Annual banned-substance review: Analytical approaches in human sports drug testing 2019/2020.

Analytical chemistry-based research in sports drug testing has been a dynamic endeavor for several decades, with technology-driven innovations continuously contributing to significant improvements in various regards including analytical sensitivity, comprehensiveness of target analytes, differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds, assessment of alternative matrices for doping control purposes, and so forth. The resulting breadth of tools being investigated and developed by anti-doping researchers has allowed to substantially improve anti-doping programs and data interpretation in general. Additionally, these outcomes have been an extremely valuable pledge for routine doping controls during the unprecedented global health crisis that severely affected established sports drug testing strategies. In this edition of the annual banned-substance review, literature on recent developments in anti-doping published between October 2019 and September 2020 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified the World Anti-Doping Agency's 2020 Prohibited List

Support of a laboratory-hosted Athlete Biological Passport Management Unit (APMU) to the anti-doping organisations

The athlete biological passport (ABP) is an established means for longitudinal monitoring of selected individual biomarkers of an athlete to obtain indirect but potentially long-term indications of the use of substances or methods prohibited in sport. Along the change from population-based reference values to individual profiling, the ABP aims at triggering follow-up investigations concerning the potential use of endogenous substances with doping potential, which might be difficult either to identify with the existing analytical methods or to interpret based only on the results of a single biological sample. The ABP program has been on-going within the World Anti-Doping Agency (WADA) management since 2009, when the hematological module was officially established to discover blood doping practices, such as administration of erythropoietin (EPO) or application of blood transfusion. Since 2014, the ABP has been complemented by the steroid module, with the aim of targeting the prohibited use of testosterone and other endogenous anabolic androgenic steroids with performance enhancing or masking capability. Although the main objective is to guide and assist the anti-doping organizations in their test distribution plans, the ABP may also be used to proceed with a case to an anti-doping rule violation. Evaluation of biological markers, especially in distinguishing between doping from other confounding factors, requires high level and diversity of expertise, which is coordinated by the athlete biological passport management unit (APMU). Since 2019, the WADA accredited anti-doping laboratories are defined as the host organizations for the APMUs. The benefit of such a structure is to obtain a fully anonymous evaluation process for the passports and an additional level of expertise for the interpretation of analytical results as well as to have a fluent communication line with the analyzing laboratories when further details are needed for the analytical testing and documentation

Analysis of anabolic steroids in urine by gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry with chlorobenzene as dopant

Corrigendum to J. Chromatogr. A 1312 (2013), p. 111-117: Support by one project was inadvertently not mentioned in the Acknowledgements. The Acknowledgement should read:Funding from the Academy of Finland (projects 251575 and 257316) and the Finnish Funding Agency for Technology and Innovation(project 1291/31/084) is acknowledged. L. Hintikka is grateful to the CHEMSEM graduate school for financial support. Dr. Ville Saarela isthanked for the design and fabrication of the microchips. J. Chromatogr. A 1448 (2016) p. 128.Peer reviewe

Retesting the anti-doping samples: Best tool for deterrence?

Long term storage of the anti-doping samples and their reanalyses becomes today more and more a trend in the anti-doping community. The procedure has been implemented by the anti-doping authorities for the samples of the Tour de France and for the Olympic Games since Athens 2004 and has been always presented as a good tool to deter doping habits in top level sport. Recently, the World Anti-Doping Code introduced the possibility for anti-doping organizations to store the athletes’ samples up to ten years. The anti-doping authorities may ask to reanalyze the samples at any time during that period of time as a function of the implementation of new methods or instruments in the accredited laboratories allowing the detection of prohibited substances or their metabolites at a much lower concentration or for a larger detection window. The most significant technological advances for the detection of doping substances have been done in the characterization of various long-term metabolites of anabolic androgenic steroids. This allowed for increasing the time of detection by even a factor of four

Automation of RNA-based biomarker extraction from dried blood spots for the detection of blood doping.

Aim: Transcriptomic biomarkers originating from reticulocytes measured in dried blood spots (DBSs) may be reliable indicators of blood doping. Methods/results: Here, we examined changes in the expression levels of the erythropoiesis-related ALAS2, CA1 and SLC4A1 genes in DBS samples from elite athletes and volunteers of clinical study with recombinant erythropoietin dose. Conclusion: By comparing the mean intraday coefficients of variation for ALAS2L, ALASLC, CA1 and SLC4A1 between manual and automated RNA extractions, an average improvement was observed, whereas the assessment of interday variability provided comparable results for both manual and automated approaches. Our results confirmed that RNA biomarkers on DBS support are efficient to detect blood doping

A fast screening method for the detection of CERA in dried blood spots.

Continuous erythropoietin receptor activator (CERA) is a third-generation erythropoiesis-stimulating agent that was developed for the treatment of anemia. However, misuse of CERA for doping in endurance sports has been reported. Previous studies have shown blood as the matrix of choice for the detection of CERA, due to its high molecular weight. The use of dried blood spots (DBSs) for anti-doping purposes constitutes a complementary approach to the standard urine and venous blood matrices and could facilitate sample collection and increase the number of blood samples available for analysis due to reduced costs of sample collection and transport. Here, we investigated whether CERA could be indirectly detected in extracts of single DBSs using an erythropoietin-specific immunoassay that is capable of providing results within approximately 2 h. Reconstituted DBS samples were prepared from mixtures of red blood cell pellets and serum samples. The samples were collected in a previous clinical study in which six healthy volunteers were injected with a single, 200 μg dose of CERA. Using a commercially available ELISA kit, CERA was detected in the DBSs with a detection window of up to 20 days post-injection. Furthermore, in order to demonstrate the fitness-for-purpose, three authentic doping control serum samples, which were identified as containing CERA, were analyzed by the presented methodological approach on DBS. The testing procedure described here could be used as a fast and cost-effective method for the detection of CERA abuse in sport

Steroid profiling by UHPLC-MS/MS in dried blood spots collected from healthy women with and without testosterone gel administration.

The quantification of a large panel of endogenous steroids in serum by LC-MS/MS represents a powerful clinical tool for the screening or diagnosis of diverse endocrine disorders. This approach has also demonstrated excellent sensitivity for the detection of testosterone misuse in the anti-doping field, especially in female athlete population. In both situations, the use of dried blood spots (DBS) could provide a viable alternative to invasive venous blood collection. Here, the evaluation of DBS sampling for the quantification of a panel of endogenous steroids using UHPLC-MS/MS is described. The UHPLC-MS/MS method was validated for quantitative analysis of eleven free and eight conjugated steroids and was then used for the analysis of DBS samples collected in 14 healthy women during a normal menstrual cycle (control phase) followed by a 28-days testosterone gel treatment (treatment phase). Results were compared with those obtained from serum matrix. Satisfactory performance was obtained for all compounds in terms of selectivity, linearity, accuracy, precision, combined uncertainty, stability as well as extraction recovery and matrix effects. In control phase, high correlation was observed between DBS and serum concentrations for most compounds. In treatment phase, higher testosterone concentrations were observed in capillary than in venous DBS, suggesting a possible interference resulting from testosterone contamination on finger(s) used for gel application. Steroid profiling in capillary DBS represents a simple and efficient strategy for monitoring endogenous steroid concentrations and their fluctuation in clinical context of steroid-related disorders, or for the detection of testosterone abuse in anti-doping

Erythroferrone as a sensitive biomarker to detect stimulation of erythropoiesis.

Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to erythropoietin stimulation. ERFE suppresses the hepatic synthesis of the master iron-regulatory hormone, hepcidin. The impact of erythropoiesis stimulation on ERFE secretion in humans is poorly understood. This paucity of information is due in part to the lack of available means for ERFE quantification in serum samples. The present study tested a new sensitive sandwich immunoassay for human ERFE. This assay was used to demonstrate that injection of various erythropoiesis stimulating agents (ESAs) increased the blood ERFE levels in healthy volunteers. After exogenous stimulation of erythropoiesis, ERFE increased up to 8-fold with a detection window of 13 days. The impact of one unit of blood withdrawal on erythropoiesis stimulation of ERFE was also tested. ERFE significantly increased after blood withdrawal in subjects injected with both iron and saline solution, suggesting that iron supplementation did not mask the ERFE increase after blood withdrawal. The effects of exercise-induced muscle damage on ERFE was assessed by comparing ERFE levels with creatine kinase levels in samples from subjects with heavy exercise loads, and determined that this was not a confounder. The ERFE assay is a sensitive means to investigate the connection between iron metabolism and erythropoiesis in humans, and to detect ESA abuse in the antidoping field