Prevalence of carboxy-Δ8 -tetrahydrocannabiniol in antidoping samples.

Δ9 -Tetrahydrocannabinol (Δ9 -THC) is usually the primary psychoactive agent in cannabis preparations. Recently, products containing another isomer, Δ8 -tetrahydrocannabinol (Δ8 -THC), have become available for sale. Δ8 -THC exists naturally in the cannabis plant at very low concentrations; hence, the Δ8 -THC present in most of the above-mentioned products is likely to be manufactured synthetically. A surge in popularity of these products, coupled with little oversight to ensure purity and potency, has led to reports of adverse events. Workplace drug testing programs as well as many sporting organizations prohibit the use of cannabinoids. Carboxy-Δ9 -THC (Δ9 -THC-COOH) is the targeted urinary metabolite for detection of cannabis use. The proliferation of products containing Δ8 -THC, which metabolizes to Δ8 -THC-COOH, presents analytical complexity with respect to separation and quantification of the individual isomers as well as legal complexity with respect to lack of clarity around the legal status of Δ8 -THC. This study aims to estimate the prevalence of Δ8 -THC use in the athlete community by monitoring for Δ8 -THC-COOH in samples collected for antidoping. A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was utilized to resolve Δ8 and Δ9 -THC-COOH. One thousand samples with a presumptive Δ9 -THC-COOH finding in routine screening were analyzed by the above LC-MS/MS method. Approximately 12% of samples contained Δ8 -THC-COOH at relative abundances between 5% and 100% of total carboxy-THC content.

Influence of multiple human chorionic gonadotropin administrations on serum and urinary steroid Athlete Biological Passport profiles in males.

The Athlete Biological Passport (ABP) is a longitudinal tool used in anti-doping to monitor biological parameters known to change with performance-enhancing drug use. The ABP consists of multiple modules, including two aimed at detecting the use of endogenous anabolic androgenic steroids: the urinary and serum steroid modules. Human chorionic gonadotropin (hCG) is a protein hormone potentially abused by male athletes to increase the production of endogenous testosterone. To date, no studies have investigated the impact of extended hCG administration on the urinary and serum steroid modules of the ABP. The goal of this study was to identify the impact of multiple hCG administrations on the parameters tracked as part of the urinary and serum steroid modules of the ABP. Ten recreationally active, healthy male individuals self-administered seven 250 µg hCG injections over 3 weeks. Serum and urine samples were collected before, during, and 2 weeks following the final injection. All ABP parameters were quantified in the respective matrix, and steroid profiles were created with Anti-Doping Administration and Management System adaptive model upper and lower limits for both matrices. In both serum and urine profiles, testosterone increased; however, the testosterone/epitestosterone ratio in urine and the testosterone/androstenedione ratio in serum showed minimal changes. Additionally, serum luteinizing hormone (LH) was quantified using an immunoassay, and a serum testosterone/LH ratio was generated. Serum LH values decreased during administration causing large increases in the serum T/LH ratio, indicating this ratio may be a more sensitive parameter for detecting hCG abuse than urinary testosterone/epitestosterone or serum testosterone/androstenedione.

Identification and synthesis of (Z)-3'-hydroxy clomiphene as a new potential doping-relevant metabolite of clomiphene.

A recent study addressed the possibility of unintentional ingestion of clomiphene through residues in chicken eggs. The method developed here helped distinguish between microdose intake of (E/Z)-clomiphene citrate and consumption of clomiphene-containing eggs by the urinary pattern of four mono-hydroxylated clomiphene metabolites. However, reanalyses of doping-control samples, which showed an adverse analytical finding for clomiphene, revealed a hydroxy clomiphene (HC) isomer that was not found after microdose intake or after consumption of clomiphene-containing eggs and could not be assigned to any of the available reference compounds. The aim of the present follow-up study was to identify this HC isomer and to characterize this metabolite with respect to its potential properties as long-term metabolite in doping controls. METHODS: (E/Z)-3'-HC and (E/Z)-4'-HC were synthesized involving the McMurry reaction. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and optimized after a derivatization step with dansyl chloride to separate eight HC isomers. Using this method, urine samples from a controlled clomiphene administration study were analyzed, in which male study participants received therapeutic doses of clomiphene for 30 days and collected urine samples for up to 8 months. Thus, isomer-specific HC elimination profiles could be monitored. RESULTS: The metabolite previously found in doping-control samples was identified as (Z)-3'-HC. The elimination profiles of the different HCs confirmed previous results, with (Z)-3-HC being the most abundant urinary hydroxy metabolite shortly after administration. A new finding was that the data suggest that (Z)-3'-HC is excreted at higher relative concentrations only several weeks after drug intake. CONCLUSION: These findings might be of particular importance in sport drug testing as they can assist in the decision-making process to distinguish between intentional doping and inadvertent exposure to clomiphene via food contamination.

Factors associated with antibody titer levels among an occupational cohort of fully vaccinated individuals and subsequent risk of COVID-19 infection: A cohort study.

This study (1) determined the association of time since initial vaccine regimen, booster dose receipt, and COVID-19 history with antibody titer, as well as change in titer levels over a defined period, and (2) determined risk of COVID-19 associated with low titer levels. This observational study used data from staff participating in the National Football League COVID-19 Monitoring Program. A cohort of staff consented to antibody-focused sub-study, during which detailed longitudinal data were collected. Among all staff in the program who received antibody testing, COVID-19 incidence following antibody testing was determined. Five hundred eighty-six sub-study participants completed initial antibody testing; 80% (469) completed follow-up testing 50-101 days later. Among 389 individuals who were not boosted at initial testing, the odds of titer < 1000 AU/mL (vs. ≥1000 AU/mL) increased 44% (odds ratio [OR] = 1.44, 95% confidence interval [CI]: 1.18-1.75) for every 30 days since final dose. Among 126 participants boosted before initial testing with no COVID-19 history, 125 (99%) had a value > 2500 AU/ml; 86 (96%) of 90 tested at follow-up and did not develop COVID-19 in the interim remained at that value. One thousand fifty-seven fully vaccinated (330 [29%] boosted at antibody test) individuals participating in the monitoring program were followed to determine COVID-19 status. Individuals with titer value < 1000 AU/mL had twice the risk of COVID-19 as those with >2500 AU/mL (HR = 2.02, 95% CI: 1.28-3.18). Antibody levels decrease postvaccination; boosting increases titer values. While antibody level is not a clear proxy for infection immunity, lower titer values are associated with higher COVID-19 incidence, suggesting increased protection from boosters.

Impact of Biotin Supplementation on Human Chorionic Gonadotropin Immunoassays Utilizing Biotin-Streptavidin Binding Methods in Urine.

BACKGROUND: Human chorionic gonadotropin (hCG) detection is indicative of pregnancy and can be indicative of some forms of cancerous tumors. The hCG drug itself, however, is a performance enhancing substance used by male athletes to increase testosterone production. Antidoping testing for hCG is conducted in urine, often on immunoanalyzer platforms, many of which utilize biotin-streptavidin dependent immunoassays in which the presence of biotin in samples is a known confounding factor. While biotin interference in serum has been well-studied, the extent of biotin interference in urine has not. METHODS: Ten active male individuals underwent a 2-week hCG administration protocol concurrent with supplementation with biotin (20 mg/day) or placebo. Urine and serum samples were collected throughout the study and analyzed for hCG and biotin concentrations. RESULTS: Urinary biotin levels in the hCG + biotin group increased 500-fold over baseline and 29-fold over corresponding serum biotin levels after biotin supplementation. When using a biotin-dependent immunoassay, the hCG + placebo group produced hCG-positive results (hCG ≥ 5 mIU/mL) in 71% of urine samples, while the hCG + biotin group produced positive results in only 19% of samples. Both groups had elevated hCG values in serum measurements by a biotin-dependent immunoassay and in urine when using a biotin-independent immunoassay. Urinary hCG measurements and biotin levels from the hCG + biotin group showed a negative correlation (Spearman r = -0.46, P < 0.0001) when measured using a biotin-dependent immunoassay. CONCLUSIONS: Biotin supplementation can severely suppress urinary hCG values in assays utilizing biotin-streptavidin binding methods and therefore these types of assays are not recommended for use in urine samples containing high levels of biotin. Clinicaltrials.gov Registration Number: NCT05450900.

δ13 C values of urinary 19-norandrosterone in antidoping samples and potential for adverse findings from boar offal consumption.

19-Norandrosterone (19NA) is the preferred urinary target compound to identify doping with nandrolone or related 19-norsteroids. At concentrations between 2.5 and 15 ng/mL, isotope ratio mass spectrometry (IRMS) is required to establish exogenous origin of urinary 19NA. An absolute difference of 3‰ between urinary 19NA and an endogenous reference compound (ERC) constitutes a finding for exogenous origin of 19NA. Over the last 3 years, 77 samples containing urinary 19NA between 2.5 and 15 ng/mL were analyzed at our laboratory. The measured δ13 C values for 19NA ranged from -29.5‰ to -16.8‰. In comparison, the δ13 C values for the corresponding urinary ERCs ranged from -22.4‰ to -16.2‰. Due to the considerable overlap in values between the target compound and the natural range of urinary ERCs, it can be challenging to distinguish between endogenous and exogenous origins of urinary 19NA. In addition, it is well known that consumption of offal from non-castrated pigs can produce 19NA in urine. To determine whether this could cause a positive IRMS finding under the current IRMS positivity criteria, meat from non-castrated boars fed a mixture of corn and soy was consumed by 13 volunteers. Two volunteers produced 19NA findings above 2.5 ng/mL, and the measured isotope values, while inconsistent with documented 19-norsteroid preparations, did meet IRMS positivity criteria. However, these increases in 19NA urinary concentrations were short-lived due to rapid elimination. Timely follow-up collections may help support a claim for dietary exposure when low urinary concentrations of 19NA with pseudo-endogenous isotope values are observed.

Anatomy of an American football game: Player-to-player contact before, during and after an NFL game in context of the 2020 COVID-19 pandemic.

OBJECTIVES: To quantify levels of potential exposure to SARS-CoV-2 surrounding a typical professional American football game, with a focus on interactions on-field between teammates and opposing players before, during, and immediately after competition. METHODS: We examined across-Club consecutive interactions ≥2 minutes within 6 feet [1.8 meters] between athletes on opposing Clubs for all 2020 NFL regular season games (n = 256). Cumulative interaction was measured for a representative subset (n = 119; 46%) of games. Wearable proximity tracking devices (Kinexon) were used to measure distance and duration of interactions; these data were combined with game schedule and Club rosters for analyses. Frequency and per-game mean, median, interquartile range for consecutive interactions ≥2/≥5 minutes and cumulative interactions ≥5/≥15 were described overall and stratified by pre-game, in-game, and post-game. RESULTS: Of the 1964 distinct player-to-opponent contacts ≥2 minutes in NFL regular season games, the majority (n = 1,699; 87%) were fewer than 5 minutes in consecutive length. Among the mean 7.7 distinct contacts ≥2 minutes with opponents each game (median = 4; IQR = 2, 8), very few were ≥5 consecutive minutes at any point (mean = 1.0; median = 0; IQR = 0, 0). Most (n = 849; 43.2%) distinct contacts were pre-game, 546 (27.8%) were during competition, and 569 (29%) were post-game. In games where cumulative interactions were analyzed, there was an average of 17.1 player/opponent interactions with cumulative exposure ≥5 minutes (median = 12; IQR = 4, 30), almost all of which occurred during competition. CONCLUSION: There is limited and short contact between and among competing players in professional American football. In the setting of infectious disease such as the COVID-19 pandemic, a robust prevention program integrating masking, distancing, hygiene, and ventilation when off-field can be created to minimize on- and off-field exposures, which effectively reduces transmission risk in outdoors and/or well-ventilated stadium settings.

EPO and the athlete biological passport: Hematological results from a placebo-controlled, boosting and microdose EPO administration in male recreational athletes.

Hematological results in the context of the Athlete Biological Passport (ABP) from a placebo-controlled EPO administration study are provided here. Twelve participants administered eight subcutaneous boosting doses of epoetin alfa (at 40 IU/kg) over the course of 20 days. After a 10-day washout period, the same volunteers administered six microdoses (900 IU), intravenously, over 13 days. A blinded placebo cohort followed the same dosing pattern, administering saline instead of EPO. All participants supplemented with oral iron, daily, throughout the entirety of the study. In the EPO cohort, as expected, significant changes from baseline were identified in IRF, RET#, RET%, RDW, HCT, HGB, and RBC. No meaningful changes were identified in the placebo cohort population. From the ABP perspective, atypical passport findings (ATPF) were identified in 49% of the samples collected during the boosting and initial washout phases, and 24% of the samples during the microdosing and final washout phases. ATPFs from this cohort were flagged as late as Day 70, the final day of the study. Only a single ATPF was identified from all samples collected from the placebo cohort. ABPs from all volunteers in the study are provided as an avenue to visually convey differences in magnitude and timing of the hematological changes caused by EPO on the individual level. These data are expected to provide important content for Athlete Passport Management Units and ABP expert panels alike.

Detection of erythropoiesis stimulating agent Luspatercept after administration to healthy volunteers for antidoping purposes.

Luspatercept (Reblozyl®) is a newly approved anti-anemic drug prohibited by the World Anti-Doping Agency. It promotes erythropoiesis by limiting apoptosis of immature erythroblasts and the risk of misuse by athletes for doping is high. Proposed detection methods have been published recently but only evaluated in vitro. The objective of this study was to perform the first administration of luspatercept in healthy volunteers for antidoping purpose and to evaluate the detectability in serum, dried capillary blood spots (DBS, collected using TASSO M20 device), and urine. Indirect detection was also evaluated by analyzing hematological parameters for the Athlete Biological Passport. Four volunteers (two males, two females) received one subtherapeutic dose of luspatercept (0.25 mg/kg) followed 3 weeks after by a second dose. Samples were collected from before administration until 7 weeks after the second dose. After immunopurification, electrophoretic separation SDS-/SAR-/IEF- polyacrylamide gel electrophoresis (PAGE), and immunodetection, luspatercept was detected at high levels in serum until the end of the collection, sign of a very slow elimination and similarly detected unchanged at lower levels in urine from 2 days after the first administration until 7 weeks postadministration. DBS showed also the same long window of detection. Luspatercept effects were however of limited amplitude on hematological markers, and only two subjects presented atypical points outside the physiological limits during the study. The direct detection method was very efficient, and change of electrophoretic method and detection antibody can be used for confirmation of suspicious samples.

A Targeted Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Quantification of Peptides from the Carboxyl-terminal Region of Type III Procollagen, Biomarkers of Collagen Turnover.

BACKGROUND: The development of analytical approaches to help reduce the risk of growth hormone (GH) doping is important to fair competition and the health of athletes. However, the reliable detection of GH use remains challenging. The identification of novel biomarkers of GH administration could lead to a better understanding of the physiological response to GH, more sensitive detection of the illicit use of GH in sport, and better management of patients treated for GH disorders. METHODS: We developed a targeted liquid chromatography-tandem mass spectrometry method to simultaneously quantify the carboxyl-terminal propeptide of type III procollagen (P-III-CP) and type III collagen degradation products in human serum. Following proteolysis, we instituted a simple acid precipitation step to reduce digested sample complexity before peptide immunoenrichment, which improved the recovery of one target peptide from serum. We evaluated the concentration of each biomarker at different age ranges and after GH administration in healthy participants. RESULTS: The assay was linear over an estimated concentration range of 0.3 to1.0 nM and 0.1 to 0.4 nM for each surrogate peptide of P-III-CP and collagen fragments, respectively. Intra-day and inter-day coefficients of variation were ≤15%. Biomarker concentrations appeared to vary with age and to reflect age-specific collagen turnover. Moreover, their concentrations changed after GH administration. CONCLUSIONS: Our method quantifies the proteins belonging to the family of P-III-CP and type III collagen degradation products in human serum, which could be used to detect GH administration in athletes and better understand diseases involving GH therapy or altered type III collagen turnover.

Feasibility of microvolumetric capillary whole blood collections for usage in Athlete Biological Passport analysis.

The hematological module of the Athlete Biological Passport (ABP) represents an important tool in the pursuit to detect blood doping in athletes. Currently, collecting blood samples for ABP analysis can be cumbersome, invasive, and expensive, involving a venous blood draw performed by a trained phlebotomist followed by cold-chain monitored shipping to the analysis laboratory. Developing innovative methods to collect and transport ABP blood samples while adhering to strict preanalytical and analytical requirements has the potential to greatly increase testing frequency and, consequently, the effectiveness of the ABP program globally. The focus of this study was to compare venous blood collections with capillary blood collections to determine if capillary samples could be used for ABP analysis without sacrificing the analytical integrity required for antidoping testing procedures. In this study, capillary blood was collected using the Tasso+ EDTA device (Tasso, Inc.), a novel microvolumetric device that collects liquid, whole blood from skin capillaries on the upper arm. Excellent laboratory agreement was observed between venous and capillary blood samples for the three main ABP parameters: HGB, RET%, and OFF-Score. Additionally, the stability of capillary samples after storage at 4°C, similar to what would be required during transport, was acceptable for up to 72 h following collection. Finally, we generated individual ABP profiles using the adaptive model for 10 participants and observed excellent agreement between venous and capillary profiles. These results indicate capillary blood collection is a viable alternative to venous blood collections for ABP analysis.

Detection of insulin analogues and large peptides >2 kDa in urine.

Insulin analogues and large bioactive peptides may be used by athletes to enhance performance and are banned by the World Anti-Doping Agency (WADA). In addition to insulin analogues, the large peptides include a structurally diverse set of peptides including analogues of growth hormone releasing hormone (GHRH), insulin-like growth factor-1 (IGF-1), and mechano-growth factor (MGF). Detection of this class of peptides is difficult due to their absorptive losses and presence at very low concentrations in urine. In this report, a high throughput method is described that allows sensitive detection of four classes of large peptides in one assay. Sample extraction is performed by ultrafiltration to concentrate the urine followed by solid phase extraction in a 96-well micro-elution plate. Peptides in the urine samples are detected on a triple quadrupole mass spectrometer coupled to standard flow liquid chromatography. The method was validated and evaluated for limit of detection, limit of identification, specificity, precision, carryover, recovery, matrix interference, and post-extraction stability. The limit of detection for insulin analogues is between 5 and 25 pg/ml and between 5 and 50 pg/ml for the other peptide classes. Specificity was good with no detection of interfering peaks in blank urine samples. Carryover from a high concentration sample was not observed and the post-extraction stability was between 77% and 107%. The method was able to detect insulin analogues in three diabetic urine samples. Increased screening for this class of peptides will improve detection and deterrence.

Growth hormone isoform testing in capillary dried blood spots: Results from single and multiple dose administration studies and large-scale field collections.

A multiphase study was designed to examine the detectability of human growth hormone (GH) use in capillary dried blood spots (DBS). First, 13 subjects self-injected a single, 2-mg dose of somatropin and collected capillary DBS samples for 24 h. Next, nine subjects self-injected 2-mg somatropin, six times over the course of 11 days; DBS were collected intermittently following dosing. Finally, a nondrug, large-scale field study involved DBS collections from an athlete and staff population over 3 years. All DBS samples were self-collected using the Tasso M20 device and were analyzed for the presence of GH using the WADA-approved GH isoforms test. Following the single dose, positive detection within 12 h of dosing was 86% and 56% sensitive on Kits 1 and 2, respectively. In the multidose study, detection within 12 h was 85% and 69% sensitive on Kits 1 and 2, respectively. No positives were detected outside the 12-h window following a single dose, wherein detection was 5.6% sensitive at 24-h in the multidose study. Combining the 12-h windows from both studies, 100% of samples had measurable recombinant (REC) and pituitary (PIT) GH concentrations above the assay LoD, 0.041 ng/ml. Finally, 1213 samples were collected in the large-scale field study: 189 showed REC and PIT concentrations above the LoD; none returned positive results. GH is detectable in capillary DBS using the isoforms method for 12-24 h following use. While detection is short lived, transitioning to a DBS self-collection method can allow more frequent testing and increase deterrence to GH abuse.

Effectiveness and use of reverse transcriptase polymerase chain reaction point of care testing in a large-scale COVID-19 surveillance system.

BACKGROUND: Rapid COVID-19 testing platforms can identify infected individuals at the point of care (POC), allowing immediate isolation of infected individuals and reducing the risk of transmission. While lab-based nucleic acid amplification testing (NAAT) is often considered the gold standard to detect SARS-CoV-2 in the community, results typically take 2-7 days to return, rendering POC testing a critical diagnostic tool for infection control. The National Football League (NFL) and NFL Players Association deployed a new POC testing strategy using a newly available reverse transcriptase polymerase chain reaction (RT-PCR) rapid test during the 2020 season, and evaluated diagnostic effectiveness compared to other available devices using real-world population surveillance data. METHODS: RT-PCR POC test results were compared to NAAT results from same-day samples by calculation of positive and negative concordance. Sensitivity analyses were performed for three subgroups: (1) individuals symptomatic at time of positive test; (2) individuals tested during the pilot phase of rollout; and (3) individuals tested daily. RESULTS: Among 4989 same-day POC/NAAT pairs, 4957 (99.4%) were concordant, with 93.1% positive concordance and 99.6% negative concordance. Based on adjudicated case status, the false negative rate was 0.2% and false positive rate was 2.9%. In 43 instances, the immediate turnaround of results by POC allowed isolation of infected individuals 1 day sooner than lab-based testing. Positive/negative concordance in sensitivity analyses were relatively stable. CONCLUSION: RT-PCR POC testing provided timely results that were highly concordant with lab-based NAAT in population surveillance. Expanded use of effective RT-PCR POC can enable rapid isolation of infected individuals and reduce COVID-19 infection in the community.

Detection of testosterone microdosing in healthy females.

The ready detectability of synthetic androgens by mass spectrometry (MS)-based antidoping tests has reoriented androgen doping to using testosterone (T), which must be distinguished from its endogenous counterpart making detection of exogenous T harder. We investigated urine and serum steroid and hematological profiling individually and combined to determine the optimal detection model for T administration in women. Twelve healthy females provided six paired blood and urine samples over 2 weeks prior to treatment consisting of 12.5-mg T in a topical transdermal gel applied daily for 7 days. Paired blood and urine samples were then obtained at the end of treatment and Days 1, 2, 4, 7, and 14 days later. Compliance with treatment and sampling was high, and no adverse effects were reported. T treatment significantly increased serum and urine T, serum dihydrotestosterone (DHT), urine 5α-androstane-3α,17ß-diol (5α-diol) epitestosterone (E), and urine T/E ratio with a brief window of detection (2-4 days) as well as total and immature (medium and high fluorescence) reticulocytes that remained elevated over the full 14 posttreatment days. Carbon isotope ratio MS and the OFF score and Abnormal Blood Profile score (ABPS) were not discriminatory. The optimal multivariate model to identify T exposure combined serum T, urine T/E ratio with three hematological variables (% high fluorescence reticulocytes, mean corpuscular hemoglobin, and volume) with the five variables providing 93% correct classification (4% false positive, 10% false negatives). Hence, combining select serum and urine steroid MS variables with reticulocyte measures can achieve a high but imperfect detection of T administration to healthy females.

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.

Generic Pharmaceuticals as a Source of Diuretic Contamination in Athletes Subject to Sport Drug Testing.

This paper describes nine instances of positive anti-doping tests that could be accounted for by the use of permitted generic prescription drugs contaminated with diuretics, which are prohibited in sport at all times under the WADA Prohibited List. The contamination levels found in the medications are reported and were below FDA limits for manufacturers that are based primarily on safety considerations. These cases demonstrate that great care must be taken to identify the source of low-level anti-doping positives for diuretics reported by WADA-accredited laboratories, and possibly other prohibited substances as well, in order to avoid sanctioning innocent athletes. An evaluation of the cases in this paper supports an approach which establishes a laboratory minimum reporting level (MRL) for diuretics found most commonly in medications. A global consensus after extensive review of similar anti-doping cases has resulted in implementation of a recently announced solution regarding potential diuretic contamination cases.

Tracking immature reticulocyte proteins for improved detection of recombinant human erythropoietin (rhEPO) abuse.

Athletes abuse recombinant human erythropoietin (rhEPO) and erythropoiesis stimulating agents to increase hemoglobin mass and improve performance. To evade detection, athletes have developed sophisticated blood doping regimens, which often include rhEPO micro-dosing. Detection of these methods requires biomarkers with increased sensitivity and a sample matrix that is more amenable to frequent testing in the field. We have developed a method to measure two immature reticulocyte proteins, CD71 and ferrochelatase (FECH), and one total erythrocyte protein, Band 3, in dried blood spots (DBS). This method was tested in response to rhEPO administration after low doses, 40 IU/kg, micro-doses, 900 IU, or saline injection in 20 healthy subjects. During administration of low-dose rhEPO, the mean CD71/Band 3 and FECH/Band 3 ratio increased by 412 ± 197% and 250 ± 44%, respectively. The mean response for the current biomarker, RET%, increased by 195 ± 35%. During administration of rhEPO micro-doses, CD71/Band 3 increased to 127 ± 25% on day 35 and 139 ± 36% on day 39, while no increase was observed in RET%. After rhEPO administration, during the washout phase, mean values decreased to a minimum of 64 ± 4% and 64 ± 11% for CD71/Band 3 and RET%, respectively. However, CD71/Band 3 remained below 75% of baseline for at least 4 weeks after rhEPO injection, while RET% returned to baseline levels. The results demonstrate that immature reticulocyte proteins have a larger response to rhEPO administration than the current biomarker, RET%, and can be monitored in the DBS matrix.

Measurement of Immature Reticulocytes in Dried Blood Spots by Mass Spectrometry.

BACKGROUND: Immature reticulocytes (IRC) are the first cells to respond to changes in erythropoiesis. For antidoping applications, measurement of IRC may improve detection of blood doping practices. Unfortunately, this small cell population has limited stability in liquid blood samples and is difficult to measure with optimal precision. We developed a method to measure 3 IRC membrane proteins in dried blood spots (DBS) to monitor changes in erythropoiesis. METHODS: DBS spots were washed with buffers to remove soluble proteins, membrane proteins remaining in the spot were digested with trypsin, and one peptide for each protein was measured by LC-MS/MS. IRC protein concentration was determined using a DBS single point calibrator. RESULTS: Intraassay precision for IRC proteins was between 5%-15%. IRC proteins were stable in DBS for 29 days at room temperature. In a longitudinal study of 25 volunteers, the mean intraindividual variation for 3 IRC proteins was 17%, 20%, and 24% from capillary blood DBS. In comparison, the mean longitudinal variation for IRC counts measured on an automated hematology analyzer was 38%. IRC protein concentration from capillary blood DBS correlated well with venous blood DBS protein concentrations. CONCLUSIONS: Measurement of IRC proteins in DBS samples provides a method to measure changes in erythropoiesis with improved analytical sensitivity, stability, and precision. When combined with the inherent advantages of capillary blood collection in the field, this method may substantially improve the detection of blood doping practices.

Evaluation of blood parameters by linear discriminant models for the detection of testosterone administration.

The steroidal module of the Athlete Biological Passport (ABP) has been used since 2014 for the longitudinal monitoring of urinary testosterone and its metabolites to identify samples suspicious for the use of synthetic forms of Endogenous Anabolic Androgenic Steroids (EAAS). Multiple recent studies have suggested that monitoring of blood parameters may provide enhanced detectability of exogenous testosterone administration. Transdermal and intramuscular testosterone administration studies were carried out in 15 subjects, and the effect on blood steroidal levels, hematological parameters, and gonadotropins was evaluated. Serum testosterone and dihydrotestosterone levels increased while gonadotropin levels were suppressed after administration. A modest increase in reticulocytes was also observed. The blood parameters that were responsive to the administrations were combined into several linear discriminant models targeting both administration (on) and washout (off) phases. The models were effective in detecting the large dose intramuscular administration but were less successful in the detection of the lower dose transdermal application. The blood profiling models may provide complementary value but do not appear to be substantially more advantageous than longitudinal urinary profiling.