The 41^st Manfred Donike workshop on doping analysis

After 2 years of virtual Manfred Donike Workshops,1 the 41st annual edition of this scientific event was conducted in-person again from February 26 until March 2, 2023. With 140 anti-doping scientists from 29 nations and all continents attending, a fantastic program was composed, and presentations of outstanding breadth and quality were contributed. The collection of articles compiled in this special issue of Drug Testing & Analysis reflects a selection of core topics of 2022/2023, including the identification of current and (potential) future issues in sports drug testing as well as the presentation of solutions as to how joint and interdisciplinary efforts have addressed these. Central topics of the 2023 conference can be largely subsumed under analytical aspects concerning improved testing strategies for steroidal compounds, erythropoiesis-stimulating agents, as well as substances of peptidic structure with growth promoting/anabolic properties. Furthermore, new data on how to approach the analytical challenge of differentiating naturally occurring analytes from those administered for doping purposes were presented, including, for example, studies on the β2-agonist higenamine or the metabolic product of the stimulant meclofenoxate (and likewise the acetylcholine biosynthetic pathway) 2-(dimethylamino)ethan-1-ol (deanol),2 and new high throughput options supporting the authentication of doping control samples were presented.3 Investigating and revisiting the metabolic fate of doping agents is vital for comprehensive sports drug testing methods as well as subsequent result interpretation. Both in vitro and in vivo approaches assisting in studying biotransformation reactions have been shown to provide particularly useful tools as demonstrated by means of 3D-cultivated HepG2 cells and rat administration studies using the anabolic-androgenic steroids (AAS) metandienone4 and boldenone.5 With sulfo-conjugated phase-II metabolites of these and other AAS receiving growing attention in routine doping controls, investigating their analytical behavior on existing testing platforms has become essential, particularly by assessing the applicability of analytical platforms commonly available in anti-doping laboratories to such metabolic products.6 Naturally occurring AAS such as nandrolone and testosterone necessitate complementary analytical strategies in sports drug testing on initial testing procedure as well as confirmatory level, and new information, for example, on δ13C values on nandrolone metabolites observed after dietary exposure in human urine7 was reported. With regard to testosterone and its metabolites and their monitoring in the context of the athlete biological passport with its steroidal module, effects of thyroid hormone administrations8 and human chorionic gonadotrophin injections9 were investigated and contextualized for result interpretation. Also, the necessity and approaches for testosterone/epitestosterone ratio corrections in routine anti-doping analyses were revisited, especially concerning the use of either gas chromatographic-mass spectrometric or liquid chromatographic-mass spectrometric approaches.10 Detecting the manipulation of erythropoiesis or oxygen-transport capacities in general has been the subject of continued investigations also in 2022/2023. An alternative method facilitating the quantification of cobalt as erythropoiesis-stimulating substance in human urine was presented, employing coordination chemistry and liquid chromatography-tandem mass spectrometry (LC–MS/MS),11 and the detection of a hemoglobin S polymerization inhibitor, which was added to the World Anti-Doping Agency (WADA) Prohibited List in 2023,12 was studied with regard to its in vitro metabolism to offer adequate target analytes for routine doping controls.13 Also, probing for the presence of a hemoglobin-based oxygen carrier derived from lugworm by LC–MS/MS was successfully conducted using diagnostic signature peptides determined with established sample preparation and analysis protocols.14 Further, new data on the identification of recombinant human erythropoietin when administered to c.577del variant carriers were provided,15 demonstrating that especially the N-deglycosylated erythropoietin analyzed in blood offers superior specificity and sensitivity. Also, options of detecting homologous blood transfusions by means of DNA analysis were assessed, indicating an inferior window of opportunity when compared to established flow cytometric methods, which were shown to allow for detection windows of up to 50 days post-transfusion.16 Doping with peptidic drugs/drug candidates or their releasing factors has required growing attention in routine doping controls over the past decade, and the applicability of a novel test method employing a quenchbody sensor was assessed concerning growth hormone administrations.17 While the technology proved principally useful, improvements in sensitivity are still required. Conversely, the optimized sensitivity of analytical approaches regarding the growth hormone secretagogue capromorelin proved capable of detecting trace amounts of the drug in a doping control urine sample, which was attributable to a contamination scenario that an athlete was exposed to when administering the drug to a dog,18 thus presenting another facet of the athlete's exposome.19 Advancing the knowledge concerning peptide-based drug metabolites for sports drug testing purposes was conducted using in vitro and animal in vivo experiments, contributing new information for thymosin-β4,20 the insulin-mimetic peptides S519 and S597,21 and myostatin inhibitory peptides (e.g., DF-3 and DF-25).22 Finally, and particularly relevant for result management and in avoiding unintentional anti-doping rule violations, higenamine excretion profiles in urine after ingestion of higenamine-containing fruit as well as enriched dietary supplement were investigated, suggesting that concomitantly observed markers of benzylisoquinoline alkaloid biosynthetic pathway in distinct abundance ratios are particularly valuable for a differentiation of diet-related exposure and doping scenarios.23 Such a differentiation might become necessary also with regards to ecdysterone, a substance currently included in WADA's Monitoring Program,24 for which however the metabolism has not (yet) revealed a unique signature indicative for diet-related exposure and doping scenarios, also not when investigating the metabolism with most modern approaches utilizing stable isotope labeling and isotope-ratio mass spectrometry.25 Taken together, this special issue article collection outlines the multifaceted nature of research and development in doping controls and related topics as pursued in 2022/2023 and presented at the 41st Cologne Workshop on Doping analysis. The continuous strive for optimizing the global anti-doping work remains vital, to the clean athlete and to fair sport.