Ultra-fast retroactive processing by MetAlign of liquid chromatography/high-resolution full-scan Orbitrap mass spectrometry data in WADA Human Urine Sample Monitoring Program.

RATIONALE: The World Antidoping Agency (WADA) Monitoring program concentrates analytical data from the WADA Accredited Laboratories for substances which are not prohibited but whose potential misuse must be known. The WADA List of Monitoring substances is updated annually, where substances may be removed, introduced or transferred to the Prohibited List, depending on the prevalence of their use. Retroactive processing of old sample datafiles has the potential to create information for the prevalence of use of candidate substances for the Monitoring List in previous years. MetAlign is a freeware software with functionality to reduce the size of liquid chromatography (LC)/high-resolution (HR) full-scan (FS) mass spectrometry (MS) datafiles and to perform a fast search for the presence of substances in thousands of reduced datafiles. METHODS: Validation was performed to the search procedure of MetAlign applied to Anti-Doping Lab Qatar (ADLQ)-screened LC/HR-FS-MS reduced datafiles originated from antidoping samples for tramadol (TRA), ecdysterone (ECDY) and the ECDY metabolite 14-desoxyecdysterone (DESECDY) of the WADA Monitoring List. Searching parameters were related to combinations of accurate masses and retention times (RTs). RESULTS: MetAlign search validation criteria were based on the creation of correct identifications, false positives (FPs) and false negatives (FNs). The search for TRA in 7410 ADLQ routine LC/HR-FS-MS datafiles from the years 2017 to 2020 revealed no false identification (FPs and FNs) compared with the ADLQ WADA reports. ECDY and DESECDY were detected by MetAlign search in approximately 5% of the same cohort of antidoping samples. CONCLUSIONS: MetAlign is a powerful tool for the fast retroactive processing of old reduced datafiles collected in screening by LC/HR-FS-MS to reveal the prevalence of use of antidoping substances. The current study proposed the validation scheme of the MetAlign search procedure, to be implemented per individual substance in the WADA Monitoring program, for the elimination of FNs and FPs.

Hyperhydration using different hydration agents does not affect the haematological markers of the athlete biological passport in euhydrated volunteers.

Athlete Biological Passport (ABP) is an indirect approach, implemented by WADA, aimed at detecting blood manipulation based on abnormal changes in haematological markers. Cases report the use of hyperhydration as masking method during anti-doping urine sample collection which could potentially mask suspicious fluctuations on ABP profiles. This study investigated the hyperhydration effect on haemoglobin concentration, reticulocyte percentage and OFF-hr score (an algorithm based on haemoglobin concentration and reticulocyte percentage), with and without recombinant human erythropoietin (rHuEPO) administration. A five-week clinical study performed; Baseline and rHuEPO Phase. Water and a sports drink were used as hyperhydration agents. To examine the hyperhydration effect on the normal ABP profile per volunteer, hyperhydration was implemented at 0, 24 and 48 hours during the baseline. During the rHuEPO phase, volunteers received Epoetin beta (3000 IU) with hyperhydration to be implemented at 0, 24 and 48 hours after drug administration. Blood and urine samples were collected and analysed according to WADA guidelines. No significant effect on ABP markers was observed due to hyperhydration at any time during the study. Pre- and post-hyperhydration data were not statistically different compared to individual baseline data. In conclusion, hyperhydration does not affect the ABP haematological markers under the examined conditions.

Ultra-fast retroactive processing of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data in anti-doping screening of human urine.

RATIONALE: Retroactive analysis of previously tested urine samples has become an important sports anti-doping tool. Retroactive reprocessing of old data files acquired from a generic screening procedure can reveal detection of initially unknown substances, like illegal drugs and newly identified metabolites. METHODS: To be able to efficiently search through hundreds to thousands of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data files of anti-doping samples, a combination of MetAlign and HR_MS_Search software has been developed. MetAlign reduced the data size ca 100-fold making possible local storage of a massive volume of data. RESULTS: The newly developed HR_MS_Search module can search through the reduced data files for new compounds (mass or isotope pattern) defined by mass windows and retention time windows. A search for 33 analytes in 940 reduced data files lasted 10 s. The output of the automatic search was compared to the standard manual routine evaluation. The results of searching were evaluated in terms of false negatives and false positives. The newly banned b2-agonist higenamine and its metabolite coclaurine were successfully searched in reduced data files originating from a testing period for which these substances were not banned, as an example of retroactive analysis. CONCLUSIONS: The freeware MetAlign software and its automatic searching module HR_MS_Search facilitated the retroactive reprocessing of reduced full-scan high-resolution liquid chromatography/mass spectrometry screening data files and created a new tool in anti-doping laboratories' network.

Effect of hyperhydration on the pharmacokinetics and detection of orally administered budesonide in doping control analysis.

The aim of the present study was to investigate if hyperhydration could influence the excretion and subsequent detection of budesonide (BDS) and its main metabolites (6ß-hydroxy-budesonide and 16α-hydroxy-prednisolone) during doping control analysis by leading to concentrations below the WADA reporting level (30 ng/mL). The influence of hyperhydration on the plasma and urinary pharmacokinetic (PK) profiles of BDS and metabolites was also examined. Seven healthy physically active non-smoking Caucasian males participated in a 15-day clinical study. BDS was administered orally at a single dose of 9 mg on Days 1, 7, and 13. Hyperhydration was applied in the morning on two consecutive days, that is, 0 and 24 hours after first fluid ingestion. Water and a commercial sports drink were used as hyperhydration agents (20 mL/kg body weight). Results showed no significant difference (P > 0.05, 95% CI) on plasma or urinary PK parameters under hyperhydration conditions for all the analytes. However, significant differences (P < 0.05, 95% CI) due to hyperhydration were observed on the urinary concentrations of BDS and metabolites. To compensate the dilution effect due to hyperhydration, different adjustment methods were applied based on specific gravity, urinary flow rate, and creatinine. All the applied methods were able to adjust the concentration values close to the baseline ones for each analyte; however, specific gravity was the optimum method in terms of effectiveness and practicability. Furthermore, no masking of the detection sensitivity of BDS or its metabolites was observed due to hyperhydration either in plasma or urine samples.

Metabolic profiling of elite athletes with different cardiovascular demand.

Intensive exercise of elite athletes can lead to physiological alterations in the cardiovascular system in response to increased stroke volume and blood pressure, known collectively as cardiovascular demand (CD). This study aimed to compare metabolic differences in elite athletes with high vs low/moderate CD and to reveal their underlying metabolic pathways as potential biomarker signatures for assessing health, performance, and recovery of elite athletes. Metabolic profiling of serum samples from 495 elite athletes from different sport disciplines (118 high CD and 377 low/moderate CD athletes) was conducted using non-targeted metabolomics-based mass spectroscopy combined with ultra-high-performance liquid chromatography. Results show that DAGs containing arachidonic were enriched in high CD together with branched-chain amino acids, plasminogens, phosphatidylcholines, and phosphatidylethanolamines, potentially indicating increased risk of cardiovascular disease in the high CD group. Gamma-glutamyl amino acids and glutathione metabolism were increased in low/moderate CD group, suggesting more efficient oxidative stress scavenging mechanisms than the high CD group. This first most comprehensive metabolic profiling of elite athletes provides an evidence that athletes with different CD show a unique metabolic signature that reflects energy generation and oxidative stress and potentially places the high CD group at a higher risk of cardiovascular disease. Further studies are warranted for confirmation and validation of findings in other sport groups in light of potential confounders related to limited available information about participants.

Hyperhydration-Induced Decrease in Urinary Luteinizing Hormone Concentrations of Male Athletes in Doping Control Analysis.

Low urinary luteinizing hormone (LH) values have been discussed as a marker to detect steroid abuse. However, suppressed LH concentrations related to highly diluted urine samples could be a misleading indication of anabolic steroid abuse. One aim of the present study was to examine the effect of hyperhydration on the interpretation of LH findings during doping control analysis and to investigate different possibilities to correct volume-related changes in urinary LH concentrations. Seven healthy, physically active, nonsmoking White males were examined for a 72-hr period, using water and a commercial sports drink as hyperhydration agents (20 ml/kg body weight). Urine samples were collected and analyzed according to the World Anti-Doping Agency's technical documents. Baseline urinary LH concentrations, expressed as the mean ± SD for each individual, were within the acceptable physiological range (7.11 ± 5.42 IU/L). A comparison of the measured LH values for both hyperhydration phases (Phase A: 4.24 ± 5.60 IU/L and Phase B: 4.74 ± 4.72 IU/L) with the baseline ("normal") values showed significant differences (Phase A: p < .001 and Phase B: p < .001), suggesting the clear effect of urine dilution due to hyperhydration. However, an adjustment of urinary LH concentrations by specific gravity based on a reference value of 1.020 seems to adequately correct the hyperhydration-induced decrease on the LH levels.

Comparison of gas chromatography/quadrupole time-of-flight and quadrupole Orbitrap mass spectrometry in anti-doping analysis: I. Detection of anabolic-androgenic steroids.

RATIONALE: The World Anti-Doping Agency (WADA) encourages drug-testing laboratories to develop screening methods that can detect as many doping substances as possible in urine. The use of full-scan high-resolution acquisition (FS/HR) with gas chromatography/mass spectrometry (GC/MS) for the detection of known and unknown trimethylsilyl (TMS) derivatives of anabolic-androgenic steroids (AAS) provides anti-doping testing bodies with a new analytical tool. METHODS: The AAS were extracted from urine samples by generic liquid-liquid extraction, after enzymatic hydrolysis, and TMS derivatization. The extracted urine was analyzed by GC/Q-TOF and GC/Q-Orbitrap to compare the performance of the two instrument types for the detection of 46 AAS in human urine. The quantitation of endogenous anabolic steroids and the ability of the two analytical platforms to comply with the requirements for testing as part of the WADA Athlete Biological Passport (ABP) were also assessed. RESULTS: The data presented show that the analytical performance for both instruments complies with the WADA specifications. The limits of detection (LODs) for both instruments are well below the WADA 50% Minimum Required Performance Levels. The mass errors in the current study for the GC/Q-Orbitrap platform are lower than those obtained for the GC/Q-TOF instrument. CONCLUSIONS: The data presented herein proved that both molecular profiling platforms can be used for antidoping screening. The mass accuracies are excellent in both instruments; however, the GC/Q-Orbitrap performs better as it provides higher resolution than the GC/Q-TOF platform.

New long-standing metabolites of 17α-methyltestosterone are detected in HepG2 cell in vitro metabolic model and in human urine.

Novel metabolites of the anabolic androgenic steroid 17α-methyltestosterone have been detected in HepG2 cell in vitro metabolic model and in human urine. Their detection was accomplished through targeted gas chromatography-(tandem) mass spectrometry analysis that has been based on microscale synthesized standards. The related synthesis and the gas chromatography-(tandem) mass spectrometry characterization of the analytical standards are described. All newly presented metabolites have a fully reduced steroid A-ring with either an 17,17-dimethyl-18-nor-Δ13 structure or they have been further oxidized at position 16 of the steroid backbone. Metabolites with 17,17-dimethyl-18-nor-Δ13 structure may be considered as side products of phase II metabolic sulfation of the 17ß-hydroxy group of methyltestosterone or its reduced tetrahydro-methyltestosterone metabolites 17α-methyl-5ß-androstane-3α,17ß-diol and 17α-methyl-5α-androstane-3α,17ß-diol that produce the known epimeric 17ß-methyl-5ß-androstane-3α,17α-diol and 17ß-methyl-5α-androstane-3α,17α-diol metabolites. The prospective of these new metabolites to increase detection time windows and improve identification was investigated by applying the World Anti-doping Agency TD2021IDCR criteria. The new metabolites, presented herein, complement the current knowledge on the 17α-methyltestosterone metabolism and in some cases can be used as additional long-term markers in the frame of sport doping drug testing.

The use of RNA-based 5'-aminolevulinate synthase 2 biomarkers in dried blood spots to detect recombinant human erythropoietin microdoses.

The hematological module of the Athlete Biological Passport (ABP) is used for indirect detection of blood manipulations; however, the use of this method to detect doping, such as with microdoses of recombinant human erythropoietin (rhEPO), is problematic. For this reason, the sensitivity of ABP must be enhanced by implementing novel biomarkers. Here, we show that 5'-aminolevulinate synthase 2 (ALAS2) mRNAs are useful transcriptomic biomarkers to improve the indirect detection of rhEPO microdosing. Moreover, the sensitivity was sufficient to distinguish rhEPO administration from exposure to hypoxic conditions. Levels of mRNAs encoding carbonate anhydrase 1 (CA1) and solute carrier family 4 member 1 (SLC4A1) RNA, as well as the linear (L) and linear + circular (LC) forms of ALAS2 mRNA, were monitored for 16 days after rhEPO microdosing and during exposure to hypoxic conditions. ALAS2 mRNAs increased by 300% compared with the baseline values after rhEPO microdosing. Moreover, ALAS2 mRNAs were not significantly increased under hypoxic conditions. By contrast, CA1 mRNA was increased after both rhEPO microdosing and hypoxia, whereas SLC4A1 mRNA did not significantly increase under either condition. Furthermore, the analyses described here were performed using dried blood spots (DBSs), which provide advantages in terms of the sample collection, transport, and storage logistics. This study demonstrates that ALAS2 mRNA levels are sensitive and specific transcriptomic biomarkers for the detection of rhEPO microdosing using the hematological module of the ABP, and this method is compatible with the use of DBSs for anti-doping analyses.

Untargeted Metabolomics Identifies a Novel Panel of Markers for Autologous Blood Transfusion.

Untargeted metabolomics was used to analyze serum and urine samples for biomarkers of autologous blood transfusion (ABT). Red blood cell concentrates from donated blood were stored for 35−36 days prior to reinfusion into the donors. Participants were sampled at different time points post-donation and up to 7 days post-transfusion. Metabolomic profiling was performed using ACQUITY ultra performance liquid chromatography (UPLC), Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The markers of ABT were determined by principal component analysis and metabolites that had p < 0.05 and met ≥ 2-fold change from baseline were selected. A total of 11 serum and eight urinary metabolites, including two urinary plasticizer metabolites, were altered during the study. By the seventh day post-transfusion, the plasticizers had returned to baseline, while changes in nine other metabolites (seven serum and two urinary) remained. Five of these metabolites (serum inosine, guanosine and sphinganine and urinary isocitrate and erythronate) were upregulated, while serum glycourdeoxycholate, S-allylcysteine, 17-alphahydroxypregnenalone 3 and Glutamine conjugate of C6H10O2 (2)* were downregulated. This is the first study to identify a panel of metabolites, from serum and urine, as markers of ABT. Once independently validated, it could be universally adopted to detect ABT.

Stabilization of human urine doping control samples: a current opinion.

Transportation of doping control urine samples from the collection sites to the World Anti-doping Agency (WADA) Accredited Laboratories is conducted under ambient temperatures. When sample delivery is not immediate, microbial contamination of urine, especially in summer, is a common phenomenon that may affect sample integrity and may result in misinterpretation of analytical data. Furthermore, the possibility of intentional contamination of sports samples during collection with proteolytic enzymes, masking the abuse of prohibited proteins such as erythropoietin (EPO) and peptide hormones, is a practice that has already been reported. Consequently, stabilization of urine samples with a suitable method in a way that protects samples' integrity is important. Currently, no stabilization method is applied in the sample collection equipment system in order to prevent degradation of urine compounds. The present work is an overview of a study, funded by WADA, on degradation and stabilization aspects of sports urine samples against the above threats of degradation. Extensive method development resulted in the creation of a mixture of chemical agents for the stabilization of urine. Evaluation of results demonstrated that the stabilization mixture could stabilize endogenous steroids, recombinant EPO, and human chorionic gonadotropin in almost the entire range of the experimental conditions tested.

Olympic anti-doping laboratory: the analytical technological road from 2016 Rio De Janeiro to 2021 Tokyo.

The summer Olympic Games is the major mega sports event since the first modern era Olympiad, held in Athens, Greece in 1896. International Olympic Committee (IOC) has the responsibility of the organization of the summer and winter Games ensuring the broadcast in all corners of earth. The World Anti-Doping Agency (WADA) is the responsible organization of the fight against doping in sports. IOC and WADA support the event's country WADA Accredited Laboratory to incorporate the maximum of the new analytical technologies to become applicable during the event's antidoping testing. The current study reviewed the last 5 years progresses of the antidoping system with emphasis on the laboratory field.

Integrating Whole Blood Transcriptomic Collection Procedures Into the Current Anti-Doping Testing System, Including Long-Term Storage and Re-Testing of Anti-Doping Samples.

Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., ‒80°C) storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes. Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments. Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

Horseradish-peroxidase-conjugated anti-erythropoietin antibodies for direct recombinant human erythropoietin detection: Proof of concept.

Antidoping testing for recombinant human erythropoietin (EPO) is routinely performed by gel electrophoresis followed by western blot analysis with primary and secondary antibodies. The two antibody steps add more than 24 h to the testing time of a purified sample. The aim of this study was to test the concept of using directly horseradish-peroxidase (HRP)-conjugated anti-EPO primary antibody, without the need for a secondary antibody, to reduce the analysis time and eliminate non-specific cross-reactivity with secondary antibodies. An in-house, periodate coupling (R&D systems, clone AE7A5) and three commercially available anti-human EPO-HRP conjugates from Genetex, Novus Biologicals and Santa Cruz were evaluated for specificity and sensitivity, using recombinant human EPO standards, negative human urine samples and urine samples from an EPO excretion study. The in-house anti-EPO-HRP conjugate was performed as well as the current two-step application of unconjugated primary and secondary antibodies used in routine analysis, with comparable specificity and sensitivity. The analysis time was markedly reduced for purified samples from 25 h with the routine method down to 7 h with the in-house HRP conjugate. Of the three commercially available conjugates tested, only the Santa Cruz anti-EPO-HRP conjugate showed comparable specificity but had lower sensitivity to both the in-house and the antibody combination currently applied routinely. The other two commercially available conjugates (Genetex and Novus Biologicals) did not show any visible bands with the EPO standards. The results clearly demonstrate the potential utility of a directly HRP-conjugated anti-EPO antibody to reduce analysis time for EPO in doping control.

Biosafety Level 2 cabinet UV-C light exposure of sports antidoping human urine samples does not affect the stability of selected prohibited substances.

The current study examined the stability of several antidoping prohibited substances analytes in urine after 15-min exposure to UV-C light in a Biosafety Level 2 cabinet. The urine matrices were exposed within the original antidoping bottles with the aim to destroy DNA/RNA and possible SARS CoV-2. The analytes small molecules Phase I and Phase II metabolites and peptides, in total 444, endogenous, internal standards, and prohibited substances, pH, and specific gravity in urine were studied. The accredited analytical methods were used by Anti-Doping Laboratory Qatar for the comparison of data of the same urine samples analyzed with and without UV-C exposure. In the study conditions, no problems of stability were detected in the substances spiked in the urine samples exposed in the UV-C irradiation.

Stabilization of human urine doping control samples: IV. Human chorionic gonadotropin.

The presence of proteolytic enzymes in urine samples, coming from exogenous or endogenous sources, enhances the cleavage of human chorionic gonadotropin (hCG). Moreover, elevated temperatures occurring occasionally during the delayed transportation of sport urine samples, favor the nicking of the hCG molecule. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in athletes' urine samples to chemically inactivate proteolytic enzymes coming from exogenous or endogenous sources so as to prevent the degradation of hCG. The stabilization mixture applied, already tested for the stabilization of endogenous steroids and recombinant erythropoietin (rEPO), was a combination of antibiotics, antimycotic substances, and protease inhibitors. Incubation experiments were conducted in the presence or absence of the stabilization mixture in urine aliquots spiked with six proteases (first series of experiments) and one microorganism associated with urinary tract infections (UTI) (second series of experiments). Intact hCG levels were evaluated by using the EIAgen Total hCG kit. In the first series of experiments, hCG levels were reduced in the untreated aliquots following incubation at 37 degrees C. The addition of the chemical stabilization mixture prevented degradation of hCG induced by four of the proteases applied. In the second series of experiments, no significant difference was found in urine inoculated with E. coli, between aliquots treated with chemical mixture and the untreated aliquots. The addition of the proposed chemical stabilization mixture improves the quality of athletes' urine samples against possible deterioration due to high temperatures or attempts of proteolytic manipulation.

Detecting - the 2004 Athens Olympic Games.

A summary of the preparations and the operation of the Athens Doping Control Laboratory for the 2004 Olympic Games is presented. The preparations involved an upgrade of the laboratory infrastructure: staff, equipment, space, new analytical methodologies. The operation of the laboratory during the Games created a major number of events in agreement with the International Olympic Committee's commitment to doping-free "clean" Games. This summary was created by the director of the Athens Doping Control Laboratory and reflected the author's outlook 15 years later.

Assessment of Serum Cytokines and Oxidative Stress Markers in Elite Athletes Reveals Unique Profiles Associated With Different Sport Disciplines.

OBJECTIVES: Circulating cytokines and oxidative stress markers vary in response to different exercise regimens. This study aims to compare the immune-inflammatory and oxidative stress profiles of elite athletes from different sport disciplines as potential biomarkers of muscle damage, and cardiovascular demand. METHODS: Serum samples from 88 consented elite male athletes from different sports disciplines (aquatics, n = 11, athletics, n = 22, cycling, n = 19, football, n = 28 and weightlifting, n = 8) collected at the anti-doping lab in Italy were screened for 38 cytokines and oxidative stress markers. Comparisons were made between different level of power, cardiovascular demand (CD) and endurance, as well as among the sport types. RESULTS: The anti-inflammatory interleukin (IL)-10 was higher (p = 0.04) in moderate power compared with the high power group. Conversely, superoxide dismutase (SOD; p = 0.001) and malondialdehyde (MDA; p = 0.007) levels were greater in the higher power groups compared with the lower power counterpart. Among athletes who belong to different CD ranks, IL-1ß and monocyte chemoattractant protein-1(MCP1) levels were higher (p = 0.03) in the low CD-rank group compared with high CD counterpart, whereas, SOD levels were higher (p = 0.001) in high and moderate CD-rank groups compared to low counterpart. For endurance groups, IL-10 and macrophage inflammatory protein (MIP)-1beta were increased (p = 0.03) in low/moderate endurance compared with the high endurance group. Finally, MIP1-beta, SOD and catalase varied significantly among the sports groups. CONCLUSION: Specific markers of inflammation and oxidative stress are associated with different sports disciplines and could be utilized as potential biomarkers of athletes' health, performance, and recovery from injury.

Alternative markers for Methylnortestosterone misuse in human urine.

Methylnortestosterone is a progestin and synthetic androgenic anabolic steroid, prohibited by WADA. Methylnortestosterone misuse is commonly detected by monitoring the parent compound and its main metabolites, 17α-methyl-5α-estrane-3α, 17ß-diol (M1) and 17α-methyl-5ß-estrane-3α, 17ß-diol (M2), in the glucuronide fraction. In the current study, a direct detection of methylnortestosterone sulfo-conjugated metabolites after ethyl acetate extraction and analysis by LC/Q/TOF-MS in negative ionization mode was performed, detecting two main sulfate metabolites (S1, S2). For the characterization of metabolites, samples from the excretion study, were additionally analyzed by GC-MS, after solvolysis and per TMS derivatization. RT and MS data collected, were compared with RT and MS data from metabolites of 17z-methyl-5α/ß-estrane-3α/ß, 17z-diols structures with prefixed stereochemistry at 3 and 5 positions, synthesized through Grignard reaction from 19-noretiocholanolone, 19-norandrosterone and 19-norepiandrosterone. Confirmed sulfate metabolites were S1, 17α-methyl-5α-estrane-3α, 17ß-diol 3α sulfate (detected up to 72 h) and S2, 17α-methyl-5ß-estrane-3α, 17ß-diol 3α sulfate (detected up to 192 h). Furthermore, applying targeted analysis based on RT and MS data of the synthesized metabolites two additional metabolites M3, 17ß-methyl-5ß-estrane-3α, 17α-diol and M4, 17ß-methyl-5α-estrane-3α, 17α-diol were detected in the glucuronide fraction and one more metabolite (S3) 17ß-methyl-5ß-estrane-3α, 17α-diol was detected in the sulfate fraction in lower abundance until the end of the excretion study (192 h). Interestingly, S2 could also be detected after the direct analysis of non-hydrolyzed steroid by GC-MS/MS as artifact, following normal ProcIV anabolic steroid procedure and using diethylether as extraction solvent.

Author Correction: Metabolic GWAS of elite athletes reveals novel genetically-influenced metabolites associated with athletic performance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.