About Doping PowerPoint Presentation

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Doping Christof Münch and Alexander Gogos 29. 22. 2011

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2/50 Content Doping: definition, history, substances and effects Doping control: History and Institutions International standards for laboratories Analytical strategy: Challenges, Instruments and Methods Questions 1& 2: Urine and blood matrix Questions 3 & 4: Alternative fluids and urine analysis for cocaine and cannabis □ doping □ WADA □ analysis □ blood & urine □ alternative fluids

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3/50 Definition of Doping The word doping is probably derived from the Dutch word dop, the name of an alcoholic beverage made of grape skins used by Zulu warriors in order to enhance their prowess in battle The practice of enhancing performance through foreign substances or other artificial means ■ doping □ WADA □ analysis □ blood & urine □ alternative fluids

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4/50 Timeline ■ doping □ WADA □ analysis □ blood & urine □ alternative fluids …a long way until the founding of the World Anti-Doping Agency (WADA) in 1999!

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5/50 Famous cases in the past – example Tour de France 1998: Festina 2000: Fuentes 2006: Landis ■ doping □ WADA □ analysis □ blood & urine □ alternative fluids Source: Wikipedia

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6/50 Classes of prohibited substances and activities ■ doping □ WADA □ analysis □ blood & urine □ alternative fluids Alcohol Beta-blockers 6 groups (see next slide) Enhancement of oxygen transfer (Blood doping) Chemical and physical manipulation (Tampering with samples) Gene doping Methods Substances Substances prohibited in particular sports Source: Badoud et al. (2010)

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7/50 Classes of doping agents ■ doping □ WADA □ analysis □ blood & urine □ alternative fluids Source: WADA (World anti-doping agency), Figures: Wikipedia Prohibited at all times (in- and outside competition)

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8/50 Classes of doping agents Many many more… ■ doping □ WADA □ analysis □ blood & urine □ alternative fluids

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10/50 Doping control and Institutions criminal prosecution in some countries (Italy, France and others) World anti-doping agency (WADA) set up in 1999 World anti-doping code: harmonization of worldwide efforts Includes also „non-analytical“ rule violations! Governmental representations in WADA executive committee Code accepted by Olympic movement Government funded organizations Some institutions out of the olympic movement □ doping ■ WADA □ analysis □ blood & urine □ alternative fluids

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□ doping ■ WADA □ analysis □ blood & urine □ alternative fluids 11/50 International standard for laboratories

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Source: WADA 2009 12/50 International standard “International Standards for different technical and operational areas within the anti-doping program will be developed in consultation with the Signatories and governments and approved by WADA. The purpose of the International Standards is harmonization among Anti-Doping Organizations responsible for specific technical and operational parts of the anti-doping programs. Adherence to the International Standards is mandatory for compliance with the Code. The International Standards may be revised from time to time by the WADA Executive Committee after reasonable consultation with the Signatories and governments. Unless provided otherwise in the Code, International Standards and all revisions shall become effective on the date specified in the International Standard or revision.” □ doping ■ WADA □ analysis □ blood & urine □ alternative fluids

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Source: WADA 2009 13/50 International standard – implications for laboratory Methodical setting for Laboratories Aim for a technically competent operating with an effective quality management Assure the competence to produce forensically valid results □ doping ■ WADA □ analysis □ blood & urine □ alternative fluids

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■ doping □ WADA □ analysis □ blood & urine □ alternative fluids Source: WADA 2009 14/50 International standard – codes related to analysis Code Article 3 PROOF OF DOPING §3.2.1 WADA-accredited laboratories are presumed to have conducted Sample analysis and custodial procedures in accordance with the International Standard for Laboratories. Code Article 6 ANALYSIS OF SAMPLES §6.1 …Samples shall be analyzed only in WADA-accredited laboratories or as otherwise approved by WADA. §6.2 Samples shall be analyzed to detect Prohibited Substances and Prohibited Methods identified on the Prohibited List and other substances…

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Source: WADA 2009 15/50 International standard – definitions Non-Threshold Substance: A substance listed on the Prohibited List for which the documentable detection of any amount is considered an anti-doping rule violation. Threshold Substance: A substance listed on the Prohibited List for which the detection and quantification of an amount in excess of a stated threshold is considered an Adverse Analytical Finding. Minimum Required Performance Level (MRPL): concentration of a Prohibited Substance or Metabolite of a Prohibited Substance or Marker of a Prohibited Substance or Method that a doping Laboratory is expected to reliably detect and confirm in the routine daily operation of the Laboratory. See Technical Document Minimum Required Performance Levels for Detection of Prohibited Substances. □ doping ■ WADA □ analysis □ blood & urine □ alternative fluids

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Source: WADA 2009 16/50 validation of the methods (ISO/IEC 17025) Specifity Identification capability Robustness Carry over Matrix interferences Standards Limit of quantifications (LOQ) Linearity □ doping ■ WADA □ analysis □ blood & urine □ alternative fluids

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Source: WADA 2009 17/50 International standard – MRPL □ doping ■ WADA □ analysis □ blood & urine □ alternative fluids

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Source: WADA 2009 18/50 Analysis of urine and blood (ISO/IEC 17025) □ doping ■ WADA □ analysis □ blood & urine □ alternative fluids take the sample transport & inspection controll for irregularities A sample B sample aliquot preparation initial testing procedure shall detect the prohibited substances or its metabolites methods?? confirmation procedure accumulate additional information to support the adverse analytical finding LC-MS GC-MS HPLC-MS(-MS) Immunoassays protein chipes .. freezing ‚B‘ sample confirmation

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Source: Badoud et al. (2010) 19/50 Analytical Instruments - Challenges Great diversity of doping agents Numerous technical approaches are needed Complex matrices Sport events last often not more than 24 – 48 hours -> “in-competition testing”, Rapid, widely applicable and automated devices WADA defines the minimal required performance limit (MRPL) High-throughput techniques to screen: a single method, a large set of different compounds Including two steps (screening & selective confirmation) Sometimes also characteristic drug metabolites relevant for confirmation □ doping □ WADA ■ analysis □ blood & urine □ alternative fluids

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Source: 1Mazzarino and Botre (2006) 20/50 General analytical methods The „usual suspects“: Extraction: Solid phase (SPE), liquid-liquid (i.e. tert-butyl-methyl ether and urine)1 Chromatography: GC-MS / LC-MS-MS, TLC, CE Nitrogen-specific detectors such as N-FID/NPD (for amphetamines and others) Immunoassays (ELISA) Protein chips (comparable to ELISA) … new techniques in development (i.e. EESI) □ doping □ WADA ■ analysis □ blood & urine □ alternative fluids

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Source: Badoud et al. (2010) 21/50 Analytical Instruments - Overview □ doping □ WADA ■ analysis □ blood & urine □ alternative fluids

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Source: Badoud et al. (2010) 22/50 Analytical instruments – An exemplary hyphenated technique UHPLC: + Times are 5- to 10-fold shorter than with conventional LC-Methods - Peaks become very narrow, an adapted detection device is required (Acquisition time) QTOF-MS(-MS) + Ideal detector for a UHPLC (Acquisition time approx. 3s) + Can handle hundreds of compounds (i.e. 103 compounds in Badoud et al. 2010) + Mass accuracy both for screening and confirmation (<2 ng/L in urine) □ doping □ WADA ■ analysis □ blood & urine □ alternative fluids

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23/50 Matrizes The „traditionals“: Urine Blood Alternative fluids … ? □ doping □ WADA □ analysis ■ blood & urine □ alternative fluids

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24/50 Urine □ doping □ WADA □ analysis ■ blood & urine □ alternative fluids „Archive“ Easy to get Calibration with standard addition High volume Detection of Amphetamines Detection of conjugated metabolites „Archive“ Interferes with Privacy Manipulation is easy Matrix (Salts) “omics” are difficult

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25/50 Urine matrix - History 1970-80s : preferential fluid Concentrations of parent compound or metabolites quiet high shortly after administration In-competition testing of amphetamines 1980s: steroid doping during training periods Need for out-of-competition testing „wash-out“ of the substances before competition □ doping □ WADA □ analysis ■ blood & urine □ alternative fluids

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26/50 Analysis in urine - UHPLC-QTOF-MS Confirmatory analysis of Atenolol (β-blocker) □ doping □ WADA □ analysis ■ blood & urine □ alternative fluids Source: Badoud et al. (2010)

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27/50 Blood samples 1990s: EPO and other peptide hormones Problem: unlike LMWC such as amphetamines, peptides are only present in very low concentrations in the urine Allow the collection of haematological and hormonal profiles These build the basis for biological passports „Omics“ such as Metabolomics: metabolite patterns □ doping □ WADA □ analysis ■ blood & urine □ alternative fluids Source: Saugy et al. (2009)

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28/50 Variables influencing quality of blood samples Exercise Altitude The position of the athlete during blood collection The characteristics of blood drawing Temperature and time of transportation prior to laboratory analysis Importance of pre-analytical quality and standardizations □ doping □ WADA □ analysis ■ blood & urine □ alternative fluids

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29/50 Conclusion Urine vs. Blood Blood has many advantages (simple and fast sample collection, „omics“-potential) Urine is still a matrix of choice: non-invasive, easy sample preparation and extraction □ doping □ WADA □ analysis ■ blood & urine □ alternative fluids Excretion- /Metabolic behavior of each prohibited substance must be known to ensure the proper matrix selection EPO, HMWC -> Blood Conjugated metabolites -> Urine

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30/50 Question 3 Would you propose an alternative matrix as body fluid which reach the same purpose of sensitivity and specificity as defined in the international standards for anti-doping laboratories ? □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids

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31/50 Alternative fluids – practical challenges □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Diversity of body fluids is limited Fluid should be part of the doping metabolism and always be available Taking the fluid should not damage the athlete Time scale of the doping components has to be considered Matrix effects

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32/50 Alternative fluids – possibilities □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Tear Cerebrospinal fluid Lymph Saliva Sweat Not realizable (small amounts, athletes may be harmed) Advantages over blood and urine: Manipulations on the part of the athletes are difficult Sampling is simple

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33/50 Saliva □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Produced from different salivary glands 98% water Contains many important substances, including electrolytes, mucus, antibacterial compounds and various enzymes. Source: Wikipedia

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34/50 Saliva - Analysis □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Agents appear in oral fluid by passive diffusion from blood Previous investigations using thin-layer chromatographic didn’t work2 Successful identification with GC-MS for - Stimulants 3, 4 - Cannabis 3 - Narcotics 3 - … In the meantime, a multitude of studies are dealing with drug-/doping screening in saliva Source: 2Vapaatalo (1984); 3Cone (2007); 4Strano-Rossi (2008)

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35/50 Saliva □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Source: Cone (2007); Strano-Rossi (2008) Some advantages over urine as a test matrix Elimination of drugs form oral fluids is much faster than from urin -> Saliva could be alternative matrix in doping tests!

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37/50 Sweat □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Excretion for thermoregulation Small amount (0.2–1%) of solute besides water Sweat form eccrine and apocrine sweat glands differs Source: Wikipedia

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38/50 Sweat – General problems □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Results are depending on the skin area The total amounts are mainly influenced by mobilization physiological and external factors Degradation induced by bacteria, selective excretion processes or skin biotransformation Interaction of doping agents with sweat? Source: De Martinis (2007), Thième (2003)

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39/50 Sweat – Analysis □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Has been investigated as an alternate biological matrix for monitoring drug Relatively little research on sweat for doping purposes Is normally collected with sweat patches (cellulose) Steroids can be identified in sweat by gas chromatography– high resolution mass spectrometry (GC–HRMS) In some cases longer detection window than plasma or urine External uncertainities could not be excluded, therefore, interpretation was mainly based on relative concentration ratios of metabolites Source: Thième (2003)

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40/50 Sweat – Analysis □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids In some cases longer detection window than plasma or urine Relatively low analyte concentrations Lack of information about dose–response relationships Patch as the sample volume is not compatible Reproducibility can hardly be established and evaluated External uncertainties can not be excluded -> Sweat as a alternative matrix for doping tests is not accecptable Source: De Martinis (2007), Thième (2003)

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42/50 Conclusions □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Analysis of oral fluid could be successfully and provide complement data for ‘in competition’ tests. Saliva and sweat allow both non-invasive collection. External uncertainties do not allow the use of sweat as an alternate matrix for doping tests.

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43/50 Question 4 □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids In the present situation, do you think that the urine analyses for cocaine and cannabis, as defined by the ISL, by athletes are in accordance to the definition of In competition forbidden substances?

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44/50 Cocaine and cannabis – Special case □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Are typically used as recreational drugs outside of sports A positive finding leads to both sports-specific and criminal sanctions The use of doping agents is prohibited specifically during the sport event Exhibit very long windows of detection The time of administration is extremely difficult to establish Source: Saugy (2009)

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45/50 Cocaine and cannabis – Implications □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Urine exhibits very long windows of detection Determination of the initial time is very difficult in urine To evaluate the time period, the matrix of choice must certainly be blood instead of urine No logistical problems with blood Source: Saugy (2009)

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46/50 Conclusion □ doping □ WADA □ analysis □ blood & urine ■ alternative fluids Especially for cannabinoids, blood is a much better matrix for ‘in-competition’ tests than urine.

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Literature: Avois L, Robinson N, Saudan C, Baume N, Mangin P, Saugy M. (2006) Central nervous system stimulants and sport practice. Br J Sports Med. 40 Suppl 1:i16-20. Review. Badoud F, Grata E, Perrenoud L, Saugy M, Rudaz S, Veuthey JL. (2010) Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole timeof- flight mass spectrometry. II: Confirmatory analysis. J Chromatogr A. 18;1217(25):4109-19. Cone E. J. and M. A. Huestis (2007) Interpretation of Oral Fluid Tests for Drugs of Abuse. Ann N Y Acad Sci. 2007 March; 1098: 51–103. De Martinis B. S. , A. J. Barnes, K. B. Scheidweiler, and M. A. Huestis (2007) Development and validation of a disk solid phase extraction and gas chromatography–mass spectrometry method for MDMA, MDA, HMMA, HMA, MDEA, methamphetamine and amphetamine in sweat, Journal of Chromatography 852: 450-458, Follador M. J. D., M. Yonamine, R. L. de Moraes Moreau and O. A. Silva (2005) Detection of cocaine and cocaethylene in sweat by solid-phase microextraction and gas chromatography/mass spectrometry, Journal of Chromatography 811: Pages 37-40. Saugy M, Robinson N, Saudan C. (2009) The fight against doping: back on track with blood.Drug Test Anal.;1(11-12):474-8. Strano-Rossi S. S. Colamonici and F. Botrè (2008) Parallel analysis of stimulants in saliva and urine by gas chromatography/mass spectrometry: Perspectives for “in competition” anti-doping analysis. Analytica chimica acta 606: 217–222. Thième D, Anielski P., J. Grosse, H. Sachs and Mueller R. K. (2003) Identification of anabolic steroids in serum, urine, sweat and hair: Comparison of metabolic patterns. Analytica Chimica Acta 483: 299–306. Vapaatalo H., Kärkäinen S. and Senius KE. (1984) Comparison of saliva and urine samples in thin-layer chromatographic detection of central nervous stimulants. Int J Clin Pharmacol Res. 4(1):5-8. WADA (2004) Technical Document TD2004MRPL (Minimum required performance limits for the detection of prohibited substances), World Anti-Doping Agency, Montreal, Canada. WADA (2010) The world anti-doping code. International Standard for Laboratories. World Anti-Doping Agency, Montreal, Canada.. 47/50 □ doping □ WADA □ analysis □ blood & urine □ alternative fluids References

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Figures: http://4.bp.blogspot.com/-2I5GNYxStbE/ToRG7UjKiWI/AAAAAAAABZw/U0MskiLPRyw/s1600/DopingSprinter.jpg http://www.google.ch/imgres?q=fuentes&um=1&hl=de&sa=N&biw=1024&bih=499&tbm=isch&tbnid=ltTU6_dkS4P_2M:&imgrefurl=http://www.nzz.ch/nachrichten/sport/aktuell/rad_dopingarzt_fuentes_erneut_verhaftet_1.8613104.html&docid=p-SgrglCZiFpKM&imgurl=http://www.nzz.ch/images/eufemiano_fuentes_fullSize_1.8613277.1292307145.jpg&w=960&h=630&ei=S7PLTpCbL9CesAbPn-XZDA&zoom=1&iact=hc&vpx=461&vpy=174&dur=1807&hovh=182&hovw=277&tx=160&ty=78&sig=115013639728651934603&page=1&tbnh=128&tbnw=175&start=0&ndsp=10&ved=1t:429,r:2,s:0 http://www.blick.ch/sport/rad/artikel41241 http://www.20min.ch/diashow/diashow.tmpl?showid=8482 http://en.rian.ru/images/15806/45/158064572.jpg http://pubs.acs.org/cen/news/84/i47/8447notw3.html http://www.blick.ch/news/neuer-doping-test-an-eth-entwickelt-38742 http://www.google.ch/imgres?q=sweat+gland+electron+microscopy&hl=de&biw=1024&bih=499&tbm=isch&tbnid=pn-F-uNZ_uo_NM:&imgrefurl=http://webecoist.com/2010/01/11/biological-photography-magnificent-microscopic-ultraminiature-photos/&docid=7gACvO-26TjrMM&imgurl=http://cdn.webecoist.com/wp-content/uploads/2010/01/human-sweat-gland1.jpg&w=468&h=412&ei=ZGXTTvGhCMGtsAbEybDNDA&zoom=1&iact=hc&vpx=412&vpy=174&dur=2164&hovh=211&hovw=239&tx=145&ty=108&sig=115013639728651934603&page=2&tbnh=126&tbnw=147&start=11&ndsp=10&ved=1t:429,r:2,s:11 http://www.google.ch/imgres?q=speichel+mikroskopieren&num=10&hl=de&biw=1024&bih=499&tbm=isch&tbnid=zwx38O5JplHx5M:&imgrefurl=http://www.welt.de/wissenschaft/article1174481/Wie_die_Spucke_den_Menschen_schuf.html&docid=hykLZvSPRKQ9ZM&imgurl=http://www.welt.de/multimedia/archive/00378/speichel_DW_Wissens_378184p.jpg&w=484&h=322&ei=xHzTTvO4J9Snsgbt77mFCA&zoom=1&iact=hc&vpx=275&vpy=155&dur=3046&hovh=183&hovw=275&tx=125&ty=48&sig=115013639728651934603&sqi=2&page=1&tbnh=119&tbnw=179&start=0&ndsp=10&ved=1t:429,r:1,s:0 48/50 □ doping □ WADA □ analysis □ blood & urine □ alternative fluids References