Annual banned-substance review: analytical approaches in human sports drug testing

International anti-doping efforts are harmonized and regulated under the umbrella of the World Anti-Doping Code and the corresponding Prohibited List, issued annually by the World Anti-Doping Agency (WADA). The necessity for a frequent and timely update of the Prohibited List (as the result of a comprehensive consultation process and subsequent consensual agreement by expert panels regarding substances and methods of performance manipulation in sports) is due to the constantly growing market of emerging therapeutics and thus new options for cheating athletes to illicitly enhance performance. In addition, 'tailor-made' substances arguably designed to undermine sports drug testing procedures are considered and the potential of established drugs to represent a doping substance is revisited in light of recently generated information. The purpose of the annual banned substance review is to support doping controls by reporting emerging and advancing methods dedicated to the detection of known and recently outlawed substances. This review surveys new and/or enhanced procedures and techniques of doping analysis together with information relevant to doping controls that has been published in the literature between October 2010 and September 2011.     

Annual banned‐substance review: analytical approaches in human sports drug testing

The annual update of the list of prohibited substances and doping methods as issued by the World Anti‐Doping Agency (WADA) allows the implementation of most recent considerations of performance manipulation and emerging therapeutics into human sports doping control programmes. The annual banned‐substance review for human doping controls critically summarizes recent innovations in analytical approaches that support the efforts of convicting cheating athletes by improved or newly established methods that focus on known as well as newly outlawed substances and doping methods. In the current review, literature published between October 2008 and September 2009 reporting on new and/or enhanced procedures and techniques for doping analysis, as well as aspects relevant to the doping control arena, was considered to complement the 2009 annual banned‐substance review. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Annual banned‐substance review: analytical approaches in human sports drug testing

Within the mosaic display of international anti‐doping efforts, analytical strategies based on up‐to‐date instrumentation as well as most recent information about physiology, pharmacology, metabolism, etc., of prohibited substances and methods of doping are indispensable. The continuous emergence of new chemical entities and the identification of arguably beneficial effects of established or even obsolete drugs on endurance, strength, and regeneration, necessitate frequent and adequate adaptations of sports drug testing procedures. These largely rely on exploiting new technologies, extending the substance coverage of existing test protocols, and generating new insights into metabolism, distribution, and elimination of compounds prohibited by the World Anti‐Doping Agency (WADA). In reference of the content of the 2014 Prohibited List, literature concerning human sports drug testing that was published between October 2013 and September 2014 is summarized and reviewed in this annual banned‐substance review, with particular emphasis on analytical approaches and their contribution to enhanced doping controls. Copyright © 2014 John Wiley & Sons, Ltd.     

Annual banned‐substance review – Analytical approaches in human sports drug testing

Within the complex construct of today's antidoping work, continuously updated routine doping controls, as well as advancements in sampling and analysis have been of particular relevance and importance. New analytes of existing classes of prohibited substances are frequently included into sports drug testing assays, analytical approaches are optimized to allow for better sensitivities, selectivity, and/or faster turnaround times, and research dedicated to addressing analytical issues concerning scenarios of both (potentially) inadvertent doping and new emerging doping agents is constantly conducted. By way of reviewing and summarizing, this annual banned‐substance review evaluates the literature published between October 2018 and September 2019 offering an in‐depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2019 Prohibited List.     

Optical coherent tomography for in vivo determination of changes in hair cross section and diameter during treatment with glucocorticosteroids--a simple method to screen for doping substances?

Eighty percent of hair follicles are in the growing phase. They grow approximately 0.3 mm/day. The hair follicles are surrounded by a close network of capillaries, which supplies them with nutrients. It is well known that substances which influence the metabolic processes of humans also influence hair growth. Steroids, which are used for doping in sport, are among these substances. In the present paper, optical coherent tomography is used for the analysis of changes in the hair structure during the application of steroids for the treatment of patients suffering from auto-immune diseases. Significant differences in the hair cross section could be detected during treatment, while the shape of the hairs was not influenced. It could be demonstrated that optical coherence tomography is a suitable, non-invasive and low-cost measuring technique that can be applied for doping control and screening. As a result of this screening process, only those athletes who show abnormalities in hair parameters would need to be investigated by classical analytical methods. The results presented in this study are not only important for doping controls, but also for several clinical applications, such as therapy and compliance control in cases where the applied substances induce changes in the hair structure.     

Doping detection in animals: A review of analytical methodologies published from 1990 to 2019

Despite the impressive innate physical abilities of horses, camels, greyhounds, or pigeons, doping agents might be administered to these animals to improve their performance. To control these illegal practices, anti-doping analytical methodologies have been developed. This review compiles the analytical methods that have been published for the detection of prohibited substances administered to animals involved in sports over 30 years. Relevant papers meeting the search criteria that discussed analytical methods aiming to detect and/or quantify doping substances in animal biological matrices published from 1990 to 2019 were considered. A total of 317 studies were included, of which 298 were related to horses, demonstrating significant advances toward the development of doping detection methods for equine sports. However, analytical methods for the detection of doping agents in sports involving other species are lacking. Due to enhanced accuracy and specificity, chromatographic analysis coupled to mass spectrometry detection is preferred over immunoassays. Regarding biological matrices, blood and urine remain the first choice, although alternative biological matrices, such as hair and feces, have been considered. With the increasing number and type of drugs used as doping agents, the analytes addressed in the published papers are diverse. It is very important to continue to detect and quantify these drugs, recognizing those that are most frequently used, in order to punish the abusers, protect animals' health, and ensure a healthier and genuine competition.     

Confiscated black market products and nutritional supplements with non-approved ingredients analyzed in the Cologne Doping Control Laboratory 2009

Doping control laboratories are frequently confronted with new substances that may be misused by athletes. Besides new pharmaceuticals, where method development for their detection is dependent on the availability of the substance and corresponding administration studies, some professional and amateur athletes are using illicit 'black market' products, which either differ from known pharmaceuticals but cause similar effects or still are undergoing clinical trials and are therefore rarely available to doping control laboratories. In the Cologne Doping Control Laboratory, different confiscated products and legally obtained nutritional supplements were analyzed in 2009, and various findings were reported including GH-labelled injection vials without any pharmacologically active content; combinations of products indicating the attempt to mask growth hormone abuse; unpurified long-R(3) -IGF-1; nutritional supplements containing the growth hormone releasing peptide-2 (GHRP-2); and ampoules containing the selective androgen receptor modulator Andarine (S-4). This review provides an overview on the substances that were analyzed in 2009. Ingredients relevant for doping control were identified by means of liquid chromatography and mass spectrometry methods. The awareness of new products on the black market and in nutritional supplements is of utmost importance for laboratories to develop detection methods accordingly and screen for new substances as early as possible.     

The androgen receptor and its use in biological assays: looking toward effect-based testing and its applications

Steroid abuse is a growing problem among amateur and professional athletes. Because of an inundation of newly and illegally synthesized steroids with minor structural modifications and other designer steroid receptor modulators, there is a need to develop new methods of detection which do not require prior knowledge of the abused steroid structure. The number of designer steroids currently being abused is unknown because detection methods in general are only identifying substances with a known structure. The detection of doping is moving away from merely checking for exposure to prohibited substance toward detecting an effect of prohibited substances, as biological assays can do. Cell-based biological assays are the next generation of assays which should be utilized by antidoping laboratories; they can detect androgenic anabolic steroid and other human androgen receptor (hAR) ligand presence without knowledge of their structure and assess the relative biological activity of these compounds. This review summarizes the hAR and its action and discusses its relevance to sports doping and its use in biological assays.     

Annual banned‐substance review: the Prohibited List 2008—analytical approaches in human sports drug testing

The list of prohibited substances and methods of doping issued by the World Anti‐Doping Agency is updated and modified annually based on most recent developments and scientific data. Compounds and methods are maintained, added, or removed from the list, or they are placed in so‐called monitoring programmes that have been established to obtain reliable data on the prevalence of particular substances and methods in‐ and/or out‐of‐competition. Consequently, doping control laboratories continuously update, modify and optimize existing screening and confirmation assays to ensure utmost comprehensiveness in detecting the prohibited and monitored substances as well as chemically and pharmacologically related analogs. The annual banned‐substance review for human sports drug testing critically summarizes recent innovations in analytical approaches supporting the detection of established and newly outlawed substances and methods of doping. Literature from January 2007 through September 2008 as indexed in Medline and Web of Science was screened and articles on detection methods for substances and methods of doping in humans were compiled according to the 2008 Prohibited List of the World Anti‐Doping Agency. Few new approaches were presented for individual doping agents and the majority of reports demonstrated new options for increasing the comprehensiveness of existing doping control assays. In addition, new techniques in separation and/or ionization of analytes complementary to commonly used procedures were described, which, so far, did not meet all requirements of sports drug testing. Copyright © 2009 John Wiley & Sons, Ltd.     

Tetrahydrogestrinone analysis and designer steroids revisited

Anabolic steroids are the main abused class of prohibited substances in doping control. These steroids are associated with enhancement of muscular mass and aggressiveness, resulting in increased performance. Chromatography and MS have a key role among methods developed to detect anabolic steroids in doping control laboratories. However, the classical analytical approach fails in detection of the so-called 'designer steroids'. This review focuses on the rise of tetrahydrogestrinone, a drug that became synonymous with designer steroids. The reasons why classical methods fail in tetrahydrogestrinone detection are discussed and how the detection was implemented is shown. Alternative strategies for detection of new drugs designed to cheat current analytical methodology are highlighted. Concern for the abuse of veterinary designer drugs and supplements is also acknowledged.     

A role for metabolomics in the antidoping toolbox?

Evidence of the continuous rise of novel doping agents and novel doping strategies calls for the development of more accurate multi‐target screening methods. Direct multi‐target screening approaches are restricted to the targeted substances and their turnover. The development of effective “indirect” screening methods requires a priori a deep understanding of the metabolism of the substance. The biological passport has been demonstrated to be very effective, but it is limited to about 20 indirect parameters. The standard antidoping analytical methods are hence targeted and do not aim directly to identify unknown substances. Also, the detection of doping agents is limited by the excretion of the substance. This study considers metabolomics for the screening of performance enhancing hormone abuse by athletes, based on the following pieces of evidence: (1) hormones have a strong influence on human metabolism, changing several parameters in many tissues, organs, and bio‐fluids; (2) metabolomics has been demonstrated to be a very accurate tool to depict the metabolic status of several organisms, tissues, and for several human diseases, including hormone deficiencies; (3) metabolomics has been demonstrated to be able to distinguish hormone‐treated animals from controls in many species, without the need for a priori knowledge of the metabolism for the specific substance. The literature shows that metabolomics could be an appropriate tool to detect hormone abuse, keeping in mind the strength and the limitation of such an approach.     

Annual banned‐substance review: analytical approaches in human sports drug testing

The aim of improving anti‐doping efforts is predicated on several different pillars, including, amongst others, optimized analytical methods. These commonly result from exploiting most recent developments in analytical instrumentation as well as research data on elite athletes' physiology in general, and pharmacology, metabolism, elimination, and downstream effects of prohibited substances and methods of doping, in particular. The need for frequent and adequate adaptations of sports drug testing procedures has been incessant, largely due to the uninterrupted emergence of new chemical entities but also due to the apparent use of established or even obsolete drugs for reasons other than therapeutic means, such as assumed beneficial effects on endurance, strength, and regeneration capacities. Continuing the series of annual banned‐substance reviews, literature concerning human sports drug testing published between October 2014 and September 2015 is summarized and reviewed in reference to the content of the 2015 Prohibited List as issued by the World Anti‐Doping Agency (WADA), with particular emphasis on analytical approaches and their contribution to enhanced doping controls.     

Annual banned‐substance review: Analytical approaches in human sports drug testing

Several high‐profile revelations concerning anti‐doping rule violations over the past 12 months have outlined the importance of tackling prevailing challenges and reducing the limitations of the current anti‐doping system. At this time, the necessity to enhance, expand, and improve analytical test methods in response to the substances outlined in the World Anti‐Doping Agency's (WADA) Prohibited List represents an increasingly crucial task for modern sports drug‐testing programs. The ability to improve analytical testing methods often relies on the expedient application of novel information regarding superior target analytes for sports drug‐testing assays, drug elimination profiles, alternative test matrices, together with recent advances in instrumental developments. This annual banned‐substance review evaluates literature published between October 2016 and September 2017 offering an in‐depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2017 Prohibited List.     

Annual banned‐substance review: Analytical approaches in human sports drug testing

A number of high profile revelations concerning anti‐doping rule violations over the past 12 months have outlined the importance of tackling prevailing challenges and reducing the limitations of the current anti‐doping system. At this time, the necessity to enhance, expand, and improve analytical test methods in response to the substances outlined in the World Anti‐Doping Agency (WADA) Prohibited List represents an increasingly crucial task for modern sports drug testing programs. The ability to improve analytical testing methods often relies on the expedient application of novel information regarding superior target analytes for sports drug testing assays, drug elimination profiles, and alternative sample matrices, together with recent advances in instrumental developments. This annual banned‐substance review evaluates literature published between October 2017 and September 2018 offering an in‐depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2018 Prohibited List.     

Doping agents and their analytical control

Doping is defined as the use of substances and/or forbidden methods for the artificial performance-enforcement of an athlete during competition or in the preparation period. A survey is given on the misuse of doping agents in recent years, with special emphasis on stimulants, beta blockers, diuretics, exogenous and endogenous steroids and peptide hormones. The introduction of new doping substances including perfluorocarbons, ecdysteroids, synthetic hemoglobin and peptide hormones (e.g. growth-hormone) precursors is described. Hyphenated separation techniques achieved great progress in doping analysis and control, of which some of the recent methods such as LC-MS, GC-MS CE-MS are briefly reviewed here.     

Annual banned-substance review: Analytical approaches in human sports drug testing 2020/2021

Most core areas of anti-doping research exploit and rely on analytical chemistry, applied to studies aiming at further improving the test methods' analytical sensitivity, the assays' comprehensiveness, the interpretation of metabolic profiles and patterns, but also at facilitating the differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds and comprehending the athlete's exposome. Further, a continuously growing number of advantages of complementary matrices such as dried blood spots have been identified and transferred from research to sports drug testing routine applications, with an overall gain of valuable additions to the anti-doping field. In this edition of the annual banned-substance review, literature on recent developments in anti-doping published between October 2020 and September 2021 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified in the World Anti-Doping Agency's 2021 Prohibited List.     

Drugs of abuse and abuse of drugs in sportsmen: the role of in vitro models to study effects and mechanisms

A particular field of analytical chemistry applied to forensic toxicology is represented by the anti-doping analysis, where biological samples (urine and in some instances blood) collected, either “in competition” or “out of competition”, from athletes ruled for national/international sport federations, are analyzed to detect the putative use of prohibited substances and methods. Together with the official anti-doping activity to test the athletes (i.e. who engages in competitive sport) for the non-physiological enhancement of sport performance, it is mandatory to activate a new strategy of doping control, that should necessarily comprise a deep and exhaustive toxicological evaluation of the entire spectrum of doping substances and methods. An outline of the present status and of the future trends of the antidoping research is here presented, showing that most of the new tasks could greatly benefit from an approach based on in vitro methods, ranging from specific toxicity studies to the possible detection of new forms of doping.     

Plasma volume expanders: use in medicine and detecting misuse in sports

Plasma volume expanders comprise a heterogeneous group of substances used in medicine that are intravenously administered in cases of great blood loss owing to surgery or medical emergency. These substances, however, can also be used to artificially enhance performance of healthy athletes in sport activities, and to mask the presence of others substances. These practices are considered doping, and are therefore prohibited by the International Olympic Committee and the World Antidoping Agency. Consequently, drug testing procedures are essential. The present work provides an overview of plasma volume expanders, assembling pertinent data such as chemical characteristics, physiological aspects, adverse effects, doping and analytical detection methods, which are currently dispersed in the literature.     

Erythropoiesis‐stimulating agents: development,detection and dangers

Epoetin alfa, the first member of the family of erythropoiesis stimulating agents (ESAs), was introduced to the market in 1989. Since then development has progressed to epoetins of the third generation. Currently drugs that use alternative approaches to stimulate erythropoiesis are under development. Uptake of all available ESAs into doping has occurred rapidly after their introduction. A multitude of dangers to health are associated with the illicit use of these substances. Different approaches to detect ESAs in doping control have been developed to comply with the very diverse nature of the compounds used. Future developments in the field of ESA require the development of new techniques in doping analysis. This review gives an overview of the development of ESA and its detection methods as well as future developments. [Correction made here after initial online publication] Copyright © 2009 John Wiley & Sons, Ltd.