Principal Investigators:
Dr. Marilyn Huestis; National Institute on Drug Abuse (NIH)
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Principal Investigators:
Dr. Mario Thevis; German Sport University Cologne
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Dr. David Cowan; King’s College London Drug Control Centre
Dr. Anthony Butch; UCLA Olympic Analytical Laboratory
Dr. Andy Hoofnagle; University of Washington
Center of Excellence (Lab Equipment) Grants
Principal Investigators:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Dr. Anthony Butch; UCLA Olympic Analytical Laboratory
Principal Investigators:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Dr. Anthony Butch; UCLA Olympic Analytical Laboratory
Principal Investigators:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Dr. Anthony Butch; UCLA Olympic Analytical Laboratory
2022 Grants
Principal Investigator:
Dr. Fong Ha Liu, National Measurement Institute, Australia
Principal Investigator:
Dr. Fong Ha Liu, National Measurement Institute, Australia
Principal Investigator:
Dr. Jacob Bedjer, University of Copenhagen
2021 Grants
Principal Investigator:
Dr. Andy Hoofnagle, University of Washington
Funded: 2021
Principal Investigator:
Dr. Geoffrey Miller, SMRTL
Funded: 2021
Principal Investigator:
Dr. Mario Thevis, German Sport University Cologne / Institute of Biochemistry
Funded: 2021
Principal Investigator:
Dr. Bradley Johnson, Texas Tech University
Funded: 2021
Principal Investigator:
Dr. John Eiler, California Institute of Technology
Funded: 2021
Principal Investigator:
Dr. Nikos Ntoumanis, University of Southern Denmark
2020 Grants
Principal Investigator:
Dr. Mario Thevis, German Sport University Cologne
Research Summary:
LGD-4033 is a selective androgen receptor modulator (SARM) that is prohibited in sporting competitions by the World Anti-Doping Agency (WADA), and as an anabolic agent suspected and proven as a substance being misusede.g.in strength-focused sports. As contaminated dietary supplements pose a risk for athletes of unknowingly ingesting LGD-4033, methods for differentiating between abuse and unintentional doping by consuming contaminated supplements are urgently needed. The objective of this project was to investigate the elimination and metabolism behavior of LGD-4033,to support anti-doping authorities in differentiating between scenarios of doping and contamination. For this, human micro-dose elimination studies at three levels and with two application schemes were conducted with approval of the responsible ethics committee and analyzed using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Based on this data, elimination profiles of LGD-4033 and its metabolites were generated and the detection time ranges determined. A peculiarity of the metabolism of LGD-4033 discovered during this project is the time-dependent shift in metabolite ratio between pairs of stereoisomeric metabolites. Of main interest here was the pair of LGD-4033 and M1. By synthesizing the compound M1, its structure could be unambiguously determined to be the epimer of LGD-4033. As the M1/LGD-4033 ratio increases over time after ingestion of LGD-4033, the generated data from this project can be used to estimate the time point of ingestion when analyzing athlete’s samples. Additionally, an approximate dose of the ingested LGD-4033 can be assessed.
This is an important first step in gaining more insight into the way LGD-4033 is metabolized and excreted to ensure best-possible result management following adverse analytical findings involving the SARMLGD-4033
Principal Investigator:
Dr. Herb Tobias, Dell Pediatric Institute
Principal Investigator:
Dr. Christian Reichel, Seibersdorf Labor GmbH Doping Control Laboratory
Principal Investigator:
Dr. Michael Zimmerman, European Molecular Biology Laboratory
Research Summary:
The synthesis of novel performance-enhancing drugs and their detection by analytical laboratories is an arms race, in which timely detection and identification of novel performance-enhancing compounds are central to efficient anti-doping control. In anti-doping laboratories, chromatography-coupled mass spectrometry-based methods have become the standard technique to detect and quantify prohibited substances in the urine. Specifically, LC-MS has been shown to offer specificity, precision, and limits of quantitation, enabling high-throughput analysis with minimal sample preparation. Several untargeted mass spectrometry-based approaches have been developed to specifically pinpoint novel, unknown androgenic anabolic steroids (AAS). However, extensive liver metabolization of steroids leads to the fact that only a fraction of unchanged AAS in found in urine, adding analytical challenges to detection and quantification. In the present project, we have tested the feasibility of a novel approach to identify unknown AAS through a combination of MS/MS prediction and molecular networking of simulated and experimental data acquired with high-mass resolution UHPLC-qTOF systems. Results have shown that the chemical structures of controlled AAS share high structural similarity with known steroids. Hence, molecular networking (MN) could cluster these structures according to the structural features of their steroidal core, classifying these analytes into six chemical classes. Strikingly, the presence of double bonds in rings A and B, the carbonyl group in position 3 and the side chain in position 17 seem to have a high impact on fragmentation. Using the data gathered from MN, we have extracted the most conserved ions from each chemical class of steroids and have used this information to improve the predicted MS/MS fragmentation patterns of these steroids. Predicted MS/MS data were also treated to remove fragments lower than m/z 50, as well as low normalized intensities. These treatments have improved MS/MS prediction by 16%, significantly increasing the efficiency of the detection. Lastly, we have also compiled a theoretical database of over 1,400 steroids, including their molecular characteristics, predicted metabolic products, and MS/MS spectra (of steroid compounds and their known and predicted metabolites) in three collision energies. This comprehensive dataset will help to better identify and annotate previously undescribed steroids.
Principal Investigator:
Dr. Aviv Amirav, Tel Aviv University
Principal Investigator:
Dr. Jen-Tsan Chi, Duke University
Principal Investigator:
Dr. Christopher Chouinard, Florida Institute of Technology/Clemson University
Research Summary:
The World Anti-Doping Agency (WADA) Prohibited List bans anabolic androgenic steroid use by athletes at all times. The list contains over 60 compounds which are most commonly detected and quantified using chromatography and mass spectrometry-based methods. One of the challenges to detection of current and new substances is the presence of isomers, compounds with identical molecular weight that often cannot be differentiated even by tandem mass spectrometry(MS/MS). However, in addition to those compounds explicitly named by WADA’s Prohibited List, also banned are “other substances with a similar chemical structure or similar biological effect(s).” As such, there is an urgent need to develop new methods capable of identifying previously undetected species, especially in the presence of potential endogenous or exogenous isomers. The project proposed a novel method by which to combine experimental approaches (collision cross section measurements and structurally specific reactions) with computational modeling and machine learning to create a predictive database that can be used with LC-IM-MS methods for identification of new compounds.
The proposed work first involved accurate measurement of collision cross section (CCS) for more than half of the WADA prohibited anabolic steroids. This data, coupled with chromatographic retention time accurate mass, and MS/MS fragmentation pattern can be used to increase confidence of identification in a targeted analysis. Furthermore, measurement of this group also provided structural trends that might be used for identification of future unknowns. These results were presented in a recent publication (Velosa et al. JASMS, 2022, 33, 54-61). We have also started applying these methods to other classes of WADA prohibited substances including glucocorticoids (Neal et al. JMSACL, 2022, 24, 50-56). Next, several reactions were investigated for their improvements to IM resolution and structural characterization. Initial reactions were based on our previous work involving ozonolysis (Maddox et al. JASMS, 2020, 31, 411-417) and Paternò-Büchi reactions (Maddox et al. JASMS, 2020, 31, 2086-2092), but have now expanded to include reactions with Girard’s Reagent P and 1,1,-carbonyldiimidazole. These results demonstrate improved IM separations and structural identification and are the subject of a recently submitted publication (Velosa et al. 2022, In Revision). Finally, the current experimental results are being used to with computational modeling to develop a machine learning algorithm to be used for prediction of theoretical CCS.
Principal Investigator:
Dr. Jean-Francois Naud, Laboratiore de Controle du Dopage
2019 Grants
Principal Investigator:
Prof. Francesco Botré, Federazione Medico Sportiva Italiana
Principal Investigator:
Dr. Alexandre Marchand, French Anti-Doping Agency
Research Summary:
Growth Hormone (GH) use has been prohibited by the World Anti-Doping Agency (WADA) for many years, however detecting doping with GH has proven to be very difficult. Two methods have however been validated by WADA to identify GH doping in serum samples:
1-the GH isoforms test consists of 2 immuno lumino metric assays (ILMA) that measures the full length22KDa form of recombinant GH (recGH) and the isoforms of GH produced by the pituitary gland (pitGH). The ratio of rec GH/pit GH increases after an injection of recombinant GH. 2-an indirect test that is based on the measurements of two biomarkers, IGF-I and P-III-NP, that increase in circulation following GH administration. IGF-I can be measured by an automated immuno-assay and/or by LC-MS/MS, P-III-NP by another automated immuno-assay and/or a radio-immunoassay.
The recent development of Multiplex array technology combining the detection of multiple targets in a single well of ELISA-plate opens new possibilities to simplify the detection of GH doping. Meso Scale Discovery (MSD) is one of the commercial leaders in this field. Assays can be processed in a few hours using protocols that are similar to those used in ELISA assays, although typically with fewer steps. Our aim was to use MSD technology to produce a customized multiplex assay dedicated to GH detection for doping controls. This array could include GH proteins (recGH and pitGH) , GH biomarkers (IGF-I, P-III-NP) and potential new biomarkers: fibronectin1 (FN1) and apolipoprotein 1 (APOL1) and Vitamin D binding protein (VDBP). The development effort included:
– screening available antibodies and selecting the best antibody pairs for each target (specificity, sensitivity, linearity, reproducibility)
– multiplexing analytes in as few MSD assays as possible.
– a final comparison of analytes concentrations in 50 serum samples provided by the French Anti-doping Laboratory (AFLD), including positive controls (serums resulting from GH administration) tested either with MSD multiplex assay(s) and with WADA approved techniques (IGF-I kit on IDS-Isys from Immuno Diagnostic Systems (IDS), P-III-NP kit on Advia Centaur (Siemens), hGH ILMA kits from CMZ-Assay GmbH for recGh and pitGH) or ELISA kits for the potential new biomarkers FN1 and APOL1.
The project only reached part of its goals as more technical issues than anticipated were identified during the development. A single multiplex to detect at once all 7 analytes was not possible. Despite extensive search for antibodies, no P-III-NP antibody pair tested allowed detection of P-III-NP in serum and VDBP assay was not evaluated, as more financial resources were needed for other analytes. IGF-IAssay need a specific acidic pre-treatment that enabled its multiplexing. However two duplex assays were validated : one for detection of recGH+pitGH (with 2 possibilities for pitGH) and one for detection of FN1+APOL1.
APOL1 did not show evidence of increase in the serum samples from GH administration and seem nota good biomarker for GH. On the contrary FN1 is more promising and showed higher values in GH positive controls than in the athlete population tested. Additional studies with more samples are needed to see if FN1 could be added to the recognized GH biomarkers.
Two pit GH assays were validated and both worked successfully with the recGH assay as a duplex. The results indicated low rec/pit values for serum from athletes and higher values for positive controls (samples from GH administration study or GH-spiked serum). RecGH+pitGH duplex assays might represent a good alternative to the ILMA from CMZ assay GmbH: working in 96-well microplates with fewer manipulations and with only 50μL serum. The recGH/pitGH ratios were however lower than those obtained with ILMA assays (≈55-70%) due to higher concentrations obtained for pitGH, but the decision limits could be reevaluated after more extensive studies of serum samples from athletes and GH-administration studies.
Principal Investigator:
Dr. Michaël Polet, Ghent University
Principal Investigator:
Dr. Federico Ponzetto, University of Turin
Principal Investigator:
Dr. Robert Roach, University of Colorado-Denver
Principal Investigator:
Dr. Fred Schaufele, XCellAssay
Research Summary:
This project aimed at developing a cell-based nuclear receptor translocation bioassay has largely achieved the projected goals. Cell lines stably expressing fluorescent-labeled AR, PR, GR and an internal control receptor(ICR)binding epitestosterone have been generated. A large number of natural and synthetic ligands, including AAS, androgen antagonists, progestins, glucocorticoids, estrogens, vitamin Ds have been tested in this bioassay, with dose-response curves and specificities established. The effect of mixtures of multiple ligands combined on bioassay performance has been determined, and the performance of the ICR as intended has been examined. Initial data with a panel of standardized sera have been obtained.
Principal Investigator:
Dr. Daniel Eichner, Sports Medicine Research And Testing Laboratory
Principal Investigator:
Dr. Christopher Chouinard, Florida Institute of Technology
Principal Investigator:
Dr. Andrew Jones, Activated Research
Research Summary:
This partnership between the PCC and Activated Research Company (ARC) was initiated with the goal to design a low-cost exhaled breath sample collection device using selected adsorbents tested in a laboratory setting. The project can be broken down into three objectives. Objective one concerns the development of adsorbents for the capture and thermal release of banned substances. Samples and adsorbents have been selected and tested on a gas chromatograph system with mass spectrometry (GC/MS) using a multimode GC inlet (MMI) and thermal desorption unit (TD). Initial results gathered on the MMI-GC/MS system led to the production of temperature programmed desorption curves used to calculate activation energies for all compounds on all adsorbents. Results from this testing indicated the following adsorbents could be used to cover a range of adsorption characteristics in a multi-adsorbent packed tube: deactivated quartz wool, silica sand, and alumina. These adsorbents were tested on the TD-GC/MS system, and desorption characteristics were consistent with those measured on MMI-GC/MS while also showing increased sensitivity.
The second and third objectives concerned the design of a low-cost exhaled breath sample collection device and the detection of banned substances in simulated exhaled breath samples. The breath collection device design involved a custom bag with a check valve installed for sampling and a vacuum pump interface for a TD tube for sample extraction from the bag. Simulated exhaled breath was created using an atomizer paired with mass flow control to approximate moisture content in human breath and human breathing flow rates. This atomizer setup was used to deposit samples into the bag for extraction using a vacuum pump and further evaluation using a the TD-GC/MS system. Recoveries for the sample collection device were studied by comparing direct GC-MS results, direct injection onto a TD tube, and finally the extraction from the bag. Recoveries varied depending on the compound, but the bag was shown to be a viable option for sampling drugs of abuse, and the vacuum extraction device was suitable for extracting the contents of the bag onto the selected adsorbent.
Principal Investigator:
Dr. Soledad Rubio Bravo, University of Cordoba
Research Summary:
Cubosomic supramolecular solvents (SUPRASs) have been designed and synthesized directly in urine, by spontaneous processes of self-assembly and coacervation of the amphiphile 1,2-hexanediol in the presence of sodium sulfate. These SUPRASs, here synthesized for the first time, consist of square and rounded cubosomes, with a size range of 140−240 nm, that are made up of 1,2-hexanediol, salt, and a high water content (36−61%, w/w).It has been proved that these cubosomic nanostructures are highly efficient to extract multiclass prohibited substances in human sport drug testing owing to their large hydrophilicity and surface area.
The applicability of cubosomic SUPRASs for the development of high throughput matrix-and compound-independent sample treatment platforms has been proved by the extraction of around a hundred of prohibited substances, including highly polar and nonpolar ones, belonging to the 10categories of the WADA list. The method involves the addition of 142 mg of sodium sulfate and 200μL of 1,2-hexanediol to 1 mL of hydrolyzed urine, the vortexing of the mixture for 5 min and centrifugation for 10 min. Then, the SUPRAS extract is directly analyzed by LC-ESI-(Q-IT)MS/MSorLC-ESI-QTOF. Chromatographic separation is carried out in a pentafluorophenyl stationary phase, which has proved the best performance in a comprehensive study on the performance of different retention mechanisms in sport drug testing by liquid chromatography-mass spectrometry.
The SUPRAS-LC-ESI (Q-IT)MS/MS and SUPRAS-LC-ESI-QTOF methods have been validated. Around82−95% of drugs were efficiently extracted (recoveries 70−120%) in urine samples, and 81−92% did not present matrix effects. The capability of extraction and interference removal of cubosomic SUPRAS was proved superior to those of other eleven SUPRASs and conventional organic solvents. The proposed SUPRAS-based sample treatment is transferrable to any WADA-accredited lab sincere agents are commercially available and operations do not require special equipment. This approach is as simple as QuEChERS, but the distinctive features of cubosomes confer them high capability in multiclass determinations.
Principal Investigator:
Dr. Owen Davies, Loughborough University
Principal Investigator:
Univ-Prof. Dr. Mario Thevis, German Sport University Cologne
Principal Investigator:
Dr. Daniel Eichner, Sports Medicine Research And Testing Laboratory
Principal Investigator:
Dr. Yvette Dehnes, Norwegian Doping Control Laboratory
Principal Investigator:
Dr. Michaël Polet, Ghent University
Research Summary:
In doping control, to confirm the exogenous origin of exogenously administered anabolic androgenic steroids (AAS), a gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) analysis is performed. Work published in 2017 (Piper et al., 2017), suggests that epiandrosterone sulfate (EpiAS) is a promising IRMS target compound for the detection of AAS, capable of prolonging the detection window. However, EpiAS is only excreted in urine in its sulfoconjugated form, while all other IRMS target compounds are excreted glucuronidated, meaning that EpiAS cannot be incorporated in the existing IRMS methods. The main issue was that it was difficult to in corporate this compound in the current doping control procedures and if you did, it remained very costly, laborious and time consuming (up to 3 days). A separate extensive sample preparation needed to be performed on this compound with a different hydrolysis and extraction procedure and a different liquid chromatography (LC) clean-up.
The current project developed a new, fast, and easy to implement EpiAS IRMS method. The approach was based on the direct GC analysis of non-hydrolyzed EpiAS, making the solid phase extraction, hydrolysis, and acetylation step redundant. Sample preparation consisted of a simple liquid–liquid extraction, followed by LC fraction collection. By applying this completely different approach and methodology, we were able to develop an easy and fast (i.e., 1 day) procedure that could be rapidly implemented in doping control laboratories worldwide.
A population study was performed to check compliance with the criteria drafted by the World Anti-Doping Agency (WADA). To verify the applicability of the developed approach, the method was applied to the samples of four administration studies (i.e., dehydroepiandrosterone (DHEA), testosterone gel (T gel), androstenedione (ADION), and intramuscular testosterone undecanoate). In contrast to previously published data, the strength of EpiAS as target compound and the prolongation of the detection window in comparison with the conventional IRMS target- compounds was less pronounced. In fact, we proved that, in the vast majority of cases, EpiAS is not capable of prolonging the detection time. With this work, we provided data to prevented that numerous costly and in fact useless EpiAS analyses would be requested and conducted in doping control laboratories worldwide. At a later stage, our findings were confirmed by a second independent study performed at SMRTL (Salt Lake City, USA; publication: Nair et al. 2020).
Principal Investigator:
Dr. Jason Whitfield, The University of Queensland
Principal Investigator:
Prof. Ajit Shah, Middlesex University London
Principal Investigator:
Dr. John Higgins, Massachusetts General Hospital
Principal Investigator:
Dr. Nikolai Nordsborg, University of Copenhagen
Principal Investigator:
Dr. Jen-Tsan Chi, Duke University
Principal Investigator:
Dr. Cleo Van Diemen, University of Groningen
Research Summary:
After optimization of the library prep similar fractions of EPO were in obtained in Ghent for 1%EPO, 0.1% EPO and 0.01% EPO spike in respectively. In the following experiments, the sensitivity of the test was successfully challenged by including 0.001% EPO samples while reducing the sequencing reads per sample, increasing the throughput per sequencing run (number of samples). Furthermore, the analysis pipeline of the NGS data has been modified and improved from a manual to a semi-automated process. As it is not feasible to count millions of reads manually for each test, the bioinformatics team in Ghent evaluated different strategies to reproduce manual read counting. Since the data analysis needed to mimic the manual read counting, different parameters were adjusted and evaluated to establish maximal sensitivity of the test.
After the successful transfer of the wet lab and bioinformatics methodology, the Ghent laboratory is now fully equipped to implement the following adjustments in the next design of the panel:
1. the addition of probes directed to other potential doping genes and plasmid or virus-derived sequences.
2. to allow detection of sample swapping during processing of samples. With the design optimized now, it will be easy to include probes covering a personal genomic barcode. The NGS lab has experience with a 25 nucleotide signature that is included in capturing experiments to identify patients in routine diagnostic testing. The signature will be included in the new extended design together with the new potential doping genes targets
Principal Investigator:
J.L. Clark
Principal Investigator:
Dr. Daniel Eichner, Sports Medicine Research And Testing Laboratory
Principal Investigator:
Dr. Amanda Patrick, Mississippi State University
Research Summary:
This report summarizes the goals, activities, outcomes, and next steps forward with respect to the recently-concluded Pilot Project, titled “Infrared Ion Spectroscopy and Ion Mobility Spectrometry forBeta2-Agonist Stereoisomers”, funded by the Partnership for Clean Competition. This exploratory project was executed by myself (PI, Amanda Patrick), a graduate research assistant (Matt Carlo), and multiple undergraduate researchers. While infrared ion spectroscopy does not seem promising for differentiating salbutamol stereoisomers, it does seem to hold some promise for elucidating gas-phase dissociation pathways and may prove useful for future applications, such as identifying the structures of novel designer drugs. These areas may prove fruitful for future research endeavors. Ion mobility spectrometry with drift gas modifiers has shown some promise for characterizing stereoisomers, but separation has not yet been achieved. Likewise, isobar differentiation (of compounds with differing structures) may also be a useful application of this method. The most promising result of this pilot project—toward development of a translatable method—arose from a sub-aim investigating complex formation consisting of alternative metal charge carriers and chiral reference ligands. This approach may allow for rapid differentiation of stereoisomers on a mass spectrometer. This work has resulted in one accepted manuscript, with another manuscript and a review article currently submitted/under review, and multiple presentations, including one presentation at the national meeting of the American Chemical Society and two accepted abstracts for poster/oral presentations in the near future at ASMS and SERMACS. A proposal to continue work started during this pilot project period has also been submitted as a grant proposal to the PCC, which is currently under consideration.
Principal Investigator:
David R. Goodlett, University of Victoria
Research Summary:
In response to a request for proposals initiated by the Partnership for Clean Competition organization, namely “Single Cell HbA1c Fraction Distribution Assay”, we proposed to develop a single cell proteomics (sc-proteomics) assay to analyze sub-milliliter volumes of whole blood, to precisely measure the percentage of glycated hemoglobin (%HbA1c) in single erythrocytes.
The status of the stated deliverables and aims of the project are summarized below:
The deliverable from Aim 1 was stated as; an automated method for single erythrocyte processing for LC-MS/MS analysis.
This aim was partially delivered. Single erythrocyte processing for quantitative proteomics was achieved but FACS could not be used for automated isolation of single erythrocytes in the required (low) volume range. For automated single erythrocyte isolation the CellenONE (cellenion, Lyon, France) instrument was tested and preliminary results indicated this instrument can be used for automated single erythrocyte isolation for downstream proteomic analysis.
The deliverable from Aim 2 was stated as; a validated isotope dilution LC-MS/MS assay capable of measuring Hb and HbA1c concentrations in individual erythrocytes.
This aim was partially delivered. Validated assays were developed for both targets and Hb measurements were in the expected endogenous concentration range but the concentration of HbA1c, which was measured as the N-terminal carboxymethylated valine of beta subunit (CMV-Hb), was unexpectedly lower than the predicted range of 0.2-15% (of Hb). CMV-Hb was measured at 0.01-0.7% (CMV-Hb/Hb x 100) and while the assay sensitivity was such that Hb was robustly measured in single erythrocytes, at least 5 erythrocytes were needed to measure CMV-Hb.
The deliverable from Aim 3 was stated as an automated and quantitative MALDI-MS/MS assay for measuring the %HbA1c (measured as %CMV-Hb and henceforth referred to as such) distribution in erythrocytes.
This aim was attempted but not successfully delivered. Experiments demonstrated MALDI-TOF lacked both sensitivity and specificity required for measuring endogenous proteolytic peptides when targeting Hb and CMV-Hb using single erythrocytes.
The deliverable from Aim 4 intended to assess the predictive value of the SE-MALDI assay for distinguishing between the distribution of %CMV-Hb in erythrocytes in non-diabetic and diabetic individuals.
This aim is delayed due to participant recruitment. At the time of submitting this report 7 participants (the aim is to recruit a total of 10 participants) have donated blood specimens for this project, and of those 6 specimens have been analyzed. Additionally, this aim will be partially delivered, since the assay lacked the sensitivity required for measuring CMV-Hb in single erythrocytes and SE-MALDI could not be used to enhance throughput (see aim 2 and 3). Isotope dilution targeted (PRM) LC-MS/MS was used to measure Hb and CMV-Hb in 5-20 erythrocytes in 10 independent measurements per participant which demonstrated the distribution of CMV-Hb can be determined with low cell numbers, mitigating the need to make measurements in single cells. The predictive value of the assay will be determined only when all 10 specimens have been analyzed.
Conclusion and next steps: A workflow has been developed that can achieve single cell protein quantification in a reproducible and accurate manner. The significance of this result should not be overlooked because it has demonstrated LC-MS protein quantification can be used for reproducible quantification in single cells and can be applied for studying analyte distribution across a cell population. However, targeting the carboxymethyl N-terminal valine of HbA1c showed this specific proteoform may not be an ideal target for measuring %HbA1c (in single erythrocytes) since the occurrence of this modification is far below the expected range of HbA1c; ~0.01-0.7%(CMV-Hb) compared to the expected 0.2-15% (HbA1c). However, as the intended application of this assay was to measure the distribution of %CMV-Hb across an erythrocyte population, we showed the assay can be used for repeated measurements using small numbers of erythrocytes (5-20 erythrocyte input per measurement) for %CMV-Hb distribution profiling. Taken together, we see these results in a positive light; the results cannot be used to reject the hypothesis that %CMV-Hb may serve as a diagnostic test for blood doping. In parallel, we predict targeting an alternate (higher concentration) HbA1c proteoform would mitigate the sensitivity limitation for measuring this proteoform in single erythrocytes, which may provide a higher diagnostic value for detecting blood doping.
Principal Investigator:
Martin Bidlingmaier, MD; Endocrine Laboratory, Medizinische Klinik und Poliklinik IV, Klinikum der UniversitätMünchen, Munich, Germany
Research Summary:
Background: Soluble alpha klotho (sαKL) is a circulating protein, present in blood, urine and liquor. Smaller studies had suggested that sαKL concentrations are elevated in acromegaly, a disease with growth hormone (GH) excess. It was also suggested that sαKL concentrations might be reduced in GH deficiency (GHD) and increase after recombinant human GH (rGH) therapy. We therefore hypothesized that sαKL in blood and urine could be a biomarker suitable for detecting doping with rGH. Methods: We first evaluated analytical performance of the sαKL assay and pre-analytical stability of sαKL concentrations in blood and urine. Then, we analyzed sαKL concentrations in adult patients with GH deficiency (AGHD) which were longitudinally followed without and with rGH therapy (n = 22), and compared them to concentrations of IGF-I, IGFBP 3 and P-III-NP. As control groups we cross-sectionally evaluated patients with pituitary disease without GHD (non-functioning pituitary adenoma (NFPA, n = 20)), healthy subjects and individuals from a population based study (n =890). We established reference intervals, and investigated the impact of biological variables (age, BMI) on sαKL concentrations. Results: In blood, sαKL exhibits excellent stability under different storage conditions. In contrast, its concentrations in urine were significantly reduced after storage at -20°C. Interestingly, sαKL presented acceptable stability in fresh urine samples at ambient temperature, and in urine samples stored at -80°C. Within individual AGHD patients, sαKL concentrations in blood increased significantly during rGH therapy. However, they never exceeded the concentration range seen in healthy subjects and individuals from a population based study. Sex, age and BMI had only minor impact on sαKL concentrations. sαKL strongly correlated to IGF-I, tended to correlate to IGBFP3, but was not associated with P-III-NP. Conclusion: SαKL is a high stable biomarker in blood. It also can be measured in urine, but urine samples must be fresh or storedat-80°C. During longitudinal evaluation, sαKL concentrations increased within individual AGHD patients during rGH therapy. However, in contrast to patients with chronic GH excess (acromegaly), where sαKL is greatly elevated, in treated AGHD patients it remains within the normal range also seen in normal subjects and NFPA patients. Biological variables such as sex and BMI have negligible impact on sαKL, and the age associated decrease is much smaller than that seen for IGF-I. Our data confirm that sαKL concentrations increase with rGH therapy, but remain within the comparably broad reference interval seen in healthy subjects. To detect doping with rGH, it might be helpful for longitudinal monitoring within the athletes biological passport. In this context, the robustness against biological variables like sex and BMI, and the marginal impact of age, might provide an advantage.
2018 Grants
Principal Investigator:
Dr. Christoph Borchers, McGill University
Principal Investigator:
Dr. Marijke Grau, German Sport University Cologne
Principal Investigator:
Dr. David Handelsman, ANZAC Research Institute, University of Sydney
Principal Investigator:
Dr. Nicholas Leuenberger, Lausanne University Hospital
Principal Investigator:
Dr. Carsten Lundby, University of Zurich
Principal Investigator:
Dr. Alexandre Marchand, AFLD
Principal Investigator:
Dr. Mario Thevis, Sport University Cologne
Research Summary:
Immunopurification of doping control samples is a mandatory necessity in EPO analysis during a Confirmation Procedure; moreover, it has become common practice to also immunopurify samples for the Initial Testing Procedure. Typically used materials (e.g. Stemcell purification plate, MAIIA purification kit) rely on anti-EPO antibodies for purification. Also, the detection of EPO after electrophoretic separation and Western blotting is based on a monoclonal anti-EPO antibody, clone AE7A5, directed against a 26amino acid sequence of the N-terminal region of human EPO. While the electrophoretic separation and blot transfer can be efficiency monitored with reference standards and quality control samples, it is presently not possible to monitor the functionality of the entire sample preparation procedure. The reliance on antibodies for both purification and detection has complicated the implementation of an internal standard (ISTD). In this study, customized EPO-polyethylene glycol (PEG)-conjugates were synthesized as potential ISTDs and assessed as to their compatibility with existing sample preparation procedures for urine and blood sample analysis using the most common immunopurification techniques. Moreover, probing for the impact of the ISTD on SAR-PAGE-based EPO analysis concerning potential interference with target analytes was conducted. The presented data demonstrate that a 12 kDa PEG residue attached to human erythropoietin represents a particularly useful construct to serve as internal standard for ERA analysis. The conjugate is applicable to both urine and blood testing using the commonly employed purification techniques, supporting and improving result interpretations especially concerning specimens where the natural abundance of human EPO is low.
Principal Investigator:
Dr. Herbert Tobias, University of Texas at Austin
Research Summary:
Confirmation of doping with endogenous anabolic androgenic steroids relies on the measurement of urinary steroid carbon isotope ratios (CIR, 13C/12C) by gas chromatography combustion isotope ratio mass spectrometry (GCC-IRMS). Currently, methods used are constrained by long measurement run times and suffer from issues- relating to the fragility of high-temperature metal oxide reactors used to convert steroids toCO2 for CIR in conventional GCC-IRMS instrumentation. ARC-i (ARC-isotope) reactors are novel low temperature catalytic combustion reactors. They have been successfully applied to traditional GCC-IRMS in a previous project funded by PCC (2016R30000236G). The goal here was to improve dimensions and catalyst combustion capacity in smaller volumes in order to preserve fast GC peak shapes and widths to achieve faster analysis. Work was to involve evaluation and the application to fast GC and GC×GC separations. Due to delays and time constraints, GC×GC separations were not evaluated, only fast GC. These ARC-I reactors operate with combustion volumes consisting of transition metal catalysts and operate at 300°C lower combustion temperatures.
Here a fast cryofocus GCC-IRMS system was developed featuring an integrated ARC “HyperX” reactor. Cryofocusing was achieved by a modified pneumatic thermal modulator. In place of a conventional metal oxide reactor, the system was configured with a purpose built, low dead volume, low temperature catalytic reactor, reducing operating temperatures by 300°C. Successful baseline resolution of complex steroid mixtures was achieved in a 7.5 min analytical run, about one third less time compared to conventional GC methods. Performance testing of various catalyst formulations with certified steroid reference material, that were developed in a previously funded PCC project, yielded CIR measurements with average standard deviations SD(δ13C)< 0.5‰ across a range of 5 ng to 100 ng of injected analyte. Average accuracy and measurement linearities approached those of conventional GCC-IRMS systems for test steroids. These results demonstrate that an alternative combustion strategy may support robust fast cryofocus GCC-IRMS systems in anti-doping laboratories increasing testing throughput and capacity and reduce operating costs.
Principal Investigator:
Dr. Michael Polet, University of Ghent
Research Summary:
In anti-doping, a high number of classes of substances are prohibited and laboratories need to detect these at low urinary concentrations. Traditionally, testing is done using complimentary liquid chromatography mass spectrometry and gas chromatography mass spectrometry. High resolution mass spectrometric acquisition has some important advantages over triple quadrupole instruments (e.g., open screening due to full scan high resolution data acquisition with retrospectivity, compatibility with libraries and a straightforward and effortless addition and validation of new compounds in the future). Doping samples can be stored for 10 years and retrospective data analysis can be used to re-evaluate previously acquired data (e.g., searching for prohibited (designer) substances that were unknown at the initial moment of analysis). During the past decade, these advantages have led to the wide-scale transfer of liquid chromatography triple quadrupole mass spectrometry screening to liquid chromatography high resolution mass spectrometry screening for doping control purposes. Up to now, for gas chromatography a similar switch to high resolution screening has not yet occurred, because so far no method has been developed that combines sufficient sensitivity with wide-scale drug detection. In this work, the current gas chromatography triple quadrupole mass spectrometry screening method for human doping control purposes was successfully converted into an equivalent and complete gas chromatography high resolution acquisition screening method. This new screening method on a gas chromatography quadrupole time-of-flight mass spectrometer has been developed and validated. The method is compliant with the World Anti-Doping Agency requirements and allows the detection of 294 target compounds (and 14 internal standards), including diuretics, stimulants, narcotics, beta-2-agonists, beta-blockers, hormone modulators, anabolic agents and the quantification of 14 endogenous steroids in a single fast run (14.1 min).
Principal Investigator:
Dr. Laura Garvican, Australian Catholic University
2017 Grants
Identification of New Testosterone Biomarkers by an Untargeted Steroidomic Profiling in Blood Matrix
Principal Investigator:
Dr. Raul Nicoli; Swiss Anti-Doping Laboratory
Research Summary:
An untargeted steroidomic protocol was employed for the analysis of serum samples collected for a clinical trial involving 19 male volunteers administered with both transdermal and oral testosterone (T). After an SPE-based sample preparation, full scan (FS) MS data were acquired by UHPLC–HRMS analyses in both positive and negative ionization modes, with the aim of detecting endogenous steroids both in their free form and a sphase II metabolites. Automatic annotation of detected steroid features was carried out using Dynasti online platform, and the obtained results were carefully reviewed by taking the advantage of the newly developed three-step work flow to limit erroneous annotations mainly due to in-source fragmentation of interfering conjugated steroids. Analysis of variance multiblock orthogonal partial least squares (AMOPLS) multivariate statistical model allowed ranking features according to their response to T administration, highlighting 12 unambiguously identified steroid hormones as promising markers of oral intake. The increase of serum concentrations after oral administration was more pronounced for phase II steroid metabolites rather than for free steroids, with a particularly strong response for glucuronide-conjugated androgen metabolites and their hydroxylated derivatives. The individual longitudinal monitoring of the most responsive markers provided significant improvements for the detection of doping with T, in particular in terms of overall detection time and sensitivity regarding individuals bearing the del/del genotype for UGT2B17 enzyme.
This first application of serum steroidomics in the antidoping context, demonstrated its usefulness in investigating alterations in steroid metabolism caused by the misuse of T. The exploratory study allowed for selecting prominent serum biomarkers, which together with serum T and 5α-dihydrotestosterone could complement the current urinary steroidal module of the athlete biological passport.
Principal Investigator:
Dr. Zach Gagnon; John Hopkins University
Principal Investigator:
Dr. Jentsan Chi; Duke University
Comprehensive Steroid Detection in Urine Via Targeted Clean-Up and Fully Automated GCxGC-MS Analysis
Principal Investigator:
Dr. Nicholas Turner; The Open University
Principal Investigator:
Dr. Corinne Buisson; AFLD – Agence française de lutte contre le dopage
Research Summary:
The GH-OMIC project aimed to use metabolomics-based methodologies to track the intake of micro-doses of growth hormone in healthy (well-trained) volunteers. In the course of this project, we performed all the metabolomics, lipidomics and stereoidomics analysis on the urine and plasma samples of our cohort of 16 volunteers, to evaluate whether their metabolomics, lipidomics or stereoidomics profiles could highlight growth hormone intake. The metabolomics and lipidomics analysis of the urine and plasma samples were performed first. After samples injection in LC-HRMS instruments, the raw data has been acquired and refined to obtain clean datasets that underwent singularly to statistical analysis to identify treatment specific profiles. Quite surprisingly, the results did not show the possibility to discriminate the two groups according to their profiles. The lack of a group specific profile, related to hormonal intake, exists probably because the effect of the micro-doses is somehow subject-specific.
A second data treatment has been applied, using a longitudinal approach: the longitudinal tracking of the volunteers’ profiles showed a specific response to the treatments; this specific response could be used to spot the different treatments using both urine and plasma samples. We demonstrated that these results allow the construction of predictive models to spot different treatments in our cohort, and that this is valid in any lipidomics and metabolomics dataset. The biological interpretation of the results, reinforced our findings, giving promising markers for each specific treatment. Due to the necessity to perform a longer and deeper data analysis of the datasets, the stereoidomics experiment has been performed lately and the data obtained is still under refinement and evaluation.
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Principal Investigator:
Dr. Jentsan Chi; Duke University
Principal Investigator:
Dr. Christiane Ayotte; INRS Santé
Principal Investigator:
Dr. Paul Armishaw; National Measurement Institute
Research Summary:
New carbon isotope delta values have been successfully assigned to ten steroid analytes in thecurrentMX017 urine CRM under the PCC funding. The new δ13CVPDB values for T, epi T, A, Etio, 5αAdiol, 5βAdiol, PD, 11βHA, 11OE and 16-enwill assist WADA laboratories in validating their methodology for carbon isotope delta measurement by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for confirmatory analysis[1].
The reconstituted MX017 urine CRM was hydrolyzed using β-glucuronidase (E.coli) to free steroids which were extracted into hexane. Steroids of interest were purified using a two dimensional preparative high-performance liquid chromatography (2D-HPLC) procedure. Four steroid analytes (11βHA, A, Etio and 16-en) were isolated in the first dimension using Kinetex Phenyl Hexyl 5μmcolumn (250mmx 4.6 mm) utilizing an aqueous acetonitrile gradient. The remaining six steroids (epiT,- 5αAdiol, 5βAdiol, T, 11OE and, PD) were further purified by Merck Chromolith column (RP-18e 50 x 4.6 mm) in the second dimension using an aqueous methanol gradient. All purified extracts were solvent exchanged to cyclohexane/2-propanol (4:1) and screened by GC-MS prior to analysis by GC-C-IRMS. A multi-point isotopic bracketing calibration approach was adopted to normalize all measured δ-values to the VPDB standard through the NMIA MX018steroid isotopic CRM[2]. Samples and calibration solutions analyzed under similar conditions by the instrument were expressed relative to the internal reference CO2gas. The reference δ13CVPDB(CRM) values (y-axis) were plotted against δ13Cmeas value (x-axis) resulting in a linear equation for scale normalization following published method[3]. Frequent bracketing of sample with the MX018 CRM mixtures during analysis allowed instrument drift to be accounted for.
The certification of carbon isotope delta values in MX017 was carried out in accordance withISO17034[4]. The assigned δ13CVPDB values of were determined using 49 bottles of MX017 analyzed over several batches during method development, homogeneity and stability testing, and confirmatory analyses. Potential factors contributing to measurement uncertainties in the reference values were identified from the sample transformation procedure, matrix effects, calibration procedure and the random variability between batches of analyses. No significant inhomogeneity was detected between units of the CRM as shown by analysis of variance (ANOVA). Short-term stability testing was carried out at room temperature up to four weeks to estimate uncertainty associated with exposure to transport conditions. The measurement uncertainties were determined at a level of confidence of 95%.
2016 Grants
Principal Investigator:
Dr. Nicolas Voelcker; Monash University
Principal Investigator:
Dr. James Harynuk; University of Alberta
Dr. Fred West; University of Alberta
Principal Investigator:
Dr. Emad Kiriakous; Queensland University
Research Summary:
As novel doping agents and evasive doping strategies are being employed by athletes, there is an increasing demand to conduct more frequent, faster and situationally responsive tests that align with major sporting events. Mass spectrometry is still the gold standard technique suitable for discriminating a variety of doping agents but conventional methods require time and specific laboratory settings to conduct tests-leading to a limit in the throughput of samples tested and responsiveness to a sporting event, e.g. on-site analysis. Nanomaterial-interfaced mass spectrometry could provide a fit-for-purpose solution to rapid testing and be adaptable to mobile/field-based testing. Our research aimed to develop nanostructured substrates that interface with laser desorption/ionization-mass spectrometers, which are inherently engineered for automated, high-throughput analysis. Using pure silicon wafers, we fabricated a range of mass spectrometry substrates for sensitive chemical profiling by a technique called surface-assisted laser desorption ionization-mass spectrometry (SALDI-MS). Through precise control of the nanostructured topography and surface chemical functionalization, we created three candidate surfaces for SALDI-MS of doping agents. One particular surface, F17 DIOS, was further optimized for the detection of derivatized androgenic-anabolic steroids (AAS). F17 DIOS surfaces detected AAS, including testosterone and androsterone, allowing sensitive detection down to 0.18 and 0.77 ng/ml, respectively, from urine. Using those surfaces we were also able to detect growth hormone releasing peptide-6, and stimulants, morphine and oxycodone, from biological fluids down to low ng/ml concentrations. SALDI-MS has an analytical run-time of 2 seconds, which provides the capacity to automate high-throughput analysis of biological samples. Larger instrumentation has MS/MS capabilities that match structural fragmentation data from leading triple quadrupole LC-MS/MS, providing confirmatory data. Finally, LDI-MS instruments are now available in robust benchtop models, opening up opportunities in MS analysis at sporting events using the developed nano fabricated substrates funded by this grant. Overall, nanofabricated substrates show considerable promise in rapid and dynamic athlete testing. The developed substrates open up the dynamic range of LDI-MS instruments from small molecule drugs to large intact proteins.
Principal Investigator:
Dr. Fred Schaufele; Xcell Assay
Research Summary:
The objective of the project was to create sensors for one of two potential internal doping controls, epitestosterone (epiT) or 17-hydroxyprogesterone (OH-P), though mutagenesis respectively of the androgen receptor (AR) or progesterone receptor (PR). The internal control measurement would be a variant of our previously developed ‘total androgen’ BioAssay of AR nuclear translocation. The goal is to create BioAssay measurements of total androgen/epiT or total androgen/OH-P ratios for athlete samples that would be sufficiently inexpensive to apply for amateur athletes, including youth athletes who self-report on androgen abuse at a high rate both in the US and in Europe.
A total of 27 mutants were introduced into the PR. The goal was to create a mutant sufficiently sensitive (down to 0.6 nM) to respond to the normal range of OH-P in males when a serum sample is diluted 5-fold into assay buffer. All mutants examined required > 200 nMOH-Pto half-maximally activate (EC50) the nuclear translocation. The transient methods, necessary to survey the large number of mutants in this project, introduce considerable measurement noise originating with variable level expression and brightly fluorescent dead cells. But even considering that EC50s obtained by the transient methods were determined to be 16-fold less sensitive than in measurement-quality stable cell lines, no OH-P mutant was sufficient to pursue further.
57 ‘epiT-AR’ mutants were examined, the most successful of which had a detection limit (that where the amounts of AR present in the nucleus and cytoplasm are equal) of 15 nM epiT. That remains above a detection limit of 0.5 nM desirable to measure ‘normal’ epiT levels in diluted blood. With funds beyond that provided by the grant, we created an additional 32 AR mutants to examine additional structural theories; some of those AR mutants had- detection limits of 3-5 nM epiT. In ‘Section C. Plans to Address Obstacles’, we outline ongoing approaches (also paid to date from internal funds) to potentially obtain the 0.5 nM detection limit desired for blood analysis.
By contrast, urine contains epiT at higher concentrations (25–105 nM in males) detectable with the currently created mutants, even following a 10x dilution in assay buffer. The next steps to be discussed with the PCC are outlined in ‘Section E: Anticipated Next Steps’ and include a concrete path for establishing a BioAssay measurement of the androgen/epiT for urine samples from males.
Principal Investigator:
Dr. Gaetano Cairo; University of Milan
Research Summary:
The erythropoietin (Epo)-erythroferrone (ERFE)-hepcidin axis coordinates erythropoiesis and iron homeostasis. In particular, mouse studies established that ERFE synthesized in response to Epo impairs hepcidin transcription. The development of a validated assay for human ERFE has demonstrated that accelerated erythropoiesis stimulates ERFE production in humans, as ERFE was shown to be increased in patients with β-thalassemia and in response to blood donation or administration of high doses of recombinant human erythropoietin (rhEpo) (1). To investigate whether ERFE could be used as an alternative or complementary marker to detect the abuse of rhEpo for doping, a randomized double-blind controlled study was conducted in which 24 healthy males received six injections (every second/third day) of i) saline (placebo), ii) Epo 20 UI/kg (micro-dose) or iii) Epo 50 UI/kg (low-dose), according to a protocol used by athletes to evade doping controls. Total hemoglobin mass (Hbmass) significantly increased after Epo low-dose injections, but not aftermicro-dose, when compared to placebo. Serum ERFE levels, measured by a validated assay, were enhanced by both Epo doses, remaining significantly above placebo levels for 48 h (micro-dose) or 72 h (low-dose) and decreased thereafter. The pattern of ERFE concentrations mirrored that of serum Epo. Conversely, hepcidin levels showed a significant decrease when Epo and ERFE arose, before any changes in serum iron parameters occurred. Therefore, the results of this project show that in healthy humans ERFE responds even to low Epo levels which are not associated with expansion of Hbmass, a sensitive marker of erythropoietic response. Since our data did not show alterations in iron saturation of transferrin (Tfsat) and ferritin, hepcidin repression after the first rhEpo injection appears to be iron-independent, thus suggesting that ERFE is a sensitive and direct inhibitor of hepcidin in response to Epo. Notably, ERFE flags even micro-dose Epo and thus holds promise as a novel anti-doping biomarker, in particular for the detection of very small hormone quantities, which is still a challenge.
Principal Investigator:
Dr. Christiane Ayotte; INRS Santé
Research Summary:
The main objective of this study was to implement a new test based on an automated capillary-electrophoresis western system from the company Protein Simple for the analysis of ESAs in urine and blood samples. This emerging technology integrates all the instrumental steps associated with traditional electrophoresis and western blotting but delivers them in a cost-effective and high throughput scheme. To achieve this principal goal, several parameters needed to be improved.
The principal aims were to optimize the operating conditions, to optimize the specific signal for ESAs while decreasing the cross-reactivity to other proteins, to demonstrate that this method could detect all types of ESAs, to determine if the method could be used with urine and blood samples, to compare this new method to the actual accredited methods and finally to put in place and validate a testing procedure that could be transferred to other anti-doping laboratories.
During the period of funding support, more than 75 runs were performed with the system, 17 primary antibodies and 6 secondary antibodies were screened, 3 different loading buffers were evaluated and different blocking agents were also used. More than 100 individual urine samples from athletes were evaluated. The major breakthrough was the discovery of a biotinylated polyclonal goat anti-EPO antibody (BAF959, RnD Systems), that used in combination with a proprietary streptavidin-HRP conjugate, allowed this technique to be sensitive enough to be used for initial testing procedure.
Currently, only LOD established from the sequential dilution of various ESAs standards were determined for all ESAs. Reproducible EPO detection was possible for urine samples containing nominal huEPO concentration of 1 mIU/μL. Those LODs are based on the concentrations in the 5 μL samples deposited in the Wes plates. Since only 40 nL is injected on the capillary, is the determined LODs are close to the one of the SAR-PAGE method although the latest is more sensitive. The other parameters like reproducibility, precision, robustness and others were not completely addressed.
Based on the LOD obtained during this study, it is estimated that more than 85% of all urine samples received for anti-doping control analysis would give a signal for ESA with this automated capillary-electrophoresis western system. Additionally, all past positive samples were correctly identified indicating that this method could be used as a screening procedure. Simple Western could help to considerably reduce the time for ESAs analysis from one and a half day, using traditional Western blotting techniques, to less than three hours. Indeed, depending on the automated system used, 25 samples (WES system) up to 96 samples (Sally system) can be processed, analyzed and quantitated at the same time. This would reduce considerably the cost per sample for ESAs screening, allowing to target more athletes, more frequently, thereby increasing the deterrence against ESAs abuse. The fast analysis rate of an automated system could also be of high interest during major sports events such as the Olympic Games where a high number of samples need to be analyzed in a short period of time.
We consider that this new high-throughput analytical strategy should definitely be considered to complement the actual testing approach.
Principal Investigator:
Dr. Judith Su; University of Arizona
Research Summary:
Human chorionic gonadropin (hCG) stimulates the production of testosterone in men and is banned by the World Anti-Doping Association (WADA) for male athletes. In this one-year pilot project, we demonstrated the feasibility of using microtoroid optical resonator sensing technology for ultra-sensitive and rapid detection of human chorionic gonadropin (hCG) in patient urine samples. We were able to demonstrate one femtomolar detection of hCG in synthetic urine as well as measure femtomolar concentrations of hCG in the urine of pregnant women in different trimesters of pregnancy in under 30 seconds. In terms of sensitivity, this represents a three order of magnitude improvement over standard mass spectrometry’s limit of detection for hCG which is ~ 3 picomolars. This is also the allowable concentration level of hCG by WADA. We expect the signal-to-noise ratio of our data to be even better in plasma as hCG is present in significantly higher (1000 times) concentrations in plasma than in urine. Testing via urine as opposed to plasma/serum is preferred by athletes as it is non-invasive. As a control, the urine of non-pregnant women and men was tested as well. These samples did not show an increase in signal from our sensor. Results from the control experiments further support the fact that our sensors can operate with patient samples. Unlike mass spectrometry, which may require involved sample preparation such as immunoextraction our sensor can work directly with urine samples with minimum sample handling. Our future steps include incorporating these devices with microfluidics for improved sample handling and translation to other laboratories and clinics. We anticipate that our technology will enable a non-invasive, sensitive, and rapid means to detect hCG and other performance enhancing drugs in the clinic or by the track.
Principal Investigator:
Dr. Nikolai Nordsborg; University of Copenhagen
Research Summary:
The development and validation of new sample collection methods, shipment procedures and analytical strategies for doping control purposes are warranted to reduce costs and protect the athletes’ integrity, as well as expand possibilities for large-scale testing. A promising step forward, from reliance solely on venous blood puncture and urine samples, is exploration of the minimal invasive fingertip puncture based dried blood spot (DBS) and dried plasma sport (DPS).
The primary aim of the project was to evaluate whether abuse of insulin lispro and four testosterone esters (Sustanon 250) can be detected at relevant concentration levels in samples obtained using Dried Blood Spot (DBS) and Dried Plasma Spot (DPS) technology.
In the testosterone ester study, 19 eugonadal men received two intramuscular injections of either 1mL of250 mg Sustanon, a blend of four testosterone esters, or 1 mL saline, in a double-blind, placebo-controlled, randomized design. The injections were separated by three weeks. Finger-prick DBS samples, urine samples and venous blood samples were collected two days pre-injection, at least 5days apart, and on day 1, 3, 5, 10 and 14 after each injection. The samples were analyzed at the Norwegian Doping Control Laboratory in Oslo. Additionally, the potential acute ergogenic effect of a single testosterone injection on neuromuscular performance was evaluated 24 hrs after the first injection. The DBS based screening method of the four testosterone esters was proofed applicable for detection of intramuscularly injected testosterone esters.
In the insulin study, eight healthy men received one subcutaneous injection of 0.15 IU/kg body weight insulin lispro. Finger-prick DBS samples werecollected0,1, 2, 4 and 6 hrs after injection. Additionally, serum was collected right before injection, every 30 minutes the first 6 hrs post-injection, as well as at 24 hrs post-injection. Urine samples were collected by the subjects themselves right before insulin lispro administration and subsequently at every urination the first 24 hrs post-injection. Analysis of the DBS samples is ongoing and expected complete in September 2019.
Principal Investigator:
Dr. Mario Thevis; German Sport University Cologne
Research Summary:
Sports drug testing is largely performed by analyzing urine, serum, or whole blood samples. These biological matrices allow a reliable and sensitive detection of a variety of performance-enhancing drugs as well as a standardized sample collection and transport. However, alternative matrices such as dried blood/plasma spots (DBS/DPS), hair, oral fluid (OF), and exhaled breath (EB) can be advantageous with regard to the invasiveness and intrusiveness of the sampling procedure, the stability of the analytes, and the effort and costs for sample transportation and storage. In forensic and clinical chemistry, many of these matrices are well established and they could consequently be promising alternatives for doping control purposes.
In this project that followed on a pilot study aiming at exploiting EB as an alternative test matrix for doping controls, the capability of EB to allow for expanding the analytical breadth was assessed and a test method was validated. Relevant and representative analytes from the classes of stimulants/new psychoactive substances (10 analytes), narcotics (4 analytes), synthetic cannabinoids (5 analytes), corticoids (3 analytes), and beta-blockers (5 analytes) were used to establish an initial testing method targeting specifically those drugs prohibited in-competition only. Method characteristics were determined including assay precision, limit of detection, recovery, and specificity, and the method was applied to authentic breath samples collected after therapeutic drug use (patient samples) or in the course of drug elimination studies (volunteer samples). The unchanged active principle (i.e. the unmetabolized drug) was detected in cases of stimulants including methylhexanamine, dimethylbutylamine, pseudoephedrine, and sibutramine for up to 48 h, with sibutramine being detected also in the form of its desmethylated metabolite. Also, in case of the betablocker bisoprolol, a sample collected 1 h post-ingestion was found to contain the unmodified substance. Corticoids such as dexamethasone, methylprednisolone, and prednisolone were detected in post-administration study samples up to 7 h following a single therapeutic oral dose, whilst prednisone was not observed in a patient’s EB samples.
In order to facilitate future exhaled breath elimination studies, options for an animal model supporting studies of EB were assessed. A preliminary system was composed of the SensAbues collection device mounted onto a breathing mask fitting a beagle dog’s face. A potential partner capable of conducting animal administration studies was identified, and next steps would include a pilot study indicating whether this model is a viable route to studying drug EB elimination features of doping agents.
In addition, future investigations into additional target analytes, interindividual differences in EB drug concentrations, effects of smoking on drugs in EB, effects of different types of membranes / collection cartridges, stability of drugs in collection devices, etc. are warranted in order to increase the knowledge concerning this convenient sampling method and the extent of its utility in sports drug testing programs.
Principal Investigator:
Dr. Herb Tobias; Dell Pediatric Research Institute
Research Summary:
ARC reactors were adapted and characterized for carbon isotope ratio (CIR) analysis of steroids using gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) for detection of synthetic steroid use in doping control. Reactors were custom designed to operate with combustion volumes consisting of a catalyst to promote complete combustion of steroids to CO2molecules, from which the13C/12C isotope ratio was measured by IRMS. Metal oxide-filled reactors constructed with ceramic tubes or fused silica capillary are widely used for combustion in gas chromatography combustion isotope ratio mass spectrometry (GCC-IRMS). However, they tend to be easily cracked or broken and prone to leaks at operating temperatures of ~950°C. We modified a commercially available catalytic combustion/reduction methanizer (PolyARC) to quantitatively convert organics toCO2for δ13C analysis while retaining chromatographic resolution. These modified ARC reactors operate with a transition-metal catalyst that require a flowing O2gas (such as high purity Zero Air or He/1%O2blendat ~0.3 ml/min mixed into the He carrier gas) to enable complete conversion to CO2at lower temperature (620°C) with acceptable reactor life, reduced complexity, and improved robustness. Performance of two versions of the ARC reactors with different combustion volumes were characterized by analysis of methane, steroid and n-alkane isotopic standard materials. Linearity of steroid isotopic standards (CU/PCC 40-1, 41-1, 42-1, 44-1) ranged from 0.02 to 0.60 ‰/Vover25 to 200 ng of each steroid injected. Precisions and accuracies of measurements for steroids and alkanes, had average standard deviations of SD ( δ13C) less than ± 0.18 ‰ and average accuracy of better than 0.19‰ δ13CVPDB. Peak width expansion within both devices were comparable to that in traditionally used metal oxide reactors used for normal GC separations. However, further work is required to enable their use in fast GC applications. The modular design reduces complexity and addresses flow connection issues associated with the high temperature metal oxide reactors. These data demonstrate for the first time that novel combustion schemes enable operation at lower temperatures to yield high precision δ13C data with GCC-IRMS of steroids and alkanes
2015 Grants
Principal Investigator:
Dr. Jack Henion; Q2 Solutions
Research Summary:
A book-type dried plasma card (DPS) card, which consists of seven-layers of materials where each layer has its function to support a successful filtration of red blood cells (RBC) from whole blood to generate plasma which will be absorbed and dried in the paper substrate, has been developed. The card can produce up to 4 plasma spots per card. The functionality of the card was evaluated through 1) visual evaluation of the generated plasma spots to ensure that the card is capable of producing hemolysis-free plasma spots from whole blood across a wide range of hematocrit (Hct) levels, 2) validation of the quantitative analysis of multiple classes of drugs, and 3) compatibility for fully automated analysis operation.
Results showed a fully functional card through a successful generation of hemolysis-free plasma spots from whole blood ranging from 30% to 60% Hct. Bioanalytical validation was performed by employing a fully automated flow-through elution method coupled with on-line SPE-LC-ESI-MS/MS for the quantitative determination of four representative opioids (Morphine, Codeine, Oxycodone, Hydrocodone) and five stimulants (Amphetamine, Methamphetamine, 3, 4-Methylenedioxymeth-amphetamine (MDMA), Phentermine, and Mephedrone) in one method using their corresponding deuterium-labeled analogues as internal standards. Method validation results showed good linearity (R2≥0.9963) ranging from 5 to 1000 ng/mL. Intra-day and inter-day precision and accuracy were within the acceptable limits at four quality control (QC) levels. Extraction recovery was ≥87.9% at both the lower limit of quantitation (LLOQ) and the upper limit of quantitation (ULOQ) along with acceptable selectivity and sensitivity. DPS on-card short-term stability was compound-dependent and storage-dependent.
In summary, the benefits of this validated book-type DPS card include a wider applicability range of Hct (30% to 60%), on-line analysis compatibility, a higher plasma volume yield, and a feature of flexible volumetric sampling.
Principal Investigators:
Dr. Peter Van Eenoo; University of Ghent Doping Control Laboratory
Dr. Michael Polet; University of Ghent
Research Summary:
The main aim of the project was to drastically increase the sensitivity of the GC-MS/MS screening method by using chemical ionization (CI) instead of electron impact (EI). At a later stage, a comparable GC-CI-MS/MS method will be set up by the doping laboratory in Salt Lake City.
The second aim was to search for new metabolites by using this increased sensitivity and the typical fragmentation patterns associated with CI.
Detailed objectives of the first part of this project:
1. Development of a CI GC-MS/MS screening method that includes a high number of doping substances currently featured on the WADA prohibited list (minimum the same as in the previous published EI method).Compounds of different classes will be included: anabolic steroids, beta-2-agonists, diuretics, hormone antagonists and modulators, narcotics, stimulants and cannabinoids.
2. The developed method in point 1 should fulfill the criteria set in WADA’s technical documents on required performance limits as well as on the identification criteria regarding chromatography and mass spectrometry. Multiple internal standards shall be used to evaluate the efficiency of every analytical step, as well as a quality control sample spiked at the MRPL with all (available) reference compounds.
3. The method shall be able to quantify all threshold substances and substances relevant for the steroid profile and comply with WADA’s technical documents on decision limits and criteria in chromatography and mass spectrometry. To allow for a high qualitative quantitation of an extended number of endogenous steroids, six point calibration curves and a minimum of 8 deuterated internal standards will be used. At least3 internal standards will be glucuronidated. The correct quantification will additionally be verified at two levels using quality control urines.
The objectives of the second part of this project are aimed at searching for new metabolites. As in EI, CI results in specific fragmentation patterns typical for a specific compound class. These are however entirely different for CI than for EI and this opens a complete new window in the search for metabolites.
Principal Investigator:
Dr. Herbert Tobias; Cornell University
Research Summary:
A Cryogenic-Focusing Fast Gas Chromatography Combustion Isotope Ratio Mass Spectrometry(Cryo-Focus Fast GCC-IRMS or Cryo IRMS) system was developed to enable faster analysis with less sample. Although the intention was to also extend capabilities to minor metabolites, they were not directly studied in the final system due to issues discussed in sections (b.) and (d.). Well characterized standard materials were used so that both precision and accuracy can be directly addressed. Significant increase in S/N was not achieved due to losses at the inlet; however, major reduction inrun times were possible (<5-6min) at down to 10ng sample. The post-column interface was optimized with minimal peak broadening using a programmable temperature vaporization (PTV) injector, and narrow-bore versions of a continuous capillary combustion reactor, cryogenic water trap, transfer lines, and open split to the IRMS. PTV injection was performed with Cryo-Focusing before the steroids enter the column (a 1m x 0.1mm i.d. x0.1um ZB1-ms column isothermal at 270oC). The fastest ramp rate procedure possible (i.e. Semi-Fast) using the normal G Coven with the fast GCC-IRMS setup was also used without Cryo-Focusing as a comparison. We characterized the analytical figures of merit of steroids from 100ng down to 2ng on column, with the ideal range of steroid mass injected at approximately 10-20ngwhen analyzing mixtures with closely eluting peaks, with greater mass injections also possible for well separated peaks. Peak widths of various steroids were characterized by Cryogenic-Focusing (with a measured N2 gas flow at~90-110oC), released using an N2 gas flow jet at350oC, and then separated by an isothermal fast GC method (at 270oC) coupled post-column to a fast quadrupole MS (Cryo qMS), the Cryo-Focusing isothermal method (at 270oC) coupled to IRMS (Cryo IRMS), and a Semi-Fast ramp rate method (without cryogenic focusing) coupled to IRMS (Ramp IRMS), with averages of 548±140ms, 802±84ms, and 1066±35ms FWHM, respectively for 10ng steroids injected. In this setup, the focusing reduced peak widths by approximately a half or more when compared to traditional GC for a suite of anti-doping carbon isotope ratio (CIR) test Target Compounds (TC), E, A, 5βA, 5αA, and Endogenous Reference Compound (ERC), 5βP, steroids (Abbreviations: see Appendix). Average FWHM widths of all the steroids investigated (802ms) was wider than the goal of <500ms, while CIR quantification limit was 10ng, instead of the goal 2ng, due to minimal increase in S/N using the Cryo-Focuser combined with~72% recovery at PTV. The precision normally met the goal of SD(δ13C)≤±0.5mUr, but also ranged up to SD(δ13C)±1.0 mUr in some cases. More work is required to identify sources of imprecision & inaccuracies at low sample mass levels; the potential sources are discussed. The results indicate the Cryo-Focus Fast GCC-IRMS system would be of use to the Anti-Doping Laboratories due to the much faster analysis (<5-6min) using less material (10ng-20ngas opposed to >50-100ngusually analyzed for normal GCC-IRMS steroid CIR).
Principal Investigator:
Dr. Eleftherios Diamandis; Mount Sinai Hospital, Toronto
Research Summary:
We here describe a method to assess hyper androgenicity in female athletes. We conducted a cross-sectional study of 106 female athletes and 114 sedentary age-matched controls. Serum from these women was analyzed for complexed prostate specific antigen (cPSA) and free prostate specific antigen (fPSA), by fifth generation assays with limits of detection of around6 and 140 fg/mL, respectively. A panel of estrogens, androgens and progesterone in the same serum was also quantified by tandem mass spectrometry. Results: Both components of serum PSA (cPSA and fPSA) were lower in the elite athletes vs the control group (P=0.033 and 0.013, respectively). Furthermore, estrone (p=0.003) and estradiol (p=0.004) were significantly lower, and dehydroepiandrosterone (p=0.095) and 5-androstene-3β, 17 β-diol (p=0.084) tended to be higher in the athletes vs controls. Oral contraceptive use was similar between groups and significantly associated with increased cPSA and fPSA in athletes (p= 0.046 and 0.009, respectively). PSA fractions were not significantly associated with progesterone changes. The Spearman correlation between cPSA and fPSA in both athletes and controls was 0.75 (P < 0.0001) and 0.64 (P < 0.0001), respectively. Conclusions: Elite athletes have lower complexed and free PSA, higher levels of androgen precursors and lower levels of estrogen in their serum than sedentary control women.
Principal Investigator:
Dr. Paul Gregorevic; Baker IDI and Diabetes Institute
Principal Investigator:
Dr. Laura Garvican-Lewis; Australian Institute of Sport
Research Summary:
Introduction: The Athlete’s Biological Passport (ABP) is a valuable tool in the fight against doping; its integrity is underpinned by understanding the magnitude of normal fluctuations of its biomarkers in response to various environmental stimuli or medical conditions e.g. altitude training or iron deficiency. The combination of intravenous iron supplementation and altitude training has a number of potentially important ramifications for the anti-doping field. Intravenous iron supplementation, via bolus injection, is currently not banned under WADA guidelines. However its potential to enhance performance via facilitating erythropoiesis warrants further investigation. Secondly, since altitude training and the treatment of iron deficiency present two potentially confounding factors to the passport, a thorough understanding of the magnitude by which the ABP can be altered when these are combined is warranted. Lastly, the combination of intravenous iron supplementation and altitude training creates an opportunity to model the end result of erythropoietic doping practices within natural and legal constraints. This allows relevant data to be collected from high-level athletes, which can be used to validate a number of novel and emerging analysis methods for doping detection.
Hypothesis: The erythropoietic response to 3 weeks of altitude training was assessed in endurance trained athletes supplemented with either oral or intravenous (IV) iron. We hypothesized that the increased bioavailability of iron via intravenous supplementation would accelerate the time-course of adaptation; therein creating a model for maximal hematological perturbations within WADA regulations, with potential for significant performance enhancement. Further, we hypothesized that the IV treated group would exhibit greater erythropoietic gains in hemoglobin mass (Hb mass) following altitude exposure, resulting in greater increases in maximal aerobic power (VO2max). Hb mass and ABP hematological parameters were measured during both the “ON” and “OFF phase” of altitude training. The sensitivity and specificity of the current ABP algorithms during these time periods was then assessed using the ABP profiles created for each athlete. We hypothesized that the expected greater enhancements in the IV treated group would result in red flags in the ABP profiles of these athletes.
Methods: The combined impact of altitude and iron supplementation on the ABP was evaluated in endurance-trained athletes (n=34) undertaking 3-weeks of simulated live-high: train-low (14 h.d-1,3000m). Athletes received either oral (ferrous sulphate), intravenous (IV, ferric carboxymaltose) or placebo iron supplementation, commencing two weeks prior and continuing throughout hypoxic exposure. Hb mass, ABP parameters and markers of iron regulation were assessed at baseline (day−14), immediately prior to (day 0), weekly during (days 8, 15), and immediately, 1, 3 and 6 weeks after the completion of altitude exposure (days 22, 28, 42 and 63). VO2max was assessed in the week prior to and week post altitude exposure. Individual ABP thresholds for hemoglobin concentration ([Hb]), reticulocyte percentage (%retic), and OFF score were calculated using the adaptive model and assessed at 99% and 99.9% specificity. Finally, a newly developed plasma volume (PV) blood test was applied to assess the possible efficacy of reducing the influence of PV contractions on the volumetric ABP markers; hemoglobin concentration ([Hb]) and the OFF-score. Serum was analyzed for Transferrin, Albumin, Calcium, Creatinine, Total Protein, and Low-Density Lipoprotein. The PV blood test (consisting of the serum markers, [Hb] and platelets) was then applied to the ABP adaptive model and new reference predictions were calculated for [Hb] and the OFF-score, thereby reducing the PV variance component.
Results: Hb mass significantly increased following altitude in IV (Mean%, [90% CI]: 3.7%, [2.8, 4.7]) and oral (3.2%, [2.2, 4.2]), and remained elevated at 7 days post-altitude in oral (2.9%, [1.5, 4.3]) and 21 days post in IV (3.0%, [1.5, 4.6]). Hb mass was not significantly higher than baseline at any timepoint in placebo. Eleven athletes returned values outside of the calculated reference ranges at 99%, with 8 at 99.9%. The percentage of athletes exceeding the thresholds in each group was similar, but IV returned the most individual occurrences. A similar frequency of abnormalities occurred across the three biomarkers, with abnormal [Hb] and OFF score values arising mainly during-, and %retic values mainly post-altitude. Removing samples collected during altitude from the model resulted in ten athletes returning abnormal values at 99% specificity, two of whom had not triggered the model previously. The PV correction refined the ABP reference predictions. The number of atypical passport findings (ATPFs) for [Hb] was reduced from 7 ATPFs of 5 subjects to 6 of 3 subjects. The OFF-score ATPFs frequency increased with the PV correction (from 9 to 13, 99% specificity); most likely the result of more specific reference limit predictions combined with the altitude-induced increase in red cell production. Importantly, all abnormal biomarker values were flagged identified by a low confidence value by the PV model’s weighting function.
Conclusions: Iron supplementation appears necessary for optimal erythropoietic adaptation to altitude exposure. However, intravenous iron supplementation during three weeks of simulated LHTL altitude training offered no additional benefit in terms of the magnitude of the erythropoietic response for non-anemic endurance athletes compared to oral supplementation. The abnormalities observed in the ABP, in response to iron supplementation and hypoxia, were not systematic and mostly in line with expected physiological adaptations. They do not represent a uniform weakness in the ABP. Nevertheless, altitude training and iron supplementation should be carefully considered by experts evaluating abnormal ABP profiles. Finally, although the multifaceted, individual physiological response to altitude confounded some results, the PV model appears capable of reducing the impact of PV fluctuations on [Hb].
Principal Investigator:
Dr. Nikolai Nordsborg; University of Copenhagen
Research Summary:
The primary aim of this project was to evaluate whether a non-targeted metabolomics analysis is capable of detecting the use of autologous blood transfusion in either blood or urine samples.
In a randomized, double-blinded, placebo-controlled cross over design (3 month wash-out), twelve trained male subjects donated 900 ml blood or were sham phlebotomized followed by a transfusion of the stored RBCs four weeks later. Urine and plasma samples were collected 2 h, 1, 2, 3, 5 and 10days after transfusion and analyzed by an ultraperformance liquid chromatography system coupled to a quadrupole time-of-flight mass spectrometer.
Models of unique metabolites reflecting autologous blood transfusion was derived by partial least squares regression discriminant analysis. For plasma samples, the numerically lowest misclassification rate (29.2 %) and highest specificity (73.8 %) was observed 2 h post transfusion. The remaining time points had a misclassification error of ~30-65% and a specificity of ~35-70%. For the urine samples, the numerically lowest misclassification rate (6.3 %) and highest specificity (98.8 %) was observed 2 h post transfusion. For the remaining time points, the misclassification rate ranged from~20-50 % while the specificity ranged from~35-80 %.
No plasma metabolites included in the developed models were able to clearly discriminate between transfusion and placebo samples at the specifically investigated time point. Thus, no metabolites were identified within the plasma samples. For the urine samples we identified five DEHP metabolites associated with autologous blood transfusion, specifically glucuronide conjugates of MEHP, 5OH-MEHP, 5cx-MEPP, 2cx-MMHP and 5oxo-MEHP. The metabolites were all included in the developed separation model 2 h following transfusion, while only the latter three and two metabolites were present in the models developed for 1 day and 2 days following reinfusion, respectively.
The present study demonstrate that plasticizers of urine is the strongest metabolite for detection of autologous blood transfusion and anti-doping authorities should further consider to use it as a biomarker. Furthermore, the study demonstrates that an autologous blood transfusion does not appear to cause a metabolic trace in plasma useful for detection. A future aim is to investigate the metabolome of the red blood cells following autologous transfusion to evaluate whether the cells provide a sensitive biomarker.
Principal Investigator:
Dr. Paul Armishaw; National Measurement Institute
Research Summary:
ACRM has been prepared providing a suite of steroids certified for their stable carbon isotope ratios. This material has been designed to assist anti-doping laboratories to validate their calibration method for stable carbon isotope measurements and to ensure accuracy and traceability in compliance with WADA Technical Document TD2016IRMS. The CRM has been packaged as a set of three ampoules (MX018-1, MX018-2 and MX018-3) containing, between them, thirteen steroids in dried form with certified carbon isotope ratios (δ13CVPDB-LSVEC) ranging from-13 ‰ to-32 ‰. The composition of steroids in each ampoule has been optimized for separation on two GC capillary columns (DB17ms and VF5ms).
EA-IRMS was employed as the primary reference method to assign δ-value for pure steroid starting materials due to the low uncertainty associated with this technique. Calibration was performed using at wo-point normalization approach2 which employed two calibration standards IAEA-CH-6 and IAEA-CH-7, permitting traceability to the internationally recognized carbon isotope reference standards VPDB and LSVEC. A second reference method was developed using the GC-C-IRMS to facilitate separation of any potential impurities from the steroid mixtures for δ-value evaluation after ampoule preparation. The target steroids were evaluated using two GC capillary columns (DB17ms and VF5ms) to reveal any potential bias in theδ-values obtained between the two reference techniques. Calibration and normalization of the GC-C-IRMS results were performed using eight steroid standards certified in-house by EA-IRMS with traceability to the reference scale, VPDB-LSVEC. The results from both the reference techniques were comparable and an estimate of the maximum potential bias due to undetected impurities was incorporated in the final measurement uncertainty budget. A homogeneity assessment was carried out using ten randomly selected ampoules and the stability of the CRM was verified by analysis of randomly selected ampoules after storage at +4oC and +40oC for period up to 6 months.
Principal Investigator:
Dr. Stephen Davies; National Measurement Institute
Research Summary:
In consultation with WADA Laboratory Directors, NMI Australia (NMIA) has developed a list of 43 new target analytes of interest to the sports doping control community. The majority of compounds listed were long term steroid metabolites and beta blockers. During a two year program NMIA successfully synthesized 15 target compounds and assessed the purity using the mass balance approach and/or quantitative nuclear magnetic resonance spectroscopy. This approach to purity determination is in line with world best practice. The synthesis and purity assignment of each compound has been reviewed by a committee of Australian academic experts in synthesis and chemical metrology to ensure the quality of the prepared certified reference materials. All WADA accredited laboratories have been provided with a suitable quantity (e.g. 1 mg) of each certified reference material produced, accompanied with a certificate of analysis. To date (7th June 2018) the project is underspent to the value of US$26,088. Work will continue on three projects detailed below during the month of June and possibly longer to complete these projects.
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Principal Investigator:
Dr. Lore Geldof; University of Ghent
Principal Investigator:
Dr. Chris Harrison; San Diego State University
Principal Investigator:
Dr. Christian Reichel; Seibersdorf Labor GmbH Doping Control Laboratory
Research Summary:
Erythropoietin (EPO) is a hormone, which stimulates the production of red blood cells. Due to its performance enhancing effect, it is prohibited by the World Anti-Doping Agency (WADA). In order to reduce the detection window of EPO-doping, athletes have been applying low doses of recombinant EPO (e.g. < 10 IU/kg body weight, daily or every second day) instead of larger doses twice or more per week (e.g. 30 IU/kg).
Microdoses of Retacrit (epoetin zeta), an EPO biosimilar, were administered intravenously and subcutaneously to human males and females. Urine and serum samples were collected and analysed applying the new biotinylated clone AE7A5 EPO-antibody and a further optimized SAR-PAGE protocol. With the improved protocol, microdosed Retacrit (7.5 IU/kg BW) was detectable for at least 52 hours after intravenous administration. Detection windows were approximately the same for serum and urine and doubled after subcutaneous administration (ca.104 hours). Previous studies applying different electrophoretic techniques and the not further optimized SAR-PAGE protocol revealed considerably shorter detection windows for rhEPO-microdoses. Since the new biotinylated antibody performed significantly more sensitive than the non-biotinylated version, the new protocol will improve the sensitivity and hence detectability of recombinant EPO in doping control.
Principal Investigator:
Dr. Richard Yost; University of Florida
Research Summary:
Drift tube ion mobility (IM) is shown to have significant potential for improving the separation of isomeric steroid species that are challenging to resolve using current analysis methods. Ion mobility was investigated for the rapid separation of 16 groups of isomeric endogenous and exogenous anabolic androgenic steroids. All analyzed compounds are shown in Table 1. While ion mobility was not able to separate protonated steroid adducts formed by APCI, nearly baseline resolution was observed for the sodiated dimer complexes of many isomeric steroids formed by ESI. To improve the mobility separation of unresolved isomeric steroids, other cations were investigated, including group I, group II, and transition metals. These IM separation strategies were then incorporated with current LC-MS methods for steroid analysis. LC-IM-MS successfully separated 16 steroids, which compose 6 isomeric groups, in under 6minutes. Furthermore, alternative cations have been successfully integrated with the LC method, improving the separation of some isomeric steroids. Continued work is planned to optimize these LC-IMS-MS methods and to evaluate them for urine analysis.
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Principal Investigator:
Dr. Nicolas Voelcker; Monash University, Australia
2014 Grants
Principal Investigator:
Dr. Jack Henion; Q2 Solutions
Research Summary:
Bioanalytical procedures to detect compounds relevant in anti-doping control (represented by opioids, beta blockers, stimulants, cannabinoids and anabolic agents) in Dried Blood Spots (DBS) were developed. A fully automated approach was realized by on-line coupling of flow-through desorption of a DBS card, sample clean-up by solid-phase extraction (SPE) and detection by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).
When applied to opioids and stimulants (five model compounds for each class), this DBS-SPE-LC-MS/MS workflow provided reliable quantification of sub-therapeutic, therapeutic and toxic levels within five to ten minute analysis times after placing a DBS card into the card rack of the Spark Holland system. The fully validated methods were successfully applied to real-life samples obtained directly from finger pricks following oral administration of selected drugs. The finger prick sample collection was done either via a pipette or an easy-to-operate microfluidic-based sampling device. This device allows practical, yet volumetric collection of bloodspots, enabling whole spot analysis instead of partial spot analysis, and thus overcoming possible inconsistencies within spots (e.g. caused by a different hematocrit values). Both sampling approaches accentuate the major benefit of the DBS technique: minimally invasive sample collection that can be performed by inexperienced individuals. Our data confirmed increased stability in the dried matrix compared to liquid blood, but also demonstrated that this aspect is compound-dependent and that, contrary to popular belief, certain DBS shipment/storage requirements such as cooling may be required.
While the fully automated integrated approach reduces manual workload and possible manipulation errors, certain limitations for multi-analyte assays were observed. Due to the on-line nature, changing one parameter affects several processes at the same time, restricting the available options for each step of the DBS-SPE-LC-MS/MS process. Detection of five representative beta blockers simultaneously was achieved. However, this was not accomplished within pre-set accuracy criteria that were easily met for opioids and stimulants, as the chemical characteristics of the studied beta blockers turned out to be too diverse for coverage by one single set of DBS-SPE-LC-MS/MS parameters.
The data obtained for the detection in of opioids, stimulants and beta blockers in DBS demonstrate that modern state-of-the art analytical techniques such as LC-MS/MS are capable of dealing with sensitivity and selectivity challenges posed by the micro sampling of finger prick blood and the DBS matrix complexity. However, due to below-average MS ionization behavior and signal suppression by co-eluting interferences (that could not be reduced efficiently due to limited on-line sample clean-up options), the DBS-SPE-LC-MS/MS approach in its current approach could not provide the sensitivity required to measure the lowest relevant blood levels of cannabinoids and some anabolic agents.
Principal Investigator:
Dr. Kara Lynch; University of California San Francisco
Principal Investigator:
Dr. Benjamin Levine; UT Southwestern Medical Center
Research Summary:
In late 2012, the scientific community became aware that Russian athletes were allegedly breathing xenon, a known anesthetic agent, for short periods of time to increase their athletic performance. At that time, there was no credible information about what dosages or inhalation times were being used. Moreover, as no experiments had been performed in humans, it was not clear if this procedure was even effective. Thus, our group took a broad approach to document the safety, detection and efficacy of xenon inhalation. In the context of clean competition, the take home messages were that while short exposures to xenon does not produce anesthesia, sedation is striking with individuals being unable to operate the breathing apparatus needed to deliver xenon. Moreover, with multiple dosages of xenon per week, a number of participants complained of severe sleep psychosis. Detecting acute xenon doping is quite difficult if athletes use an open circuit breathing apparatus, with a high probability for detection only possible for up to ~3 hours after dosing in blood and urine samples. A single dose of xenon did have a small erythropoietic effect (elevated erythropoietin concentrations). If xenon was given every day for one week, plasma volume was expanded, but not red cell mass. However, given the tremendous expense of medical grade xenon, we doubt many athletes would consider or be able to afford this doping regime for longer periods of time to try an increase red cell mass. Thus, we gave participants xenon 3 days per week for one month and saw no changes in erythropoietin, plasma volume, red cell mass, exercise capacity (maximal oxygen consumption) or exercise performance (3k time trial). Ultimately using xenon to try and improve athletic performance carries a high level of risk without a trained anesthesiologist, yet xenon given every day did increase plasma volume and thus may improve performance independent of oxygen carrying capacity. In contrast, a more practical dosing regimen with an interval between dosing days seems to have no beneficial effect on any aspect of exercise capacity or athletic performance.
Principal Investigators:
Sports Medicine Research and Testing Laboratory (SMRTL) and UCLA Olympic Analytical Laboratory WADA Accredited Labs
Principal Investigator:
Dr. Mike Ashenden; Science and Industry Against Blood doping (SIAB)
Research Summary:
Among the many substances that can be used for doping in sport, pharmaceuticals used for the treatment of anaemias, such as recombinant human erythropoietin (EPO), are prevalent because they increase oxygen transport to the muscles and thus athletic performance. Historically, athletes have gone to great lengths to disguise their use of EPOs. For example, soon after the advent of are liable test by Lasne in 2000, athletes dabbled with adding proteases to denature any EPO present in urine controls. This progressed to what athletes misunderstood to be ‘undetectable’ second-generation Aranesp during the era of the 2002 Salt Lake City Olympics, then later Dynepo because athletes believed it was indistinguishable from natural EPO. Eventually third-generation products like CERA were adopted because athletes mistakenly believed its molecular structure was too large to be filtered by the kidney and thus invisible to urine controls.
The contemporary athlete’s tactic to evade detection seems to be ‘microdosing’. This entails reducing the dosage but increasing the frequency of EPO injections–preferably taken in the evening when anti-doping authorities are prohibited from collecting samples. The paradigm under pinning this tactic is that drug traces will be eliminated before the window for doping control re-opens at 6AM. Similarly, drinking a large volume of water is thought to increase the rate of elimination and furthermore dilute the amount of EPO in urine, thus rendering identification of recombinant EPOs more difficult.
Our aim was to gather evidence to indicate how best to identify the presence of EPO assuming these contemporary masking strategies. Our research focused on microdosing and water intake to dilute the substance, and our findings indicate that only the former can meaningfully subvert EPO controls. Yet the timing of tests is crucial. Whilst we found no evidence to support that itis necessary to collect samples during the night, a collection in the early morning enhances the likelihood of detecting reportable levels of rapidly cleared drugs like EPO. Specifically, provided samples are collected as early as possible in the morning, current anti-doping methodologies can be 100% successful in detecting drug remnants from a microdose injection taken during the previous evening. On the other hand, if testing is delayed until the afternoon then it may well prove a futile exercise, because depending on the type of sample collected as few as 8% of samples had reportable levels of EPO by 5PM.
In terms of whether anti-doping authorities should focus on the collection of blood or urine samples if they are targeting an athlete suspected of microdosing, our findings lend strong support to the collection of urine. Urine had equal or better sensitivity to plasma in the mornings, but superior sensitivity at both noon and 5PM.
Another important finding was that drinking large volumes of water did not mask the presence of EPO. Our findings indicate that the methods employed in anti-doping laboratories to detect EPO are robust in the face of dilutional strategies.
Principal Investigator:
Dr. Richard Holt; University of Southampton
Research Summary:
Insulin-like growth factor-I (IGF-I) has anabolic and metabolic effects that make it attractive as a performance-enhancing drug, either in addition to or instead of growth hormone (GH). As GH misuse detection methods have developed, athletes may have turned to IGF-I as a mediator of the effects of GH. IGF-I is included in the WADA List of Prohibited Substances because of its potential to improve performance and the risks of harm to the athlete if misused.
Recombinant human IGF-I (rhIGF-I) is structurally identical to endogenous IGF-I and so the main challenge in detecting rhIGF-I misuse is to distinguish exogenous from endogenous IGF-I. The GH-2000 and GH-2004 research teams have developed a method to detect GH misuse based on changes in GH-sensitive serum peptides (GH-2000 biomarker method). We have investigated the detection of IGF-I misuse using similar principles and reported that serum IGF-I increases in recreational athletes after administration of rhIGF-I/rhIGF binding protein-3 (rhIGFBP-3) complex for 28 days. The aim of this project was to assess whether measuring other GH-sensitive markers could improve the sensitivity and specificity of a test for detecting IGF-I misuse.
We had stored serum samples from 26 female and 30 male recreational athletes who had been randomised to 28 days’ treatment with rhIGF-I/rhIGFBP-3 complex or placebo, followed by 56 days’ washout. The following serum peptides were measured in these samples using commercial immunoassays: IGF-II, IGFBP-2, IGFBP-3, ALS, osteocalcin, PICP and ICTP.
Serum IGFBP-2 increased and serum IGF-II decreased significantly in response to rhIGF-I/rhIGFBP-3administrationin both women and men. We combined these markers with serum IGF-I to create four “IGF scores” that could discriminate between athletes who had administered rhIGF-I/rhIGFBP-3 and those in the placebo group. We then measured these markers in serum samples from 250 elite athletes and calculated their IGF scores. We have proposed decision limits for each of these scores to determine whether an elite athlete is misusing rhIGF-I/rhIGFBP-3.
In order to implement a test to detect IGF-I misuse in anti-doping laboratories, it will be necessary to decide which combination of serum markers is optimal and which analytical assays should be used. These markers must then be measured in a larger population of elite athletes to improve there liability of the score decision limits
Principal Investigator:
Dr. David Chen; University of British Columbia
Research Summary:
We used the pilot grant to explore the feasibility of using capillary electrophoresis coupled to mass spectrometry to for the characterization of glycans in EPO. Even though it took us longer than expected due to delays in signing the contract and looking for qualified personnel we are starting to see significant progress in reaching the proposed goals for differentiating the glycans of different origin, in order to distinguish between EPO produced in human vs. those produced through genetic engineering.
We have recently published a paper entitled “Improved Sensitivity by Post‐Column Chemical Environment Modification of CE‐ESI‐MS Using a Flow-Through Microvial Interface” in the journal Electrophoresis. This paper is a direct result of this PCC Pilot Project. We have just obtained the university approval to study human EPOs from plasma samples, and more results will be forth coming in the near future.
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Research Summary:
Athletes can enhance performance by increasing oxygen delivery to working muscles. This may be achieved through several forms of blood doping including abuse of recombinant erythropoietin, erythropoiesis stimulating agents, and allogeneic/autologous blood transfusions. These blood doping practices are often difficult or impossible to detect by direct methods. However, most forms of blood doping can be detected using the Athlete Biological Passport (ABP) program. The ABP uses an indirect method which monitors longitudinal measurements of hemoglobin concentration [Hb], reticulocyte% (RET%) and the OFF-score, defined as ([Hb]-60√RET%). Due to the success of this method, athletes have adopted increasingly complex blood doping strategies to evade detection. Measurement of additional markers of erythropoiesis and iron homeostasis may increase the sensitivity of the test. One promising biomarker, hepcidin, is a 25 amino acid peptide secreted from the liver which is a key regulator of iron metabolism. It is primarily regulated by body iron stores, circulating iron, and erythropoiesis. In Specific Aim 1, a method was developed and validated to measure hepcidin in human plasma by mass spectrometry. The method was developed in a 96-wellplate format to allow high throughput screening of samples. In Specific Aim 2, plasma hepcidin concentrations were measured in 181 athlete samples. The mean hepcidin concentration was 7.3 nM with a range from 0.6–27.0 nM. The large inter-individual variation in hepcidin concentrations suggests that establishment of individual thresholds, rather than population-based, is important. In Specific Aim 3, hepcidin concentrations were measured in plasma samples from an autologous transfusion study of 34 healthy volunteers. Additional markers, including serum iron, ferritin, soluble transferrin receptor (sTFR), and erythroferrone were measured in the same samples to determine their utility for the detection of blood transfusion. In contrast to previous reports, serum iron did not demonstrate a significant change after blood transfusion. However, hepcidin, ferritin, and sTFR changed significantly. Intra-individual baseline variability was largest for hepcidin. Hepcidin may be most useful for prediction of when additional testing by alternative methods is needed and as supporting data. Ferritin and sTFR values were stable and could be useful markers if incorporated into a new statistical model for detection of blood doping.
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Principal Investigator:
Dr. Peter Van Eenoo; Doping Control Laboratory, University of Ghent
Research Summary:
In 2012 REV-ERB agonists SR9009 and SR9011 were described by the SCRIPPS Research institute as promising drug candidates to treat several metabolic disorders [1-3]. Because of the enhancing effects of these REV-ERB agonists on exercise endurance in animal models their fraudulent use as doping agent can be anticipated.
Although no pharmaceutical preparations are available yet, illicit use of SR9009 and SR9011 for doping purposes can be anticipated, especially since, SR9009 is marketed in illicit products. Moreover, soon these compounds were discussed as ‘exercise in a pill’ compounds [4-6]. Therefore the aim of this project was to perform preventive anti-doping research to ensure effective (doping) control for SR9009 and SR9011. The main objectives included the development of an LC-(HR) MS method and the identification of potential diagnostic metabolites via in vitro metabolism studies.
The presence of SR9009 was demonstrated in a black market product obtained via the Internet. This finding highlights the real threat for the misuse of these potentially performance-enhancing substances. In addition reference material for both SR9009 and SR9011 was obtained. A LC-(HR)MS/MS method was developed and our routine screening method was validated for the detection of SR9009 and SR9011. Via human liver microsomal metabolic assays eight metabolites were detected for SR9009 and fourteen metabolites for SR9011 by LC-HRMS. Structure elucidation was performed for all metabolites by LC-HRMS product ion scans in both positive and negative ionization mode.
Furthermore, a retrospective data analysis was performed to evaluate the incidence of SR9009, SR9011 and their metabolites in 1511 doping control samples previously analyzed in our laboratory. SR9009 and SR9011 were also included in our routine screening method for monitoring. So far, the presence of neither the parent compound nor the metabolites could be detected in routine urine samples. However, to further discourage use of these potential harmful compounds incorporation of SR9009 and SR9011 into screening methods is highly recommended. To allow other doping control laboratories to implement SR9009 into their initial testing procedures SR9009 was distributed to all WADA laboratories.
Principal Investigator:
Dr. David Cowan; King’s College London Drug Control Centre
Principal Investigator:
Dr. Mario Thevis; German Sport University Cologne
Research Summary:
The family of growth hormone releasing hormones is prohibited in sport due to performance enhancing properties, attributed to enhanced endogenous growth hormone production and/or secretion. Most prominent candidates include the single-chain peptidic drugs/drug candidates Geref (Sermorelin), Tesamorelin, CJC-1295 and CJC-1293. Effective doping control analysis is featured by sensitive determination of the most reliable target metabolites in the respective biological fluid (urine or blood). Thus, the knowledge about the metabolism of a prohibited substance is crucial. In this study, detailed analytical information about the target peptides and their potential metabolites after in vitro-and in-vivo experiments (administration to rats) and literature review was obtained. Most promising analyte candidates for doping control purposes were synthesized and characterised by mass spectrometry. Several sample preparation strategies were evaluated using solid phase extraction, protein precipitation, ultrafiltration prior to immunoaffinity purification (with magnetic beads or MSIA) and liquid chromatographic-mass spectrometric detection. All developed methods showed sufficient sensitivity to cover the estimated concentrations after administration, and the implementation into existing procedures for other prohibited peptides (insulins, synacthen, etc.) is enabled. The procedures were validated for qualitative result interpretation and considered “fit for purpose” for doping control analysis.
Principal Investigator:
Dr. Pauline Rudd; NIBRT
Research Summary:
In 2011, funding was initially received from the PCC to evaluate “Structural differences as a biomarker of EPO use”, and this enabled N-glycan profiling of 15 biosimilar recombinant α erythropoietin’s using a 2-AB label. Limited amounts of sample coupled with the low sensitivity of LC-MS analysis of 2-AB labelled glycans unfortunately meant we were unable to perform orthogonal structural analysis. We proposed to utilise procainamide (PA), a label that has higher fluorescence intensity and ionisation efficiency to analyse 9 biosimilars from the original project and also the human urinary(hu)EPO standard by Liquid Chromatography Mass Spectrometry (LC-MS). Preliminary experiments using PA labelling of Eprex and analysis by HILIC UPLC and LC-MS demonstrated that 100 μg quantities was needed to identify glycan variants of interest such as acetylated variants or N-glycolyneuraminic acid containing variants. Due to the low remaining quantity (5 μg) of biosimilar and the inability to source more, we were unable to characterise them using LC-MS. This was also the case for huEPO. The huEPO standard is a crude urinary preparation containing many other glycoproteins. We purified EPO from this preparation using MAIIA diagnostics anti-EPO affinity purification kit. The standard is estimated to contain 80 ng of EPO and due to the sensitivity of PA it was not possible to characterise this. SDS-PAGE analysis of the biosimilars revealed the presence of a number of contaminant proteins. Extraction of these gel bands followed by trypsin digestion and analysis by LC-MS identified a number of human serum proteins that are present in the final formulations. It is proposed that these contaminants are introduced by the use of human serum albumin in the formulation. We collated all of our findings from both projects and created a manuscript entitled “Challenges and potential of using glycan analysis for the detection of doping with recombinant erythropoietin biosimilars” to Drug Testing and Analysis in September 2016. Unfortunately this was found to be not suitable for publication in its current form in November 2016.
2013 Grants
Detecting bio-molecules by nanotechnology & Raman spectroscopy: Rapid Screening kit for EPO in urine
Principal Investigator:
Dr. Emad Kiriakous; Queensland University
Research Summary:
The purpose of this project was to deliver a proof of concept for the detection of bio-active compounds in biological samples by means of functionalized nanoparticles and surface-enhanced Raman spectroscopy. The new method has been demonstrated for Erythropoietin (EPO)as a prototype molecule. The developed method is applicable to the detection of many proteins and peptides such as insulin, human growth hormone (somatotropin) and insulin-like growth factors (IGFs).
Recombinant human erythropoietin (rHuEPO) is widely used in endurance sports as a doping agent. Current methods for EPO detection are lengthy, laborious and relatively insensitive to low concentrations of the drug. In this research we developed a rapid method for the selective and rapid extraction of rHuEPO followed by the subsequent screening of the protein by surface enhanced Raman spectroscopy. For these purposes, we aimed to develop gold nanoparticles that are functionalized rHuEPO-specific antibodies and used them as a nano-sensor to selectively bind with EPO in solution within 30 minutes. The nanoparticles bearing the protein are then removed from solution and submitted to screening by surface enhanced Raman spectroscopy (SERS). Since the nanoparticles have a gold surface, they act as signal amplifier device to enhance the Raman signal intensity by many orders of magnitude and, therefore they enabled the detection of ultra-trace amounts of rHuEPO down to of 3.5×10-13 molar (1.4 mIU/ml) detection limit. The method demonstrated in this project report detects EPO by monitoring the conformational changes caused by the binding between the antibody and the protein. The significant changes in the Raman spectrum of the antibody-functionalized nano-sensor before and after binding with rHuEPO are used to identify the presences of rHuEPO in a sample. The developed EPO detection kit can be used in combination with handheld Raman device for the rapid in-field screening of EPO doping. The detailed synthesis of the nanoparticles and their subsequent functionalization with rHuEPO antibodies followed by their use to detect rHuEPO are presented in this report. The outcomes of the research have been disseminated in a research article and a master’s thesis. Also a new article has been communicated to Nano research journal (currently under review) and another two articles are being drafted.
Another significant outcome of this project is the discovery of a new selective and very sensitive approach for the rapid detection and quantification of proteins in field by aptamer-functionalized disposable strips and combined handheld SERS/electrochemistry. The new approach will allow for the detection of the protein/hormone own finger print directly on disposable strips as well as the precise their quantification in field. The new approach will significantly reduce the analytical cycle at the lab by ruling out negative samples from being sent for complete analysis. The discovered approach will be presented to PCC on a new project application towards the development and optimization of SERS disposable stripes for the direct finger printing and quantification of ultra-trace amounts of proteins in field.
Principal Investigator:
Dr. Ruprecht Keller; Cologne Central Hospital Lab.
Research Summary:
A new method that can be used to alleviate the negative impact of a supervised urination procedure and which can also identify urine as coming from a specific athlete is the urine marker. Monodisperse low molecular weight polyethylene glycols are given orally prior to urination. Urine samples can be traced to the donor by analysis of the polyethylene glycols previously given. The work supported by Partnership for Clean Competition presents three studies investigating the use of the urine marker during urine doping controls. It investigates athletes’ acceptance of this new method via two questionnaire studies (n total =253). Furthermore, a third study (n=91) investigates whether ingestion of the marker can identify the urine as coming from a specific person and whether the maker impacts the doping analytic procedure. The results indicate that this new method finds wide acceptance both from athletes that have only read about the procedure and those that have actually tested the new procedure. Furthermore, the marker can identify urine as coming from a specific person and thereby makes supervised urination obsolete. The marker does not impact the doping analysis.
Principal Investigator:
Dr. Rodrigo Aguilera
Research Summary:
Isotope Ratio Mass Spectrometry (IRMS) analysis of endogenous and exogenous androgens urinary steroids has been used for several years to detect doping abuse in sports. The GC/C/IRMS technique and the use of δ13C values obtained are now recognized as the gold standard in differentiating exogenous from endogenous steroids in urine.
A significant variation in the13C/12C ratio of a steroid represents an undeniable and unambiguous evidence of the use of pharmaceutical (synthetic) steroid(s). Nonetheless, the potentially high precision of these measurements from the GC/C/IRMS does not always lead to highly accurate results. The potential inaccuracy depends upon several factors: 1) The quality and efficiency of sample extraction, 2) Analysis of free or underivatized steroids, and 3) Instrumentation-chromatographic separation, and related issues such as insufficient sensitivity to detect small changes in δ13C values, small amounts of steroids present in urine samples, kinetic and/or enzymatic fractionation occurring in sample preparation.
Several different approaches have been reported from different research groups regarding the measurement of a) underivatized vs. derivatized steroids, and b) androsterone & etiocholanolone vs. androstanediols. Even within the same methodology large ambiguities remain regarding the baseline of δ13C values used in establishing reference ranges. Several hypotheses have arisen concerning different baseline populations, which include effects of diet, biological and physiological variation.
In order to establish or confirm these discrepancies, urines were analyzed from several populations. Urine samples were collected from different countries and analyses were performed using one method on one instrument. The data was classified according to ethnicity, sex, and age. Consequently, any variations will not be dependent to the method, steroids assayed and instrument. Through some previous work, it was established that there were two distinct subgroups of males showing variability in the natural13C/12C ratios of steroids.
Principal Investigator:
Dr. Anthony Butch; UCLA Olympic Analytical Laboratory
Research Summary:
This project is designed to develop a rapid electrochemical biosensor detection system to quantify the 22 kDa and 22 kDa isoforms of human growth hormone (hGH) in whole blood samples. Previous work with electrochemical biosensors has demonstrated that the technique is sensitive down to femtogram concentrations, and is a rapid (~30 minutes) and robust method for detection of biomarkers in biofluids such as serum and saliva. The ultimate goal of the project is the quantification of hGH isoforms in either dried blood or dried serum/plasma samples that have been spotted onto filter paper. In this pilot study project, experiments were designed to determine if growth hormone isoforms could be detectable in whole blood samples at concentrations comparable to those achieved using the commercially available hGH isoform test.
For this project we initially utilized a 16 array gold electrode chip to develop an electrochemical method to detect the 22 kDa isoform of hGH in whole blood samples. Unfortunately, we found unacceptably high variability both within and between assays using the 16 array gold electrode chip. This prompted us to switch to a96-well gold electrode system about 6 months into the project. Using the 96-well electrochemical sensor format we were able to produce an assay that was linear (R2=0.985) and could detect the 22 kDa isoform of hGH with an analytical sensitivity around 50 pg/mL. Using antibodies specific for the 20 kDa isoform of hGH a second electrochemical sensor assay was configured that was linear (R2= 0.982) with an analytical sensitivity around 38 pg/mL.
These findings indicate that the electrochemical sensor technology can be used to detect hGH isoforms in whole blood samples. Additional studies are needed to further improve the lower limit of detection for both the 22 kDa and 20 kDa isoforms of hGH in order to develop a viable test to detect doping with hGH. A further extension of this project will be to utilize the electrochemical sensor to detect hGH isoforms in either dried whole blood or serum/plasma samples with adequate analytical sensitivity for doping control purposes. This approach has the potential to significantly reduce the shipping costs associated with the transportation of whole blood samples to laboratories at refrigerated temperatures.
Principal Investigator:
Dr. David Cowan; King’s College London Drug Control Centre
Principal Investigator:
Dr. Tom Brenna; Cornell University
Research Summary:
High precision carbon isotope ratio analysis of urinary steroids by gas chromatography combustion isotope ratio mass spectrometry (GCC-IRMS) is a recognized test to detect illicit doping of the endogenous steroid testosterone; however, there are limited amounts of universal steroid isotopic standards (SIS) available for instrument calibration. Our group created some of the first SIS, CU/USADA 33-1, 34-1 & 34-2, and 35-1, that contain mixtures of 3 to 4 steroids as acetates or free steroids at ~400g each per ampoule. These have been widely distributed to colleagues at anti-doping laboratories for inter-laboratory comparison. Here we created and characterized new carbon stable isotopic standards. This includes a new mixture, coded CU/PCC 44-1 that contains ~400g each per ampoule of androsterone-AC, 5A-diAC, 5-cholestane, and 5P-diAC. These range in isotopic values from -32.73‰ to -21.16‰. We also created and characterized three new high volume single steroid SIS coded CU/PCC 40-1, CU/PCC 41-1, & CU/PCC 42-1 that contain ~20 mg of androsterone, androsterone-AC, and5-cholestane, respectively. These range in isotopic valuesfrom-25.03to-32.82‰. In addition, we made and characterized additional batches of previous SIS mixtures CU/USADA 33-1, and CU/USADA 34-1/34-2, now coded CU/PCC 33-2 and CU/PCC 34-3. For all these SIS, we used our previously developed protocol to calibrate the isotopically uniform steroids against the NIST isotopic standard natural gases methane and ethane in NIST RM 8559 that is traceable to the international standard Vienna Pee Dee Belemnite (VPDB).
Principal Investigator:
Dr. David Cowan; King’s College London Drug Control Centre
Principal Investigator:
Dr. Detlaf Thieme; IDAS Dresden
Principal Investigator:
Dr. Christoph Borchers; University of Victoria
Research Summary:
Testosterone doping is currently determined by steroid profiling; however, this approach may produce false negatives when steroid profiles are only marginally affected. Therefore, we proposed an alternative method based on the hypothesis that doping with testosterone produces detectable and systemic changes in an athlete’s blood proteome. Our assay was developed using dried blood spot (DBS) sampling to provide a minimally invasive approach for sample collection and multiple reaction monitoring mass spectrometry (MRM‐MS) for highly specific protein quantitation. The final assay quantified a total of 97 proteins (represented by 169 peptides) that spanned almost 5 orders of magnitude ‐‐ from serum albumin (P02768) at 18.0mg/mL down to cholinesterase (P06276) at 190ng/mL. The average intra‐assay and inter‐assay precision for 6 biological samples were 6.1‐7.5% CV and 9.5‐11.0% CV, respectively. The majority of the concentrations of the peptide targets were stable after 154 days at storage temperatures from -20°C to 37°C. This assay represents the highest degree of multiplexing yet achieved for targeted protein quantification in DBS samples.
We then applied this novel DBS‐MRM assay to a 26‐day clinical study investigating testosterone doping. This study was performed under the direction of Dr. Mario Thevis (German Sport University, Cologne, Germany). Four volunteers donated DBS samples before, during, and after controlled testosterone administration (25 mg/day, transdermal gel). Our results indicated that 15 proteins showed a measurable change in abundance based on a difference of one standard deviation or more. We have observed 3 main patterns based on the rate of change in the concentrations of these 15 proteins and their recoveries to their original values: 1) proteins that resume their original concentrations by the end of the study, 2) those showing the onset of recovery, and 3) those for which there was no noticeable start of a recovery to their original values (probably due to the short duration of the study (26 days). Although the low number of participating individuals (n=4) limited the statistical significance of the results, the results show a very clear indication of detectible changes in the concentrations of these 15 proteins that can be determined using DBS and LC‐MS/MS.
2012 Grants
Principal Investigator:
Dr. James Watkins; Norwich Ventures
Principal Investigator:
Dr. Frank Suhr; German Sport University Cologne
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Research Summary:
Current venous blood collection and expensive shipment methods for the hematological module of the athlete biological passport (ABP) program have several disadvantages that limit its utility. The current project will improve this method by moving to a less invasive, finger-prick blood collection that can easily be performed by a doping control officer. Secondly, the finger-prick blood will be spotted on to dried blood spot (DBS) cards which will improve sample stability and decrease the cost of shipping. The new method will significantly increase the frequency of un-announced blood testing that can be performed and allow blood collection in any location or venue, thus improving detection and deterrence. The goal of this project was to develop methods that would allow conversion of the current method to a DBS method. The current ABP method measures hemoglobin, reticulocytes and red blood cells in venous blood using an automated hematology analyzer. The DBS method will measure hemoglobin using a simple colorimetric method and will count blood cell types by measuring cell-specific proteins by mass spectrometry. In the first Specific Aim, proteins were screened for their correlation with blood cell counts measured on the Sysmex. Using this method one cell-specific protein was identified for each cell type: RBC, WBC, platelets, reticulocytes, and immature reticulocytes. Conditions for extraction and digestion of the DBS spot, and LC-MS/MS conditions were optimized. This data was used to make a mass spectrometry method for all of the cell types and hemoglobin in a single assay which can be performed on a single DBS spot. In Specific Aim 2 and 3, this method was further tested on several batches of human samples and a partial method validation was completed. The data obtained demonstrate that the proposed method, to accurately count cell types in dried blood spots, is possible and this is the first reported method to demonstrate the concept. Good correlation and method performance was observed for measurement of hemoglobin, RBC and WBC. Preliminary data suggest accurate measurement of immature reticulocytes and platelets is possible and future work will continue to test and validate this method. Since a protein with good correlation to total reticulocytes was not yet identified, the new DBS method may initially replace measurement of total reticulocytes with measurement of immature reticulocytes. Additional work is need to identify reticulocyte-specific proteins that are amenable to the DBS method.
Principal Investigator:
Dr. Mike Ashenden; Science and Industry Against Blood doping (SIAB) Australia
Principal Investigator:
Dr. Jenny Schulze; Karolinska Institutet
Research Summary:
The endocrine basis for employment of the same doping tests in women as in men has not been validated in relation to the large endocrine differences between sexes. This project aimed to study- the urinary steroid profiles in relation to hyperandrogenism, menstrual disturbances and polycystic ovaries, i.e. polycystic ovary syndrome (PCOS), hormonal contraceptive use and genetic variation in female populations. The scientific report is divided in three studies:
Study 1) The use of Hormonal Contraceptives in Female Athletes
Study 2) Emergency Oral Contraceptives, Menstrual Cycle and Urinary Steroid Profile in Female Volunteers.
Study 3)The Urinary Steroid Profile in women diagnosed with PCOS
Taken all the results together it seems that, of all urinary androgens, epitestosterone glucuronide excretion is most affected by both hormonal contraceptives (significantly lower), the menstrual cycle (highest during the end of the menstrual cycle) and PCOS (significantly higher). As epitestosterone is one of the major biomarker in the biological passport this is important information when interpreting longitudinal steroid data.
The genetic polymorphisms that were previously shown to significantly affect the male androgen disposition have a less pronounced effect in women. For example, it was previously clearly shown that the CYP17 polymorphism was associated with urinary epitestosterone concentration and T/E ratio in males, however, in females the CYP17 polymorphism only affected the urinary epitestosterone concentration in the group that used hormonal contraceptives. This is most likely due to the menstrual cycle dependent epitestosterone excretion, which is non-existent in the hormonal contraceptive users. This variation is larger than the polymorphic-dependent variation.
The 5-alpha reduced steroids were significantly increased in women with a PCOS diagnosis, which affected the A/Etio and the 5a/5b diol ratios in the ABP. The urinary testosterone concentration was also increased, although not to the same extent as the urinary epitestosterone concentration. The PCOS women did not have significantly different T/E ratios compared to the control group. The control group was not matched for BMI and age to the PCOS group and the results will be re-evaluated when we have finished the sample collection from the matched control group.
The emergency contraceptives did not affect the steroid profile to any significant extent. As continuous use of oral doses of HC seem to be necessary to decrease the urinary concentrations of androgens could mean that other mechanisms rather than a direct effect on HPA axis may be involved.
In all, we believe that the publications resulting from this study will be beneficial to everyone evaluating steroid profiles in athletes. There is an urgent need of published data of androgen disposition and urinary steroid profiles in women that may be consulted when evaluating deviating longitudinal profiles from female athletes.
Principal Investigator:
Dr. Philip Smith; University of North Carolina at Chapel Hill
Research Summary:
The urinary testosterone/epitestosterone (T/E or T/EpiT) ratio is a primary assay for monitoring illicit use of testosterone (T) in sports. UGT2B17 is the major enzyme for T glucuronidation and it is thought that in its absence, or in subjects with low expression, the value obtained in a urinary T/E test would be low, thus avoiding the detection of T abuse. A UGT2B17 polymorphism is known to occur. The main UGT2B isoform thought to be involved in epitestosterone (EpiT) glucuronidation is UGT2B7. In this study we developed an assay to assess the in vitro glucuronidation rates (catalysis) of T and EpiT in recombinant UGT enzyme preparations and in human liver microsomes (HLMs). We used the method to test a series of recombinant isoforms, including UGTs 2B17 and 2B7, and then a library of 60 HLM samples in which we had previously quantified UGT content (14 isoforms) using a targeted quantitative proteomic nanoUPLC-MS/MS method developed in our lab. In the recombinant samples we found only UGT2B17 to glucuronidate T and only UGT2B7 to glucuronidate EpiT. Treatment of results/data focused on evaluating the relationship, in the HLMs, between the T and EpiT glucuronidation rates and the UGTs 2B17 and 2B7 content. The rate of EpiT-glucuronide (EpiT-G) catalysis was greater than that of T-glucuronide (T-G) catalysis in all of the HLMs. T-G catalysis was found to be highly correlated with UGT2B17 content in the samples(Spearman, r = 0.89, n = 60). T-G/EpiT-G ratios were also found to correlate highly with UGT2B17 content (r = 0.85). For those samples containing low or no UGT2B17 there was little correlation between T-G catalysis and UGT2B17 content (r = 0.32, n= 19) and no correlation between T-G/EpiT-G ratios and UGT2B17 content (r =-0.06). For the remaining samples (n = 41), containing appreciable amounts of UGT2B17, the corresponding correlations were 0.88 and 0.84. For EpiT-G catalysis vs. UGT2B7 content the correlation values determined were 0.59 (all HLMs, n = 60), 0.89 (samples containing low or no UGT2B17 content, n= 19) and 0.47 (samples containing high UGT2B17content, n = 41). This is the first report relating T and EpiT glucuronidation to UGT2B17 and UGT2B7 content in HLMs. Previously used techniques have included, for example, genotyping. UGT2B17 influences the T-G/EpiT-G ratio in vitro, as shown by the Spearman rank correlation testing. Thus in the absence of UGT2B17, as occurs with a well-known (*2/*2) polymorphism, the urinary T/E ratio will decrease. Since absence or low expression of UGT2B17 results in no correlation with the T-G/EpiT-G ratio, this ratio, whether in vitro or in a urine sample, would likely not identify T abuse by subjects who have the polymorphism.
2011 Grants
Principal Investigator:
Dr. Barbara Daniel; King’s College London
Research Summary:
Introduction
Blood transfusion is prohibited by WADA. Although the detection of homologous transfusion is possible, to date, no method for the detection of autologous blood transfusion has been recognized. The practice of autologous blood transfusion entails the removal of several units of blood from the athlete’s own body. The blood is treated to isolate the red blood cells(RBCs), which are then stored ready to be re-introduced into the athlete prior to a race/event. This practice will boost the oxygen carrying capacity of blood thereby enhancing performance. The athlete needs to have time to recover from the initial blood loss. This means the separated red cells have to be stored for several weeks prior to their re-transfusion. Sometimes stored blood can cause adverse reactions in recipients and hematologists have largely investigated the storage-dependent changes (collectively known as ‘storage lesion’) affecting blood preparations. By comparing fresh and stored blood they found differences in the level of specific proteins with some showing an increase in concentration. Red blood cells do not contain DNA, but recent studies haves hown the presence of RNA (molecules that direct proteins synthesis in cells). It is therefore possible that differences in the protein content are reflected in the RNA profile. These translational differences may be exacerbated by storage triggering other processes that affect the relative levels of RNA molecules.
Hypothesis
In this project we have evaluated the possibility to detect autologous blood transfusion by looking at changes of RNA species occurring during storage. We have been able to test our hypothesis by taking advantage of powerful high-throughput analysis techniques (i.e. microarrays) as well as validating the results through independent experimental verification(i.e. real-time polymerase chain reaction, RT-PCR).
Results
The results of our study showed that RBC preparations contain both messenger RNA (mRNA) and microRNA (miRNA) and a reliable method for their co-extraction has been developed and tested. Microarray analysis revealed significant changes to the RBC transcriptome following four weeks storage. A list of transcripts (miRNA sand RNAs) susceptible to storage has been created and an insight into the nature of changes detected between fresh and stored RBC transcriptomes has been gained. A sub-set of candidate markers has been further validated through RT-PCR (performed on 10 donors). Analysis of the data confirmed the presence of storage susceptible markers (a total of 7) which were able to distinguish between fresh and stored RBCs and could be integrated in a test for autologous blood transfusion detection.
Principal Investigator:
Dr. Richard Snyder; University of Florida
Principal Investigator:
Dr. Andy Hoofnagle; University of Washington
Principal Investigator:
Dr. Marijke Grau; German Sport University Cologne
Research Summary:
Autologous blood doping is undetectable with the current methods but, regarding the current debate, its detection is more important than ever. Thus, new techniques to detect autologous blood doping are needed. During storage, red blood cells (RBC) become more susceptible to hemolysis and it has been shown that RBC deformability, which is influenced by RBC nitric oxide synthase (RBC-NOS) activity, decreases during blood storage. Therefore, the recent study aimed to determine RBC deformability, RBC parameters (RBC count, hemoglobin concentration, hematocrit, mean cellular volume), free hemoglobin concentration, L-arginine concentration, RBC-NOS activation and HbNO concentration in blood from 15 male volunteers. Basal anthropometric parameters of the subjects were as follows (mean ± SD): age [years]: 25.1 ± 2.38, height [m]: 1.84 ± 0.05, weight [kg]: 78.1 ± 6.24, body mass index [kg/m2]: 23.02 ± 1.67. Blood was sampled, anticoagulated using Citrate-Phosphate -Dextrose-Adenine (CPDA) solution (Sigma-Aldrich, Germany), leuko-reduced and stored as either packed RBC (hematocrit: 69.44 ± 1.81%) or RBC diluted in Saline-Adenine-Glucose-Mannitol (SAGM) (hematocrit :45.11 ± 0.37%) for eight weeks at 4°C. Aliquots were drawn on days 0, 3, 7, 10, 21, 28, 35, 42, 49 and 56 to measure the abovementioned parameters. Additionally, fresh blood was drawn at each investigation day from the respective volunteers to measure the described parameters. Data from stored samples were compared to data obtained from the freshly drawn blood. Finally, stored and freshly drawn blood were mixed to obtain 5%, 10%, 20% and 50% stored blood in fresh blood. RBC deformability and a RBC parameter were measured in these mixtures.
Results revealed decreasing RBC deformability during blood storage with packed RBC showing a higher decrease compared to RBC diluted in SAGM. The decrease in deformability was related to increased mean cellular volume and increased cell lysis but also to a decrease in RBC-NOS activation. The changes were more pronounced in concentrated RBC than in RBC diluted with SAGM suggesting that the storage method affects the quality of blood. The concentration of the RBC-NOS substrate, L-arginine, was not the limiting factor because the concentration remined unaltered during storage. HbNO concentration decreased in both storage conditions and it turned out that this parameters is not suitable to complement RBC deformability measurements.
Reduced RBC deformability was also detectable in mixtures containing both stored and fresh blood. A minimum of 10% stored packed RBC and 50% stored RBC+SAGM were required to detect differences to fresh blood. The same applies to the RBC parameters measured. Mixtures containing stored packed RBC showed altered RBC parameters compared to fresh blood while those parameters remained almost unaltered in stored RBC+SAGM.
In conclusion, RBC deformability is a very stable parameter show less than 5% day-to-day variation. Blood storage impairs RBC deformability due to impairments of the NO signaling pathway. A reduced deformability is observed in stored blood but also in blood samples containing 10% stored packed RBC and 90% fresh blood. The data thus indicated that RBC deformability represents a promising marker to detect autologous blood doping. Further studies are now needed to test how other storage conditions affect RBC deformability.
Principal Investigator:
Dr. Donald Siegel; University of Pennsylvania
Research Summary:
Blood doping by athletes through surreptitious transfusion of either autologous or allogeneic red blood cells (RBCs) not only provides an unfair endurance enhancement, but also poses potentially serious short-and long-term health risks to the individuals participating in these illicit programs. Developing analytical tools for detecting this activity will serve to deter some athletes who are contemplating transfusion as well as to identify those athletes who have succumbed to their own temptation and/or to manipulation by others. Identification of the latter group of athletes would not only disqualify them from participation in sport, but allow one to better understand the full extent of the problem and the effects of a dangerous transfusion practice that appears to be carried out without proper pre-transfusion testing, blood storage, blood administration, and monitoring of recipients for potential post-transfusion adverse effects. The goal of the proposed research was to test the hypothesis that the expression of “neo-antigens” which develop on the surface of stored RBCs can be exploited to develop immunological reagents that can identify the presence of allogeneic or autologous transfused blood in an athlete. Initially, we employed an approach that was completely “agnostic” in that no assumptions were made regarding the molecular nature of the neoantigen(s) (e.g., altered membrane phospholipid, carbohydrate or protein composition, etc.). Our methods used antibody phage display to create recombinant antibody libraries from the immune repertoires of laboratory animals (two chickens, a cynomolgus macaque, and a llama)- immunized with stored human RBCs. Isolation of antibodies specific to stored RBCs were sought by using simultaneous positive/negative cell surface panning–positively selecting on stored RBCs and negatively selecting on fresh RBCs from the same blood donor. After carrying out these selections with two different chicken scFv antibody fragment libraries, a macaque Fab antibody fragment library, and a llama single-domain nanobody library, the polyclonal libraries as well as numerous individual monoclonal antibodies were assayed for their ability to differentiate stored vs. fresh RBCs. Unfortunately, though RBC-specific antibodies were obtained from all libraries, none of them differentiated fresh from stored RBCs. In an alternative experimental approach, antibody libraries were first selected on N(ε)-(carboxymethyl)lysine (CML), a major antigenic advanced glycation end-product known to form on the surface of RBCs as cells age. Libraries enriched in anti-CML antibodies were then subjected to the positive/negative RBC selection process utilized initially. Though numerous anti-CML RBC-binding antibodies were obtained, none were able to distinguish between fresh and stored RBCs. As described below, we hope to achieve our original goals in future studies that will employ screening a non-immune/synthetic phage display library that does not depend on the immunogenicity in animals of stored vs. fresh human RBCs, nor is it subject to potential immune tolerance self-editing mechanisms that would remove desired specificities.
Principal Investigator:
Dr. Thomas Piper; Deutsche Sporthochschule Koln
Research Summary:
Carbon isotope ratio (CIR) analysis has been routinely and successfully used in sports drug testing for many years to uncover the misuse of endogenous steroids. One limitation of the method is the availability of steroid preparations exhibiting CIRs equal to endogenous steroids. To overcome this problem, hydrogen isotope ratios (HIR) of endogenous urinary steroids were investigated as a potential complement; results obtained from a reference population of 67 individuals are presented herein. An established sample preparation method was modified and improved to enable separate measurements of each analyte of interest where possible. From the fraction of glucuronidated steroids; pregnanediol, 16-androstenol, 11-ketoetiocholanolone,androsterone (A), etiocholanolone (E), dehydroepiandrosterone (D), 5α-and 5β-androstanediol, testosterone and epitestosterone were included. In addition, sulfate conjugates of A, E, D, epiandrosterone and 17α-and 17β androstenediol were considered and analyzed after acidic solvolysis. The obtained results enabled the calculation of the first reference-population-based thresholds for HIR of urinary steroids that can readily be applied to routine doping control samples. Proof-of-concept was accomplished by investigating urine specimens collected after a single oral application of testosterone-undecanoate. The HIR of most testosterone metabolites were found to be significantly influenced by the exogenous steroid beyond the established threshold values.
Additionally, one regular doping control sample with an extraordinary testosterone/epitestosterone ratio of 100 without suspicious CIR was subjected to the complementary methodology of HIR analysis. The HIR data eventually provided evidence for the exogenous origin of urinary testosterone metabolites. Despite further investigations on HIR being advisable to corroborate the presented reference-population-based thresholds, the developed method proved to be a new tool supporting modern sports drug testing procedures.
The HIR of body water and, therefore, of all endogenously synthesized compounds in humans, is mainly affected by the HIR of ingested drinking water. As a consequence, the entire organism and all of its synthesized substrates will reflect alterations in the isotope ratio of drinking water, which depends on the duration of exposure. To investigate the effect of this change on endogenous urinary steroids relevant to doping-control analysis the hydrogen isotope composition of potable water was suddenly enriched from-50 to 200 ‰ and maintained at this level for two weeks for two individuals. The steroids under investigation were pregnandiol, 16-androstenol, A, E, 5α-and 5β-androstanediol (excreted as glucuronides) and E, A and D (excreted as sulfates). The HIR of body water was estimated by determination of the HIR of total native urine, to trace the induced changes. The hydrogen in steroids is partly derived from the total amount of body water and cholesterol-enrichment could be calculated by use of these data. Although the sum of changes in the isotopic composition of body water was 150 ‰, shifts of approximately 30 ‰ were observed for urinary steroids. Parallel enrichment in their HIR was observed for most of the steroids, and none of the differences between the HIR of individual steroids was elevated beyond recently established thresholds. This finding is important to sports drug testing because it supports the intended use of this novel and complementary methodology even in cases where athletes have drunk water of different HIR, a plausible and, presumably, inevitable scenario while traveling.
2010 Grants
Principal Investigator:
Dr. Jason Dragoo; Leland Stanford Junior University
Principal Investigator:
Dr. David Bruns; University of Virginia
Research Summary:
Growth hormone is likely one of the most abused substances in athletic competitions. It has been shown that recombinant human growth hormone (rhGH) use increases lipolytic activity and has anabolic properties, thus being an attractive substance for athletes looking for an unfair advantage over their competition.
When the human body is treated with hormones, changes in gene expression may occur. One such change reflects the ability of the body to sense the administered hormone and turn off its synthesis within the body. The hypothesis of the initially proposed research is that if an individual is administered rhGH or substances that mimic GH, the levels of endogenous GH messenger RNA (mRNA) that is needed to make more GH will fall.
By designing experiments to detect levels of GH mRNA in bodily fluids, we will be able to determine if athletes have been exposed to rhGH or its mimics. It has been shown previously that testing for the primary doping substance, rhGH, may prove very challenging in part due to the short half-life of rhGH in circulation. However, by testing for the body’s response to a stimulus, we may be able to identify and thus limit usage of not only rhGH but also GH-boosting substances.
We have developed a sensitive and specific quantitative PCR-based method to detect GH mRNA in blood. We have collected samples from many individuals with pituitary disorders that either produce too much growth hormone or necessitate growth hormone replacement therapy. We have collected samples over the period of nearly one year from a single individual to observe any intra-individual variability with time.
We believe this initial research has achieved the pilot-project objective; we have developed an assay that is capable of quantifying circulating GH mRNA in all obtained samples. The assay can now be applied to samples collected prior and subsequent to starting rhGH therapy and this can be pursued with continued support.
Principal Investigator:
Dr. Pauline Rudd; National Institute for Bioprocessing Research and Training
Principal Investigator:
Dr. Andrew Kicman; King’s College London
Principal Investigator:
Dr. Richard Holt; University of Southampton
Principal Investigator:
Dr. Monica Mazzarino; Labortio anti doping FMSI
Research Summary:
The project formally started on June 1st 2011. This final report provides an overview on the experimental activities carried out in the twelve-month period of the project. The specific objectives of the project were defined as follows:
A. Development of synthesis methods for the preparation of toremifene metabolites as reference substances.
B. In vitro metabolic profile of toremifene, as assessed in the presence of selected CYP enzyme.
C. Development and validation of LC-MS/(MS) methods according to ISO 17025 requirements.
All the objectives above were addressed in the twelve months project. More specifically:
Objective “A”: an effective synthesis method has been designed and developed to obtain toremifene metabolites to be used as reference compounds in anti-doping analysis. The synthesis method is based on the same strategy already developed for the synthesis of tamoxifen metabolites. Specifically, two of the most representative toremifene metabolites were synthesized (carboxy-tamoxifen, originally indicated as“M5”or “metabolite A” and N-demethyl-carboxy-tamoxifen originally indicated as “M3”or “metabolite B”).
Objective “B”: the “in vitro” study of toremifene metabolism with human liver microsomes (HML) and with expressed cytochrome P450s (CYP3A4/5, CYP2D6, CYP2C8/9/19) and UDP-glucuronosyltransferases (UGT1A4, UGT2B7, UGT2B17,UGT2B15, UGT1A2) was carried out. The results of the study were compared with those obtained “in vivo”, as assessed by urinary excretion studies (five males and five females) following oral administration of a single dose of toremifene.
Objective “C”: an LC-MS/MS method for the analysis of toremifene metabolites, identified following both “in vitro” and “in vivo” studies, was developed. The method was validated according to ISO 17025 and to the WADA requirements for the accredited laboratories (as detailed in the WADA International Standard for Laboratories and related technical documents) and does not require last-generation instrumental set up and could be implemented also by any other WADA-accredited anti-doping laboratory.
Principal Investigator:
Dr. Daniel Eichner; Sports Medicine Research and Testing Laboratory (SMRTL)
Principal Investigator:
Dr. Nicolas Leuenberger; Swiss Laboratory for Anti-Doping Analyses
Research Summary:
It is well documented that several molecular changes occur in stored red blood cells, referred to as the “storage lesion”. Moreover, it has been shown by mRNA microarray profiling that autologous transfusion triggers immune reaction within the T-lymphocytes of the recipient. However, due to its low stability and its sensitivity to RNases, utilization of mRNA as biomarkers in blood doping detection is contested.
In this project, we hypothesized that circulating microRNAs could be very specific and sensitive for detection of autologous transfusion. Previously, circulating miRNAs have been found to be very stable and protected to RNase and harsh conditions. Indeed, this high stability precludes circulating miRNA as having an important interest in doping control.
MiRNAs have been demonstrated to play a key role in apoptosis and immune reaction. Thus, miRNAs could be promising biomarkers to detect these “molecular signatures” involved in autologous transfusion, where premature apoptosis of newly infused red cells has been shown to occur.
To this end, we analyzed miRNA in plasma by miRNAs by quantitative real-time PCR (qRT-PCR). This high-throughput profiling method has allowed to us to investigate the quantity of all microRNAs inhuman.
To investigate the role of miRNAs in autologous transfusion we collaborated with the Prof. Y.O. Schumacher and Dr. T. Pottgiesser, experts and authors of many publications on different aspects of autologous transfusion and blood manipulation in sport.
Their group provided us with blood samples obtained during several published longitudinal clinical studies, involving autologous transfusion in different settings in healthy individuals but also controlled no n-transfused volunteers.
Genomic data analyses was performed by Dr. S. Pradervand from the Genomic Technology Facility of the University of Lausanne (GTF), Switzerland. Dr. S. Pradervand is Head of the bioinformatics unit in GTF and is an expert in large genomic data analysis and interpretation.
Principal Investigator:
Dr. Svetlana Appolonova; Moscow Anti-Doping Laboratory
Principal Investigator:
Dr. Andrew Phillips; Yale University
2009 Grants
Principal Investigator:
Dr. Fred Schaufele; UC San Francisco
Principal Investigator:
Dr. Peter Van Eenoo; University of Ghent Doping Control Laboratory
Research Summary:
In this project a chimeric mouse (uPA+/+-SCID mouse with a humanized liver) was used as a model system to test anabolic steroid preparations which are inappropriate for human studies, and for which other animal models are inadequate.
This was a unique project that had a great deal of applicability to anti-doping efforts. Ethical considerations often prevent giving black-market anabolic steroids to human volunteers. By utilizing chimeric mice to perform excretion studies, the time needed to detect designer anabolic steroids could be markedly reduced. Preliminary data with this model showed the potential of this model as a good alternative for human excretion studies and to increase the efficiency of detecting AAS.
In the framework of doping control, accredited labs must be able to detect steroids in human urine. However the urinary detection of (designer) steroids is not only based on the parent but often on metabolites formed in the human body. Therefore the performance of excretion studies with healthy human volunteers is a necessary tool to be able to predict what could be expected in an athlete’s urine after intake of steroids. However experiments with (designer) steroids are difficult in healthy human volunteers, since ethical constraints hamper the execution of such studies.
Therefore the chimeric uPA+/+-SCID mouse, developed by CEVAC (center for vaccinology, Ghent University), could be applied to assist in optimizing screening methods since the same matrix (urine) is used. In this mouse model, human liver cells are transplanted and have developed to partially replace the mouse liver and could therefore support the identification of metabolites of those steroids that can be used for detection in human urine. By administration of several steroid compounds to the chimeric mouse model the metabolic profile could be investigated with GC or LC techniques coupled to mass spectrometric detection. This work may facilitate the synthesis of reference materials for the testing laboratories and provide guidance for development of methods of detection.
Principal Investigator:
Dr. Michael Collins; National Measurement Institute, Australia
Research Summary:
There is concern amongst the anti-doping fraternity that custom synthesis of testosterone to produce carbon-13 enriched substrates may circumvent the current Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) test. Further to the reported studies of legitimate pharmaceutical grade testosterone products and border-level seizures within Australia, a detailed analysis of illicit materials obtained from different regions around the world was required to obtain intelligence concerning trends in 13C value of “black market” testosterone.
Testosterone was liberated from 163 sample esters supplied by laboratories in Germany (n=70), Switzerland (n=33), United States of America (n=29) and Australia (n=31) using acid hydrolysis followed by liquid-liquid extraction at pH 9.8 into hexane before GC-C-IRMS analysis to determine the 13C value (μ = -28.5 ‰,σ= 1.8 ‰, range =−23.4 ‰ to−32.9 ‰). Most important for doping control, 9 testosterone preparations (5.5 %) were confirmed to have 13C values within the reference intervals (13C =−17.3 ‰ to−25.8 ‰) reported for endogenous urinary androgen metabolites.
Concentration of the testosterone extracts (5:1) enabled GC-Thermal Conversion (TC)-IRMS analysis to determine the 2H value of testosterone contained in the preparations. However due to circumstances beyond the control of the Australian Sports Drug Testing Laboratory, access to a suitable IRMS instrument was terminated during the course of the project and a complete data set was unable to be obtained. Therefore the preliminary 2H results obtained have been omitted from this final report. It was envisaged that this information could have been used incurrent and future studies validating the use of 2H measurements for urinary steroid metabolites to enable source discrimination.
Principal Investigator:
Dr. Diana Wilkins; University of Utah
Longitudinal evaluation of urinary endogenous steroid concentrations in the NFL drug testing program
Principal Investigator:
Dr. Doug Rollins; University of Utah
Principal Investigator:
Dr. Anthony Butch; UCLA Olympic Analytical Laboratory
Principal Investigator:
Dr. Vanessa Agon; National Measurement Institute, Australia
Research Summary:
Glycoprotein hormones and their recombinant counterparts are abused by athletes seeking to gain a competitive edge in sports. The predicament that anti-doping laboratories face is that these hormones are naturally produced in the body and the distinction against their recombinant analogues is not possible using conventional immunoassays. However, most peptide hormones undergo post translational modification, i.e. glycosylation, and there are significant differences in the nature of the attached carbohydrates between natural and recombinant hormones as shown by previous studies. As recombinant proteins are produced from technology using non-human cells, the resulting glycosylation profile will lead to differences in isoform distribution. Therefore, this project aims to analyze the differences between recombinant and natural hormones through derivatization of their respective carbohydrates.
Experiments were performed using commercially available glycan standards to evaluate labelling techniques using a variety of chemical derivatives. Experiments were also performed on N-glycans following deglycosylation of glycoprotein hormones such as human Chorionic Gonadotropin (hCG). The labelled carbohydrates were analyzed by LC/MS/MS and compared with unlabeled glycans and glycan standards to reveal structural information on the sugar groups as well as isoform composition
Principal Investigator:
Dr. Diana G. Wilkins; University of Utah
Research Summary:
In this study, we originally proposed to determine the viability of oral fluid as a matrix for measuring endogenous steroid concentrations and to compare it to established urine protocols for determining the endogenous steroid profile. We hypothesized that oral fluid represents a viable, non-invasive biological matrix to measure and monitor the endogenous steroid profile in elite athletes. To test this hypothesis, three specific aims (objectives) were proposed as described below:
Specific Aim One: Develop and validate analytical testing methods for relevant endogenous steroids in oral fluid. Aim-Specific Hypothesis: Methods for the analysis of endogenous steroids relevant to anti-doping can be developed and validated using oral fluid as the biological matrix.
Specific Aim Two: Compare endogenous steroid concentrations in urine and oral fluid from human volunteers. Aim-Specific Hypothesis: An endogenous steroid profile can be established in oral fluid.
Specific Aim Three: Compare endogenous steroid concentrations in urine and oral fluid from human volunteers administered a low dosage of testosterone. Aim-Specific Hypothesis: Low dose administration of testosterone to human volunteers will be detected by changes in the concentrations of endogenous steroids excreted in oral fluid (i.e. oral fluid steroid profile).
2008 Grants
Principal Investigator:
Dr. Tom Brenna; Cornell University