Hans Geyer’s research while affiliated with Deutsche Sporthochschule Köln and other places

The 17th edition of the annual banned‐substance review on analytical approaches in human sports drug testing is dedicated to literature published between October 2023 and September 2024. As in previous years, focus is put particularly on new or enhanced analytical options in human doping controls as well as investigations into the metabolism and elimination of compounds of interest, which represent central (while not exclusive) cornerstones of the global anti‐doping mission. New information published within the past 12 months on established doping agents as well as new potentially relevant substances are reviewed and discussed in the context of the World Anti‐Doping Agency's 2024 Prohibited List. Thereby, analytical challenges, especially with regard to the continuously growing number of target compounds and potentially relevant drug classes as well as the exigency (and consequences) of utmost analytical retrospectivity, are thematized and contextualized. Investigations especially into anabolic agents, peptide hormones, and strategies for the detection of gene doping were identified as core areas of anti‐doping research in the reviewed period.

Roxadustat (FG‐4592), an orally active hypoxia‐inducible factor prolyl hydroxylase stabilizer, has been shown to enhance erythropoiesis by increasing endogenous erythropoietin. It is indicated for the treatment of anemia and chronic kidney disease and is approved for clinical use in several countries, including the European Union, Japan and others. Due to its reasonably anticipated performance‐enhancing effect in athletes, roxadustat is prohibited for use in sports at all times. A few cases of adverse analytical findings in routine doping controls have been reported worldwide, some of which were claimed to be the result of contaminated dietary supplements. The present study offers new data demonstrating the long‐term excretion pattern of roxadustat. Even after a single‐dose administration, roxadustat can remain detectable in urine for 8 months, albeit at very low concentrations (<10 pg/mL). Following three times a week treatment with 70 to 100 mg of roxadustat, the drug was still detectable in the urine of anemic patients for between 9 and 18 months after treatment was discontinued. Lastly, an athlete who admitted use of roxadustat for almost a year (50 mg 3 to 5 times a week) has now tested positive multiple times over the course of 15 months (the first test being 12 months after the drug was discontinued), with an estimated concentration of roxadustat between 3 and 8 pg/mL. Altogether, these findings indicate the unusually prolonged terminal excretion kinetics of roxadustat, a property that testing authorities should consider in their results management process.

Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called Adverse Analytical Finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offences and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from non-athletes were analysed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e. samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the non-approved PPARδ agonist GW1516) in a total of 81 samples, with 91 % of these suspected cases being verified by the confirmation method. Besides the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. Significance Statement This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing.

Doping practices in sports by unscrupulous athletes have been an important societal issue for several decades. Recently, sample swapping has been raised as a potential practice performed by athletes to swap their doped samples with clean samples to evade the positive doping test. So far, the only proven method to detect such cases is by performing DNA analysis on samples. However, it is expensive and time-consuming, which goes beyond the budgetary limits of anti-doping organisations when implementing to all the samples collected during sports events. Therefore, in this paper, we propose a self attention-based convolutional neural network (SACNN) that incorporates both spatial and temporal behaviour of the longitudinal profile and generates embedding maps for solving the fraud detection problem in sports. We conduct extensive experiments on the real-world datasets. The result shows that SACNN outperforms other state-of-the-art baseline models for sequential anomaly detection. Moreover, we conduct a study with domain experts on real-world profiles using both DNA analysis and our proposed method; the result demonstrates the effectiveness of our proposed method and the impact it could bring to the society.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in‐ and out‐of‐competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C‐terminal peptide (CTP) of the human chorionic gonadotropin (hCG) β‐subunit, with current WADA‐approved doping control assays for hGH and hCG was investigated. For that purpose, cross‐reactivity tests and a somatrogon administration study were conducted, and only “Kit 2” of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post‐administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG β‐subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10 ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)‐conjugated magnetic beads, proteolytic digestion, and liquid chromatography high‐resolution tandem mass spectrometry (LC‐HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post‐administration serum samples were successfully analyzed as proof‐of‐concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only “Kit 2” of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

Purpose Inadvertent and/or unknowing exposure to drugs and drug residues has been frequently debated in situations of so-called adverse analytical finding (AAF) in the context of sports drug testing programs. Transfer of drug residues via unprotected intercourse is a conceivable scenario but scientific data and authentic case reports are scarce. Herein, investigations into two AAFs with the peroxisome proliferator-activated receptor delta (PPARδ) agonist GW1516 are reported and discussed. Methods To probe for a contamination scenario involving sexual intercourse, two assays were used to determine semenogelin in human urine, with one employing an immunochromatographic lateral flow approach and another based on liquid chromatography–tandem mass spectrometry. Further, drug-residue testing using patients’ ejaculate was conducted by utilizing liquid chromatography in conjunction with a triple quadrupole mass spectrometer, followed by re-analysis of suspect samples (i.e., samples indicating the presence of relevant compounds) using high resolution/high mass accuracy mass spectrometry. Results In one case, but not the other, the possibility of intimate contact as the source of the AAF was confirmed after a thorough investigation of potential contamination scenarios. Subsequent research revealed analytical evidence for the presence of seminal fluid in one of the female athlete’s doping control urine samples, and the analysis of clinical ejaculate specimens provided first data on an authentic concentration level of GW1516 and its metabolites in human seminal fluid. Conclusions The combined facts substantiate the possibility of an AAF caused by unprotected sexual intercourse and the plausibility of the case-related arguments.

Hypoxen, a poly(dihydroxyphenylene) thiosulfonate‐based drug, has been investigated concerning its effect on mitochondrial respiration and the utilization of lactate, especially in the context of strenuous exercise. Since 2023, patterns of use regarding hypoxen amongst the athletic population are monitored by the World Anti‐Doping Agency (WADA) and its accredited anti‐doping laboratories, necessitating information on suitable urinary markers indicative of the administration of hypoxen. In this exploratory study, urine samples collected post‐administration of 1.5 and 2.0 g of hypoxen were analyzed by means of liquid chromatography‐high resolution/high mass accuracy (tandem) mass spectrometry, which allowed for the identification of eight analytes that were plausibly attributable to metabolites of hypoxen. The identified species were assigned to the unconjugated species of S‐(2,2′,5,5′‐tetrahydroxy‐[1,1′‐biphenyl]‐3‐yl) sulfurothioate and its glucuronide and additional tentatively identified analytes comprising a mercaptobenzene core structure. Including the identified markers into routine doping control analytical procedures enabled the detection of hypoxen use in athletes' doping control samples, thus contributing relevant information to WADA's monitoring program.

In this 16 th edition of the annual banned‐substance review on analytical approaches in human sports drug testing, literature on recent developments in this particular section of global anti‐doping efforts that was published between October 2022 and September 2023 is summarized and discussed. Most recent additions to the continuously growing portfolio of doping control analytical approaches and investigations into analytical challenges in the context of adverse analytical findings are presented, taking into account existing as well as emerging challenges in anti‐doping, with specific focus on substances and methods of doping recognized in the World Anti‐Doping Agency's 2023 Prohibited List. As in previous years, focus is put particularly on new or enhanced analytical options in human doping controls, appreciating the exigence and core mission of anti‐doping and, equally, the conflict arising from the opposingly trending extent of the athlete's exposome and the sensitivity of instruments nowadays commonly available in anti‐doping laboratories.

Higenamine is prohibited in sports as a β2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of cmax values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.