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Doping

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Doping

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  1. Doping ChristofMünch and Alexander Gogos 29. 22. 2011

  2. □ doping□ WADA □ analysis □ blood & urine □ alternative fluids 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 2/50

  3. ■ doping□ WADA □ analysis □ blood & urine □ alternative fluids 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 3/50

  4. ■ doping□ WADA □ analysis □ blood & urine □ alternative fluids Timeline …a longwayuntilthefoundingofthe World Anti-Doping Agency (WADA) in 1999! 4/50

  5. ■ doping□ WADA □ analysis □ blood & urine □ alternative fluids Famouscases in thepast – exampleTour de France • 1998: Festina • 2000: Fuentes • 2006: Landis 5/50 Source: Wikipedia

  6. ■ doping□ WADA □ analysis □ blood & urine □ alternative fluids Classes of prohibited substances and activities Methods • Enhancementofoxygentransfer (Blood doping) • Chemical andphysicalmanipulation (Tamperingwithsamples) • Gene doping • 6 groups (see next slide) Substances • Alcohol • Beta-blockers Substancesprohibited in particularsports 6/50 Source: Badoud et al. (2010)

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

  8. ■ doping□ WADA □ analysis □ blood & urine □ alternative fluids Classesofdopingagents Many many more… 8/50

  9. □ doping■ WADA □ analysis □ blood & urine □ alternative fluids Doping controlandInstitutions • 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 acceptedby • Olympic movement • Government funded organizations • Some institutions out of the olympic movement 10/50

  10. □ doping■ WADA □ analysis □ blood & urine □ alternative fluids International standardforlaboratories 11/50

  11. □ doping■ WADA □ analysis □ blood & urine □ alternative fluids 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 orrevision.” 12/50 Source: WADA 2009

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

  13. ■ doping □ WADA □ analysis □ blood & urine □ alternative fluids 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. §6.2Samples shall be analyzed to detect Prohibited Substances and Prohibited Methods identified on the Prohibited List and other substances… 14/50 Source: WADA 2009

  14. □ doping■ WADA □ analysis □ blood & urine □ alternative fluids 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. 15/50 Source: WADA 2009

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

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

  17. □ doping■ WADA □ analysis □ blood & urine □ alternative fluids Analysis of urine and blood(ISO/IEC 17025) takethe sample A sample B sample freezing transport & inspection ‚B‘ sample confirmation controllforirregularities aliquot preparation methods?? shalldetecttheprohibitedsubstancesoritsmetabolites initialtestingprocedure accumulate additional informationtosupporttheadverseanalyticalfinding LC-MS GC-MS HPLC-MS(-MS) Immunoassays proteinchipes .. confirmationprocedure 18/50 Source: WADA 2009

  18. □ doping □ WADA■analysis □ blood & urine □ alternative fluids 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 19/50 Source: Badoud et al. (2010)

  19. □ doping □ WADA■analysis □ blood & urine □ alternative fluids General analyticalmethods • 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) 20/50 Source: 1Mazzarino andBotre(2006)

  20. □ doping □ WADA■analysis □ blood & urine □ alternative fluids Analytical Instruments - Overview Chromatographic System (Usually LC) UHPLC(ultra-high –pressure liquid chromatography) Monolithiccolumns HTLC (high-temperature liquid chromatography) ->Acceleratetheanalyticalprocess Interface ESI(electrosprayionization) -> Ionizesbothlow- andhighmolecularweightcompounds Mass spectrometry QTOF (Hybrid quadrupole time-of-flight) (TOF (time-of-flight)) -> High reproducibility, Mass accuracy 21/50 Source: Badoud et al. (2010)

  21. □ doping □ WADA■analysis □ blood & urine □ alternative fluids 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) 22/50 Source: Badoud et al. (2010)

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

  23. □ doping □ WADA □ analysis■blood & urine □ alternative fluids Urine • „Archive“ • Easy toget • Calibrationwithstandardaddition • High volume • DetectionofAmphetamines • Detectionofconjugatedmetabolites • „Archive“ • Interfereswith Privacy • Manipulation is easy • Matrix (Salts) • “omics” are difficult 24/50

  24. □ doping □ WADA □ analysis■blood & urine □ alternative fluids 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 25/50

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

  26. □ doping □ WADA □ analysis■blood & urine □ alternative fluids 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 27/50 Source: Saugy et al. (2009)

  27. □ doping □ WADA □ analysis■blood & urine □ alternative fluids 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 28/50

  28. □ doping □ WADA □ analysis■blood & urine □ alternative fluids 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 Excretion- /Metabolicbehaviorofeachprohibitedsubstance must beknowntoensurethe proper matrixselection EPO, HMWC -> Blood Conjugatedmetabolites -> Urine 29/50

  29. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids 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 ? 30/50

  30. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids Alternative fluids – practicalchallenges • 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 31/50

  31. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids Alternative fluids – possibilities • Tear • Cerebrospinal fluid • Lymph • Saliva • Sweat Not realizable (smallamounts, athletesmaybeharmed) • Advantages overbloodandurine: • Manipulations on the part of the athletes are difficult • Sampling is simple 32/50

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

  33. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids Saliva - Analysis • 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 34/50 Source: 2Vapaatalo (1984); 3Cone (2007); 4Strano-Rossi (2008)

  34. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids Saliva • 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! 35/50 Source: Cone (2007); Strano-Rossi (2008)

  35. 36/50

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

  37. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids Sweat – General problems • 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? 38/50 Source: De Martinis (2007), Thième (2003)

  38. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids Sweat – Analysis • 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 39/50 Source: Thième(2003)

  39. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids Sweat – Analysis • 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 40/50 Source: De Martinis (2007), Thième (2003)

  40. 41/50

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

  42. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids Question 4 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 competitionforbiddensubstances? 43/50

  43. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids Cocaine and cannabis – Special case • 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 44/50 Source: Saugy(2009)

  44. □ doping □ WADA □ analysis□ blood & urine■ alternative fluids Cocaine and cannabis – Implications • 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 45/50 Source: Saugy(2009)

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

  46. □ doping□ WADA □ analysis □ blood & urine □ alternative fluids References 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 analysisofdoping agents in urine by ultra-high-pressure liquid chromatography-quadrupoletimeof- 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 ofAbuse. Ann N Y AcadSci. 2007 March; 1098: 51–103. De Martinis B. S. , A. J. Barnes, K. B. Scheidweiler, and M. A. Huestis (2007) Development andvalidationof a disk solid phaseextractionand gas chromatography–mass spectrometrymethodfor MDMA, MDA, HMMA, HMA, MDEA, methamphetamineandamphetamine in sweat, Journal ofChromatography 852: 450-458, Follador M. J. D., M. Yonamine, R. L. de Moraes Moreau and O. A. Silva (2005) Detectionofcocaineandcocaethylene in sweatby solid-phasemicroextractionand gas chromatography/mass spectrometry, Journal ofChromatography 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. Colamoniciand F. Botrè (2008) Parallel analysisofstimulants in salivaandurineby gas chromatography/mass spectrometry: Perspectivesfor “in competition” anti-dopinganalysis. Analyticachimica acta 606: 217–222. Thième D, Anielski P., J. Grosse, H. Sachs and Mueller R. K. (2003) Identificationofanabolicsteroids in serum, urine, sweatandhair: Comparisonofmetabolicpatterns. AnalyticaChimica Acta 483: 299–306. Vapaatalo H., Kärkäinen S. andSenius KE. (1984) Comparisonofsalivaandurinesamples in thin-layerchromatographicdetectionofcentralnervousstimulants. Int J ClinPharmacol 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

  47. □ doping□ WADA □ analysis □ blood & urine □ alternative fluids References 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