Doping Control Samples Research Articles

Discovery of c.577del in EPO: Investigations into endogenous EPO double-band detected in blood with SAR-PAGE.

Recently, some athletes were repetitively found to have rEPO positive results, including a characterized double-band pattern in blood samples, in routine doping analysis. In contrast to previous findings from excretion studies, this double-band pattern showed the same relative intensity even when the samples were collected weeks (/months) apart. We therefore suspected that these "positive" doping control samples were related with a novel pathway of endogenous EPO production. Thus, follow-up investigations were warranted to characterize the origin of such analytical test results and to avoid the issuing of adverse analytical findings in the absence of rEPO by identifying the root cause of these "constantly positives." In this study, we designed and conducted a series of causal studies, including population screening of EPO profiles, exploration of EPO de-N-glycosylation, single nucleotide polymorphism (SNP) browsing in EPO, sequencing of EPO exons, genealogical analysis of the c.577del EPO variant, and finally expression and investigation of mutant EPO. In summary, we found that these "constantly positives" were related to endogenous EPO production associated with the c.577del EPO variant. The frequency of this variant was 0.39% in our Chinese population pool. The mutant EPO encoded by this variant is 27 amino acids longer than the wild-type. The molecular weight of this mutant EPO is approximately the same as that of rEPO, exhibiting a similar electrophoretic behavior. To prevent charges against carriers of the c.577del variant, a revised rEPO testing strategy has been implemented in the new version of TD EPO.

Emergence of the less common cannabinoid Δ8 -Tetrahydrocannabinol in a doping sample.

Δ8 -Tetrahydrocannabinol (Δ8 -THC) as isomer of the well-known Δ9 -THC has a similar mode of action, and the potency was estimated to be two thirds compared with Δ9 -THC. Content of Δ8 -THC in plant material is low, but formulations containing Δ8 -THC in high concentrations are gaining popularity. Δ8 -THC is to be regarded as prohibited substance according to the Prohibited List of the World Anti-Doping Agency (WADA). Contradictory results between initial testing procedure and confirmatory quantitation for 11-Nor-9-carboxy-Δ9 -tetrahydrocannabinol (Δ9 -THC-COOH) of a doping control sample gave rise for follow-up testing procedures. After alkaline hydrolysis and liquid-liquid extraction, the sample was analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) using isocratic elution instead of gradient elution, which is used for standard procedure. Isocratic elution resulted in two peaks instead of one using gradient elution. Both peaks showed same fragmentation. Using certified reference materials, one peak could be assigned to Δ9 -THC-COOH and the other one with higher intensity to the less common 11-Nor-9-carboxy-Δ8 -Tetrahydrocannabinol (Δ8 -THC-COOH) in a concentration of approximately 1200 ng/ml. As complementary method, gas chromatography tandem mass spectrometry (GC-MS/MS) can also be used for identification. Here Δ8 - and Δ9 -THC-COOH can be distinguished by chromatography and by fragmentation. Additional investigations of doping control samples containing Δ9 -THC-COOH revealed the simultaneous presence of Δ8 -THC-COOH in low concentrations (0.22-8.91 ng/ml) presumably due to plant origin. Percentage of Δ8 -THC-COOH varies from 0.05 to 2.83%. In vitro experiments using human liver microsomes showed that Δ8 -THC is metabolized in the same way as Δ9 -THC.

In vitro metabolism of the REV-ERB agonist SR-9009 and subsequent detection of metabolites in associated routine equine plasma and urine doping control samples.

SR-9009 is a synthetic compound widely available to purchase online as 'supplement' products due to its potential performance-enhancing effects, presenting a significant threat with regard to doping control in sport. In vitro metabolism with equine liver microsomes was performed to identify potential targets for detection of SR-9009. Six metabolites were identified, with the most abundant consisting of N-dealkylated metabolites (M1-M3). The addition of the identified metabolites to high-resolution accurate mass databases resulted in a positive finding for the N-dealkylated metabolite M1 of SR-9009 in an associated plasma and urine doping sample. Liquid chromatography-high-resolution mass spectrometry was used to verify the presence of the N-dealkylated metabolite (M1) in both matrices, with a low concentration of the parent compound and additional N-desalkyl metabolites (M2 and M3) detected in the plasma sample as supporting evidence of administration. To the best of the authors' knowledge, this is the first report of an adverse analytical finding in an equine sample for SR-9009 or its metabolites in equine doping control.

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 5days, 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.

First Steps toward Uncovering Gene Doping with CRISPR/Cas by Identifying SpCas9 in Plasma via HPLC-HRMS/MS.

The discovery of the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system as a programmable, RNA-guided endonuclease has revolutionized the utilization of gene technology. Because it enables the precise modification of any desired DNA sequence and surpasses all hitherto existing alternatives for gene editing in many ways, it is one of the most frequently used tools for genome editing. However, these advantages also potentially facilitate the illicit use of the CRISPR/Cas system in order to achieve performance-enhancing effects in sporting competitions. This abuse is classified as gene doping, which is banned in sports according to the Prohibited List of the World Anti-Doping Agency (WADA). Therefore, there is a pressing need for an adequate analytical method to detect the misuse of the CRISPR/Cas system by athletes. Hence, the first aim accomplished with this study was the identification of the exogenous protein Cas9 from the bacterium Streptococcus pyogenes (SpCas9) in plasma samples by means of a bottom-up analytical approach via immunoaffinity purification, tryptic digestion, and subsequent detection by HPLC-HRMS/MS. A qualitative method validation was conducted with three specific peptides allowing for a limit of detection of 25 ng/mL. Additionally, it was shown that the developed method is also applicable to the detection of (illicit) gene regulation through the identification of catalytically inactive Cas9. A proof-of-concept administration study employing an in vivo mouse model revealed a detection window of SpCas9 for up to 8 h post administration, confirming the suitability of the test strategy for the analysis of authentic doping control samples.

Evaluation of erythropoietin biosimilars Epotin™, Hemax® and Jimaixin™ by electrophoretic methods used for doping control analysis and specific N-glycan analysis revealed structural differences from original epoetin alfa drug Eprex®

Recombinant human erythropoietin (rEPO) biosimilars are copies of epoetin drugs developed after the first patents ended. However differences in the process of production can result in small structural differences when compared to the reference product. Differences in N-glycosylation profiles are of particular importance for rEPOs, because they can drastically impact the half-life in circulation and activity. Changes of structure can also impact electrophoretic profiles that are used to reveal the presence of a rEPO in a doping control sample. In this study three not well characterized biosimilars were evaluated (Jimaixin™ authorized in China, and Hemax® and Epotin™ authorized in Algeria). As these products could be used for doping, first their EPO profiles were determined using the antidoping methods (electrophoretic separation by the charge (isolectric focusing, IEF-PAGE) or the molecular weight (SDS-PAGE) and specific EPO immunodetection). Compared to the original epoetin alfa Eprex®, it revealed more basic isoforms for Epotin™ and Jimaixin™ after IEF-PAGE and a slightly lower molecular weight after SDS-PAGE in particular for Hemax®. To better understand the reason for these differences, EPO specific N-glycans were evaluated using two complementary approaches: MALDI-TOF mass spectrometry (MS) and hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection. All three biosimilars presented a significant decrease in the major glycan forms of Eprex® along with an increase in less complex forms. Jimaixin™ and Epotin™ presented also a lower amount of fully sialylated forms. HILIC method also showed that O-acetylation level of sialic acid residues might vary from one rEPO to the other.

Research on spiking rat EPO as internal standard in doping control samples for detection of EPO using SAR-PAGE analysis with biotinylated primary antibody.

According to the current Technical Document (TD) for erythropoietin (EPO), SAR-PAGE is the most commonly applied method for both screening and confirmation procedures. Although this method is effective and robust, it lacks an internal standard (IS) to monitor the efficiency of analysis for each sample covering every step of the whole procedure, including preparation, immunopurification, and western blotting. This internal standard needs to be recognized by both anti-EPO antibodies used for immunopurification and western blotting, respectively. Besides that, the band of IS could not be allowed to interfere with the recognition of all types of targeted EPO and analogs. To meet these two principles, rat EPO was selected. In this study, rat EPO was used to spike both urine and blood samples at the beginning of analysis. After preparation and immunopurification, single blotting was performed with biotinylated AE7A5 as the primary antibody, followed by incubation with streptavidin-coupled HRP. Based on the comparison of different immunopurification methods, the AB-286-NA antibody coupled to M-280 magnetic beads was the better choice for urine samples, whereas the MAIIA column was suitable for blood samples. All these methods were validated for selectivity, repeatability, and sensitivity. The modified method in this study could not only eliminate the cross-reactivity between antibodies but also monitor the whole procedure of the analysis of EPO with spiked rat EPO. Besides that, rat EPO could also be used as an indicator for monitoring the presence of protease(s) in urine samples.

Determination of anabolic androgenic steroids as imidazole carbamate derivatives in human urine using liquid chromatography-tandem mass spectrometry.

Anabolic androgenic steroids are widely abused substances in sports doping. Their detection present limitations regarding the use of soft ion sources such as electrospray or atmospheric pressure chemical ionization by liquid chromatography-tandem mass spectrometry. In the current study, a novel derivatization method was developed for the ionization enhancement of selected anabolic androgenic steroids. The proposed method aims at the introduction of an easily ionizable moiety into the steroid molecule by converting the hydroxyl groups into imidazole carbamates using 1,1'-carbonyldiimidazole as derivatization reagent. The proposed method was applied to water and urine samples spiked with exogenous anabolic androgenic steroids in various concentration levels. Steroid imidazole carbamate derivatives have shown intensive [M+H]+ signals under electrospray ionization and common fragmentation patterns in tandem mass spectrometry mode with [M-CO2 +H]+ and [M-ΙmCO2 +H]+ as major ions with low collision energy. The obtained results showed that the majority of steroids were detectable at concentrations equal or lower to their minimum required performance level according to the World Anti-Doping Agency technical document. The proposed method is sensitive with a preparation procedure that could be easily applied to the analysis of doping control samples.

Detecting the misuse of 7-oxo-DHEA by means of carbon isotope ratio mass spectrometry in doping control analysis.

The misuse of 7-oxo-DHEA (3β-hydroxyandrost-5-ene-7,17-dione) is prohibited according to the World Anti-Doping Agency (WADA) code. Nevertheless, it is easily available as a dietary supplement and from black market sources. In two recent doping control samples, significant amounts of its main metabolite 7β-OH-DHEA were identified, necessitating further investigations. As both 7-oxo-DHEA and 7β-OH-DHEA are endogenously produced steroids and no concentration thresholds applicable to routine doping controls exist, the development and validation of a carbon isotope ratio (CIR) mass spectrometry method ha been desirable. Excretion studies encompassing 7-oxo-DHEA, 7-oxo-DHEA-acetate, and in-house deuterated 7-oxo-DHEA were conducted and evaluated with regard to urinary CIR and potential new metabolites of 7-oxo-DHEA. Numerous urinary metabolites were identified, some of which have not been reported before, while others corroborate earlier findings on the metabolism of 7-oxo-DHEA. The CIRs of both 7-oxo-DHEA and 7β-OH-DHEA were significantly influenced for more than 50 h after a single oral dose of 100 mg, and a novel metabolite (5α-androstane-3β,7β-diol-17-one) was found to prolong this detection time window by approximately 25 h. Applying the validated method to routine doping control specimens presenting atypically high urinary 7β-OH-DHEA levels clearly demonstrated the exogenous origin of 7-oxo-DHEA and 7β-OH-DHEA. As established for other endogenously produced steroids such as testosterone, the CIR allows for a clear differentiation between endo- and exogenous sources of 7-oxo-DHEA and 7β-OH-DHEA. The novel metabolites detected after administration may help to improve the detection of 7-oxo-DHEA misuse and simplify its detection in doping control specimens.

Analysis of luteinizing hormone (LH): Validation of a commercial ELISA kit for LH analysis and quantification in doping control samples.

Luteinizing hormone (LH) is a dimeric glycoprotein produced and secreted by the pituitary gland, with a molecular weight of approximately 30 000 Da. The main clinical use for exogenous LH-administration is typically linked to the treatment of infertility, in both men and women. The desired effect of LH misuse in sport is due to the enhancement of testicular production of testosterone. Elevated LH levels may also indicate the usage of gonadotropin-releasing factors or estrogen blockers. Therefore, LH is listed by the World Anti-Doping Agency (WADA) as a prohibited substance in male athletes, and according to the WADA technical document, laboratories should determine the the total LH concentrations in urine. The TD lists two different assays that are suitable for measuring total LH in urine, Delfia and Siemens Immulite. Other fit-for-purpose assays can be used, as long as they are capable of detecting total LH in urine. In case an assay not listed in the TD will be used, population-based reference values have to be determined in the validation procedure. In this study a new immunoassay was validated for the measurement of LH in urine. The assay (AccuBind ELISA Microwells, Luteinizing Hormone, Monobind Inc.), originally intended for serum, showed adequate sensitivity and was proven fit-for-purpose in routine doping control. Population-based distribution of the assay was in good agreement with the results of Delfia and Immulite assays, for which the method-specific cut-off-values are 40 IU/L (Delfia) and 60 IU/L (Siemens Immulite).

Studies on the in vivo metabolism of methylstenbolone and detection of novel long term metabolites for doping control analysis.

The anabolic-androgenic steroid methylstenbolone (MSTEN; 2α,17α-dimethyl-17β-hydroxy-5α-androst-1-en-3-one) is available as a so-called designer steroid or nutritional supplement. It is occasionally detected in doping control samples, predominantly tested and confirmed as the glucuronic acid conjugate of methylstenbolone. The absence of other meaningful metabolites reported as target analytes for sports drug testing purposes can be explained by the advertised metabolic stability of methylstenbolone. In 2013, a first investigation into the human metabolism of methylstenbolone was published, and two hydroxylated metabolites were identified as potential targets for initial testing procedures in doping controls. These metabolites were not observed in recent doping control samples that yielded adverse analytical findings for methylstenbolone, and in the light of additional data originating from a recent publication on the in vivo metabolism of methylstenbolone in the horse, revisiting the metabolic reactions in humans appeared warranted. Therefore, deuterated methylstenbolone together with hydrogen isotope ratio mass spectrometry (IRMS) in combination with high accuracy/high resolution mass spectrometry were employed. After oral administration of a single dose of 10 mg of doubly labeled methylstenbolone, urine samples were collected for 29 days. Up to 40 different deuterated methylstenbolone metabolites were detected in post-administration samples, predominantly as glucuronic acid conjugates, and all were investigated regarding their potential to prolong the detection window for doping controls. Besides methylstenbolone excreted glucuronidated, three additional metabolites were still detectable at the end of the study on day 29. The most promising candidates for inclusion into routine sports drug testing methods (2α,17α-dimethyl-5α-androst-1-ene-3β,17β-diol and 2α,17α-dimethyl-5α-androst-1-ene-3α,17β-diol) were synthesized and characterized by NMR.

Targeting the administration of ecdysterone in doping control samples

PurposeThe phytosteroid ecdysterone was recently reported to enhance performance in sports and may thus be considered as a substance of relevance in anti-doping control. To trace back an administration of ecdysterone from urine samples, analytical properties have been investigated to assess its integration into initial testing procedures (ITP) in doping control laboratories.MethodsAnalytical properties of ecdysterone were evaluated using gas chromatography-quadrupole/time-of-flight-mass spectrometry (GC–QTOF–MS) and liquid chromatography (LC)–QTOF–MS. Its metabolism and elimination in human were studied using urines collected after administration.ResultsThe detectability of ecdysterone by GC–MS (after derivatization) and/or LC–MS(/MS) has been demonstrated and sample preparation methods were evaluated. Dilute-and-inject for LC–MS(/MS) or solid phase extraction using Oasis HLB for GC–MS or LC–MS were found most suitable, while liquid–liquid extraction was hampered by the high polarity of ecdysteroids.Most abundantly, ecdysterone was detected in the post administration urines as parent compound besides the metabolite desoxy-ecdysterone. Additionally, desoxy-poststerone was tentatively assigned as minor metabolite, however, further investigations are needed.ConclusionAn administration of ecdysterone can be targeted using existing procedures of anti-doping laboratories. Ecdysterone and desoxy-ecdysterone appeared as suitable candidates for integration in ITP. Using dilute-and-inject a detection of the parent compound was possible for more than 2 days after the administration of a single dose of ~ 50 mg.

The Use of Samples Originating From Doping Control Procedures for Research Purposes: A Qualitative Study.

Doping control samples may be used for research purposes by the World Anti-Doping Agency (WADA)-accredited laboratories after their compulsory storage period has expired. This study investigates opinions of stakeholders toward the governance of antidoping research on these samples and to evaluate the current framework. Semistructured interviews were conducted with stakeholders in antidoping research. The distinction between research and quality assurance in the International Standard for Laboratories (ISL) is neither well-understood nor interpreted uniformly by WADA-accredited labs. Most laboratories would not seek ethics approval for research on doping control samples. Interviewees considered that athletes should be better informed on what antidoping research can entail. A consistent and uniform approach toward the consent should be employed worldwide. Standards and safeguards should be implemented to reduce the risk of reidentification. Centralization of the Informed Consent Form in the ADAMS (Anti-Doping Administration & Management System) database would facilitate providing more information and allow the implementation of the right to withdraw.

Assessing serum albumin concentration following exercise-induced fluid shifts in the context of the athlete biological passport.

The hydration status of an athlete at the time of a doping control sample collection is an important factor to consider when reviewing athlete biological passports (ABPs). Dehydration results in a reduction of the circulating plasma volume (PV), which may lead to artificially high values of some blood parameters. This study aimed to identify whether serum albumin could serve as a single marker of fluid shifts, which are not currently accounted for in the hematological passport. An additional marker could be used to assist experts when interpreting irregularities in the ABP. Twelve subjects underwent multiple controlled exercise trials designed to induce varying levels of PV shifts. Pre-exercise blood samples were collected to establish baseline values for individual passports. During exercise interventions, blood samples were collected before the start of exercise and at 10 minute, 1 hour, 2 hours, and 24 hours following exercise. Significant increases in hematological parameters - hemoglobin [Hb], hematocrit (HCT), albumin (ALB), and calculated OFF-score - were identified at varying time points following fluid shift-inducing exercise. Changes in ALB correlated strongly with changes in [Hb] (r=0.753) and with estimated PV shifts (r=-0.764). In analyzing ABPs, the resulting increases in Hb did not trigger any atypical findings at 99% specificity. Monitoring changes in ALB longitudinally may assist experts when reviewing PV shifts in the biological passport.

Urine Caffeine Concentration in Doping Control Samples from 2004 to 2015.

The ergogenic effect of caffeine is well-established, but the extent of its consumption in sport is unknown at the present. The use of caffeine was considered “prohibited” until 2004, but this stimulant was moved from the List of Prohibited Substances to the Monitoring Program of the World Anti-Doping Agency to control its use by monitoring urinary caffeine concentration after competition. However, there is no updated information about the change in the use of caffeine as the result of its inclusion in the Monitoring Program. The aim of this study was to describe the changes in urine caffeine concentration from 2004 to 2015. A total of 7488 urine samples obtained in official competitions held in Spain and corresponding to athletes competing in Olympic sports (2788 in 2004, 2543 in 2008, and 2157 in 2015) were analyzed for urine caffeine concentration. The percentage of samples with detectable caffeine (i.e., >0.1 μg/mL) increased from ~70.1%, in 2004–2008 to 75.7% in 2015. The median urine caffeine concentration in 2015 (0.85 μg/mL) was higher when compared to the median value obtained in 2004 (0.70 μg/mL; p < 0.05) and in 2008 (0.70 μg/mL; p < 0.05). The urine caffeine concentration significantly increased from 2004 to 2015 in aquatics, athletics, boxing, judo, football, weightlifting, and rowing (p < 0.05). However, the sports with the highest urine caffeine concentration in 2015 were cycling, athletics, and rowing. In summary, the concentration of caffeine in the urine samples obtained after competition in Olympic sports in Spain increased from 2004 to 2015, particularly in some disciplines. These data indicate that the use of caffeine has slightly increased since its removal from the list of banned substances, but urine caffeine concentrations suggest that the use of caffeine is moderate in most sport specialties. Athletes of individual sports or athletes of sports with an aerobic-like nature are more prone to using caffeine in competition.

Case Study: Atypical δ13 C values of urinary norandrosterone.

Isotope ratio mass spectrometry (IRMS) has been established in doping control analysis to identify the endogenous or exogenous origin of a variety of steroidal analytes including the 19-norsteroid metabolite norandrosterone (NorA). NorA can be found naturally in human urine in trace amounts due to endogenous demethylation or in situ microbial degradation. The administration of nortestosterone (nandrolone) or different prohormones results in the excretion of urinary NorA. Usually, this can be detected by IRMS due to differing δ13 C values of synthetic 19-norsteroids compared to endogenous reference compounds. The consumption of uncastrated pig edible parts like offal or even meat may also lead to a urinary excretion of NorA. In order to determine the δ13 C values of such a scenario, urine samples collected after consumption of a wild-boar-testicle meal were analyzed. IRMS revealed highly enriched δ13 C values for urinary NorA, which could be related to the completely corn-based nutrition of the animal. Isotopic analysis of the boar's bristles demonstrated a dietary change from C3 -based forage, probably in winter and spring, to a C4 -based diet in the last weeks to months prior to death. These results supported the interpretation of an atypical test result of a Central European athlete's doping control sample with δ13 C values for NorA of -18‰, most probably caused by the consumption of a wild boar ragout. As stated before, athletes should be fully aware of the risk that consumption of wild boar may result in atypical or even adverse analytical findings in sports drug testing.

Multiple incidence of the prescription diuretic hydrochlorothiazide in compounded nutritional supplements.

Diuretic agents are prohibited in sports in- and out-of-competition according to the regulations of the World Anti-Doping Agency (WADA) because of their possible masking effects on other doping agents in urine samples, and their ability to produce fast acute weight losses. Despite previous studies reported adverse analytical findings (AAFs) resulting from contaminations at ppm level (μg/g) of medicinal products, and recommended to introduce reporting limits for diuretics in doping controls, these are not adopted in analyses performed by WADA-accredited laboratories. We report the case of an athlete with two AAFs for hydrochlorothiazide (HCTZ) at low urinary concentrations (<10ng/mL), who declared the use of nutritional supplements prepared in a compounding pharmacy. His nutritional supplements were analyzed revealing HCTZ presence in different concentrations, at the ppm level (μg/g and ng/mL). With the aim of testing the plausibility of the observed urinary HCTZ concentrations with the nutritional supplement ingestion, a urinary excretion study with three healthy volunteers was performed. HCTZ-contaminated powder (6.4μg/g of HCTZ) was administered to each subject in different dosages, reproducing the possible ingestion pattern occurred. Urine specimens were collected before and after ingestion of the powder, up to 24hours, and underwent liquid-liquid extraction and liquid chromatography-tandem mass spectrometry determination. Post-administration specimens were found to contain HCTZ at concentrations of 5-230ng/mL, which supported the accidental inadvertent intake of the prohibited substance by the athlete. This study makes the argument that the introduction of reporting limits for diuretics are warranted in doping control samples, in order to protect against inadvertent AAFs due to contaminated products.

Combined immuno-purification and detection of recombinant erythropoietins and activin receptor type II-Fc fusion proteins by isoelectric focusing for application in doping control.

Iso-electric focusing (IEF) was the first method established to discriminate endogenous and recombinant erythropoietins (rEPOs). It is still approved by the World Anti-Doping Agency (WADA) as an initial testing procedure to detect erythropoiesis stimulating agents (ESAs) in doping control samples. However EPO-Fc, one of the prohibited rEPOs designated by WADA, is not detectable with the actual IEF conditions. Other newly developed ESAs - luspatercept and sotatercept, both activin receptor type II-Fc fusion proteins (ActRII-Fc) - are also now prohibited and could be used in combination with rEPOs. Methods of identification of ActRII-Fc in blood by SAR/SDS-PAGE have been described, but not by IEF. Here we detail improvements in blood sample preparation and IEF analysis: A combined immuno-purification of EPOs and ActRII-Fc proteins in a single procedure, an appropriate isoforms separation for all proteins using new pre-loading and gel conditions, and a single detection of all rEPOs and ActRII-Fc proteins after successive incubation with anti-EPO and anti-ActRII antibodies. With these changes, distinctive profiles for all the ESAs were obtained by IEF. Therefore, IEF could be used as a screening method to detect a wide spectrum of prohibited ESAs in blood samples prior to specific confirmation for the identified rEPO or ActRII-Fc.

Detection of prohibited substances in equine hair by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry - application to doping control samples.

The detection of drugs in human hair samples has been performed by laboratories around the world for many years and the matrix is popular in disciplines, such as workplace drug testing. To date, however, hair has not become a routinely utilised matrix in sports drug detection. The analysis of hair samples offers several potential advantages to doping control laboratories, not least of which are the greatly extended detection window and the ease of sample collection and storage. This article describes the development, validation, and utilisation of a sensitive ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS) method for the detection of 50 compounds. This provides significantly improved coverage for those analytes which would be of particular interest if detected in hair, such as anabolic steroid esters and selective androgen receptor modulators (SARMs). Qualitative validation of the method resulted in estimated limits of detection as low as 0.1pg/mg for the majority of compounds, with all being detected at 2pg/mg or below. The suitability of the method for the detection of prohibited substances in incurred material was demonstrated by the successful detection of several compounds, such as stanozolol, boldenone undecylenate, clenbuterol, and GW-501516, in genuine equine hair samples. Estimated concentrations of the detected substances ranged from 0.27 to 8.6pg/mg. The method has been shown to be fit-for-purpose for routine screening of equine hair samples by the analysis of over 400 genuine hair samples.

Analytical challenges in sports drug testing.

Analytical chemistry represents a central aspect of doping controls. Routine sports drug testing approaches are primarily designed to address the question whether a prohibited substance is present in a doping control sample and whether prohibited methods (for example, blood transfusion or sample manipulation) have been conducted by an athlete. As some athletes have availed themselves of the substantial breadth of research and development in the pharmaceutical arena, proactive and preventive measures are required such as the early implementation of new drug candidates and corresponding metabolites into routine doping control assays, even though these drug candidates are to date not approved for human use. Beyond this, analytical data are also cornerstones of investigations into atypical or adverse analytical findings, where the overall picture provides ample reason for follow-up studies. Such studies have been of most diverse nature, and tailored approaches have been required to probe hypotheses and scenarios reported by the involved parties concerning the plausibility and consistency of statements and (analytical) facts. In order to outline the variety of challenges that doping control laboratories are facing besides providing optimal detection capabilities and analytical comprehensiveness, selected case vignettes involving the follow-up of unconventional adverse analytical findings, urine sample manipulation, drug/food contamination issues, and unexpected biotransformation reactions are thematized.