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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,17β-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.

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.

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.

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.

Background Measuring the seroprevalence of antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is central to understanding infection risk and fatality rates. We studied Coronavirus Disease 2019 (COVID-19)-antibody seroprevalence in a community sample drawn from Santa Clara County. Methods On 3 and 4 April 2020, we tested 3328 county residents for immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to SARS-CoV-2 using a rapid lateral-flow assay (Premier Biotech). Participants were recruited using advertisements that were targeted to reach county residents that matched the county population by gender, race/ethnicity and zip code of residence. We estimate weights to match our sample to the county by zip, age, sex and race/ethnicity. We report the weighted and unweighted prevalence of antibodies to SARS-CoV-2. We adjust for test-performance characteristics by combining data from 18 independent test-kit assessments: 14 for specificity and 4 for sensitivity. Results The raw prevalence of antibodies in our sample was 1.5% [exact binomial 95% confidence interval (CI) 1.1–2.0%]. Test-performance specificity in our data was 99.5% (95% CI 99.2–99.7%) and sensitivity was 82.8% (95% CI 76.0–88.4%). The unweighted prevalence adjusted for test-performance characteristics was 1.2% (95% CI 0.7–1.8%). After weighting for population demographics, the prevalence was 2.8% (95% CI 1.3–4.2%), using bootstrap to estimate confidence bounds. These prevalence point estimates imply that 53 000 [95% CI 26 000 to 82 000 using weighted prevalence; 23 000 (95% CI 14 000–35 000) using unweighted prevalence] people were infected in Santa Clara County by late March—many more than the ∼1200 confirmed cases at the time. Conclusion The estimated prevalence of SARS-CoV-2 antibodies in Santa Clara County implies that COVID-19 was likely more widespread than indicated by the number of cases in late March, 2020. At the time, low-burden contexts such as Santa Clara County were far from herd-immunity thresholds.

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.

“Doping” is as old as the ancient Olympics (776 BC to 394 AD) when athletes used herbal medications, animal organs (organotherapy was common to cure diseases and to improve vitality), stimulants, and hallucinogenic mushrooms to improve athletic performance. We discuss the issues of proper medical use of prohibited drugs (therapeutic use exemption) and the concept of the athlete’s biological passport to highlight disordered physiology rather than necessarily the chemical iasdentification of a banned substance. The growth hormone/IGF-1 system is used as an example of both the detection of the drugs and the consequences upon multiple analytes with the ongoing validation of a peptides module, just as hematologic and steroid modules have been used.

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.

On January 30, 2020, the World Health Organization (WHO) declared SARS-CoV-2 a global pandemic, based on a high infection rate and a high case fatality rate (CFR). The combination of these two points led WHO to forecast a high expected mortality rate of approximately 2% of the population. The phenomenon of Simpson's paradox teaches us that we should be careful when we combine two variables together. Indeed, despite the high mortality rate in several places, this forecast seems to have collapsed. We believe one of the reasons for the erroneous forecasts is that combining the above points ignored a confounding variable - many of the virus carriers are asymptomatic and therefore not diagnosed.

The steroidal module of the Athlete Biological Passport (ABP) has been used since 2014 for the longitudinal monitoring of urinary testosterone and its metabolites in order to identify samples suspicious for the use of synthetic forms of endogenous anabolic androgenic steroids (EAAS). Samples identified by the module may then be confirmed by isotope ratio mass spectrometry (IRMS) to establish clearly the exogenous origin of testosterone and/or metabolites in the sample. To examine the detection capability of the steroidal ABP model, testosterone administration studies were performed with various doses and three routes of administration - transdermal, intramuscular, and subcutaneous with 15 subjects for each route of administration. Urine samples were collected before, during, and after administration and steroid profiles were analyzed using the steroidal ABP module in ADAMS. A subset of samples from each mode of administration was also analyzed by IRMS. The steroidal ABP module was more sensitive to testosterone use than population-based thresholds and with high dose administrations there was very good agreement between the IRMS results and samples flagged by the module. However, with low dose administration the ABP module was unable to identify samples where testosterone use was still detectable by IRMS analysis. The testosterone/epitestosterone (T/E) ratio was the most diagnostic parameter for longitudinal monitoring with the exception of low testosterone excretors for whom the 5 alpha-androstane-3 alpha, 17 beta-diol/epitestosterone (5 alpha Adiol/E) ratio may provide more sensitivity.

Identification and evaluation of long-term markers is crucial in prolonging the detection window for anabolic steroid abuse in sport. Recently, sulfoconjugated epiandrosterone was identified as a potential long-term marker for the abuse of certain endogenous anabolic agents, including testosterone, which continues to be widely used as a performance enhancing agent in sport. To evaluate the applicability of epiandrosterone sulfate as a marker for testosterone use, administration studies were conducted with multiple modes of testosterone administration - transdermal, intramuscular, and subcutaneous. A modified sample preparation method was used to collect both glucuronidated and sulfoconjugated analytes of interest. Carbon isotope ratio measurements from the administration studies are presented here. Epiandrosterone was less effective than the conventionally used target compounds for detection of the low dose application (transdermal gel). With intramuscular administration, epiandrosterone was more diagnostic than with transdermal administration, but it did not prolong the detection window more than the conventional target compounds. With subcutaneous administration, the doses administered to the subjects were varied and the effect on the epiandrosterone values was dependent on the magnitude of the dose administered. Epiandrosterone does not appear to be a useful marker in the detection of low dose testosterone administration. It is responsive to higher dose administration, but it does not provide an extension of the detection window relative to conventional target compounds.

The integrity of the athlete biological passport (ABP) is underpinned by understanding normal fluctuations of its biomarkers to environmental or medical conditions, for example, altitude training or iron deficiency. 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 , 3000 m). Athletes received either oral, intravenous (IV) or placebo iron supplementation, commencing 2 weeks prior and continuing throughout hypoxic exposure. Venous blood was sampled twice prior, weekly during, and up to 6 weeks after altitude. Individual ABP thresholds for haemoglobin concentration ([Hb]), reticulocyte percentage (%retic), and OFF score were calculated using the adaptive model and assessed at 99% and 99.9% specificity. 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 3 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 10 athletes returning abnormal values at 99% specificity, 2 of whom had not triggered the model previously. In summary, the abnormalities observed 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.

The dried blood spot (DBS) matrix has significant utility for applications in the field where venous blood collection and timely shipment of labile blood samples is difficult. Unfortunately, protein measurement in DBS is hindered by high abundance proteins and matrix interference that increases with hematocrit. We developed a DBS method to enrich for membrane proteins and remove soluble proteins and matrix interference. Following a wash in a series of buffers, the membrane proteins are digested with trypsin and quantitated by parallel reaction monitoring mass spectrometry methods. The DBS method was applied to the quantification of four cell-specific cluster of differentiation (CD) proteins used to count cells by flow cytometry, band 3 (CD233), CD71, CD45, and CD41. We demonstrate that the DBS method counts low abundance cell types such as immature reticulocytes as well as high abundance cell types such as red blood cells, white blood cells, and platelets. When tested in 82 individuals, counts obtained by the DBS method demonstrated good agreement with flow cytometry and automated hematology analyzers. Importantly, the method allows longitudinal monitoring of CD protein concentration and calculation of interindividual variation which is difficult by other methods. Interindividual variation of band 3 and CD45 was low, 6 and 8%, respectively, while variation of CD41 and CD71 was higher, 18 and 78%, respectively. Longitudinal measurement of CD71 concentration in DBS over an 8-week period demonstrated intraindividual variation 17.1-38.7%. Thus, the method may allow stable longitudinal measurement of blood parameters currently monitored to detect blood doping practices.

Autologous blood transfusion is used by athletes to enhance performance by increasing oxygen transport to muscles. It is the only form of blood doping that is currently not detectable using a direct method. Autologous blood transfusions are detected by an indirect method which monitors the

Understanding and characterizing confounding factors to the Athlete Biological Passport (ABP) is crucial for the reliable interpretation of biological profiles in the antidoping field. The physiological effects on hematological parameters and plasma volume (PV) following competition in a long-distance triathlon, as seen in the Ironman discipline, have yet to be fully described and are the focus of this study. Complete blood count blood tests were conducted on 19 Ironman triathletes before and after an Ironman triathlon to characterize changes in hematological parameters and the effect on ABP interpretation, as it was hypothesized that changes in the plasma volume may result in the presentation of atypical ABP profiles. Baseline blood samples were collected from the athletes prior to the event, and one sample was collected per day for up to 1 week following the race. Differences were observed between the male and female athletes across multiple parameters. Most importantly to the ABP, decreases in hemoglobin concentration (HGB) and hematocrit (HCT) were identified post-race, with the largest decreases identified on day +2. The average HGB returned to pre-race baseline levels on day +5. Beginning 5-6 days after the race, increases in the reticulocyte percentage (Ret%) were identified. Atypical Passport Findings were identified in 32% (6/19) of the ABPs, flagged mainly due to atypical hemoglobin concentration and one instance in which the OFF-score exceeded the adaptive model limits. These results offer a characterized timeline of hematological changes and expected shifting of plasma volume following an Ironman triathlon providing important data for the reliable interpretation of ABP profiles in this field.

The laboratory profile of intranasal testosterone gel has not been previously reported from an anti-doping perspective. Because intranasal testosterone gel is newly available as a commercial product, we sought to examine the laboratory parameters following administration of this formulation, with particular attention to anti-doping guidelines. Five healthy and active male subjects were administered testosterone intranasal gel three times daily for four weeks, using a pattern of five consecutive days on, two days off. Urine was collected after each five-day round of drug administration and analyzed using a full steroid screen and isotope ratio mass spectrometry (IRMS). Windows of detection for elevated testosterone/epitestosterone (T/E) and other steroid ratios, World Anti-Doping Agency (WADA) athlete biological passport (ABP) findings, and IRMS results were analyzed in this study. In the 0-24 h window post-administration, 70% of samples were flagged with a suspicious steroid profile and 85% were flagged as atypical passport findings according to the WADA ABP steroid module. In the 24-48 h window, 0% of samples displayed suspicious steroid profiles while 40% resulted in atypical passport findings. IRMS testing confirmed the presence of exogenous testosterone in 90% and 40% of samples in the 0-24 h and 24-48 h windows post-administration, respectively. Additionally, IRMS data were analyzed to determine commonalities in the population changes in δ13 C values of testosterone, androsterone, etiocholanolone, 5αAdiol, and 5βAdiol. Though no discernible metabolic trend of the route of administration was identified, we discovered that intranasal gel testosterone is detectable using conventional anti-doping tests. Copyright © 2016 John Wiley & Sons, Ltd.

Context Clomiphene is a performance-enhancing drug commonly abused by males in sport, but the extent to which testosterone increases in healthy males following its use is unknown. In addition, evidence suggests that clomiphene, a mixture of cis- and trans-isomers zuclomiphene and enclomiphene, is detectable in urine for months following use; the isomer-specific urinary detection window has yet to be characterized in a controlled study. Objective To determine the effect of once-daily, 30-day clomiphene treatment on serum testosterone and gonadotropin levels in the subject population studied and the urinary clearance and detection window of clomiphene isomers following administration for antidoping purposes. Participants and Design Twelve healthy males aged 25 to 38 years, representing a recreational athlete population, participated in this open-label, single-arm study. Intervention Oral clomiphene citrate (50 mg) was self-administered once daily for 30 days. Serum and urine samples were collected at baseline and at days 7, 14, 21, 28, 30, 32, 35, 37, 44, 51, and 58; urine collections continued periodically up to day 261. Results Mean testosterone, LH, and FSH levels increased 146% (SEM, ±23%), 177% (±34%), and 170% (±33%), respectively, during treatment compared with baseline. Serum drug concentrations and urinary excretion were nonuniform among individuals as isomeric concentrations varied. The zuclomiphene urinary detection window ranged from 121 to >261 days. Conclusions Clomiphene significantly raised serum testosterone and gonadotropin levels in healthy men and thus can be abused as a performance-enhancing drug. Such abuse is detectable in urine for ≥4 months following short-term use.

Purpose: Iron is integral for erythropoietic adaptation to hypoxia, yet the importance of supplementary iron compared with existing stores is poorly understood. The aim of the present study was to compare the magnitude of the hemoglobin mass (Hbmass) in response to altitude in athletes with intravenous (IV), oral, or placebo iron supplementation. Methods: Thirty-four, nonanemic, endurance-trained athletes completed 3 wk of simulated altitude (3000 m, 14 h·d−1), receiving two to three bolus iron injections (ferric carboxymaltose), daily oral iron supplementation (ferrous sulfate), or a placebo, commencing 2 wk before and throughout altitude exposure. Hbmass and markers of iron regulation were assessed at baseline (day −14), immediately before (day 0), weekly during (days 8 and 15), and immediately, 1, 3, and 6 wk after (days 22, 28, 42, and 63) the completion of altitude exposure. Results: Hbmass significantly increased after altitude exposure in athletes with IV (mean % [90% confidence interval (CI)], 3.7% [2.8–4.7]) and oral (3.2% [2.2–4.2]) supplementation and remained elevated at 7 d postaltitude in oral (2.9% [1.5–4.3]) and 21 d after in IV (3.0% [1.5–4.6]) supplementation. Hbmass was not significantly higher than baseline at any time point in placebo. Conclusions: Iron supplementation appears necessary for optimal erythropoietic adaptation to altitude exposure. IV iron supplementation during 3 wk of simulated live high–train low altitude training offered no additional benefit in terms of the magnitude of the erythropoietic response for nonanemic endurance athletes compared with oral supplementation.

In doping control laboratories, autologous blood transfusions are currently detected using an indirect method that monitors changes in an athlete's hemoglobin concentration [Hb] and reticulocyte percent (Ret%) over time. The method is limited by the need for a phlebotomist to collect venous blood and the limited blood stability which requires temperature-controlled shipment and analysis within 72 hours. These limitations significantly reduce the number of samples collected from each athlete and thus the utility of the method. We have recently developed a method to measure immature reticulocytes (IRC) and red blood cells (RBC) in dried blood spots, which could replace the current venous blood method. In the DBS method, cell-specific proteins are digested with trypsin and measured by mass spectrometry. Two proteins, CD71 and Band3, are measured to count IRC and RBC, respectively. The method was tested in an autologous transfusion study consisting of 15 subjects who received blood and 11 subjects who received saline. After transfusion, the average CD71/Band3 ratio in the blood group was statistically different from the saline group at days 5, 6, 13, and 20. The average CD71/Band3 ratio decreased to a minimum of 61 ± 8% of baseline, while Ret% decreased to 75 ± 5% of baseline. Based on experimentally defined criteria, the CD71/Band3 ratio could detect 7 out of 10 blood transfusion subjects, while Ret% could detect 3 out of 10. Thus, the DBS method could improve detection of autologous transfusion and allow increased sample collection.

Context Patients with 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH) require lifelong treatment with glucocorticoids. In growing children, the drug of choice is hydrocortisone. Commercially available hydrocortisone tablets do not conform to very low doses prescribed to infants and toddlers, and compounded hydrocortisone is often dispensed to meet therapeutic needs. However, safety, efficacy, and uniformity of compounded products are not tested. We report a case of Cushing syndrome in a child with CAH who was inadvertently receiving excessive hydrocortisone in compounded form. Design A 20-month-old girl with CAH developed growth deceleration, excessive weight for length, irritability, increased facial fat, plethora, and excess body hair while receiving hydrocortisone from a local compounding pharmacy. The signs and symptoms persisted despite decreasing hydrocortisone dose. Iatrogenic Cushing syndrome was suspected. The prescribed hydrocortisone capsules were sent for analysis to the Sports Medicine Research & Testing Laboratory, where testing revealed that each 1-mg hydrocortisone capsule contained five to 10 times the dose prescribed and listed on the label. Conclusion Physicians must be aware that errors in compounded medications may lead to unanticipated adverse effects. Iatrogenic Cushing syndrome should be suspected in any child receiving compounded glucocorticoid treatment who develops growth arrest and excess weight gain.

Background: Dried blood spot sample collection could improve detection of the misuse of IGF-1, its analogs and growth hormone. An LC–MS/MS method was developed to measure two IGF-1 peptides and one analog peptide after trypsin digestion. In addition to standard method validation parameters, the effect of hematocrit on cysteine alkylation, trypsin digestion and the selection of internal standard were evaluated. Results: Quantification of IGF-1 peptides was possible with an LLOQ of 25 ng/ml and imprecision of less than 15%. Conclusion: While the effects of hematocrit must be evaluated empirically for each method, dried blood spots are a suitable matrix for the measurement of IGF-1 and its analogs by MS.

Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method.

Current anti-doping testing is primarily conducted in urine and blood. Recently, due to confounding factors with urine and blood collections such as invasiveness, cost, and stringent shipping conditions, there has been a push for the use of alternative sample matrices to ameliorate these issues. Gaining support within the anti-doping field is the use of oral fluid, and more recently exhaled breath, as viable alternative or complementary matrices to traditional urine and blood for drug testing. Thus, we designed a first-in-field study with the purpose of investigating the utility of oral fluid and exhaled breath testing, and the preference of athlete participants, comparative to conventional anti-doping methods of urine testing. To accomplish this, 521 total matched samples, consisting of exhaled breath, oral fluid, and urine samples, were collected and analyzed, and the results compared across matrices. Participants in this study preferred the exhaled breath collection (rated 4.90 ± 0.34 out of 5, mean ± SD) over the oral fluid collection procedure (4.29 ± 0.85), and most preferred both over urine collections. Exhaled breath resulted in the shortest collection time (2.58 ± 1.00 min, mean ± SD), followed by urine (3.08 ± 1.50 min), and finally oral fluid (4.14 ± 1.94 min). Prohibited substances from the drug categories of stimulants, narcotics, cannabinoids, diuretics, glucocorticoids, beta-blockers, and others, were analyzed in this study for a comparison of testing efficacy. Of the total findings 49% were detectable in only urine, 38% in urine + oral fluid, and 9% in all three matrices. Of the unique findings 3% were detectable in only oral fluid, 1% in oral fluid + breath, and 0% of unique findings were present only in exhaled breath. The findings from this study provide a strong foundation for the future use of oral fluid and exhaled breath as viable alternative or complementary matrices for in-competition anti-doping testing.

There is significant evidence that athletes are using recombinant human growth hormone (rhGH) to enhance performance, and its use is banned by the World Anti-Doping Agency and professional sports leagues. Insulin-like growth factor-1 (IGF-1) is the primary mediator of growth hormone action and is used as a biomarker for the detection of rhGH abuse. The current biomarker-based method requires collection and expedited shipment of venous blood which is costly and may decrease the number of tests performed. Measurement of GH biomarkers in dried blood spots (DBS) would considerably simplify sample collection and shipping methods to allow testing of a greater number of samples regardless of location. A method was developed to quantify intact IGF-1 protein in DBS by liquid chromatography-tandem mass spectrometry. A step-wise acid-acetonitrile extraction was optimized to achieve a sensitive assay with a lower limit of quantification of 50 ng/mL. IGF-1 remained stable at room temperature for up to 8 days, which would allow shipment of DBS cards at ambient temperature. In a comparison between plasma concentrations of IGF-1 and concentrations measured from venous and finger prick DBS, there was good correlation and agreement, r(2) of 0.8551 and accuracy of 86-113 % for venous DBS and r(2) of 0.9586 and accuracy of 89-122 % for finger prick DBS. The method is intended for use as a rapid screening method for IGF-1 to be used in the biomarker method of rhGH abuse detection.

Importance Recent reports have described the increasing use of nonsteroidal selective androgen receptor modulators, which have not been approved by the US Food and Drug Administration (FDA), to enhance appearance and performance. The composition and purity of such products is not known. Objective To determine the chemical identity and the amounts of ingredients in dietary supplements and products marketed and sold through the internet as selective androgen receptor modulators and compare the analyzed contents with product labels. Design and Setting Web-based searches were performed from February 18, 2016, to March 25, 2016, using the Google search engine on the Chrome and Internet Explorer web browsers to identify suppliers selling selective androgen receptor modulators. The products were purchased and the identities of the compounds and their amounts were determined from April to August 2016 using chain-of-custody and World Anti-Doping Association–approved analytical procedures. Analytical findings were compared against the label information. Exposures Products marketed and sold as selective androgen receptor modulators. Main Outcomes and Measures Chemical identities and the amount of ingredients in each product marketed and sold as selective androgen receptor modulators. Results Among 44 products marketed and sold as selective androgen receptor modulators, only 23 (52%) contained 1 or more selective androgen receptor modulators (Ostarine, LGD-4033, or Andarine). An additional 17 products (39%) contained another unapproved drug, including the growth hormone secretagogue ibutamoren, the peroxisome proliferator-activated receptor-δ agonist GW501516, and the Rev-ErbA agonist SR9009. Of the 44 tested products, no active compound was detected in 4 (9%) and substances not listed on the label were contained in 11 (25%). In only 18 of the 44 products (41%), the amount of active compound in the product matched that listed on the label. The amount of the compounds listed on the label differed substantially from that found by analysis in 26 of 44 products (59%). Conclusions and Relevance In this limited investigation involving chemical analyses of 44 products marketed as selective androgen receptor modulators and sold via the internet, most products contained unapproved drugs and substances. Only 52% contained selective androgen receptor modulators and many were inaccurately labeled.