Investigations in carbon isotope ratios of seized testosterone and boldenone preparations

30 Jun 2021

Investigations in carbon isotope ratios of seized testosterone and boldenone preparations / Thomas Piper, Mario Thevis. 

  • June 2021
  • Drug Testing and Analysis
  • PMID: 34192821
  • DOI: 10.1002/dta.3120


    Abstract

    In order to detect the misuse of testosterone (T) or boldenone (Bo) in doping control analysis, the confirmation of atypical findings employing the determination of carbon isotope ratios (CIR) is mandatory for issuing adverse analytical findings. Elevated concentrations of T (or elevated T/epitestosterone ratios) may result from confounding factors such as ethanol intake, and the presence of low urinary concentrations of Bo can originate from endogenous or urinary in situ production of small amounts of the steroid. As pharmaceutical preparations of Bo and T are generally depleted in 13 C, their CIR differ significantly from the 13 C-enriched endogenous steroids. Some rare cases have been reported on pharmaceutical preparations showing 13 C-enriched isotope ratios that complicate the current application of CIR in sports drug testing. Therefore, the CIR of a subset of n = 157 T preparations and n = 39 Bo preparations seized in Switzerland and Germany between 2013 and 2018 was analyzed in order to estimate the possible impact of steroid preparations showing 13 C-enriched isotope ratios on the current approach to detect their misuse. All investigated Bo preparations showed CIR in the expected range between - 26.7 and -30.3‰. Within the T samples, 95% showed the expected values below -26‰ while six samples fall between -25 and -26‰ and one sample was indistinguishable from endogenously produced T with a CIR of -23.3‰.

    Urinary Elimination of Ecdysterone and Its Metabolites Following a Single-Dose Administration in Humans

    9 Jun 2021

    Urinary Elimination of Ecdysterone and Its Metabolites Following a Single-Dose Administration in Humans / Gabriella Ambrosio, Tasha Yuliandra, Bernhard Wuest, Monica Mazzarino, Xavier de la Torre, Francesco Botrè, Patrick Diel, Eduard Isenmann, Maria Kristina Parr. - (Metabolites 11 (2021) 6 (9 June); p. 1-15)

    • PMID: 34207569
    • PMCID: PMC8227119
    • DOI: 10.3390/metabo11060366


    Abstract

    Ecdysterone is a phytosteroid widely discussed for its various pharmacological, growth-promoting, and anabolic effects, mediated by the activation of estrogen receptor beta (ERbeta). Performance-enhancement in sports was demonstrated recently, and ecdysterone was consequently included in the Monitoring Program, to detect potential patterns of misuse in sport. Only few studies on the pharmacokinetics of ecdysterone in humans have been reported so far. In this study, post-administration urine samples in twelve volunteers (single dose of 50 mg of ecdysterone) were analyzed using dilute-and-inject liquid-chromatography-tandem mass spectrometry. Identification and quantitation of ecdysterone and of two metabolites, 14-deoxy-ecdysterone and 14-deoxy-poststerone, was achieved. Ecdysterone was the most abundant analyte present in post-administration urine samples, detected for more than 2 days, with a maximum concentration (Cmax) in the 2.8-8.5 h urine (Cmax = 4.4-30.0 µg/mL). The metabolites 14-deoxy-ecdysterone and 14-deoxy-poststerone were detected later, reaching the maximum concentrations at 8.5-39.5 h (Cmax = 0.1-6.0 µg/mL) and 23.3-41.3 h (Cmax = 0.1-1.5 µg/mL), respectively. Sex-specific differences were not observed. Cumulative urinary excretion yielded average values of 18%, 2.3%, and 1.5% for ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone, respectively. Ecdysterone and 14-deoxy-ecdysterone were excreted following first-order kinetics with half-lives calculated with three hours, while pharmacokinetics of 14-deoxy-poststerone needs further evaluation.

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

    3 Jul 2021

    The use of RNA-based 5'-aminolevulinate synthase 2 biomarkers in dried blood spots to detect recombinant human erythropoietin microdoses / Francesco Loria, Holly D, Cox, Sven C. Voss, Angela Rocca, Geoffrey D. Miller, Nathan Townsend, Costas Georgakopoulos, Daniel Eichner, Tiia Kuuranne, Nicolas Leuenberger. - (Drug Testing and Analysis (2021) 3 July)

    • PMID: 34216436
    • DOI: 10.1002/dta.3123


    Abstract

    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.

    An enzyme immunoassay to determine human chorionic gonadotropin (HCG) in serum and urine samples using an ultra-microanalytical system

    2 Jul 2021

    An enzyme immunoassay to determine human chorionic gonadotropin (HCG) in serum and urine samples using an ultra-microanalytical system / Ruben Del Valle García, Juliette M. Cazanave Mora, Nancy L. Carrazana San Martín, Orlando Zulueta Rodríguez, Antonio Melchor Rodríguez, Liliana Hernández Pérez, Raquel López Cisneros, Evelyn D. Gato Orozco, Delia Benítez Gordillo, Adriana González Quintero, Iria García de la Rosa , Remigio Coto Rodeiro. - (Journal of Pharmaceutical and Biomedical Analysis 104 (2021) 10 September; 114239)

    • PMID: 34252818
    • DOI: 10.1016/j.jpba.2021.114239


    Abstract

    The determination of Human Chorionic Gonadotropin (HCG) in biological fluids is of great interest in the early pregnancy diagnostics, the evaluation of pregnancy disorders, as a tumor marker, as a screening procedure for anti-doping control, and many other purposes. A simple sandwich-type UltraMicro Enzyme-Linked ImmunoSorbent Assay (UMELISA) has been developed for the measurement of HCG in serum and urine samples. Strips coated with a high affinity MAb directed against HCG are used as solid phase, to ensure the specificity of the assay. The HCG assay was completed in 1.5 h, with a measuring range of 0.76-400 mIU/mL. The intra- and inter-assay coefficients of variation were lower than 10 %, depending on the HCG concentrations evaluated. Recovery percentages were 96.43-97.16 % (serum) and 98.10-99.04 % (urine). The assay detected intact HCG, nicked HCG, HCG β, and nicked HCG β, and did not recognize any of the interfering molecules tested. Regression analysis showed a good correlation with Elecsys in serum (n = 1459, r = 0.952, ρc = 0.948) and urine (n = 869, r = 0.988, ρc = 0.978). A good correlation was also found with 84 RIQAS samples analyzed with the kits Elecsys (r = 0.969, ρc = 0.957), Architect (r = 0.982, ρc = 0.970), Dimension (r = 0.989, ρc = 0.977), and Bioscience (r = 0.992, ρc = 0.980), all with a p < 0.01. Comparison with transvaginal ultrasonography in early pregnancy detection showed a specificity and a sensitivity of 100 % (n = 2385, κ = 1). The analytical performance characteristics of UMELISA HCG endorse its use for the quantification of HCG in serum and urine samples. This assay will make a cost-effective diagnostic kit accessible to low-income countries and is now available in the Cuban Public Health System.

    Anti-doping and other sport integrity challenges during the COVID-19 pandemic

    13 Jul 2021

    Anti-doping and other sport integrity challenges during the COVID-19 pandemic / Giscard Lima, Borja Muniz-Pardos, Alexander Kolliari-Turner, Blair Hamilton, Fergus M. Guppy, Gerasimos Grivas, Andrew Bosch, Paolo Borrione, Alessia Di Gianfrancesco, Chiara Fossati, Fabio Pigozzi, Yannis Pitsiladis. - (Journal of Sports Medicine and Physical Fitness 61 (2021) 8 (August); p. 1173-1183)

    • PMID: 34256541
    • DOI: 10.23736/S0022-4707.21.12777-X


    Abstract

    The coronavirus disease (COVID-19) pandemic has had an unprecedent impact on the world of sport and society at large. Many of the challenges with respect to integrity previously facing competitive sport have been accentuated further during the pandemic. Threats to the integrity of sporting competition include traditional doping, issues of technological fairness, and integration of transgender and intersex athletes in elite sport. The enforced lull in competitive sport provides an unprecedented opportunity for stakeholders in sport to focus on unresolved integrity issues and develop and implement long-lasting solutions. There needs to be a concerted effort to focus on the many technological innovations accelerated by and perfected during COVID-19 that have enabled us to work from home, such as teaching students on-line, applications for medical advice, prescriptions and referrals, and treating patients in hospitals/care homes via video links and use these developments and innovations to enhance sport integrity and anti-doping procedures. Positive sports integrity actions will require a considered application of all such technology, as well as the inclusion of "omics" technology, big data, bioinformatics and machine learning/artificial intelligence approaches to modernize sport. Applications include protecting the health of athletes, considered non-discriminative integration of athletes into elite sport, intelligent remote testing to improve the frequency of anti-doping tests, detection windows, and the potential combination with omics technology to improve the tests' sensitivity and specificity in order to protect clean athletes and deter doping practices.

    Depletion of clomiphene residues in eggs and muscle after oral administration to laying hens

    15 Jul 2021

    Depletion of clomiphene residues in eggs and muscle after oral administration to laying hens / Luisa Seyerlein, Nathalie Gillard, Philippe Delahaut, Gilles Pierret, Andreas Thomas, Mario Thevis. - (Food Additives & Contaminants: Part A (2021) 15 July; p. 1-8)

    • PMID: 34266369
    • DOI: 10.1080/19440049.2021.1949497


    Abstract

    The selective oestrogen receptor modulator (SERM) clomiphene is therapeutically used to induce ovulation. While prohibited as a doping agent in sports, it is frequently detected in sports drug testing urine samples. Few reports exist on clomiphene's (illicit) use in the farming industry to increase the egg production rate of laying hens, which creates a risk that eggs as well as edible tissue of these hens contain residues of clomiphene. To investigate the potential transfer of clomiphene into eggs and muscle tissue, laying hens were orally administered with clomiphene citrate at 10 mg/day for 28 days. To determine clomiphene residues in eggs, chicken breast and chicken thigh, the target analyte was extracted from homogenised material with acetonitrile and subjected to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The test method reached a limit of quantification (LOQ) of 1 µg/kg and was characterised concerning specificity, precision, trueness and linearity. Analyses were performed on whole egg, egg white and yolk separately, and chicken muscle from breast and thigh. Clomiphene was detectable in eggs two days after the beginning of the drug administration period. The drug concentrations increased to 10-20 µg per egg within one week, and after withdrawal of clomiphene, residues decreased after 4 days, but traces of clomiphene were still detectable until the end of the study (14 days after the last administration). In the chicken's muscle tissue, clomiphene levels up to 150 µg/kg (thigh) and 36 µg/kg (breast) were found. Six days after the last dose, tissue clomiphene concentrations fell below the LOQ. Overall, these results underline the concerns that clomiphene may be transferred into animal-derived food and future research will therefore need to focus on assessing and minimising the risk of unintentional adverse analytical findings in doping controls.

    Screening for gene doping transgenes in horses via the use of massively parallel sequencing

    19 Jul 2021

    Screening for gene doping transgenes in horses via the use of massively parallel sequencing / Jillian Maniego, Bogusia Pesko, Jocelyn Habershon-Butcher, Jim Huggett, Polly Taylor, James Scarth, Edward Ryder. - (Gene Therapy (2021) 19 July)

    • PMID: 34276046
    • DOI: 10.1038/s41434-021-00279-1


    Abstract

    Throughout the history of horse racing, doping techniques to suppress or enhance performance have expanded to match the technology available. The next frontier in doping, both in the equine and human sports areas, is predicted to be genetic manipulation; either by prohibited use of genome editing, or gene therapy via transgenes. By using massively-parallel sequencing via a two-step PCR method we can screen for multiple doping targets at once in pooled primer sets. This method has the advantages of high scalability through combinational indexing, and the use of reference standards with altered sequences as controls. Custom software produces transgene-specific amplicons from any Ensembl-annotated genome to facilitate rapid assay design. Additional scripts batch-process FASTQ data from experiments, automatically quality-filtering sequences and assigning hits based on discriminatory motifs. We report here our experiences in establishing the workflow with an initial 31 transgene and vector feature targets. To evaluate the sensitivity of parallel sequencing in a real-world setting, we performed an intramuscular (IM) administration of a control rAAV vector into two horses and compared the detection sensitivity between parallel sequencing and real-time qPCR. Vector was detected by all assays on both methods up to 79 h post-administration, becoming sporadic after 96 h.

    Dealing with doping. A plea for better science, governance and education

    22 Jul 2021

    Dealing with doping. A plea for better science, governance and education / Jules A.A. Heuberger, April Henning, Adam F. Cohen, Bengt Kayser. - (British Journal of Clinical Pharmacology (2021) 22 July)

    • PMID: 34291479
    • DOI: 10.1111/bcp.14998


    Abstract

    The creation of WADA contributed to harmonization of anti-doping and changed doping behavior and prevalence in the past 22 years. However, the system has developed important deficiencies and limitations that are causing harm to sports, athletes and society. These issues are related to the lack of evidence for most substances on the Prohibited List for performance or negative health effects, a lack of transparency and accountability of governance and decision-making by WADA and the extension of anti-doping policies outside the field of professional sports. This article tries to identify these deficiencies and limitations and presents a plea for more science, better governance and more education. This should lead to a discussion for reform among stakeholders, which should cover support of a new Prohibited List by actual research and evidence and introduce better governance with accountable control bodies and regulation. Finally, comprehensive education for all stakeholders will be the basis of all future positive improvements.

    Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC-Ion Mobility-HRMS

    28 Jul 2021

    Multidimensional Separations of Intact Phase II Steroid Metabolites Utilizing LC-Ion Mobility-HRMS / Don E. Davis Jr, Katrina L. Leaptrot, David C. Koomen, Jody C. May, Gustavo de A. Cavalcanti, Monica C. Padilha, Henrique M.G. Pereira, John A. McLean. - (Analytical Chemistry 93 (2021) 31 (10 August); p. 10990-10998)

    • PMID: 34319704
    • DOI: 10.1021/acs.analchem.1c02163


    Abstract

    The detection and unambiguous identification of anabolic-androgenic steroid metabolites are essential in clinical, forensic, and antidoping analyses. Recently, sulfate phase II steroid metabolites have received increased attention in steroid metabolism and drug testing. In large part, this is because phase II steroid metabolites are excreted for an extended time, making them a potential long-term chemical marker of choice for tracking steroid misuse in sports. Comprehensive analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been used to detect and identify glucuronide and sulfate steroids in human urine with high sensitivity and reliability. However, LC-MS/MS identification strategies can be hindered by the fact that phase II steroid metabolites generate nonselective ion fragments across the different metabolite markers, limiting the confidence in metabolite identifications that rely on exact mass measurement and MS/MS information. Additionally, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is sometimes insufficient at fully resolving the analyte peaks from the sample matrix (commonly urine) chemical noise, further complicating accurate identification efforts. Therefore, we developed a liquid chromatography-ion mobility-high resolution mass spectrometry (LC-IM-HRMS) method to increase the peak capacity and utilize the IM-derived collision cross section (CCS) values as an additional molecular descriptor for increased selectivity and to improve identifications of intact steroid analyses at low concentrations.

    Simulated validation of intron-less transgene detection using DELLY for gene-doping control in horse sports

    2 Aug 2021

    Simulated validation of intron-less transgene detection using DELLY for gene-doping control in horse sports / T. Tozaki  1 , A. Ohnuma, M. Kikuchi, T. Ishige, H. Kakoi, K. Hirota, K. Kusano, S. Nagata. - (Animal Genetics (2021) 2 August)

    • PMID: 34339052
    • DOI: 10.1111/age.13127


    Abstract

    Gene doping is prohibited in horseracing. In a previous study, we developed a method for non-targeted transgene detection using DELLY, which is based on split-read (SR) and paired-end (PE) algorithms to detect structural variants, on WGS data. In this study, we validated the detection sensitivity of DELLY using artificially generated sequence data of 12 target genes. With DELLY, at least one intron was detected as a deletion in eight targeted genes using the 150 bp PE read WGS data, whereas all targeted genes were detected by DELLY using the 100 bp PE read data. The detection sensitivity was higher in 100 bp PE reads than in 150 bp PE reads, despite a lower total sequence coverage, probably because of mismatch tolerance between the mapped reads and reference genome. In addition, it was observed that the average intron size detected by SR alone was 293 bp and that detected by both SR and PE was 8924 bp. Thus, we showed that transgenes with various intron-exon structures could be detected using DELLY, suggesting its application in gene-doping control in horses.

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