Analytical and Bioanalytical Chemistry https://doi.org/10.1007/s00216-018-0934-9

FEATURE ARTICLE

Analytical challenges in sports drug testing Mario Thevis 1,2 & Oliver Krug 1,2 & Hans Geyer 1,2 & Katja Walpurgis 1 & Norbert Baume 3 & Andreas Thomas 1 Received: 12 December 2017 / Revised: 11 January 2018 / Accepted: 31 January 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Analytical chemistry represents a central aspect of doping controls. Routine sports drug testing approaches are primarily designed to address the question whether a prohibited substance is present in a doping control sample and whether prohibited methods (for example, blood transfusion or sample manipulation) have been conducted by an athlete. As some athletes have availed themselves of the substantial breadth of research and development in the pharmaceutical arena, proactive and preventive measures are required such as the early implementation of new drug candidates and corresponding metabolites into routine doping control assays, even though these drug candidates are to date not approved for human use. Beyond this, analytical data are also cornerstones of investigations into atypical or adverse analytical findings, where the overall picture provides ample reason for follow-up studies. Such studies have been of most diverse nature, and tailored approaches have been required to probe hypotheses and scenarios reported by the involved parties concerning the plausibility and consistency of statements and (analytical) facts. In order to outline the variety of challenges that doping control laboratories are facing besides providing optimal detection capabilities and analytical comprehensiveness, selected case vignettes involving the follow-up of unconventional adverse analytical findings, urine sample manipulation, drug/food contamination issues, and unexpected biotransformation reactions are thematized. Keywords Clenbuterol . Proguanil . Chlorazanil . Manipulation . Doping . Mass spectrometry

Introduction Sports drug testing is a multi-faceted endeavor that has been governed internationally by the World Anti-Doping Agency (WADA) since 2004. A major aspect of routine doping controls is the specific, sensitive, and comprehensive detection of prohibited substances and methods of

* Mario Thevis thevis@dshs–koeln.de 1

Center for Preventive Doping Research – Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany

2

European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany

3

Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Geneva and Lausanne, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Chemin des Crosiettes 22, 1066 Epalinges, Switzerland

doping as classified in WADA’s annually issued Prohibited List [1]. This list has been subject to continual review processes that resulted in a series of additions and modifications over the years, which were primarily made in order to account for the constantly growing number of drug candidates (possessing the potential for misuse in sport) but also with respect to newly generated scientific data that warranted the elimination of formerly prohibited substances from the list. Besides this dominant part of the doping control laboratories’ tasks, i.e., providing routine analytical data for doping controls, they frequently address a variety of challenges that are not immediately perceived as part of the anti-doping laboratories’ assignment and activity. These challenges include for example investigations into emerging non-approved drug candidates or designer substances and their implementation into routine tests, the clarification of the origin of a prohibited substance and related adverse analytical findings (AAFs) to support result management, or the characterization of samples provided as an individual’s doping control specimen, which yielded analytical results that indicate manipulation.

Thevis M. et al.

Frequency

SARMs findings 2010 - 2016 45 40 35 30 25 20 15 10 5 0 2010

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ostarine

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A frequently suspected and occasionally proven challenge in doping controls has been the misuse of new drug entities or designer drugs that have not undergone or completed clinical trials. Due to commonly limited information on these compounds’ structures and/or metabolic fate, their detection by means of routinely applied chromatographic-mass spectrometric approaches has been particularly difficult. Nevertheless, the number of case reports dealing with such findings has been increasing, supported by a substantially and continuously improving quality of analytical instruments and available test methods but also by significant intelligence contributions. Earlier examples include, e.g., the detection of bromantan in 1996 [2] and tetrahydrogestrinone (THG) in 2003 [3], and more recently cases concerning selective androgen receptor modulators (SARMs), peroxisome proliferatoractivated receptor delta (PPARδ) agonists, and hypoxiainducible factor (HIF) stabilizers were reported (vide infra). Research into the class of SARMs in the context of sports drug testing was initiated in 2005 due to the recognized potential for misuse resulting from significant muscle and bone anabolic properties of these agents. SARMs are being developed for the management and/or prevention of conditions involving the loss of muscle mass (e.g., cachexia or sarcopenia), hypogonadism, and frailty [4], but to date no drug candidate has received full clinical approval. Regardless of the lack of clinical approval, various SARMs have been found to be readily available through Internet-based suppliers, which necessitated studies enabling the inclusion of these compounds and, where available, known metabolites into routine doping controls. However, the absence of certified reference material and/ or published structures of human urinary metabolites have been substantial obstacles towards efficient doping controls. Consequently, metabolic reactions have been simulated using in vitro or animal in vivo models, and generated metabolites were studied using liquid chromatography/high-resolution/ high-accuracy mass spectrometry to provide sufficient information for subsequent chemical synthesis of target analytes suited for routine sports drug testing programs. This strategy proved justified already in 2010, when first AAFs for the drug candidate S-4 (also referred to as andarine) were reported [5]. Of note, andarine was discontinued during phase-II clinical trials in 2006 [6]; yet, the drug candidate found its way into the world of sports and numerous additional findings of other SARMs followed (Fig. 1a), primarily concerning ostarine (enobosarm, S-22), RAD140, and LGD-4033 [7]. Despite their significant structural differences, these SARMs were found to be eliminated into urine largely as the intact drug conjugated to glucuronic acid, which allowed targeting them by extending existing LC-MS/MS-based test methods with diagnostic precursor/product ion pairs. Of note, a considerable

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Year Fig. 1 Frequency of AAFs as reported by WADA-accredited doping control laboratories between 2010 and 2016 indicating the growing concern of SARMs in sports (a), the decreasing prevalence of GW1516 (b), and the low detection rate of sample manipulation (c), with the latter inherently remaining complex to uncover in doping controls

number of AAFs with ostarine was allegedly covered up in a major doping scandal revealed in 2016 [8], which further highlights the importance of preventive research including

Analytical challenges in sports drug testing

studies on metabolic biotransformation reactions and the early implementation of testing strategies for emerging doping agents while strictly ensuring the obligatory specificity of the analytical approaches [9]. The considerable heterogeneity of SARMs comprising of steroidal as well as non-steroidal substances and information concerning analytical approaches in routine doping controls has recently been summarized elsewhere [10]. A similar history as for the SARM andarine was registered for the PPARδ agonist GW1516. Referred to as an experimental Bexercise mimetic^ in 2008 (due to its capability of stimulating and synergizing with gene expressions normally associated with physical exercise) [11], GW1516 was added to WADA’s Prohibited List that came into effect in 2009. Despite the fact that the drug candidate’s development was discontinued in 2007 [12], test methods had to be established for routine doping controls [13], again (at that time) in the absence of certified reference materials and data on human metabolism and elimination profiles. Only by chemical synthesis and with the aid of liquid chromatography/ low- and high-resolution/high accuracy mass spectrometry were target analytes identified and characterized, which eventually allowed for detecting a series of AAFs between 2013 and 2016 (Fig. 1b), particularly by aiming at the sulfone metabolite of GW1516. Among new drug candidates, also the class of hypoxia-inducible factor (HIF) stabilizers, more specifically prolyl hydroxylase inhibitors, has received growing attention in anti-doping research. HIF stabilizers suc h as ro xadu stat, m olidus ta t, vad adusta t, or daprodustat have undergone advanced clinical trials in the context of renal disease-induced anemia [14], and their erythropoiesis-stimulating properties have necessitated consideration of these agents also in doping controls. The aforementioned HIF stabilizers were shown to be orally available and increase plasma concentrations of erythropoietin (EPO), which can translate into enhanced red blood cell production and oxygen transport capacities. In anticipation of their potential misuse in sport, which was unmistakably demonstrated in Internet-based chat room discussions following the public announcement of upcoming phase-I clinical trials with drug candidates, proactive studies focusing on sports drug testing method development and metabolic biotransformation were conducted [15, 16]. The market launch of approved drugs is still missing; however, already two of the drug candidates were reportedly detected in routine doping controls including roxadustat (FG4592) in 2015 [17] and molidustat in 2017 [18], corroborating once more the relevance of preventive antidoping research and industry collaborations regarding new drug entities. The approach of preventive anti-

doping research concerning new erythropoiesisstimulating agents has recently been extended to antibody- or decoy receptor-based therapeutic ligand traps such as sotatercept and luspatercept [19, 20]. Due to the proteinaceous composition of these drug candidates, combined immunological, electrophoretic, and bottom-up mass spectrometric identification strategies were suggested to allow for an unequivocal determination of these agents in doping controls. To date, no AAF with these therapeutics has been reported.

AAFs presumably caused by circumstances other than intentional use of a prohibited substance According to the World Anti-Doping Code, the presence of a prohibited substance or its metabolites or markers in an athlete’s sample constitutes an anti-doping rule violation and the principle of strict liability applies [21]. A concomitant phenomenon of AAFs has been the issuing of explanations (other than the intentional administration of a doping agent) about which situation arguably led to the finding of a prohibited substance. These explanations have occasionally been beyond belief; however, in some cases, follow-up investigations of doping control laboratories corroborated the plausibility of scenarios that were initially considered extremely unlikely. Various examples exist that demonstrate the importance of case-related research, which significantly assisted the authorities in result management process and contributed to clarifying specific anti-doping rule violations. A prominent instance of unintentional ingestion of a doping agent has been the issue of clenbuterolcontaminated meat in different countries [22], and a significant number of athletes has been affected by the presence of the anabolic agent clenbuterol in food products in the past. Circumstantial evidence including the consideration of the individuals’ whereabouts, the prevalence of meat contamination in the visited countries (or countries of residence), actual food analyses, and/or hair testing [23] has been taken into account on a case-bycase basis in the course of result management. As a consequence, numerous athletes were exonerated from the suspicion of the intentional misuse of clenbuterol, but until today, the unequivocal analytical distinction of clenbuterol misuse from an inadvertent ingestion by means of routine doping control strategies has not been accomplished [24]. While the issue of clenbuterol-contaminated food or the problem of anabolic steroid- or stimulantcontaminated dietary supplements was known for many

Thevis M. et al.

years, the presence of trace amounts of diuretics in approved over-the-counter analgesic was not reported until 2015 [25]. In the course of an inquest into an AAF concerning hydrochlorothiazide, only the detailed investigation of a reported scenario involving the use of a permitt ed non-steroidal ant i-i nflammatory drug (NSAID) enabled clarifying the origin of the banned substance in the doping control sample. A series of experiments including the analysis of the athlete’s remaining tablet and corresponding retention samples of the manufacturer, and the verification of the reported drug administration regimen allowed confirming the presence of hydrochlorothiazide contaminations in the coating of the NSAID tablets. Of note, the contamination did not exceed the permitted good manufacturing practice limit of 10 ppm. Yet, by means of placebo tablets that were enriched with 2.5 μg of hydrochlorothiazide and administered to a volunteer in accordance to the protocol of drug use provided by the athlete, urinary concentrations of hydrochlorothiazide matching those detected in the doping control sample were obtained. Overall, the body of evidence corroborated the unintentional ingestion of a pharmacologically irrelevant dose of a diuretic by the athlete, and no fault or negligence was established. Analogously, another athlete was cleared recently for a similar finding [26], highlighting the fact that such cases are not necessarily of singular occurrence. In 2014, the worldwide first AAFs concerning the diuretic chlorazanil were registered. Since the installation of the ban of diuretics in sport in 1988, not a single incidence of chlorazanil was reported, and the drug has meanwhile become obsolete in clinical practice. Regardless, two individuals were tested positive in 2014 for the presence of chlorazanil, and follow-up studies were considered warranted [27]. A common aspect of both athletes who produced the AAFs was the prior use of proguanil, a permitted drug used for malaria chemoprophylaxis. Despite a structural similarity, no misinterpretation of proguanil as chlorazanil or metabolic conversions of proguanil into chlorazanil was found or confirmed. Further, no drug impurities or contaminations with chlorazanil were found. Eventually, the in vesica biotransformation of a metabolite of proguanil, namely N-(4-chlorophenyl)-biguanide, which reacts under physiological conditions with formaldehyde, was identified as the origin of the urinary chlorazanil. Urinary formaldehyde concentrations are known to increase under dietary supplementation of creatine, which is also a common scenario in the world of sport, and the exposure of the major metabolite N-(4chlorophenyl)-biguanide to formaldehyde was identified as the most likely source of the diuretic. The scenario

was reproduced in a laboratory setting where urine was enriched with N-(4-chlorophenyl)-biguanide and formaldehyde. In addition, urine samples from patients who received proguanil-based malaria chemoprophylaxis were collected and analyzed, and some of these were also found to contain trace amounts of chlorazanil. In a situation where no misconduct by any of the involved parties (i.e., athlete, physician, or laboratory) existed, an authentic AAF was produced, which was plausibly attributed to a formerly unrecognized conversion of a permitted drug to a prohibited substance.

AAFs attributed to sample manipulation Urine sample manipulation has been a rarely detected anti-doping rule violation in the past (Fig. 1c) but was recently mentioned as a mainstay of an alleged system installed to comprehensively cover-up doping practices in Russia [8]. One approach towards identifying manipulated doping control specimens utilizes initial testing procedures commonly used in doping controls. These include, among several assays, steroid profile analysis and gas chromatography-mass spectrometry (GC-MS)/nitrogen phosphorus detection-based measurements. Human urine contains a distinct pattern of naturally occurring and endogenously produced steroidal compounds, including, e.g., testosterone, epitestosterone, androsterone, and etiocholanolone, which yield an individual’s Burinary signature^ utilized in sports drug testing. If these analytes are not detected in a doping control sample, manipulation is principally proven. However, in order to further corroborate the AAF, the identification of the sample’s surrogate is frequently requested, and a variety of analytical strategies can be applied. In the case of a urine sample substitution using alcoholfree beer [28], the specimen was characterized by proteomics methods flanked with GC-MS and GC-FID measurements. Eventually, the identification of barleyspecific proteins such as serpin-Z4, the presence of hordenine, and an ethanol content of 120 mg/kg (0.015%) supported the assumption that a brewing product rather than a urine sample was provided. A similar case (but literally of different flavor) occurred when a water sample was declared as an athlete’s doping control specimen. Again, the absence of any commonly observed human urinary steroid hormone was indicative for sample manipulation, but in contrast to the aforementioned beer, it was the lack of any organic matter (including, e.g., creatinine, urea, uric acid) and the specific gravity of 1.001 that led to the (preliminary) conclusion that not human urine but only water was in the doping control sample container. The comparison to a urine sample collected from the same athlete at a later occasion corroborated the suspicion of

Analytical challenges in sports drug testing

manipulation as the analysis of the additional sample yielded levels within commonly observed reference ranges for all tested parameters (Table 1). Scenarios different from the aforementioned urine manipulation were uncovered when identical steroid profiles were determined for urine samples presumably originating from different athletes. In one case, three team members were urine-sampled for doping controls out-of-competition, and the obtained data presented identical steroid profiles. Subsequently collected buccal cell material and blood specimens and DNA-STR analyses of both urine and buccal cell/blood samples revealed that the urine originated from none of the athletes but was apparently substituted for the athletes’ own urines with fraudulent intent [30]. In a similar case, a total of eight identical steroid profiles was obtained from arguably different athletes competing at different locations. In contrast to the aforementioned report, the only aspect common to all eight controls was the doping control officer (DCO). An inquest into the finding eventually proved that the DCO did not visit the athletes but filled all doping control sample containers with her own urine, forged the corresponding documentation, and sent the specimens to the designated laboratory for analysis [28]. As the athletes were verifiably not involved in the uncovered manipulation, only the DCO was sanctioned.

Table 1 Test results for doping control samples indicating manipulation

Tested parameter

Appearance Specific gravity pH Odor Urea Creatinine Uric acid Androsterone Etiocholanolone Testosterone Epitestosterone 5α-Androstanediol 5β-Androstanediol a

[29]

Conclusion The analytical challenges of sports drug testing are manifold. On the one hand, there is the desire and the need for utmost analytical sensitivity and comprehensiveness that enables an appropriate coverage of (new) analytes as well as periods between two doping controls, i.e., that allows for detecting the misuse of prohibited substances with sufficient retrospectivity as athletes cannot and/or should not be subjected to the procedure of doping controls too often. On the other hand, the capability of measuring trace amounts of banned substances does not necessarily enable the differentiation of the administration of pharmacologically relevant doses and intentional drug misuse from situations of inadvertent drug administration. Minute amounts of a doping agent found in a single doping control sample can be the result of a recent intake of a low dose (cf. abovementioned contamination cases) or originate from doping practices days, weeks, or even months ago, which are to be uncovered and sanctioned. Hence, thorough result management of AAFs has been of immense importance, and the consideration of numerous aspects such as the athlete’s testing frequency, whereabouts, earlier test results, and indicated medication has been shown to be critical.

Sample 1

Sample 2

Colorless liquid 1.001 7.8 No urine odor

Yellowish liquid 1.012 6.5 Urine odor

< LOD (1.2 mg/dL) < LOD (0.2 mg/dL) < LOD (0.4 mg/dL) < LOD (0.5 ng/mL) < LOD (0.5 ng/mL) < LOD (0.1 ng/mL) < LOD (0.1 ng/mL) < LOD (0.5 ng/mL) < LOD (0.5 ng/mL)

920 mg/dL 66.6 mg/dL 25.3 mg/dL 1468 ng/mL 1243 ng/mL 6.5 ng/mL 2.7 ng/mL 17.0 ng/mL 76.7 ng/mL

Commonly observed urinary values (males)

ca. 900–2430 mg/dL ca. 39–260 mg/dL ca. 21–56 mg/dL ca. 1300–5000 ng/mLa ca. 1000–4000 ng/mLa ca. 6–100 ng/mLa ca. 9–70 ng/mLa ca. 20–110 ng/mLa ca. 40–350 ng/mLa

Thevis M. et al. Acknowledgments The authors thank the Manfred-Donike-Institute for Doping Analysis (Cologne, Germany) and the Federal Ministry of the Interior of the Federal Republic of Germany (Bonn, Germany) for the support.

Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

References 1.

2.

3.

4.

5.

6.

7. 8.

9.

10.

11.

12.

13.

14. 15.

WADA. The 2018 Prohibited List. Montreal: Wolrd Anti-Doping Agency; 2017 [cited 26.10.2017]. Available from: https://www. wada-ama.org/sites/default/files/prohibited_list_2018_en.pdf. Kidd B, Edelman R, Brownell S. Comparative analysis of doping scandals: Canada, Russia, and China. In: Wilson W, Derse E, editors. Doping in elite sport. Chamaign: Human Kinetics; 2001. p. 153–88. Catlin DH, Sekera MH, Ahrens BD, Starcevic B, Chang Y-C, Hatton CK. Tetrahydrogestrinone: discovery, synthesis, and detection in urine. Rapid Commun Mass Spectrom. 2004;18(12):1245– 9. Dalton JT, Taylor RP, Mohler ML, Steiner MS. Selective androgen receptor modulators for the prevention and treatment of muscle wasting associated with cancer. Curr Opin Support Palliat Care. 2013;7(4):345–51. Grata E, Perrenoud L, Saugy M, Baume N. SARM-S4 and metabolites detection in sports drug testing: a case report. Forensic Sci Int. 2011;213(1–3):104–8. Thevis M, Volmer DA. Mass spectrometric studies on selective androgen receptor modulators (SARMs) using electron ionization and electrospray ionization/collision-induced dissociation. Eur J M a s s S p e c t r o m . 2 0 1 7 ; 2 3 . h t t p s : / / d o i . o r g / 1 0 . 11 7 7 / 1469066717731228. Cox HD, Eichner D. Detection of LGD-4033 and its metabolites in athlete urine samples. Drug Test Anal. 2017;9(1):127–34. McLaren R. The Independent Person 2nd Report: Richard McLaren; 2016 [cited 27.10.2017]. Available from: https://www. wada-ama.org/sites/default/files/resources/files/mclaren_report_ part_ii_2.pdf. Perrenoud L, Schweizer Grundisch C, Baume N, Saugy M, Nicoli R. Risk of false positive results to SARM S-4 in case of therapeutic use of antineoplastic/antiandrogen drug containing flutamide: a case study. Drug Test Anal. 2016;8(11–12):1109–13. Thevis M, Schänzer W. Detection of SARMs in doping control analysis. Mol Cell Endocrinol. 2017;in press; https://doi.org/10. 1016/j.mce.2017.01.040. Narkar VA, Downes M, Yu RT, Embler E, Wang YX, Banayo E, et al. AMPK and PPARdelta agonists are exercise mimetics. Cell. 2008;134(3):405–15. Billin AN. PPAR-beta/delta agonists for type 2 diabetes and dyslipidemia: an adopted orphan still looking for a home. Expert Opin Investig Drugs. 2008;17(10):1465–71. Thevis M, Möller I, Thomas A, Beuck S, Rodchenkov G, Bornatsch W, et al. Characterization of two major urinary metabolites of the PPARdelta-agonist GW1516 and implementation of the drug in routine doping controls. Anal Bioanal Chem. 2010;396(7): 2479–91. Schmid H, Jelkmann W. Investigational therapies for renal diseaseinduced anemia. Expert Opin Investig Drugs. 2016;25(8):901–16. Eichner D, Van Wagoner RM, Brenner M, Chou J, Leigh S, Wright LR, et al. Implementation of the prolyl hydroxylase inhibitor

Roxadustat (FG-4592) and its main metabolites into routine doping controls. Drug Test Anal. 2017; https://doi.org/10.1002/dta.2202. 16. Dib J, Mongongu C, Buisson C, Molina A, Schänzer W, Thuss U, et al. Mass spectrometric characterization of the hypoxia-inducible factor (HIF) stabilizer drug candidate BAY 85-3934 (molidustat) and its glucuronidated metabolite BAY-348, and their implementation into routine doping controls. Drug Test Anal. 2017;9:61–7. 17. Buisson C, Marchand A, Bailloux I, Lahaussois A, Martin L, Molina A. Detection by LC-MS/MS of HIF stabilizer FG-4592 used as a new doping agent: investigation on a positive case. J Pharm Biomed Anal. 2016;121:181–7. 18. CyclingNews. Kvasina positive for EPO stimulator at Fleche du Sud: CyclingNews; 2017 [cited 05–07-2017]. Available from: http://www.cyclingnews.com/news/kvasina-positive-for-epostimulator-at-fleche-du-sud/. 19. Walpurgis K, Thomas A, Vogel M, Reichel C, Geyer H, Schänzer W, et al. Testing for the erythropoiesis-stimulating agent Sotatercept/ACE-011 (ActRIIA-Fc) in serum by means of western blotting and LC-HRMS. Drug Test Anal. 2016;8(11–12):1152–61. 20. Reichel C, Gmeiner G, Thevis M. Antibody-based strategies for the detection of luspatercept (ACE-536) in human serum. Drug Test Anal. 2017; https://doi.org/10.1002/dta.2302. 21. WADA. World Anti-Doping Code. Montreal: World Anti-Doping Agency; 2015 [cited 12–04-2016]. Available from: https://wadamain-prod.s3.amazonaws.com/resources/files/wada-2015-worldanti-doping-code.pdf. 22. Thevis M, Geyer L, Geyer H, Guddat S, Dvorak J, Butch A, et al. Adverse analytical findings with clenbuterol among U-17 soccer players attributed to food contamination issues. Drug Test Anal. 2013;5:372–6. 23. Krumbholz A, Anielski P, Gfrerer L, Graw M, Geyer H, Schanzer W, et al. Statistical significance of hair analysis of clenbuterol to discriminate therapeutic use from contamination. Drug Test Anal. 2014;6(11–12):1108–16. 24. Parr MK, Blokland MH, Liebetrau F, Schmidt AH, Meijer T, Stanic M, et al. Distinction of clenbuterol intake from drug or contaminated food of animal origin in a controlled administration trial—the potential of enantiomeric separation for doping control analysis. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2017;34(4):525–35. 25. Helmlin H-J, Mürner A, Steiner S, Kamber M, Weber C, Geyer H, et al. Detection of the diuretic hydrochlorothiazide in a doping control urine sample as the result of a non-steroidal anti-inflammatory drug (NSAID) tablet contamination. Forensic Sci Int. 2016;267:166–72. 26. USADA. U.S. Volleyball Athlete Alexandra Klineman Accepts Finding of No Fault for Anti-Doping Rule Violation Colorado Springs: U.S. Anti-Doping Agency; 2017 [cited 17–11-2017]. Available from: https://www.usada.org/alexandra-klinemanaccepts-finding-no-fault/. 27. Thevis M, Geyer H, Thomas A, Tretzel L, Bailloux I, Buisson C, et al. Formation of the diuretic chlorazanil from the antimalarial drug proguanil—implications for sports drug testing. J Pharm Biomed Anal. 2015;115:208–13. 28. Thevis M, Geyer H, Sigmund G, Schänzer W. Sports drug testing: analytical aspects of selected cases of suspected, purported, and proven urine manipulation. J Pharm Biomed Anal. 2012;57:26–32. 29. Geyer H, Mareck U, Schänzer W, Donike M, editors. The cologne protocol to follow up high testosterone/epitestosterone ratios. Recent advances in doping analysis. Cologne: Sport und Buch Strauss; 1997. 30. Thevis M, Geyer H, Mareck U, Sigmund G, Henke J, Henke L, et al. Detection of manipulation in doping control urine sample collection: a multidisciplinary approach to determine identical urine samples. Anal Bioanal Chem. 2007;388:1539–43.

Analytical challenges in sports drug testing Mario Thevis is a forensic chemist and the Director of the WADAaccredited doping control laboratory of Cologne, Germany. Both routine doping controls as well as research into drug metabolism and optimization of detection methods and alternative testing matrices are conducted under his supervision.

Oliver Krug is a scientific associate at the Institute of Biochemistry/ European Centre for Emerging Doping Agents. His main field of research is the investigation of upcoming black market products and structure elucidation of emerging drugs.

Hans Geyer is Deputy Director of the WADA-accredited doping control laboratory of Cologne. His particular research interests and expertise are steroid metabolism and steroid profile analyses.

Katja Walpurgis is a postdoctoral researcher at the Center for Preventive Doping Research/ Institute of Biochemistry of the German Sport University Cologne and her work focuses on the development of novel detection methods for performanceenhancing protein drugs by using different proteomics techniques such as gel electrophoresis, western blotting, and LC-HRMS.

Norbert Baume is the Deputy Director of the Swiss Laboratory for Doping Analyses in Lausanne, Switzerland. He has been working for several years in the antidoping field and his main interests are the metabolism and detection of exogenous and endogenous anabolic steroids as well as the implementation and development linked to the Athlete Biological Passport.

Andreas Thomas has worked as a senior scientist in the field of doping control analysis for more than 12 years. His special interest is focused on identification of prohibited substances such as small molecules, peptides, and proteins by means of LC-MS.