Detection of doping substances in paired dried blood spots and urine samples collected during doping controls in Danish fitness centers.

The use of dried blood spot (DBS) in anti-doping can be advantageous in terms of collection, transportation, and storage compared with the traditional anti-doping testing matrices urine and venous blood. There could, nonetheless, be disadvantages such as shorter detection windows for some substances compared with urine, but real-life comparison of the detectability of prohibited substances in DBS and urine is lacking. Herein, we present a liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based screening method for simultaneous detection of 19 target analytes from the doping substance categories S1-S5 in a single spot. Ninety-eight urine and upper-arm DBS (Tasso-M20) sample pairs were collected from fitness centers customers notified for doping control by Anti Doping Denmark, and three sample pairs were collected from active steroid users undergoing clinical evaluation and treatment at a Danish hospital. The analytical findings were cross compared to evaluate the applicability of the developed DBS testing menu in terms of feasibility and analytical performance. To our knowledge, this is the first study to compare the detectability of prohibited substances in DBS and urine samples collected in a doping control setting. Twenty-seven of the urine samples and 23 DBS samples were positive, and we observed a very high concordance (95%) in the overall analytical results (i.e., positive or negative samples for both urine and DBS). Collectively, these results are very promising, and DBS seems suitable as a stand-alone matrix in doping control in fitness centers likely because of the high analyte concentration levels in these samples.

Serum Insulin-like Factor 3, Testosterone, and LH in Experimental and Therapeutic Testicular Suppression.

BACKGROUND: Serum insulin-like factor 3 (INSL3) is a Leydig cell biomarker, but little is known about the circulating concentration of INSL3 during hypothalamus-pituitary-testicular suppression. AIM: To study the concomitant changes in serum concentrations of INSL3, testosterone, and LH during experimental and therapeutic testicular suppression. METHODS: We included serum samples from 3 different cohorts comprising subjects before and after testicular suppression: (1) 6 healthy young men who were treated with androgens (Sustanon, Aspen Pharma, Dublin, Ireland); 2) 10 transgender girls (male sex assigned at birth) who were treated with 3-monthly GnRH agonist injections (Leuprorelinacetat, Abacus Medicine, Copenhagen, Denmark); and (3) 55 patients with prostate cancer who were randomized to surgical castration (bilateral subcapsular orchiectomy) or treatment with GnRH agonist (Triptorelin, Ipsen Pharma, Kista, Sweden). Serum INSL3 and testosterone concentrations were quantified in stored serum samples using validated liquid chromatography-tandem mass spectrometry methodologies, and LH was measured by an ultrasensitive immunoassay. RESULTS: The circulating concentrations of INSL3, testosterone, and LH decreased during experimental testicular suppression in healthy young men by Sustanon injections and subsequently returned to baseline levels after release of suppression. All 3 hormones decreased during therapeutic hormonal hypothalamus-pituitary-testicular suppression in transgender girls and in patients with prostate cancer. CONCLUSION: INSL3 resembles testosterone as a sensitive marker of testicular suppression and reflects Leydig cell function, also during exposure to exogenous testosterone. Serum INSL3 measurements may complement testosterone as a Leydig cell marker in male reproductive disorders, during therapeutic testicular suppression as well as in surveillance of illicit use of androgens.

Analysis of dried blood spots is a feasible alternative for detecting ephedrine in doping control.

Dried blood spot (DBS) testing allows fast, easy and minimally invasive collection of microvolumes of blood. In an anti-doping context, DBS testing has particular relevance for substances prohibited in-competition only such as ephedrine, which is currently detected by urine analysis, because DBS can add information about the blood drug concentrations during the in-competition period. Several collection methods and devices exist for DBS collection from different anatomical sites. Thus, agreements between concentrations of target analytes in DBS samples from different sampling sites, along with between DBS and those in conventional venous plasma samples, need to be evaluated. Herein, we collected matched upper-arm DBS, fingerprick DBS and venous plasma samples from eight healthy male subjects in an 8-h period following oral administrations of 20 mg ('low dose') and 60 mg ('high dose') of ephedrine. We show that the use of alternative sampling sites and matrices is a feasible possibility for ephedrine analysis in doping control. We observed very good agreement between collection sites and that specificity and sensitivity can be upheld despite use of an alternative collection site. However, potential concentration differences between DBS and venous plasma should be considered, and distinct threshold might be necessary if implementing both blood matrices in ephedrine analysis.

Stability and detectability of testosterone esters in dried blood spots after intramuscular injections.

While misuse of testosterone esters is widespread in elite and recreational sports, direct detection of intact testosterone esters in doping control samples is hampered by the rapid hydrolysis by esterases present in the blood. With dried blood spot (DBS) as sample matrix, continued degradation of the esters is avoided due to inactivation of the hydrolase enzymes in dried blood. Here, we have developed the method further for detection of testosterone esters in DBS with focus on robustness and applicability in doping control. To demonstrate the method's feasibility, DBS samples from men receiving two intramuscular injections of Sustanon® 250 (n = 9) or placebo (n = 10) were collected, transported, and stored prior to analysis, to mimic a doping control scenario. The presented nanoLC-HRMS/MS method appeared reliable and suitable for direct detection of four testosterone esters (testosterone decanoate, isocaproate, phenylpropionate, and propionate) after extraction from DBS. Sustanon® was detected in all subjects for at least 5 days, with detection window up to 14 days for three of the esters. Evaluation of analyte stability showed that while storage at room temperature is tolerated well for a few days, testosterone esters are highly stable (>18 months) in DBS when stored in frozen conditions. Collectively, these findings demonstrate the applicability of DBS sampling in doping control for detection of steroid esters. The fast collection and reduced shipment costs of DBS compared with urine and standard blood samples, respectively, will allow more frequent and/or large-scale testing to increase detection and deterrence.

No pain, just gain: Painless, easy, and fast dried blood spot collection from fingertip and upper arm in doping control.

This study aimed to determine and compare the perception, painfulness, and usability of the minimally invasive dried blood spot (DBS) collections from fingertip versus upper arm from different athlete populations: males and females representing sports dependent on hand/arm, sports less dependent on hand/arm and para-athletes. To accomplish this, 108 national level athletes from Denmark were recruited (♀ = 49, ♂ = 59, 25 ± 6 years; mean ± SD) and 11 Doping Control Officers (DCOs) collected manual fingerprick DBS (HemaSpot HF) and automated upper-arm DBS (Tasso-M20) from each athlete. Athletes and DCOs responded to questionnaires regarding the perception of sample collection procedures. On a 0-10 scale, the athletes reported a low pain score and a very good general experience for both sampling sites, but following upper-arm DBS collection, the associated pain was rated lower (-0.4 ± 1.6, p < 0.05), and the general experience rated better (+0.6 ± 2.3, p ≤ 0.001) than after the fingerprick DBS collection. The DCOs rated the general experience with the upper-arm DBS collection better (+1.6 ± 1.1, p ≤ 0.01) than the fingerprick DBS collection, partly because problems occurred more frequently during the DBS collection from the fingertip (28%) than from the upper arm (6%). In conclusion, it appears that DBS sampling is affiliated with minimal sensation of pain and is preferred by both DCOs and athletes, independent of gender and discipline, over conventional sample collection methods. Collection of DBS from the upper arm was preferred over fingerprick by both athletes and DCOs.

An Intramuscular Injection of Mixed Testosterone Esters Does Not Acutely Enhance Strength and Power in Recreationally Active Young Men.

Purpose: Limited data are available on the acute performance-enhancing effects of single-dose administration of testosterone in healthy humans. Studies of testosterone administrations to healthy humans are rare due to the difficult nature and necessity of close clinical monitoring. However, our unique physiological experimental facilities combined with close endocrinological collaboration have allowed us to safely complete such a study. We tested the hypothesis that an intramuscular injection of 250 mg mixed testosterone esters (TEs) enhances physical performance in strength and power exercises acutely, measured 24 h after injection. Additionally, we investigated whether the basal serum testosterone concentration influences the performance in countermovement jump (CMJ), 30-s all out cycle sprint, and one-arm isometric elbow flexion. Methods: In a randomized, double-blind, placebo-controlled design, 19 eugonadal men received either a TE (n = 9, 23 ± 1 years, 183 ± 7 cm, 83 ± 10 kg) or a PLA (n = 10, 25 ± 2 years, 186 ± 6 cm, 82 ± 14 kg) injection. Hormonal levels and the performance in CMJ, 30-s all out cycle sprint, and one-arm isometric elbow flexion were measured before and 24 h after injection. Results: Firstly, an intramuscular injection of 250 mg mixed TEs did not enhance the vertical jump height in a CMJ test, peak power, mean power, and fatigue index in a 30-s all-out cycle sprint or rate of force development and maximal voluntary contraction in a one-arm isometric elbow flexion 24 h post-injection. Secondly, baseline testosterone levels appeared not to influence performance in strength and power exercises to a large extent in healthy, recreationally active young men. Conclusion: A single intramuscular injection of 250 mg mixed TEs has no acute ergogenic effects on strength and power performance in recreationally active, young men. This novel information has implication for basic physiological understanding. Whether the same applies to an elite athlete population remains to be determined. If so, this would have implications for anti-doping efforts aiming to determine the most cost-efficient testing programs.

Single-dose administration of clenbuterol is detectable in dried blood spots.

Clenbuterol is a ß2 -agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time-consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 µg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post-ingestion, with additional urine collections on days 7 and 10. Using LC-MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post-ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7-10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 µg.

Changes in blood parameters after intramuscular testosterone ester injections - Implications for anti-doping.

Testosterone treatment stimulates the production of red blood cells and alters iron homeostasis. Thus, we investigated whether the 'haematological module' of the athlete biological passport (ABP) used by the World Anti-Doping Agency can be used to indicate misuse of testosterone. Nineteen eugonadal men received intramuscular injections of either 250 mg Sustanon®, a blend of four testosterone esters, or placebo on days 0 and 21 in a randomized, placebo-controlleddouble-blind design. Urine samples and blood samples were collected twice pre-treatment, at least 5 days apart, and on days 1, 3, 5, 10 and 14 post-injections to assess steroidal and haematological biomarkers of the ABP. The steroidal profile was flagged suspicious in all Sustanon®-treated subjects, whereas the haematological profile was flagged suspicious in six out of nine subjects. When both sensitivity and specificity were considered, reticulocyte percentage (RET%) appeared as the best marker of the haematological module for implying testosterone ester misuse. Atypical blood passport samples were used to select time points for further isotope-ratio mass spectrometry (IRMS) analysis of testosterone and its metabolites in simultaneously collected urine. In addition to the testosterone (T) to epitestosterone (E) ratio, the RET% and OFF-Score could help identify suspicious samples for more targeted IRMS testing. The results demonstrate that unexpected fluctuations in RET% can indicate testosterone doping if samples are collected 3-10 days after injection. From an anti-doping perspective, the haematological and steroidal modules of the ABP should complement each other when planning targeted follow-up testing and substantiating likely misuse of testosterone.

Autologous Blood Transfusion Enhances Exercise Performance-Strength of the Evidence and Physiological Mechanisms.

This review critically evaluates the magnitude of performance enhancement that can be expected from various autologous blood transfusion (ABT) procedures and the underlying physiological mechanisms. The review is based on a systematic search, and it was reported that 4 of 28 studies can be considered of very high quality, i.e. placebo-controlled, double-blind crossover studies. However, both high-quality studies and other studies have generally reported performance-enhancing effects of ABT on exercise intensities ranging from ~70 to 100% of absolute peak oxygen uptake (VO2peak) with durations of 5-45 min, and the effect was also seen in well-trained athletes. A linear relationship exists between ABT volume and change in VO2peak. The likely correlation between ABT volume and endurance performance was not evident in the few available studies, but reinfusion of as little as 135 mL packed red blood cells has been shown to increase time trial performance. Red blood cell reinfusion increases endurance performance by elevating arterial oxygen content (CaO2). The increased CaO2 is accompanied by reduced lactate concentrations at submaximal intensities as well as increased VO2peak. Both effects improve endurance performance. Apparently, the magnitude of change in haemoglobin concentration ([Hb]) explains the increase in VO2peak associated with ABT because blood volume and maximal cardiac output have remained constant in the majority of ABT studies. Thus, the arterial-venous O2 difference during exercise must be increased after reinfusion, which is supported by experimental evidence. Additionally, it remains a possibility that ABT can enhance repeated sprint performance, but studies on this topic are lacking. The only available study did not reveal a performance-enhancing effect of reinfusion on 4 × 30 s sprinting. The reviewed studies are of importance for both the physiological understanding of how ABT interacts with exercise capacity and in relation to anti-doping efforts. From an anti-doping perspective, the literature review demonstrates the need for methods to detect even small ABT volumes.

Coagulation competence and fluid recruitment after moderate blood loss in young men.

The coagulation system is activated by a reduction of the central blood volume during orthostatic stress and lower body negative pressure suggesting that also a blood loss enhances coagulation. During bleeding, however, the central blood volume is supported by fluid recruitment to the circulation and redistribution of the blood volume. In eight supine male volunteers (24 ± 3 years, blood volume of 6.9 ± 0.7 l; mean ± SD), 2 × 450 ml blood was withdrawn over ∼ 30 min while cardiovascular variables were monitored. Coagulation was evaluated by thrombelastography, and fluid recruitment was estimated by red blood cell count. Withdrawing 900 ml blood increased heart rate (62 ± 7 to 69 ± 13 bpm, P < 0.05; mean ± SD) and reduced stroke volume (113 ± 12 to 96 ± 14 ml, P < 0.05) leaving cardiac output, mean arterial pressure, and total peripheral resistance unchanged and, furthermore, reduced red blood cell count (4.80 ± 0.33 to 4.64 ± 0.37 × 10(12) cells l(-1), P < 0.05) indicating that 218 ± 173 ml fluid was recruited to the circulation. Withdrawing 450 ml blood reduced the time until initial fibrin formation (R: 6.5 ± 0.9 to 5.1 ± 1.0 min, P < 0.01), whereas the rate of clot formation increased after withdrawal of 900 ml blood (α-Angle: 66 ± 4 to 70 ± 3 deg, P < 0.01). Clot strength (maximal amplitude: 57 ± 4 mm), clot lysis 30 min after maximal amplitude (LY30: 0.8% [0-3.5%] (median [range])), and platelet count (218 ± 25 × 10(9) l(-1)) were unaffected. For supine males, ∼ 25% of a moderate blood loss is compensated by fluid recruitment to the circulation, which may explain the minor cardiovascular response. Yet, a blood loss of 450 ml accelerates coagulation, and this is further accentuated when blood loss is 900 ml.

Blood manipulation: current challenges from an anti-doping perspective.

The delivery of oxygen is the limiting factor during whole-body endurance exercise in well-trained individuals, so manipulating the amount of hemoglobin in the blood results in changes in endurance exercise capacity. Athletes began using novel erythropoiesis-stimulating agents well before they were approved for medical use. Older manipulation practices, such as autologous blood transfusions or the administration of first-generation recombinant human erythropoietins, are still widely abused due to challenges in their detection. More recent performance enhancement maneuvers include efforts to mask doping and to induce increased endogenous erythropoietin expression. Confessions by athletes have revealed an ongoing yet extremely sophisticated modus operandi when manipulating the blood. In this review, weaknesses in detection methods and sample collection procedures are scrutinized and strategies developed to circumvent the test system discussed.

Detection of autologous blood transfusions in athletes: a historical perspective.

Autologous blood transfusions (ABTs) has been used by athletes for approximately 4 decades to enhance their performance. Although the method was prohibited by the International Olympic Committee in the mid 1980s, no direct detection method has yet been developed and implemented by the World Anti-Doping Agency (WADA). Several indirect methods have been proposed with the majority relying on changes in erythropoiesis-sensitive blood markers. Compared with the first methods developed in 1987, the sensitivity of subsequent tests has not improved the detection of blood doping. Nevertheless, the use of sophisticated statistical algorithms has assured a higher level of specificity in subsequent detection models, which is a crucial aspect of antidoping testing particularly to avoid "false positives." Today, the testing markers with the best sensitivity/specificity ratio are the Hbmr model (an algorithm based on the total amount of circulating hemoglobin level [hemoglobin level mass] and percentage of reticulocytes, 4.51·ln(Hbmass)-√%ret) and the OFF-hr model (algorithm based on hemoglobin level concentration and percentage of reticulocytes, Hb(g/L)-60·âˆš%ret). Only the OFF-hr model is currently approved by WADA. Recently, alternative indirect strategies for detecting blood doping have been proposed. One method is based upon a transfusion-induced immune-response resulting in specific changes in gene expression related to leukocytes such as T lymphocytes. Another method relies on detecting increased plasticizer metabolite levels in the urine caused by the leakage of plasticizers from the blood bags used during the blood storage. These methods need further development and validation across different types of transfusion regimes before they can be implemented. In addition, several research projects have been funded by WADA in recent years and are now under development including "Detection of Autologous Blood Transfusions Using Activated Red Blood Cells (the red blood cells eNOS system)" and "Detection of Autologous Blood Transfusion by Proteomic: Screening to find Unique Biomarkers, Detecting Blood Manipulation from Total Hemoglobin Mass using 15-nitric Oxide as a Tracer Gas, Storage Contamination as a Potential Diagnostic Test for Autologous Blood Transfusion and Test for Blood Transfusion (Autologous/Homologous) based on Changes of Erythrocyte Membrane Protome" (WADA, WADA Funded Research Projects. http://www.wada-ama.org/en/Science-Medicine/Research/Funded-Research-Projects/. 2010). Although strategies to detect autologous blood transfusion have improved, a highly sensitive test to detect small volumes of transfused autologous blood has not yet been implemented.

[Changes in blood profiles during Tour de France 2007]. / Aendringer i blodprofiler under Tour de France 2007.

INTRODUCTION: Marked changes in different blood variables, e.g. hemoglobin concentration ([Hb]) and hematocrit (Hct), are indications of blood manipulation. Blood sampling and the development of individual, longitudinal blood profiles have therefore been implemented in doping control in different sports federations. MATERIALS AND METHODS: During the Tour de France 2007, 7 riders were randomly tested on 3 different occasions; the day before the prologue, and 12 and 19 days after the prologue. Blood was drawn into 3 mL EDTA covered tubes and kept at 4 degrees Celsius. They were analyzed within 24 hours on a Sysmex R-500. RESULTS: [Hb] and Hct were significantly lower on day 12 (p = 0.005) and day 19 (p < 0.001) compared to baseline. All 7 riders had lower [Hb] and Hct on day 19 compared to baseline, while this was the case in 6 out of 7 riders already on day 12. The [Hb] and Hct were 11.5% and 12.1% lower on day 19 compared to baseline. CONCLUSION: We observed significant decreases in [Hb] and Hct in 7 riders during Tour de France 2007. Whether or not this is due to decrease in hemoglobin mass or hemodilution, or the latter solely, increases in [Hb] and Hct during prolonged stage racing seem unphysiological and should therefore lead to further examination of the rider.

Detection of darbepoetin alfa misuse in urine and blood: a preliminary investigation.

INTRODUCTION: Darbepoetin alfa is a modified erythropoietin (EPO) molecule with a longer serum half-life than recombinant human erythropoietin (rhEPO). Because the detection period of rhEPO in urine is only 2-3 d after the last injection, blood algorithms have been developed in order to expand the detection window of rhEPO misuse. The main objectives were to establish the period of detection of darbepoetin alfa by isoelectric focusing (IEF) and examine the applicability of blood algorithms and individual variations in blood variables in an antidoping context. METHODS: Six recreationally active males and six recreationally active females had 0.78 microg.kg(-1).wk(-1) of darbepoetin alfa administered for 3 wk. Blood and urine samples were collected continuously during and after administration. Urine samples were analyzed by IEF and immunoblotting for darbepoetin alfa, and blood samples were analyzed for erythropoietic sensitive blood variables on a hematological analyzer. RESULTS: Darbepoetin alfa was detected in 8 of 12 samples at 10 d after the last injection. Ten subjects showed variations in hemoglobin concentration [Hb] > 10%, whereas only three males and one female exceeded suggested upper [Hb] limits of 17.0 and 16.0 g.dL(-1), respectively. Four subjects exceeded the 1:1000 ON- as well as the OFF-model cutoff limit. CONCLUSION: The large number of samples containing detectable amounts of darbepoetin alfa at 10 d into the washout period stipulate the possibility of a 7-d window of detection after administration, wherein a sample would be regarded as an adverse analytical finding. The marked variations in all examined blood parameters could be used for the targeting of urine samples. These preliminary findings open up for larger scale studies with more frequent urine sampling in the washout period on elite athletes.