1 Doping Valerie Waser, Jovana Teofilovic

2 Classification of substances and practices Sampling procedures Outline Introduction Classification of substances and practices Sampling procedures Analytical Strategies Analytical Methods Challenges with standard methods: new strategies and approaches Valerie Waser, Jovana Teofilovic

3 1) Introduction [1] Death of the cyclist Tom Simpson 1967 (Tour the france) lead to the elaboration of anti-doping-rules by the international cyclist union controls out-of-competions were introduced foundation of WADA (world- anti-doping agency) List of prohibited substances at olympic games 1972, in Munich. Tom Simpson at tour de france 1967, collapse and death due to excessive physical excertion after intake of alcohol and amphetamine Valerie Waser, Jovana Teofilovic

4 2) Classification of doping agents [1]Automatically included are substances with similar structures and pharmological effects. Valerie Waser, Jovana Teofilovic

5 Anabolic-androgenic stereoidsMost commonly used class of doping agents Derivatives of Testosterone Stimulation of protein-synthesis/buildup of mussle mass Side effects: Damage of cardio-vascular-system (heart attacks), liver damage, virilisation of woman, feminization of men Statistic of WADA-laboratories: Number of positive test results worldwide for anabol-andreoid-stereoids. Chemical structure of testosteron.

6 Peptidehormons Most commonly used hormons:Choriangonadotropin (hCG): pregnancy hormone of woman stimulates testostorne synthesis in man Human growth hormone (hGH): stimulation of fat- degradation, stimulation and protein synthesis Etythropoetin (EPO): stimulation of production of red blood cells in bone marrow Insulin: anabolic effects Detection is very difficult for endogenous hormons Valerie Waser, Jovana Teofilovic

8 Dieuretics Substances which stimulate the water excretion2 Effects which can be abused: decrease of body weight, beneficial for division into lower weight categories dilution of urine, manipulation of doping tests Chemical structure of furosemid (most found substance in the category of dieuretics by WADA ) Valerie Waser, Jovana Teofilovic

9 Antiestrogene Antiestrogenes: derivatives of diethylstilbestrol, blocking of estrogene-receptors Aromatase-blocking agents: blocks the aromatases involved in the transformation of testosterone to estrogens Used to suppress unwanted side effects of anabolic agents in men Chemical structure of diethylstilbestrol Valerie Waser, Jovana Teofilovic

10 Stimulants Prohibited only during competitionMost common compound: amphetamin and derivatives, effects in synapses similare to natural katecholamines (adrenalin, noradreanlin, dopamines), enlogated stimulation of target cell Chemical structure of amphetamine Chemical structure of noradrenalin Valerie Waser, Jovana Teofilovic

11 Narcotics and CannabioidsOnly prohibited during competition Narcotics: used for pain release Connabioids: used for calming effects Chemical Structure of THC (principal psychoaktive constituent of cannabis) Valerie Waser, Jovana Teofilovic

12 Glucokortikoids Immunosuppressant and anti-inflammatoryEffects on metabolism, supports the degradation of proteins to glucose (can be disadvantageous in the long term) Analgesic, relaxing effect of respiratory tract, euphorising A TUE can be submitted Structure of Triamcinolonhexacetonid, most common found compound of this class Valerie Waser, Jovana Teofilovic

13 Further doping practicesBlooddoping (enrichment of red blood cells by transfusion of autologous or homologeous blood) Mechanical practices for masking doping results (catheters etc.) Homologeous bood doping detectable since 2000 by antigene methodes Valerie Waser, Jovana Teofilovic

14 3) Sampling procedures [2]Antidoping Switzerland (missioned and financed by Swiss Government and Swiss olymipics): In and out of competition testing Valerie Waser, Jovana Teofilovic

15 Whereabout requirementsIn Competition: (executed by comissioned organisation of event) location of athletes should be known at all times Out of competition: (executed by antidoping Switzerland) pools of athletes taking part in national and international competitions, daily time windows in which tests can be carried out must be specified Extract of statues of antidoping switzerland «Ausführungsbestimmungen für Kontrollen und Ermittlungen» [2] Valerie Waser, Jovana Teofilovic

16 Choice of test individualsTargeted control of suspicious individuals (history fo doping, violation of wherabout requirements, suspicious history of perfomance etc.) Random selection Extract of statues of antidoping Switzerland «Ausführungsbestimmungen für Kontrollen und Ermittlungen» [2] Valerie Waser, Jovana Teofilovic

17 Sample collection Setup of control area on siteNotification and identification of test person, from the moment of notification, the test person must be accompanied by a dopping control officer at all times Providing a urine sample and/or blood sample (Sample A and B): Urine sample: min. 90 ml Blood sample: 13ml Sealing the probe: Containers and bottels cannot be reopenend, lids are broken off by the labaratory instantaneously before the analysis The probes are send to a WADA-accredited laboratory (if a substance of interest is found in sample A, the athlete may demand a second test on probe B) Valerie Waser, Jovana Teofilovic

18 4) Analytical strategies[4], [5] High sensitivity and selectivity in complex matrices Compounds with wide range of physico-chemical properties and molecular weight Limited sample volumes Results within 24-48h The analytical strategy must have: - high sensitivity and selectivity in complex matrices, involving mostly urine and blood samples - compounds with wide range of physico-chemical properties and molecular weight - limited sample volumes and - very fast turnaround time requirements (results within 24–48 h for important sport events). The analytical strategy is generally determined by the chemical structures of the substances and their biochemical behaviors, such as metabolism and excretion process. In anti-doping laboratories, a two-step analytical strategy is performed to fulfill WADA requirements: Screening step is conducted, which consists of detecting the highest number of relevant analytes in a complex matrix (i.e.,urine or blood).This step must be fast, selective and sensitive, while avoiding false-negative and falsepositive results. Confirmatory step must be performed to assess the presence of the suspected prohibited substance, and a quantitative estimation may be required. Valerie Waser, Jovana Teofilovic

19 WADA accredited laboratories report, 2012Samples [6] Urine Blood WADA accredited laboratories report, 2012 Challenges Complexity of the matrix Low concentration Sensitivity requirements Urine and blood (whole blood, serum, and plasma) are considered the matrices of choice for routine antidoping analysis. 1. Urine samples include its noninvasive collection and accessibility to large volumes of matrix. 2. Blood collection is still considered invasive and with a limited volume. For these reasons, the majority of antidoping controls is still carried out on urine. Since years anabolic agents are the most frequently detected doping substances in sports. This is also valid for Of 4500 adverse analytical and atypical findings reported by the World Anti-Doping Agency (WADA) accredited laboratories via the Anti-Doping Administration and Management System (ADAMS), about 50% are anabolic agents. Anabolic agents are detected in all sports. Because of the large number of compounds involved, the structural similarity of exogenous and endogenous steroids, the complexity of the urine matrix, the low steroid concentrations in urine and WADAs sensitivity requirements (MRPLs of 2–10 ng/mL), the analysis of anabolic steroids is challenging Valerie Waser, Jovana Teofilovic

20 Typical workflow of doping control analysis[4], [7] * Urine: Dilute & shoot, LLE, SPE, SLE * Blood: PP, LLE, SPE, SLE Dilute-and-shoot Generic, fast, inexpensive and does not require any specific equipment Stimulants, narcotics, diuretics, β2-agonists and β-blockers LLE Sufficient elimination of major interferences, simple, cost-efficient and provides a clean extracts Endogenous and exogenous steroids as well as glucocorticoids Direct analysis of urine is the ideal situation, but it is not feasible in the majority of cases due to matrix interferences, the effect of aqueous media in GC columns and the LODs required for these compounds. Sample preparation is a critical step due to the wide heterogeneity of the analytes and the complexity of the matrices containing salts, lipids, and proteins. Sample preparation is mandatory to achieve a sufficient level of sensitivity and selectivity and also to avoid clogging the chromatographic column and contaminating the mass spectrometer. The sample preparation procedure for doping control analysis should be as generic as possible because many illicit substances must be monitored simultaneously during the initial screening procedure. Various sample treatments (i.e., dilute and shoot, protein precipitation) or sample preparation techniques (i.e., solid-phase extraction SPE), liquid−liquid extraction (LLE), or supported-liquid extraction (SLE)) can be selected. The choice of the best sample preparation procedure should be based on the physicochemical properties of the substances and the employed analytical instrumentation. Dilute and shoot: This sample pretreatment approach is generic, fast, and inexpensive and does not require any specific equipment. However, in cases for which certain classes of forbidden substances should be monitored at trace levels in the biological fluid of interest (e.g., anabolic androgenic steroids), a preconcentration step is mandatory. LLE procedure has been historically the most widely used preconcentration technique in doping control analysis because the sensitivity of LC-MS or GC/MS platforms was limited in the past. Sufficient elimination of major interferences is obtained with LLE, and the extraction protocol is simple and cost-efficient and provides clean extracts. LLE is used for compounds such as endogenous and exogenous steroids as well as glucocorticoids. SLE affords faster sample preparation than LLE because there is no need for phase separation. SLE Alternative to the LLE Faster and better adapted for conformation procedurs of selected categories of forbidden compounds Valerie Waser, Jovana Teofilovic

21 Typical workflow of doping control analysis[4], [7] * Urine: Dilute & shoot, LLE, SPE, SLE * Blood: PP, LLE, SPE, SLE SPE Good alternative to LLE and SLE Ease of automation and simultaneous sample clean up and preconcentration Anabolic agents, β2-agonists, hormone antagonists and modulators, diuretics, stimulants, narcotics, glucocorticoids and β-blockers Superiority and more generic conditions provided by SPE. PP Blood samples Analogous to the dilute-and-shoot method for urine SPE is a good alternative to LLE or SLE. It is used for the anabolic agents, β2-agonists, hormone antagonists and modulators, diuretics, stimulants, narcotics, glucocorticoids, and β- blockers. For blood - protein precipitation (PP), which is analogous to the dilute-and-shoot method for urine. Protein precipitation is the least time-consuming sample preparation for plasma and serum. Despite the simplicity and rapidity of dilute-and-shoot and PP approaches, more selective sample preparations (e.g., LLE and SPE) are often preferred for urine and blood analyses. Valerie Waser, Jovana Teofilovic

22 SLE LLE SPE SLE: Its principles are relatively simple: a chemically inert, high-surface-area, highly purified, graded diatomaceous earth serves as a stationary vehicle for the aqueous phase of the LLE experiment. Water very easily adsorbs onto the surface (and is absorbed by the surface) of the diatomaceous earth particles. The dry solid sorbent [Figure 1(a)] is placed into a cartridge, column or well of a 96-well plate, the same devices used for solid-phase extraction (SPE). As shown in Figure 1(a), the aqueous-based sample (for example, diluted plasma or drinking water) is added to the dry sorbent and allowed to wet (disperse by capillary action and absorption) the diatomaceous earth, a process that usually takes only 5–10 min, sometimes a bit longer. Often the aqueous sample is pretreated (that is, the pH is adjusted or an ion-pairing agent or buffer is added) such that the analyte or analytes of interest are in a suitable form to be extracted into an organic solvent, just as is performed in conventional LLE. Next, a small volume of immiscible organic extraction solvent is added to the top of the cartridge and allowed to percolate by gravity (or sometimes with gentle vacuum or pressure) through the supported aqueous phase [Figure 1(c)]. For a more viscous liquid sample, a vacuum is used to pull liquid through the cartridges. Because the aqueous sample has been widely dispersed throughout the solid support, the organic solvent has intimate contact with the thin film of aqueous phase and rapid extraction (equilibration) occurs [Figure 1(c)]. As mentioned above, this intimate contact also occurs in LLE because of the vigorous shaking of the separatory funnel. This shaking causes the immiscible organic solvent to disperse into tiny droplets that provide closer contact to the surrounding aqueous solvent. The downside of this dispersion process in LLE is that emulsions can form and the two phases can take a long time to separate. The final step in the process is collection of the organic effluent containing the analytes of interest from the outlet of the SLE device [Figure 1(c), right-hand side]. The aqueous phase remains on the device. A phase-separation filter is incorporated into the outlet frit of the device to ensure that organic effluents remain uncontaminated by aqueous matrix. LLE: Liquid/liquid extraction involves adding a solvent to the sample that is immiscible, followed by selective partitioning of analytes versus contaminants between the two phases. In the interest of extraction completeness, it is necessary to use an adequate amount of extracting solvent to capture all of the analytes from the original sample. In most cases, a volume of extracting solvent equal to or less than the original sample volume is adequate, assuming reasonable analyte solubility in the solvent. This extracting solvent is added to the sample, then the two phases are agitated, by vortexing or shaking, to bring about substantial physical mixing. After agitate on, the phases are allowed to separate. SPE: SPE involves the use of a chromatographic sorbent in a column format. A sample is passed through the column bed, analytes retain on the sorbent while the sample matrix liquid passes through, then the sorbent bed is washed to remove undesired interferences, and the purified analytes subsequently eluted from the column. Valerie Waser, Jovana Teofilovic

23 Analysis Chemical (Separation techniques) Biological(Immunological tests) Two major types of analyses, chemical (separation techniques) and biological (immunological tests) are performed in anti-doping laboratories to cover all of the prohibited substances and methods. To be able to detect the current very sophisticated doping practices, these direct screening and confirmation procedures should be continuously updated, including monitoring additional substances with potential doping properties and/or new metabolites of already known compounds with increased detection windows. Valerie Waser, Jovana Teofilovic

24 Analytical Methods GC/MS(/MS)[7], [8] GC/MS(/MS) The most frequently detected doping substances worldwide are anabolics, stimulants and cannabinoids. GC-MS library - possibility of using existing mass spectral libraries. Hydrolysis and derivatization steps are required prior to the analysis of many doping agents to make them volatile and to improve their sensitivity. Hydrolysis and derivatization may induce important variablity; expensive and time-consuming. GC is interfaced with MS with either electronic impact (EI) or chemical ionization (CI) sources. GC is generally coupled to MS through electron impact (EI) ionization, and this approach has been particularly useful for toxicological laboratories due to the low interinstrument variability and the possibility of using existing mass spectral libraries. EI in full-scan mode is commonly used for anti-doping purpose in the screening step. GC–MS libraries contain many prohibited substances and their metabolites, which increase the probability to detect an unknown compound. Volatile and thermostable molecules on the WADA Prohibited List are directly compatible with GC–MS, while non-volatile compounds (e.g., anabolic steroids) can be effectively analyzed by GC if the targeted molecules are derivatized first. However, some derivatives are thermolabile and complex to analyze. When using GC, hydrolysis and derivatization steps are required prior to the analysis of many doping agents to make them sufficiently volatile and also to improve their sensitivity. These procedures are expensive and time-consuming because enzymatic hydrolysis can vary from minutes to hours depending on the incubation temperature, whereas derivatization can be performed within ∼1 h. Indeed, anabolic steroids are excreted in urine at very diverse concentrations, and there are also a large number of isomers and metabolites that are difficult to separate in a satisfactory manner. To improve the resolving power of GC, comprehensive two-dimensional GC (GCxGC) can be used to tackle the extreme complexity of samples. In addition, GCxGC is often coupled with high-resolution mass spectrometers possessing fast data acquisition rates. The presence of endogenous steroids and corticosteroids with a similar structure in the urine matrix can hamper the specific detection of the exogenous steroids. For this purpose, the selection of a specific MS2 transition is very important. Non-volatile or unstable substances, e.g. many anabolics, need to be prepared by derivatisation before they can be analysed. Valerie Waser, Jovana Teofilovic

25 GC/MS Long term metabolites[8], [9] Traditional analytical methods can detect AAS for only 5 days after administration. Long term metabolites and capable instrumentation (longer periods of time for AAS detection) Sophisticated violators of drug doping regulations know that AAS can be “safely” used in the “training” period, if their use is stopped long enough prior to a scheduled test to avoid detection. The timing between the last use of an AAS and urine sample collection for doping control is carefully adjusted by the violator for each drug to ensure negative test results. So, doping laboratories around the world are looking for ways of detecting AAS for longer periods of time after cessation of use. Typical doping control analysis for anabolic steroids and related substances in urine includes screening by gas chromatography/mass spectrometry (GC/MS). However, traditional analytical methods can typically detect AAS for only five days or so after administration. Detection of anabolic steroids for longer periods of time after drug use can be achieved by finding and monitoring longterm metabolites in urine and using instrumentation capable of detecting very low concentrations of AAS. Anabolics are present in urine either in free form or bound to sugar or sulphur moieties. Moreover, some anabolics are no longer present in their original form but have been transformed and broken down (metabolised) in the body. Metabolites are the intermediate products of metabolic reactions catalyzed by various enzymes that naturally occur within cells. Sample preparation 1. After initial cleanup (removal of urine matrix) on solid phase extraction cartridges, 2. The extract is incubated with b-glucuronidase to effectively cleave glucuronide conjugates and produce free steroids. 3. Glucoroconjugates present in urine are enzymatically hydrolyzed with beta-glucoronidase from E coli. 4. Also to cleave the sulfate conjugates, beta-glucoronidase/arylsulfatase is used. 5. Then an SPE or LLE step at basic pH, provides the purified anabolic steroids in the organic phase. 6. The liquid extract is evaporated to dryness and the residue derivatized for GC. Analytical GC-MS methods for determination of anabolic steroids in doping control require analyte derivatization to provide volatile species. Triple Quadropole GC/MS extends the detection window Triple Quadrupole GC/MS instrument can extend the detection window for anabolic steroids even further. This is due to a lack of matrix interferences and increased sensitivity over conventional GC/MS SIM analysis. Methenolone detection is possible 14 days after steroid administration using SRM, versus nine days with GC/MS SIM. After 12 days, GC/MS SIM analysis cannot distinguish the methenolone metabolite peak from the background. Analysis of the same urine samples on the Triple Quadrupole GC/MS using SRM is very selective, revealing (koji otkriva) the absence of a metabolite peak before administration due to a flat baseline, and can detect the metabolite as much as nine days after administration of the steroid. Conclusion A unique selective extraction procedure using b-Glucuronidase/Arylsulfatase provides metabolites cleaved from their sulfate conjugates and ready for derivatization and GC/MS analysis. This procedure can almost double the detection window for some anabolic steroids from five to nine days for methenolone, using GC/MS SIM analysis. Performing analysis on the Triple Quadrupole GC/MS using SRM can extend the detection time after administration from nine to 14 days for methenolone due to the elimination of matrix interferences. This instrument system also allows the monitoring of some anabolic steroids that are not easily analyzed by traditional GC/MS in the SIM mode. The Triple Quadrupole GC/MS is therefore a preferable alternative to GC/MS SIM for longterm monitoring of anabolic steroids. GC/MS Triple Quadropole GC/MS One of the used urine sample-preparation procedures prior to chromatographic identification and determination of steroids. Valerie Waser, Jovana Teofilovic

26 Analytical Methods LC/MS(/MS)[4], [5], [9] LC/MS(/MS) Screening for diuretics, gene doping substances, beta-blockers, glucocorticoids. Robust, easy and high sensitive Reduced sample pre-treatment, improved LODs Possibility of detecting thermolabile compounds and non-volatile analytes. Liquid chromatography (LC) is increasingly replacing gas chromatography (GC) as a separation method. This has the advantage that expensive sample preparation processes such as derivatisation can be omitted. Using LC–MS(/MS), sample preparation can be simple, fast and without requiring the removal of water or salts, deconjugation and derivatization. For doping control, LC–MS was initially focused on the analysis of thermolabile and non-volatile analytes that were not covered by the gold standard GC–MS method. Steroids with a 4,9,11-triene nucleus present difficulties in derivatization, and reduced thermal stability in GC analysis, so they need other methods. LC-MS2 - several advantages (e.g., reduced sample pre-treatment, improved LODs and excellent suitability for the multi-analyte screening), and is also a complementary tool to common GC-MS procedures for anabolic steroids. LC-MS-MS technique has proved to be a robust, easy and sensitive alternative to GC-MS, avoiding the derivatization step. Progress of LC-MS2 techniques and improvement in the analytical equipment have reduced the LODs for doping-control requirements. MS with triple-quadrupole instruments is a highly selective, sensitive technique for the detection of known steroids Structures of the most common 4,9,11-trien-3-one steroids Valerie Waser, Jovana Teofilovic

27 Analytical Methods Immunochemical methods (Immunoassays)[4] Immunochemical methods (Immunoassays) Antibody-antigen reaction Routinely used for detection of macromolecules in urine samples. Applied for the Initial Testing procedures and Conformation Procedures. Possibility of false-positive or false-negative results Challenging: newly marketed substances Immunoassays is usually employed for macromolecules. An immunoassay is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes). The molecule detected by the immunoassay is often referred to as an "analyte" and is in many cases a protein. Immunoassay methods are used for preliminary screening (i.e., initial screening). Since these methods are based on an antibody-antigen reaction, small amounts of the drug or metabolite(s) can be detected. Antibodies are tagged with markers such as an enzyme (enzyme immunoassay, EIA), a radio isotope (radioimmunoassay, RIA) or a fluorescence (fluorescence polarization immunoassay, FPIA) label. Valerie Waser, Jovana Teofilovic

28 Endogenous Steroidhormones[4], [5] Detection of the synthetic and natural testosterone: Determination of ratio between steriod hormone testosterone excreted in urine and a chemically similar steroide hormone epitestosterone (T/E) The natural ratios: Male: T/E= 1-2 Female: T/E< 1 Direct detection of synthetic testosterone by IRMS Detection of the bioidentical steroid hormone testosterone is relatively difficult because synthetic and natural testosterone are very similar in both structure and mass. Testosterone/epitestosterone ratio (T/E ratio), a value that is characteristic for every individual. T/E ratio In men, the natural T/E ratio is about 1 to 2; in women, the ratio is lower. After administration of testosterone, increased quantities of testosterone are present in urine while the amount of epitestosterone remains unchanged. As a result, the T/E ratio increases. If the T/E ratio in a male athlete is found to be greater than four, other investigations are carried out, such as a comparison with the T/E ratios of previous or subsequent samples (longitudinal studies). However, this procedure is very time-consuming and expensive. Neither GC–MS(/MS) nor LC–MS(/MS) are able to distinguish between endogenous analytes (e.g., testosterone) and corresponding synthetic analog. The first indication is obtained by conventional GC–MS screening after measuring the ratio of testosterone to epitestosterone (T/E). When T/E is higher than 4 or when the steroid profile is abnormal, a confirmatory procedure must be performed, and GC combustion IRMS (GC-C-IRMS) is the technique of choice to assess the intake of exogenous steroids by measuring the 13C/12C ratio. Direct detection of synthetic testosterone by IRMS An additional problem is the fact that the T/E ratio rapidly normalises after the administration of small amounts of testosterone. The ratio drops below the suspicion threshold of four within only a few hours. A more recent method makes it possible to detect synthetic testosterone directly. The method of isotope ratio mass spectrometry (IRMS) is used to determine the ratio of carbon isotopes, a value that distinguishes natural from synthetic testosterone. Valerie Waser, Jovana Teofilovic

29 6) Challenges with standard methods: new strategies and approachesDesinger drugs Endogenous substances Autologeous blood doping Inserate found on website of WADA ( ) Valerie Waser, Jovana Teofilovic

30 Non-target screening with tandem mass-spectrometryCommon fragment of mass spectra of testosterone derivatives. Non-target screening allows screening for structurally similar compounds. [10] Valerie Waser, Jovana Teofilovic

31 Biological passport and biomarkersExtract from statues of WADA «Athletes biological passport guidlines» 2017 [3] Introduced by WADA in 2009 Personal values are measured and saved in a personal profile, observation of abnormal blood profiles over time Valerie Waser, Jovana Teofilovic

32 Examples of a normal bloodprofile (upper) and with peculiar fluctuations (lower) [3]Valerie Waser, Jovana Teofilovic

33 Storage and reanalyisis [3]Practice introduced into the Code of WADA in 2015 Prioritiezed samples are stored and open to reanalyisis for 10 years to allow reanalyisis upon invention and establishment of new methods Measure for detterence Valerie Waser, Jovana Teofilovic

34 Metabolites are the intermediates and products of metabolism. Addition Why should the samples be stored up to 10 years and re-analyzed only then? Benefits and drawbacks of this approach. The timeline for storage should be a compromise between there being enough time for development of new and more sensitive detection methods and the detterence effect for the athlets still given. [11] Long term metabolites: definition, analytical strategies for identification and characterization in doping control. Metabolites are the intermediates and products of metabolism. The aim is to find transformation products of used substances in the human body after the «parent» compound is not detectable anymore. Timepoint and extend of appearance of the metabolites are determined by pharmacokinetics, which vary for different substances and individuals. Tranformation products are identified by methodologies such as fractionation and elucidation of urine samples collected from human administration studies and animal model elimination studies. [6] Valerie Waser, Jovana Teofilovic

35 Addition Inter-individual variation and retesting: how can the inter-inter-individual variations in the metabolism of prohibited substances influence the analysis results and retesting strategy. Individual aspects such as pharmaceutical, genetic and pathological variations can influence the uptake and transformation of used substances. Indirect approaches such as for example observation of the steroid profiles can take those variations into account. As the intake of androgenic-anabolic stereoids suppresses the synthesis of endegenouse stereoids, those are monitored and the information collected for each athlet within the Biological Passport. Individual references are calculated by comparison to population based reference ranges. In the case of suspicous profiles further investigation on the athlete can be carried out. [6] Valerie Waser, Jovana Teofilovic

36 Disadvantages: Instrument costAddition Analytical techniques: advantages and drawbacks of new analytical platforms in anti-doping analysis. To ensure a specific and sensitive detection in athlet’s urine, different analytical approaches were followed, such as liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry in order to detect and identify the new target analytes. [12] (See slides 24-26): GC-MS/MS Advantages: Low LOD, high sensitivity, low instrumentation variablity and possibility of using existing mass spectral libraries. Disadvantages: sample preparation (time consuming), not applicable for analysis of some substances LC-MS/MS Advantages: Expensive sample preparation process such as derivatisaion can be omitted, sample preparation is simple and fast, robust, easy and sensitive alternative to GC-MS, low LOD. Disadvantages: Instrument cost Valerie Waser, Jovana Teofilovic

37 6.) Is the retesting approach a real deterrence to athletes? Addition 6.) Is the retesting approach a real deterrence to athletes? The detterence effect of a measure is given by the likelihood of detection, severity of the penalty and speed with which sanctions will be applied. As in the restesting approach the likelihood and timepoint of getting caught is unkown, the value of this metod can be questioned. It can be argued that the dominating psychological determinant for decision making is the grade of common acceptance of practices in a sports community. [13] 7.) In your opinion, are there other alternatives/additional approaches to improve the fight against doping in the future? By improving analytical methods, imposing sanctions and deterring athles from doping, the athletes certainly get strongly limited in their scope of action. But there will probably always be methods or trials to forego the detection. Methods such as psychlogical approaches offer alternatives to this cat and mouse game. Intake of substances which are supposed to help with physical performance seem to be widespread in our culture, on amateur as well as professional levels. (continued on next slide) Valerie Waser, Jovana Teofilovic

38 Addition Continuation q7:One can try to induce processes of rethinking in a society by drastic prevention campaigns as it is done with smoking or drug abuse. For such measures to be effectful, strong evidence for the harm of doping has to be on hand, visible for the public and studies might have to be carried out. Valerie Waser, Jovana Teofilovic

39 Thank you for the attention!Two major types of analyses, chemical (separation techniques) and biological (immunological tests) are performed in anti-doping laboratories to cover all of the prohibited substances and methods. To be able to detect the current very sophisticated doping practices, these direct screening and confirmation procedures should be continuously updated, including monitoring additional substances with potential doping properties and/or new metabolites of already known compounds with increased detection windows. Valerie Waser, Jovana Teofilovic

40 References [1] Willhelm Schänzer; Mario Thevis, «Doping im Sport» in Medizinische Klinik (2007) [2] https://www.antidoping.ch/sites/default/files/downloads/2015/ab_k_e_2015_de_0.pdf ( ) [3] https://wada-main-prod.s3.amazonaws.com/resources/files/guidelines_abp_operating_v6_2017_jan.pdf ( ) [4] F. Badoud et al, Forensic Science International 213 (2011) 49-61 [5] ( ). [6] Geyer H, et al. Br J Sports Med 2014; 48: [7] Anal. Chem. 2016, 88, [8] ( ) [9] Anal. Chem. 2011, 30(5): [10] ( ) [11] Kuuranne T and Saugy M, Swiss Sports & Exercise Medicine, 64 [3], (2016) [12]S. Guddat, G. Fusshöller, S. Beuck, A. Thomas, H. Geyer, A. Rydevik, U.Bondesson, M. Hedeland, A. Lagojda, W. Schänzer, M. Thevis, Anal Bioanal Chem (2013) 405: [13] Jiri Dvorak, et. al., Br J Sports Med 2014; 48: Valerie Waser, Jovana Teofilovic