The 42nd Manfred Donike Workshop on Doping Analysis

Abstract

Between February 26th and February 29th of the Olympic Year 2024, the 42nd edition of the Manfred Donike Workshop on Doping Analysis was conducted in Cologne, Germany. A total of 179 antidoping scientists from 29 nations and all continents attended this event, which was characterized once more by over 100 contributions on antidoping research of outstanding breadth and quality—just the appropriate venue to honor the recently deceased Professor Don Hardt Catlin, one of the most distinguished antidoping scientists in the history of sports drug testing [1].

As previously [2], a collection of selected articles is compiled in a special issue of Drug Testing and Analysis on the occasion of the conference, reflecting core topics of research conducted and presented in 2023/2024. The main areas of research presented at the latest Manfred Donike Workshop can be largely subsumed under analytical considerations and methodological improvements concerning testing strategies for steroidal compounds and new target analytes on both the so-called Monitoring Program [3] as well as the Prohibited List [4] as issued by the World Anti-Doping Agency for 2024. Furthermore, new approaches to determine novel peptidic drugs, assessing elimination profiles of established target analytes, and utilizing genotyping strategies for sample authentication as well as for the detection of blood doping methods were presented. Finally, attention was also drawn to substances that are not yet considered in neither the Monitoring Program nor the Prohibited List but that might warrant a watching brief.

Optimizing the analytical approaches for steroidal compounds was the subject of several investigations, especially concerning those analytes that are naturally/endogenously produced and also available as pharmaceutical preparations, accomplished primarily by sophisticated derivatization techniques. For instance, Pfeffer et al. demonstrated that methylation of intact Phase II metabolites of testosterone and epitestosterone by trimethylsilyldiazomethane treatment substantially improves the chromatographic and mass spectrometric behavior of the analytes, offering therefore an alternative option to determining accurately urinary concentration levels [5]. Sakellariou et al. employed Girard's reagent T to complement the analytical portfolio for the quantification of intact Phase II nandrolone metabolites, specifically glucuronides and sulfates of 19-norandrosterone, 19-noretiocholanolone, and 19-norepiandrosterone [6]. The quick and straightforward method was shown to allow for a competitive performance compared to established testing strategies based on gas chromatography–mass spectrometry, which require enzymatic hydrolysis. In order to account for low sample volumes combined with low analyte concentrations in dried blood spots (DBS), Miyamoto et al. utilized methoxyamine to produce the methyloxime derivatives of a total of 28 steroid esters (including testosterone, nandrolone, and boldenone) [7]. By means of liquid chromatography–tandem mass spectrometry, limits of detection between 0.1 and 0.9 ng/mL were accomplished, enabling the detection of a single testosterone enanthate administration for up to 9 days. The importance of aiming at determining intact esters of steroidal formulations in blood or DBS was underlined by data presented by Polet et al., who illustrated the increasing prevalence of nandrolone formulations exhibiting carbon isotope signatures within commonly observed natural δ¹³C values [8]. Workplace drug testing urine samples that produced adverse analytical findings for nandrolone metabolites (and occasionally also other anabolic-androgenic steroids) were retested using isotope ratio mass spectrometry, suggesting that combined and complementary test methods as well as modifications to the result management processes are warranted.

The growing attention towards the analysis of serum steroids has further necessitated the assessment of preanalytical conditions and the comparability of samples as presented by Goodrum et al. [9]. In a comprehensive study, the agreement of test results obtained for testosterone and androstenedione (plus luteinizing hormone) determined from venous and capillary serum samples was shown, opening the opportunity of including less invasive sample collection procedures also for the blood steroid profile module of the athlete biological passport. An improved sample preparation and analysis protocol for the determination of carbon isotope signatures of serum steroids was presented by Piper and Thevis, extending the existing spectrum of target analytes by pregnenolone sulfate and 5-androstene-3β,17β-diol sulfate in support athlete biological passport–related findings [10]. Here, however, serum volumes obtained from venous blood collections are required.

Also, various new substances, under (pre)clinical investigation or recently approved, have been the subject of antidoping research. Euler et al. studied the metabolism and elimination of the fast skeletal troponin activators reldesemtiv and tirasemtiv using both in vitro (human liver microsomes as well as 3D cultivated human hepatic cells on an organ-on-a-chip model) and in vivo approaches. Eight and eleven metabolites were identified for tirasemtiv and reldesemtiv, respectively, two of which were found to represent excellent target analytes for routine doping controls [11]. Expanding initial testing procedures with regard to the number of covered target analytes requires sample preparation procedures of adequate comprehensiveness, and the utility of the so-called QuEChERS approach was shown to be suitable for sports drug testing purposes by Derwand et al. [12] who demonstrated the successful analysis of 312 low molecular mass compounds from urine. In order to determine higher molecular mass analytes such as somatrogon, a human growth hormone/human chorionic gonadotropin fusion protein, Walpurgis et al. considered a more active ingredient–specific approach to isolate the drug and its analogs from serum [13]. By means of growth hormone receptor–coated magnetic beads, somatrogon (as well as the internal standard ¹⁵N-labeled human growth hormone) was extracted and trypsinized, and diagnostic (glycosylated) peptides were determined by liquid chromatography–high-resolution mass spectrometry, allowing the detection of the administration of a single dose for up to 96 h.

New data on the (unexpectedly long) traceability of roxadustat administrations were presented by Sobolevsky et al. [14] Following up on a case investigation into a finding of roxadustat 1 year after cessation of drug use, urine samples collected from patients as well as healthy volunteers up to 17 months after drug use were analyzed, confirming the particularly long traceability of the drug at low pg/mL levels. Details as to why the drug is eliminated for such a long period of time are yet to be clarified. Also, new information on how to monitor the use of hypoxen in routine doping controls was provided by Görgens et al. [15] Using controlled elimination study urine samples, tentatively identified Phase I and II urinary metabolites of hypoxen were reported, and a test method based on a dilute-and-inject liquid chromatography–high-resolution mass spectrometry approach was presented, facilitating the detection of hypoxen use as demonstrated by first findings in routine sports drug testing samples.

Further, studies were presented on utilizing technological advances in determining genetic information from human urine and blood for doping control purposes. Specifically, Akiyama et al. reported on the value of mitochondrial DNA analyses for the authentication of doping control samples, especially when conventional short tandem repeat analysis is compromised due to nuclear DNA degradation [16]. Donati et al. described the use of single nucleotide polymorphism (SNP)–based genotyping of athletes' DBS samples to probe for indicators of homologous blood transfusion [17]. Employing four SNPs (two autosomal and two sex chromosomes), mixed blood samples were identified with as little as 1% of donor blood, a pilot study result that warrants follow-up studies.

Finally, investigations into the options as to how thyroid hormone administrations can be monitored and how therapeutic use can potentially be differentiated from misuse of these hormones were presented by Martinez Brito et al. [18, 19] Targeting an array of nine compounds included in the thyroid hormone metabolic pathway and investigating both athletes and volunteers participating in controlled administration studies, indications that ratios of triiodothyronine (T3)/thyronine, thyroxine (T4)/3,3′-diiodothyronine, and urinary concentrations of 3-iodothyronine could be markers for thyroid hormone use were generated. Concerns as to yet unknown confounding factors still need to be addressed though.

Taken together, this special issue article collection once more outlines the multifaceted nature of research and development in doping controls and related topics as pursued in 2023/2024 and presented at the 42nd Cologne Workshop on Doping Analysis. The continuous strive for optimizing the global antidoping work remains vital to the clean athlete and to fair sport.

Cologne, September 24, 2024