ISBN 978-87-7209-334-5 Sara Amalie Solheim Improving time- and cost-efficiency in doping analysis
Improving time- and cost-efficiency in doping analysis
PhD Thesis Sara Amalie Solheim
December 2019 UNIVERSITY OF COPENHAGEN FACULTY OF SCIENCE
Improving time- and cost-efficiency in doping analysis
PhD thesis Sara Amalie Solheim
This thesis has been submitted to the PhD School of the Faculty of Science, University of Copenhagen ImprovingImproving time time-- and and cost cost-efficiency-efficiency in indoping doping analysis analysis PhDPhD thesis thesis 2019 2019 © © Sara Sara Amalie Amalie Solheim Solheim / ISBN:
DepartmentPrinted by: of Nutrition, Exercise and Sports FacultyFront & of back Science cover design: Ditte Toft Clausen, Anti Doping Denmark University of Copenhagen, Copenhagen, Denmark
Department of Nutrition, Exercise and Sports, SupervisorsFaculty of Science , AssociateUniversity Professor of Copenhagen, Nikolai Baastrup Copenhagen, Nordsborg Denmark Department of Nutrition, Exercise and Sports, University of Copenhagen, Copenhagen, Denmark Supervisors
Associate Professor Nikolai Baastrup Nordsborg Dr. Jakob Mørkeberg Department of Nutrition, Exercise and Sports, Anti Doping Denmark, Brøndby, Denmark University of Copenhagen, Copenhagen, Denmark
Dr. Yvette Dehnes Dr. Jakob Mørkeberg Norwegian Doping Control Laboratory, Anti Doping Denmark, Brøndby, Denmark Oslo University Hospital, Oslo, Norway
AssessmentDr. Yvette Dehnescommittee AssociateNorwegian Professor Doping Thomas Control Elbenhardt Laboratory, Jensen (chairman) DepartmentOslo University of Nutrition, Hospital, Exercise Oslo, Norway and Sport s, University of Copenhagen, Copenhagen, Denmark Assessment committee Dr.Associate Jenny Schulze Professor Thomas Elbenhardt Jensen (chairman) SDepartmentwedish Anti- Doping,of Nutrition, Exercise and Sports, SwedishUniversity Sports of Copenhagen, Confederation, Copenhagen, Stockholm, SwedenDenmark
ProfDr. essorJenny Per Schulze Aaagaard Department of Sports Science and Clinical Biomechanics Swedish Anti-Doping, University of Southern Denmark, Odense, Denmark Swedish Sports Confederation, Stockholm, Sweden
ISBN 978-87-7209-334-5 PrintedProfessor by SLPer grafik, Aaagaard Frederiksberg, Denmark (slgrafik.dk) Front & back cover design by Ditte Toft Clausen, Anti Doping Denmark Department of Sports Science and Clinical Biomechanics 2 University of Southern Denmark, Odense, Denmark
2 Improving time- and cost-efficiency in doping analysis PhD thesis 2019 © Sara Amalie Solheim / ISBN: Table of contents
Printed by: LIST OF PAPERS ...... 5 Front & back cover design: Ditte Toft Clausen, Anti Doping Denmark ACKNOWLEDGEMENTS ...... 6
Department of Nutrition, Exercise and Sports, ABSTRACT ...... 7 Faculty of Science, RESUMÉ ...... 8 University of Copenhagen, Copenhagen, Denmark
ABBREVIATIONS ...... 9 Supervisors Associate Professor Nikolai Baastrup Nordsborg 1. INTRODUCTION ...... 10 Department of Nutrition, Exercise and Sports, 1.1 Performance-enhancing effects of testosterone ...... 10 University of Copenhagen, Copenhagen, Denmark 1.1.1 Ergogenic effects of long-term and short-term testosterone administration ...... 11 1.1.2 Acute performance-enhancing effects of testosterone ...... 12
Dr. Jakob Mørkeberg 1.2 Performance-enhancing effects of clenbuterol ...... 14 Anti Doping Denmark, Brøndby, Denmark 1.2.1 Ergogenic effects of long-term clenbuterol administration...... 14 1.2.2 Ergogenic effects of acute clenbuterol administration ...... 15 1.2.3 Summary ...... 15 Dr. Yvette Dehnes Norwegian Doping Control Laboratory, 1.3 Actual or potential health risk ...... 16 Oslo University Hospital, Oslo, Norway 1.4 Indirect detection of doping with testosterone and its synthetic analogues ...... 16 1.4.1 Steroid profile ...... 16 Assessment committee 1.4.2 Evaluation of the steroid profile ...... 19 Associate Professor Thomas Elbenhardt Jensen (chairman) 1.4.3 Future possibilities ...... 20 Department of Nutrition, Exercise and Sports, 1.5 Direct detection of doping with testosterone and its synthetic analogues ...... 21 University of Copenhagen, Copenhagen, Denmark 1.6 Detection of doping with clenbuterol ...... 22
Dr. Jenny Schulze 1.7 Biological matrices in doping analysis ...... 23 Swedish Anti-Doping, 1.7.1 Microvolume dried matrices...... 24 Swedish Sports Confederation, Stockholm, Sweden 1.7.2 Detectability of testosterone esters and clenbuterol in DBS/DPS ...... 29
1.8 Applicability to anti-doping: objectives ...... 30 Professor Per Aaagaard
Department of Sports Science and Clinical Biomechanics 2. RESULTS AND DISCUSSION ...... 33 University of Southern Denmark, Odense, Denmark 2.1 Acute performance-enhancing effects of testosterone in humans ...... 33 2.1.1 Acute ergogenic effects of testosterone administration on muscular strength ...... 33
2 2.1.2 Acute ergogenic effects of testosterone administration on sprint performance...... 34 2.1.3 Acute ergogenic effects of testosterone administration on vertical jump performance ...... 35 2.1.4 Serum testosterone and performance capacities...... 36 2.1.5 Implications for anti-doping ...... 36 2.1.6 Study limitations ...... 37
2.2 Indirect detection of testosterone esters ...... 38 2.2.1 The steroid profile...... 38 2.2.2 The hematological profile ...... 38
2.3 Direct detection of testosterone esters ...... 40 2.3.1 Further development of DBS analysis of testosterone esters ...... 40 2.3.3 Detection of testosterone ester administration in DBS ...... 42
2.4 Detection of clenbuterol ...... 43 2.4.1 Detection of clenbuterol in urine ...... 44 2.4.2 Detection of clenbuterol in DBS...... 45
2.5 Implementation of DBS in doping control...... 46 2.5.1 Sample collection procedures ...... 46 2.5.2 Transportation and storage procedures ...... 47 2.5.3 Possibilities and benefits with DBS ...... 48
3. CONCLUSION AND PERSPECTIVES ...... 49
4. REFERENCES ...... 51
PAPER I-IV ...... 63
2.1.2 Acute ergogenic effects of testosterone administration on sprint performance...... 34 List of papers 2.1.3 Acute ergogenic effects of testosterone administration on vertical jump performance ...... 35 2.1.4 Serum testosterone and performance capacities...... 36 I. Solheim SA, Mørkeberg J, Dehnes Y, Hullstein I, Juul A, Upners EN, Nordsborg NB. 2.1.5 Implications for anti-doping ...... 36 Changes in blood parameters after intramuscular testosterone ester injections – 2.1.6 Study limitations ...... 37 Implications for anti-doping. Submitted to Drug Testing and Analysis
2.2 Indirect detection of testosterone esters ...... 38 II. Solheim SA, Levernæs MC, Mørkeberg J, Juul A, Upners EN, Nordsborg NB, Dehnes Y. 2.2.1 The steroid profile...... 38 Dried blood spot analysis of testosterone esters is applicable in doping analysis. Draft. 2.2.2 The hematological profile ...... 38 III. Solheim SA, Mørkeberg J, Juul A, Freiesleben SY, Upners EN, Dehnes Y, Nordsborg NB. 2.3 Direct detection of testosterone esters ...... 40 An intramuscular injection of mixed testosterone esters does not acutely enhance strength 2.3.1 Further development of DBS analysis of testosterone esters ...... 40 and power in recreationally active young men. Submitted to European Journal of Applied 2.3.3 Detection of testosterone ester administration in DBS ...... 42 Physiology
2.4 Detection of clenbuterol ...... 43 IV. Solheim SA, Jessen S, Mørkeberg J, Thevis M, Dehnes Y, Eibye K, Hostrup M, 2.4.1 Detection of clenbuterol in urine ...... 44 Nordsborg NB. Single-dose administration of clenbuterol is detectable in Dried Blood 2.4.2 Detection of clenbuterol in DBS...... 45 Spots. Submitted to Drug Testing and Analysis
2.5 Implementation of DBS in doping control...... 46 2.5.1 Sample collection procedures ...... 46 2.5.2 Transportation and storage procedures ...... 47 2.5.3 Possibilities and benefits with DBS ...... 48
3. CONCLUSION AND PERSPECTIVES ...... 49
4. REFERENCES ...... 51
PAPER I-IV ...... 63
Acknowledgements
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Acknowledgements Abstract
testosterone by the ‘Athlete Biological Passport’ (A
also sincere Paper I-III – n n Paper III Paper IV – Paper II IV Paper IV Paper I Paper III – to
Resumé
estosteronestere og clenb terol er blan t e est an en te opingsto er i elite og otionsi r t ire te ete tion i rin og blo pr er er orb n et e bety elige pr eopsa lingso ostninger strenge ra til transport og analyse sa t h rtig ne bry ning a testosteronestere i blo enne a han ling ha e til or l at orbe re oste e ti iteten a anti opingtestning genne n ers gelse a an en elighe en a en o ple ent r pr eopsa lings atri rie bloo spots ( B og h or i t et h atologis e o l i ’the Athlete Biological Passport’ (ABP an br ges til at in i ere testosteron oping og er e orbe re ete teringen gi et testosterons sti leren e e e t p pro tionen a r e blo celler og e at gi e on rete orslag til testning baseret p sporingsti og pr stations re en e e e ter Studie I-III ble B rin og blo pr er ra n er o tog to intra s l re in e tioner a stanon (n eller placebo (n i en ran o iseret placebo ontrolleret esign analyseret or ire te og in ire te (ABP p isning a testosteronestere og ling a repro tionshor oner i ser Studie III ble pr stationen i co nter o e ent p se n ers cy elsprint og iso etris alb e le sion lt r og ti er e ter en rste stanon in e tion Studie IV ble B og rinpr er ra ras e n er o tog en en elt oral osis a g clenb terol opsa let og analyseret or p isning a clenb terol Studie II og IV iste at analyser a B an p ise oping op til age e ter en intra s l r in e tion a g stanon og i in st age e ter en oral osis a g clenb terol e speci icitet n i ere iste Studie IV at clenb terol an p ises i in st age i rin e ter en oral osis g Studie I iste at e h atologis e ar rer i ABP p ir es a testosterona inistration og at e est ar ante n ringer in er ste age e ter en in e tion Stude III iste at en en elt in e tion a testosteronestere i e orbe rer pr stationen a t i co nter o e ent p se n ers cy elsprint eller iso etris alb e le sion a let set ser et til at B basere e analyser a testosteronestere og clenb terol har tilstr elig speci icitet og sensiti itet til at nne i ple enteres i oping ontrol i elite og otionsi r t betragtning a en l ngere ete tionsti or clenb terol i rin or entes rin at ortsat re en oretr ne pr e atri til at ete tere clenb terol oping og il i ple entering a B re cere pr eopsa lingsti en og er e ligg re en h ere test re ens sa t testning a ange atleter p ort ti n i ere an n ringer i ar rer i et h atologis e o l in i ere testosteron oping og b r betragtes so et e stra r t til sporing a testosteron oping her n er lrettet testning a testosteron i e ha e nogen a t pr stations re en e e e t i styr e ra t elser il atleter san synlig is i e ha e en or el h is e a inistrerer en en elt osis testosteronestere i elbart r eller n er en on rrence i styr e ra ti r tter
Resumé Abbreviations
estosteronestere og clenb terol er blan t e est an en te opingsto er i elite og otionsi r t ire te α iol α a rosta e α β iol ete tion i rin og blo pr er er orb n et e bety elige pr eopsa lingso ostninger strenge ra til β iol β a rosta e α β iol A A rostero e transport og analyse sa t h rtig ne bry ning a testosteronestere i blo enne a han ling ha e til or l at AA A erse a al tical i i g orbe re oste e ti iteten a anti opingtestning genne n ers gelse a an en elighe en a en AA A a olic a roge ic steroi s o ple ent r pr eopsa lings atri rie bloo spots ( B og h or i t et h atologis e o l i ABP Athlete iological passport ABP A or al loo pro ile score ’the Athlete Biological Passport’ (ABP an br ges til at in i ere testosteron oping og er e orbe re A At pical i i g ete teringen gi et testosterons sti leren e e e t p pro tionen a r e blo celler og e at gi e A P At pical passport i i g on rete orslag til testning baseret p sporingsti og pr stations re en e e e ter cA P clic a e osi e o ophosphate ar o eth l cell lose Studie I-III ble B rin og blo pr er ra n er o tog to intra s l re in e tioner a B rie loo spot stanon (n eller placebo (n i en ran o iseret placebo ontrolleret esign analyseret or ih rotestostero e ire te og in ire te (ABP p isning a testosteronestere og ling a repro tionshor oner i ser Studie P rie plas a spot III ble pr stationen i co nter o e ent p se n ers cy elsprint og iso etris alb e le sion lt r pitestostero e tracell lar sig al reg late i ases og ti er e ter en rste stanon in e tion Studie IV ble B og rinpr er ra ras e n er A r thropoiesis sti lati g age t o tog en en elt oral osis a g clenb terol opsa let og analyseret or p isning a clenb terol t th l gl c ro i e Studie II og IV iste at analyser a B an p ise oping op til age e ter en intra s l r in e tion tio tiochola olo e as chro atograph ta e ass spectro etr a g stanon og i in st age e ter en oral osis a g clenb terol e speci icitet l cose tra sporter n i ere iste Studie IV at clenb terol an p ises i in st age i rin e ter en oral osis g Studie I h a chorio ic go a otropi iste at e h atologis e ar rer i ABP p ir es a testosterona inistration og at e est ar ante e atocrit B e oglo i n ringer in er ste age e ter en in e tion Stude III iste at en en elt in e tion a testosteronestere i e as chro atograph co stio isotope ratio ass spectro etr orbe rer pr stationen a t i co nter o e ent p se n ers cy elsprint eller iso etris alb e le sion i i chro atograph ta e ass spectro etr a let set ser et til at B basere e analyser a testosteronestere og clenb terol har tilstr elig tei i i g hor o e speci icitet og sensiti itet til at nne i ple enteres i oping ontrol i elite og otionsi r t betragtning a i it o etectio ea corp sc lar he oglo i en l ngere ete tionsti or clenb terol i rin or entes rin at ortsat re en oretr ne pr e atri til at ea corp sc lar he oglo i co ce tratio ete tere clenb terol oping og il i ple entering a B re cere pr eopsa lingsti en og er e ea corp sc lar ol e ligg re en h ere test re ens sa t testning a ange atleter p ort ti n i ere an n ringer i ar rer a i al ol tar co tractio a o a o scale i et h atologis e o l in i ere testosteron oping og b r betragtes so et e stra r t til sporing a P A Protei i ase A testosteron oping her n er lrettet testning a testosteron i e ha e nogen a t pr stations re en e B e loo cell e e t i styr e ra t elser il atleter san synlig is i e ha e en or el h is e a inistrerer en en elt osis B e loo cell co t etic loc te perce tage testosteronestere i elbart r eller n er en on rrence i styr e ra ti r tter ate o orce e elop e t rh P eco i a t h a er thropoieti estostero e estostero e eca oate estostero e isocaproate P estostero e propio ate PP estostero e phe lpropio ate Pea o ge pta e A A orl A ti opi g Age c ̇ 2peak VO 1. Introduction
a o the per or a ce a o i age e ha ci g r gs se athletes a o athletes all ithi the categor o lea ass il ers that e ha ce scle gro th re ce o at or a co i atio thereo peci icall estio aires i ter ie s a a ti opi g testi g ig res sho that a a olic age ts are so e o the ost pre ale t s sta ces se athletes – a people e gagi g i recreatio al e ercise trai i g – o all a erse a al tical i i gs i the i orl A ti Doping Agency’s (WADA) anti opi g a a age e t s ste A A ere s sta ces er Section 1, ‘anabolic agents’ o the Prohi ite ist his i cl es s sta ces s ch as the a a olic a roge ic steroi (AA testostero e a its s thetic a alog es as ell as the a a olic age t cle terol oreo er a ec otal a e pirical e i e ce i icate that opi g partic larl ith cle terol is a i creasi g pro le a o g e ales e ar o the e ale it ess c sto ers ho eli ere a positi e opi g test et ee a ar a teste positi e or this s sta ce hich is li el to ha e partic lar appeal to o e eca se it oes ot ca se the a roge ic si e e ects associate ith AA h s etectio o these s sta ces is o high rele a ce ithi a ti opi g o e er the c rre t etho s are challe ge the relate costs ro sa ple collectio to a al sis, as well as the athletes’ experience of the sample collection process. here ore the prese t thesis ill oc s o the rther e elop e t o oth c rre t etectio etho s a alter ati e etho s ith the ai s o i pro i g ti e a cost e icie c ithi a ti opi g a i g the process ore athlete rie l a i creasi g the li elihoo o catchi g opers
he ollo i g sectio s ill i tro ce possi le ergoge ic e ects a a erse si e e ects o testostero e a cle terol ho opi g ith these s sta ces ca e etecte c rre t challe ges or opi g co trol sa ple collectio a a al sis as ell as t re possi ilities
1.1 Performance-enhancing effects of testosterone
o g ter testostero e a i istratio has ell o ph siological e ects s ch as pro oti g s eletal scle gro th at loss a i creasi g re loo cell ( B pro ctio he erl i g echa is s o testostero e i ce s eletal scle h pertroph ha e ee e te si el s ari e earlier e phasi i g the ltiple cell lar path a s i ol e i cl i g acti atio o satellite cells ia i tercell lar a roge receptors e iate sti latio o the a a olic A path a a i hi itio o scle rea o thro gh represse ostati acti it
1. Introduction 1.1.1 Ergogenic effects of long-term and short-term testosterone administration While the physiological effects of testosterone are well explore , the n mber of well esigne a o the per or a ce a o i age e ha ci g r gs se athletes a o athletes all placebo controlle st ies in estigating the ergogenic effects of testosterone a ministration on h man 1 –1 ithi the categor o lea ass il ers that e ha ce scle gro th re ce o at or a exercise performance is limite . When controlling for the potentially confo n ing ariables co i atio thereo peci icall estio aires i ter ie s a a ti opi g testi g ig res sho that n trition an training ol me, hasin an co wor ers showe that s praphysiological oses ( a a olic age ts are so e o the ost pre ale t s sta ces se athletes – a people e gagi g i mg w ) of intram sc lar testosterone enanthate a ministere for 1 wee s increase m scle si e in recreatio al e ercise trai i g – o all a erse a al tical i i gs i the i orl A ti arms an legs an maximal strength in bench press an s at in healthy e gona al men with weight 1 Doping Agency’s (WADA) anti opi g a a age e t s ste A A ere s sta ces er lifting experience, compare with placebo. oreo er, the combination of testosterone Section 1, ‘anabolic agents’ o the Prohi ite ist his i cl es s sta ces s ch as the a a olic a ministration an strength training lea to greater strength impro ement in bench press an s at a roge ic steroi (AA testostero e a its s thetic a alog es as ell as the a a olic age t than testosterone a ministration or strength training alone. rther, 1 wee s with wee ly cle terol oreo er a ec otal a e pirical e i e ce i icate that opi g partic larl ith intram sc lar in ections of aro n mg testosterone enanthate, combine with a s per ise 1 ,1 cle terol is a i creasi g pro le a o g e ales e ar o the e ale it ess strength training program, impro e maximal bench press strength an m r nning sprint 1 c sto ers ho eli ere a positi e opi g test et ee a ar a teste positi e or performance in 11 weight traine men compare with the strength training placebo gro p (n 1 ). this s sta ce hich is li el to ha e partic lar appeal to o e eca se it oes ot ca se the nterestingly, the ma ority of strength gain too place in the initial wee s of the 1 wee s a roge ic si e e ects associate ith AA h s etectio o these s sta ces is o high rele a ce a ministration perio (1 at wee an at wee 1 ), s ggesting that short term testosterone 1 ithi a ti opi g o e er the c rre t etho s are challe ge the relate costs ro sa ple a ministration can ha e ergogenic effects. his was s pporte by the fin ing that three wee s with collectio to a al sis, as well as the athletes’ experience of the sample collection process. here ore intram sc lar in ections of mg w testosterone enanthate, b t not placebo, combine with the prese t thesis ill oc s o the rther e elop e t o oth c rre t etectio etho s a hea y strength training was s fficient to increase maximal bench press strength an total wor in a 1 alter ati e etho s ith the ai s o i pro i g ti e a cost e icie c ithi a ti opi g a i g 1 s cycle sprint in nine healthy, weight traine , yo ng men. Altho gh ne ral a aptations acco nt the process ore athlete rie l a i creasi g the li elihoo o catchi g opers for the ma or proportion of initial strength gains, s eletal m scle hypertrophy has been reporte to occ r alrea y after three wee s of resistance training in recreationally acti e in i i als.1 his early he ollo i g sectio s ill i tro ce possi le ergoge ic e ects a a erse si e e ects o hypertrophic response is li ely to be potentiate by testosterone a ministration. herefore, the testostero e a cle terol ho opi g ith these s sta ces ca e etecte c rre t challe ges reporte strength gains following short term testosterone a ministration combine with strength or opi g co trol sa ple collectio a a al sis as ell as t re possi ilities training may be attrib te to both ne ral a aptations an m scle hypertrophy.
nterestingly, 1 mg wee of testosterone propionate for three wee s was ins fficient to in ce 1.1 Performance-enhancing effects of testosterone consistent a ministration effects on lower an pper bo y iso inetic strength, in nine weight traine
o g ter testostero e a i istratio has ell o ph siological e ects s ch as pro oti g s eletal a lts ( men, 1 woman). owe er, this might be explaine by the a ministere ose, beca se the 1 , , 1 scle gro th at loss a i creasi g re loo cell ( B pro ctio he erl i g echa is s effects of testosterone a ministration on m scle strength, leg power, m scle si e in healthy, o testostero e i ce s eletal scle h pertroph ha e ee e te si el s ari e earlier yo ng men are ose an concentration epen ent, e i ent by larger changes in the an e phasi i g the ltiple cell lar path a s i ol e i cl i g acti atio o satellite cells ia i tercell lar mg w gro ps than in the gro ps recei ing wee ly in ections of either , or 1 mg of testosterone a roge receptors e iate sti latio o the a a olic A path a a i hi itio o enanthate for wee s. n this regar , it sho l be mentione that there are ethical concerns relate scle rea o thro gh represse ostati acti it to the ose an n mber of oses when a ministering testosterone to healthy s b ects, e to the suppression of the subjects’ reproductive hormone production.
urther one stud h s investi ted the effect of testosterone on runnin perform nce nd recover . ume nd co or ers h pothesi ed th t mu tip e doses of ou d enh nce the recover nd thereb the response to endur nce tr inin e din to improved endur nce perform nce. o consistent tre tment effects ere observed on runnin speed t n erobic thresho d or on biom r ers of ph sic stress in he th recre tion ctive men fo o in four ee s of combined endur nce tr inin nd or dministered p cebo n testosterone undec no te m n or nor ndrostendione m n three times per ee . ote orth serum testosterone concentr tion rem ined unch n ed durin the intervention hich su ests th t the dministered dos e s insufficient to induce n me sur b e ch n es in recover nd ph sic d pt tion to tr inin . herefore this sin e re tive sm nd short study preclude firm conclusions, especially on testosterone’s effects on endur nce perform nce. nterestin testosterone dministr tion h s been found to stimu te er thropoiesis in dose dependent m nner possib throu h combin tion of direct nd indirect ndro en effects on the bone m rro er thropoiesis. his inc udes incre sed number of er thropoietin responsive pro enitor ce s in the bone m rro incre sed ren secretion of er thropoietin nd incre sed iron v i bi it throu h suppressed hepcidin. hus theoretic it is possib e th t the testosterone dministr tion c n enh nce endur nce perform nce throu h incre sed rteri o en content nd thereb m im o en upt e re tive to bod ei ht or hich is n
import nt determin nt of endur nce perform nce. ote orth V ̇ Od t 2max su est V̇ O2peak th t four ee s dministr tion of the er thropoiesis stimu tin ent recombin nt hum n er thropoietin rh is sufficient to incre se hemo obin concentr tion rteri o en content nd
time tri perform nce in he th non th etes. herefore more studies ddressin theV̇ O 2peakeffect of testosterone dministr tion on endur nce perform nce re needed.
sed on the e istin iter ture supr ph sio o ic doses of testosterone seem to induce me sur b e incre ses in stren th nd po er but not in endur nce perform nce ho ever more empiric evidence on the tter is needed. urther er o enic effects h ve been reported fter both on term months nd short term ee s testosterone dministr tion.
1.1.2 Acute performance-enhancing effects of testosterone ote orth imited d t re v i b e on hether testosterone dministr tion h s cute seconds to hours er o enic effects on hum n e ercise perform nce. n he th men n intr muscu r injection of hum n chorionic on dotropin h incre sed the p sm testosterone eve s nd reduced the
2 cortical motor t res old to e o e a muscular response ours post in ection erefore, increased urther one stud h s investi ted the effect of testosterone on runnin perform nce nd recover . plasma le els of testosterone can potentially facilitate t e corticospinal pat ay and t ere y influence ume nd co or ers h pothesi ed th t mu tip e doses of ou d enh nce the recover nd thereb muscle acti ity urt er, stimulation it suprap ysiolo ical testosterone le els rapidly and transiently the response to endur nce tr inin e din to improved endur nce perform nce. o consistent seconds to minutes increased t e intracellular calcium concentration in cultured rat myotu es is tre tment effects ere observed on runnin speed t n erobic thresho d or on biom r ers of ph sic response as mediated trou t e inositol trip osp ate pat ay, and follo ed y increased stress in he th recre tion ctive men fo o in four ee s of combined endur nce tr inin nd p osp orylation of e tracellular si nal re ulated inases n anot er study, stimulation it or dministered p cebo n testosterone undec no te m n or nor ndrostendione di ydrotestosterone , t e most potent testosterone meta olite, increased t e force production y m n three times per ee . ote orth serum testosterone concentr tion rem ined more t an in intact isolated mice s eletal muscle fi res, accompanied y increased p osp orylation unch n ed durin the intervention hich su ests th t the dministered dos e s insufficient to of mito en acti ated protein inase urt er, in addition to t e rapid acti ation of t e t, induce n me sur b e ch n es in recover nd ph sic d pt tion to tr inin . herefore this sin e and m pat ays, testosterone stimulation of incu ated uman s eletal muscle cells as een re tive sm nd short study preclude firm conclusions, especially on testosterone’s effects on found to increase lucose transporter m e pression and protein translocation, endur nce perform nce. nterestin testosterone dministr tion h s been found to stimu te it t e potential to promote lucose upta e and reco ery in s eletal muscle cells ese results su est er thropoiesis in dose dependent m nner possib throu h combin tion of direct nd indirect t at testosterone administration, eit er directly or ia , rapidly can affect s eletal muscle ndro en effects on the bone m rro er thropoiesis. his inc udes incre sed number of er thropoietin contractions, ener y meta olism and reco ery responsive pro enitor ce s in the bone m rro incre sed ren secretion of er thropoietin nd incre sed iron v i bi it throu h suppressed hepcidin. hus theoretic it is possib e th t the estosterone t erapy rapidly causes asodilatation in vitro and mediates asodilatation and au ments testosterone dministr tion c n enh nce endur nce perform nce throu h incre sed rteri o en cardiac output in eart failure patients urt er, i testosterone le els, eit er naturally or y
– content nd thereb m im o en upt e re tive to bod ei ht or hich is n administration, increases t e competiti e dri e and a ressi e e a iour in some indi iduals or e ample, arr and co or ers demonstrated a rapid increase in a ressi e e a iour it in an our import nt determin nt of endur nce perform nce. ote orth V ̇ Od t 2max su est V̇ O2peak th t four ee s dministr tion of the er thropoiesis stimu tin ent recombin nt hum n er thropoietin follo in a sin le testosterone el administration in men it dominant or impulsi e personality rh is sufficient to incre se hemo obin concentr tion rteri o en content nd styles ased on t ese studies, acute testosterone administration can potentially increase t e
cardiorespiratory capacity, as ell as pro ide psyc olo ical ad anta es in sports time tri perform nce in he th non th etes. herefore more studies ddressin theV̇ O 2peakeffect of testosterone dministr tion on endur nce perform nce re needed. e a ility to deduce uman effects from rodent studies and in vitro studies is unclear i e ise, testosterone induced p ysiolo ical c an es may not al ays result in measura le alterations of uman sed on the e istin iter ture supr ph sio o ic doses of testosterone seem to induce me sur b e performance ence, until furt er researc on umans is conducted, t e effects of sin le dose incre ses in stren th nd po er but not in endur nce perform nce ho ever more empiric evidence administration on uman e ercise performance remain speculati e e ert eless, ased on t e e istin on the tter is needed. urther er o enic effects h ve been reported fter both on term months nd literature, it is possi le t at testosterone administration induces acute performance en ancin effects, short term ee s testosterone dministr tion. ic may pro ide testosterone dopers it an acute competiti e ed e if administerin testosterone ri t efore or durin a competition ecause restin testosterone le els are positi ely related to ertical ump ei t , and sprintin performance, t e iolo ical effects of suc sin le dose administration may 1.1.2 Acute performance-enhancing effects of testosterone depend on t e initial serum testosterone le el, causin a lunted iolo ical effect in indi iduals it ote orth imited d t re v i b e on hether testosterone dministr tion h s cute seconds to naturally i testosterone le els tudies e aluatin t ese uestions are of i rele ance ot for a hours er o enic effects on hum n e ercise perform nce. n he th men n intr muscu r injection of hum n chorionic on dotropin h incre sed the p sm testosterone eve s nd reduced the
2
1.2 Performance-enhancing effects of clenbuterol
Clenbuterol is a sympathomimetic amine classified as a β –
β
– β
β
’s human studies applying other β
1.2.1 Ergogenic effects of long-term clenbuterol administration
f β m. quadriceps
β
term β s s s s s s s V̇ O2peak s s s s s s s s s s s 1.2 Performance-enhancing effects of clenbuterol administrationV̇ O 2peakof β s s s s Clenbuterol is a sympathomimetic amine classified as a β s s – 1.2.2 Ergogenic effects of acute clenbuterol administration β s s s s s s
β s s s s s s s s s s s s
– β of several β s s s s s s s s s β s s s s s s s s s s s s s s s ’s s s s s
V ̇ O2peak s s s human studies applying other β s s s term β s s V̇ O2peak enhancing effect of acute β s s s s s s s s 1.2.1 Ergogenic effects of long-term clenbuterol administration
f β
1.2.3 Summary
m. quadriceps s s administration of β s s β s s s s s s s s s
s β s s β s s s s s s s s s s term β s 1.3 Actual or potential health risk
s s s s s s s s s s s s s s s s s s s s s s s s s s s s ss s s s s s s s s s s s s s s s s s ss s s s s s s s s s s s
1.4 Indirect detection of doping with testosterone and its synthetic analogues
1.4.1 Steroid profile s s s s s s s s s s s s s s s ‘ s profiles’ ss s s s s s s s s s s s s s s s s s s s s s 5α s 3α, 17β diol (5α diol)/5β s 3α, 17β diol (5β diol), 5α s s s s s s s
Table 1. s s s s s
5α- 5α- 5β- 5α- T E A Etio T/E A/T diol/5β- A/Etio diol diol diol/E diol s s ↑ ↓ ↑ ↑ ↑ ↑ ↑ ↓ ↑ ↑ s s – ↑ ↓ ↑ ↑ ↑ ↑ ↓ ↑ ↑ ↑ s ↓ ↑ ↑ ↑ ↑ ↓ ↑ ↓ ↓ ↑ ↑ ↓ s – ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↑ ↑
1.3 Actual or potential health risk s ill s r e i le ele e ri r o e r io s of s rise i e r io of o
– s s s s s s is e i i i or of es os ero e i is r io oreo er e re se e re io of s s s s s s s ell o r s res l of es os ero e i e s ppressio of o o ropi rele si or o e s s s s s s or l ei i i or o e ro e po l i pi i r re l is o se e l s s s s re e e o e o s pro io of eres i l s ies o e i is r io of opi l s s s s s s ss s s s s or intranasal testosterone gel have demonstrated augmented 5α diol/5β iol 5α iol io – s s s s probably due to high levels of 5α re se i e s i s s s s s s s s s ss s s s s s s s s s s s
1.4 Indirect detection of doping with testosterone and its synthetic analogues
1.4.1 Steroid profile s s s s s s s s s s s s s s s ‘ s profiles’ ss s s s s s s s s s s s s s s s s s s s s s 5α s 3α, 17β diol (5α diol)/5β s 3α, 17β diol (5β diol), 5α s s s s s s s
Table 1. s s s s s
5α- 5α- 5β- 5α- T E A Etio T/E A/T diol/5β- A/Etio diol diol diol/E diol s s ↑ ↓ ↑ ↑ ↑ ↑ ↑ ↓ ↑ ↑ s s – ↑ ↓ ↑ ↑ ↑ ↑ ↓ ↑ ↑ ↑ s ↓ ↑ ↑ ↑ ↑ ↓ ↑ ↓ ↓ ↑ ↑ ↓ s – ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↑ ↑
Figure 1. 1.4.2 Evaluation of the steroid profile the sample’s ratios and concentrations, i.e. the sample’s urinary steroid profile, screening method used for individuals tested only once; the first ABP sample in an athlete’s steroidal specificity sensitivity 65,75,76
‘ ’ T/E, A/T, A/Etio, 5α diol/5β diol, and 5α cted and incorporated in the ‘adaptive model’, the individual’s own previous test results. thresholds is termed an ‘ ’ Subsequently, a confirmatory WADA’s technical document for
Figure 1. 1.4.3 Future possibilities he implementation of the steroidal module of the ABP has improved the sensitivity and specificity for screening for testosterone doping. he superiority of using individuali ed thresholds was illustrated in a study analy ing urine samples from control su ects and samples following the administration of mg testosterone undecanoate. he use of a population ased limit of . for resulted in incorrect identifications of suspicious samples i.e. specificity and correct identifications of suspicious samples i.e. sensitivity . While the use of ABP, on the other hand, lead to two incorrect identifications of suspicious samples i.e. specificity and correct identifications of suspicious samples i.e. sensitivity . oreover, the adaptive model in the ABP steroidal module has shown promising results for doses as low as mg intramuscular testosterone enanthate and mg testosterone gel in healthy men. owever, the passport evaluation is still a difficult tas due to confounding factors such as pregnancy, the use of hormonal contraceptives and alcohol consumption, – a le along with other pharmaceutical, technical analytical and iological factors that have een e tensively reviewed previously. , or e ample, oth small and large doses of ethanol have een found to increase the ratio and reduce the A ratio in healthy volunteers. – oreover, the reliance on collection of ‘clean’ aseline samples, from which the intra individual thresholds are calculated, further complicates the passport evaluation. herefore, more research should e conducted to improve the performance of the ABP.
Because testosterone stimulates erythropoiesis and alters iron homeostasis, , , one approach might e to loo at the already e isting hematological module of the ABP, which collects information on varia les that can indicate lood doping y e.g. As or lood transfusions. he two primary physiological mar ers are the B and reticulocyte percentage , which also are com ined in the core core B g – 60*(√[RET%])). he module also consists of a multiparametric marker named the ‘a normal lood profile score’ (ABPS), which is calculated from seven hematological parameters hematocrit , B, mean corpuscular hemoglo in , mean corpuscular hemoglo in concentration , mean corpuscular volume , B count and . , A normal fluctuations in either of the primary mar ers B or core can trigger an A P , while the and the ABP , among others, are regarded as secondary mar ers. nterestingly, studies indicate that and ABP have great potential in identifying doping with As, and in some cases even superior sensitivity compared with the primary mar ers B and core. ,
2 1.4.3 Future possibilities ecent tudie ha e e ta li hed that and marker hich are inc rp rated in the he implementation of the steroidal module of the ABP has improved the sensitivity and specificity hemat l ical m dule can increa e alread ithin ne t ur da ll in m for screening for testosterone doping. he superiority of using individuali ed thresholds was te t ter ne el r an intramu cular in ecti n m te t ter ne enanthate ence it i illustrated in a study analy ing urine samples from control su ects and samples following p i le that c m inin in rmati n r m the l d pa p rt ith that in the ter id pa p rt can the administration of mg testosterone undecanoate. he use of a population ased limit of . for help e aluatin the likelih d d pin in ter idal pa p rt ca e r e ample in the pre ence resulted in incorrect identifications of suspicious samples i.e. specificity and correct c n undin act r that d n t a ect the hemat l ical m dule e er the menti ned tudie identifications of suspicious samples i.e. sensitivity . While the use of ABP, on the other hand, did n t appl the adapti e m del in the hemat l ical m dule repeated admini trati n n r a lead to two incorrect identifications of suspicious samples i.e. specificity and correct place controlled design to evaluate the biomarkers’ sensitivity over a period of time uch tudie identifications of suspicious samples i.e. sensitivity . oreover, the adaptive model in the ABP are needed t e aluate the p tential the hemat l ical pr ile a a creenin t l t indicate steroidal module has shown promising results for doses as low as mg intramuscular testosterone te t ter ne d pin and r the electi n u pici u ample r ll up te tin enanthate and mg testosterone gel in healthy men. owever, the passport evaluation is still a difficult tas due to confounding factors such as pregnancy, the use of hormonal contraceptives and alcohol consumption, – a le along with other pharmaceutical, technical analytical and 1.5 Direct detection of doping with testosterone and its synthetic analogues iological factors that have een e tensively reviewed previously. , or e ample, oth small and ll in a u pici u in le urine ample r an in the ter idal m dule a urine anal i large doses of ethanol have een found to increase the ratio and reduce the A ratio in healthy i needed t directl c n irm the ri in the ter id he principle ehind the meth d i that the car n volunteers. – oreover, the reliance on collection of ‘clean’ aseline samples, from which the intra i t pic rati the end en u l pr duced ter id hich i determined the rati individual thresholds are calculated, further complicates the passport evaluation. herefore, more the d in ur diet e rain c rn di er r m that e en u ana lic ter id i ure research should e conducted to improve the performance of the ABP. hu in anal i the the ter id in the urine ample e pre ed in unit per th u and
‰) relati e t the internati nal tandard ienna ee ee elemnite (δ are c mpared t the
, , Because testosterone stimulates erythropoiesis and alters iron homeostasis, one approach might δ end en u re erence c mp und e er tudie ha e rep rted that te t ter ne e to loo at the already e isting hematological module of the ABP, which collects information on preparati n ith i t pic c mp iti n cl e t r ithin the ran e r end en u urinar ter id varia les that can indicate lood doping y e.g. As or lood transfusions. he two primary e i t i ure he u e the e pr duct ill n t e c n irmed acc rdin t the et criteria physiological mar ers are the B and reticulocyte percentage , which also are com ined in de cri ed in the technical d cument r anal i urtherm re the u e anal i the core core B g – 60*(√[RET%])). he module also consists of a c uld c n irm d pin in nl t ut i e u ect et een and h ur a ter a in le d e multiparametric marker named the ‘a normal lood profile score’ (ABPS), which is calculated from m te t ter ne el here re urther impr ement the en iti it a ell a the seven hematological parameters hematocrit , B, mean corpuscular hemoglo in , de el pment upplementar c n irmati n meth d l ie t detect te t ter ne d pin are mean corpuscular hemoglo in concentration , mean corpuscular volume , B count arranted ecau e i a er la ri u and e pen i e techni ue e peciall the latter i hi h and . , A normal fluctuations in either of the primary mar ers B or core rele ance can trigger an A P , while the and the ABP , among others, are regarded as secondary mar ers. nterestingly, studies indicate that and ABP have great potential in identifying doping with As, and in some cases even superior sensitivity compared with the primary mar ers B and core. ,
2 2 Figure 2 Carbon isotopic composition δ in testosterone preparations humans and plants he figure has been modified from rand the international standard ienna ee ee elemnite plants vegetables fruits grains plants corn sugarcane
ue to the rapid hepatic metabolism of pure testosterone many testosterone products are provided as testosterone esters ith esterification of the hydro ygroup hich makes the molecule more hydrophobic in proportion to the length of the side chain his modification slo s do n the release of testosterone ester from the in ection site into the circulation and thus prolongs the duration of action espite rapid hydrolysis into active testosterone in the bloodstream intact testosterone esters can still be present in blood – ased on characteristic retention times and diagnostic mass to charge ratios for the precursor ions and characteristic product ions orsdahl and co orkers ere able to separate steroid esters from endogenously produced testosterone by the use of li uid chromatography coupled to tandem mass spectrometry ) he assay as proofed applicable for confirmatory doping analysis by means of an administration study here three volunteers received an intramuscular in ection of ustanon a blend of four testosterone esters t as reported that intact esters could be detected up to days post in ection depending on the length of the ester sidechain herefore hen both blood and urine samples have been collected confirmatory analysis for steroid ester abuse can be considered done in serum or plasma
1.6 Detection of doping with clenbuterol
etection of clenbuterol amino dichloro a tert butylaminomethylben yl alcohol hydrochloride) in urine is a part of routine analysis at the accredited laboratories he e creted prodrug is detectable in urine ith reliable identification by in the lo pg m range n contrast to inhaled β agonists such as salbutamol formoterol and salmeterol clenbuterol is a non threshold 22 ‘ ’ ‘ ’
Figure 2 Carbon isotopic composition δ in testosterone preparations humans and plants he figure has been modified from rand the international standard ienna ee ee elemnite plants vegetables fruits grains plants corn sugarcane n n n ue to the rapid hepatic metabolism of pure testosterone many testosterone products are provided as testosterone esters ith esterification of the hydro ygroup hich makes the molecule more hydrophobic in proportion to the length of the side chain his modification slo s do n the release of testosterone ester from the in ection site into the circulation and thus prolongs the duration of action espite rapid hydrolysis into active testosterone in the bloodstream intact testosterone esters after three days, which is likely to be detected by today’s can still be present in blood – ased on characteristic retention times and diagnostic mass to charge ratios for the precursor ions and characteristic product ions orsdahl and co orkers ere able to separate steroid esters from endogenously produced testosterone by the use of li uid chromatography coupled to tandem mass spectrometry ) he assay as proofed applicable for confirmatory doping analysis by means of an administration study here three volunteers received an intramuscular in ection of ustanon a blend of four testosterone esters t as reported that intact esters could be detected up to days post in ection depending on the length of the ester sidechain herefore hen both blood and urine samples have been collected
confirmatory analysis for steroid ester abuse can be considered done in serum or plasma 1.7 Biological matrices in doping analysis
1.6 Detection of doping with clenbuterol etection of clenbuterol amino dichloro a tert butylaminomethylben yl alcohol hydrochloride) in urine is a part of routine analysis at the accredited laboratories he e creted prodrug is detectable in urine ith reliable identification by in the lo pg m range n contrast to inhaled β agonists such as salbutamol formoterol and salmeterol clenbuterol is a non threshold 22 2 etabolites a d rodr s of ai ly lower olec lar ass lood co le e ts with the e al atio of he atolo ical ara eters, ide tificatio of hi her olec lar ass a alytes ot e creted i ri e, as well as the a tificatio of the biolo ical acti ity of dr s owe er, the collectio a d tra s ortatio of ri e a d cooled blood sa les accordi to the sta dards are lo istical challe i , e e si e a d ti e co s i rther, the low to oderate a alyte stability at roo te erat re i the c rre t atrices is a iss e of co cer oreo er, i a st dy by lbe a d erbye, o er o e third of the elite athletes co leti the web based s r ey had e erie ced stress a d diffic lties ri ati d ri do i co trol, which ill strates that the ri e sa le collectio i fro t of a o i o trol fficer ca be i ti idati e ce, de elo e t a d alidatio of ew sa le collectio , shi e t a d a alytical ethods are warra ted lter ati e, co le e tary atrices i cl di sali a, e haled breath, hair a d dried atrices s ch as dried blood s ots or dried las a s ots ha e bee s b ect of se eral st dies related to s orts dr testi he e t sectio will foc s o the se of dried atrices i do i a alysis
1.7.1 Microvolume dried matrices he abo e e tio ed challe es ca be addressed by the i le e tatio of icro ol e sa li of whole blood or las a o filter a er c rre t a licatio s, of ca illary blood is collected er s ot o a filter a er has attracted assi e research atte tio d e to the i i ally i asi e ess a d the wide a licatio ossibilities, i cl di ewbor scree i , scree i of s bsta ce ab se a d roteo e wide associatio st dies, b t f rther de elo e t a d alidatio is re ired for syste atic a licatio i s eciali ed fields s ch as a ti do i f s ecial i terest for a ti do i efforts are the red ced costs for collectio , tra s ortatio a d stora e, i cl di o eed for trai ed hleboto ists, the low i asi e ess a d disco fort, the ossibility for a to ated syste for o li e li id chro ato ra hy hi h resol tio ass s ectro etry a alysis, , as well as a hi h a alyte stability for so e co o ds co ared with li id atrices – or e a le, the sy thetic e tide hor o e y acthe has bee fo d to be stable i at roo te erat re for at least te days, d e to the dryi rocess a d the corres o di red ctio of e y atic acti ity hilst the a alyte is ra idly de raded withi ho rs i ri e a d las a ser his ote tial be efit of co ld si lify sa le tra s ortatio a d allow sa le collectio i eo ra hically re ote areas
a or li itatio of is the li ited sa le ol e a ailable, which i fl e ces the assay se siti ity other challe e relies o the co ositio of the a aly ed blood, es ecially whe a tificatio a alyses are erfor ed this re ard the of the blood is of i orta ce, as it 2 etabolites a d rodr s of ai ly lower olec lar ass lood co le e ts with the e al atio of he atolo ical ara eters, ide tificatio of hi her olec lar ass a alytes ot e creted i ri e, as well as the a tificatio of the biolo ical acti ity of dr s owe er, the collectio a d tra s ortatio of ri e a d cooled blood sa les accordi to the sta dards are lo istical challe i , e e si e a d ti e co s i rther, the low to oderate a alyte stability at roo te erat re i the c rre t atrices is a iss e of co cer oreo er, i a st dy by lbe a d erbye, o er o e third of the elite athletes co leti the web based s r ey had e erie ced stress a d diffic lties ri ati d ri do i co trol, which ill strates that the ri e sa le collectio i fro t of a o i o trol fficer ca be i ti idati e ce, de elo e t a d alidatio of ew sa le collectio , shi e t a d a alytical ethods are warra ted lter ati e, co le e tary atrices i cl di sali a, e haled breath, hair a d dried atrices s ch as dried blood s ots or dried las a s ots ha e bee s b ect of se eral st dies related to s orts dr testi he e t sectio will foc s o the se of dried atrices i do i a alysis 1.7.1 Microvolume dried matrices he abo e e tio ed challe es ca be addressed by the i le e tatio of icro ol e sa li of whole blood or las a o filter a er c rre t a licatio s, of ca illary blood is collected er s ot o a filter a er has attracted assi e research atte tio life testing to demonstrate the assays’ d e to the i i ally i asi e ess a d the wide a licatio ossibilities, i cl di ewbor scree i , scree i of s bsta ce ab se a d roteo e wide associatio st dies, b t f rther de elo e t a d alidatio is re ired for syste atic a licatio i s eciali ed fields s ch as a ti do i f s ecial i terest for a ti do i efforts are the red ced costs for collectio , tra s ortatio a d stora e, i cl di o eed for trai ed hleboto ists, the low i asi e ess a d disco fort, the ossibility for a to ated syste for o li e li id chro ato ra hy hi h resol tio ass s ectro etry a alysis, , as well as a hi h a alyte stability for so e co o ds co ared with li id atrices – or e a le, the sy thetic e tide hor o e y acthe has bee fo d to be stable i at roo te erat re for at least te days, d e to the dryi rocess a d the corres o di red ctio of e y atic acti ity hilst the a alyte is ra idly de raded withi ho rs i ri e a d las a ser his ote tial be efit of co ld si lify sa le tra s ortatio a d allow sa le collectio i eo ra hically re ote areas
a or li itatio of is the li ited sa le ol e a ailable, which i fl e ces the assay se siti ity other challe e relies o the co ositio of the a aly ed blood, es ecially whe a tificatio a alyses are erfor ed this re ard the of the blood is of i orta ce, as it 2 2 Table 2. romising dried lood s ot assays for rohi ited s stan es and methods Reference Substance/Method Class Study design o aine tim lants ntelo or hine ar oti s eal life testing of oming e et ar oti s in l ded in olydr g a sers n al odeine s onitoring rogram nt nes et al ormone and eal life testing of amo i hen meta oli mod lators atients n e tide hormones eal life testing for o et al ns lin li e gro thfa tor gro th fa tors related endogeno s n s stan es and mimeti s healthy ol nteers ani lation of lood do ing monitoring lood and lood o et al Indirectly via the biomarkers om onents eal life testing of lean CD71, Band 3, CD41, e tide hormones non athletes and athletes CD45 gro th fa tors related s stan es and mimeti s tologo s lood la e o ontrolled ani lation of o et al transf sion Indirectly via administration st dy n lood and lood the biomarkers CD71 and lood n om onents Band 3 la e o e tide hormones ontrolled erro at al rh Indirectly via the gro th fa tors related administration st dy n biomarker fibronectin-1 s stan es and mimeti s rh n ontrol ner et al irt in a ti ating on a ro ed dministration st dy om o nds s stan es ith rats n e tide hormones otater e t i ed sam les gro th fa tors related ange et al la e o ontrolled s ater e t s stan es and mimeti s administration st dy ormone and imagr ma imagr ma n n a meta oli mod lators a son o s dministration st dy n and anna tenolol eta lo ers healthy ol nteers o ima et al hedrine dministration st dy n tim lants ethyle hedrine healthy ol nteers ommers et al or hine ar oti s i ed sam les e tide hormones ller et al eginesatide gro th fa tors related i ed sam les s stan es and mimeti s estoterone nde anoate Indirectly via. blood steroid profile estoterone ro ionate eng et al dministration st dy n Indirectly via. blood steroid na oli agents healty ol nteers profile estosterone enanthate Indirectly via. blood steroid profile rotti et al eal life testing of y odone ar oti s atients n la e o ontrolled ilot e erter e tide hormones administration st dy n ran hat et al rh gro th fa tors related rh n ontrol s stan es and mimeti s dministration st dy n healhty ol nteers
2 Table 2. romising dried lood s ot assays for rohi ited s stan es and methods dministration st dy rh ith rh n Reference Substance/Method Class Study design e erter e tide hormones healthy ol nteers o aine tim lants ran hat et al gro th fa tors related eal life testing of ntelo or hine ar oti s eal life testing of s stan es and mimeti s atients for n oming e et ar oti s in l ded in olydr g a sers n al odeine s onitoring i ed sam les rogram e tide hormones nt nes et al ormone and eal life testing of osting et al amo i hen h gro th fa tors related i ed sam les meta oli mod lators atients n e tide hormones eal life testing for s stan es and mimeti s o et al ns lin li e gro thfa tor gro th fa tors related endogeno s n osting et al ormone and man ins lin i ed sam les s stan es and mimeti s healthy ol nteers meta oli mod lators dministration st dy ani lation of ani lation of ith rh n lood do ing monitoring lood and lood lood and lood healthy ol nteers o et al Indirectly via the biomarkers om onents eal life testing of lean alamin et al lood do ing Indirectly om onents la e o ontrolled CD71, Band 3, CD41, e tide hormones non athletes and athletes via the biomarker ALAS2 e tide hormones administration st dy ith CD45 gro th fa tors related gro th fa tors related lood ithdra al n s stan es and mimeti s s stan es and mimeti s tologo s lood la e o ontrolled healthy ol nteers ani lation of o et al transf sion Indirectly via administration st dy n or hine ar oti s lood and lood the biomarkers CD71 and lood n om onents ar oti s in l ded in Band 3 la e o odeine s onitoring e tide hormones ontrolled rogram erro at al rh Indirectly via the gro th fa tors related administration st dy n biomarker fibronectin-1 o aine i ed sam les s stan es and mimeti s rh n ontrol a sserea et al eal life testing of dr g ner et al irt in a ti ating on a ro ed dministration st dy m hetamine a sers n om o nds s stan es ith rats n etham hetamine e tide hormones tim lants otater e t i ed sam les gro th fa tors related ange et al la e o ontrolled s ater e t s stan es and mimeti s administration st dy ormone and imagr ma imagr ma n n a meta oli mod lators al tamol eta agonists a son o s dministration st dy n and anna tenolol eta lo ers o aine i ed sam les healthy ol nteers homas et al ‐ dministration st dy se doe hedrine tim lants se doe hedrine n o ima et al hedrine dministration st dy n tim lants m hetamine healthy ol nteer healthy ol nteers ethyle hedrine anna inoids ommers et al or hine ar oti s i ed sam les eto rolol e tide hormones iso rolol eta lo ers ller et al eginesatide gro th fa tors related i ed sam les ro ronalol s stan es and mimeti s len terol estoterone nde anoate Indirectly via. blood steroid etandienone profile ndarine na oli agents estoterone ro ionate eng et al dministration st dy n tano olol Indirectly via. blood steroid na oli agents healty ol nteers profile homas et al i ed sam les estosterone enanthate al tamol Indirectly via. blood steroid eta agonists profile ormoterol rotti et al eal life testing of nastro ole ‐ y odone ar oti s ormone and atients n lomi hene la e o ontrolled ilot meta oli mod lators emestane e erter e tide hormones administration st dy n i reti s and mas ing ran hat et al rh gro th fa tors related rh n ontrol ydro hlorothia id s stan es and mimeti s dministration st dy n agents healhty ol nteers tim lants
2 2 try hnine ethylhe aneamine ethyl henidate o aine i ethamide eso ar anna inoids e amethasone l o orti oids desonide man ins lin ns lin lis ro ns lin as art ns lin gl lisine i ed sam les homas et al ormone and dministration st dy ns lin glargine meta oli mod lators ins lin as art n ns lin detemir dia eti atient ns lin tresi a o ine ins lin or ine ins lin estosterone nde anoate estosterone de anoate estosterone henyl ro ionate i ed sam les estosterone a etate dministration st dy ret el et al na oli agents estosterone estosterone y ionate nde anoate n estosterone iso a rioate healthy ol nteer androlone henyl ro ionate ren olone enanthate androlone e tide hormones ret el et al dministration st dy n yna then gro th fa tors related healthy ol nteer s stan es and mimeti s tano olol dministration st dy n na oli agents ret el et al ehydro hlormethyl healthy ol nteers testosterone dministration st dy n se doe hedrine tim lants healthy ol nteers tim lant in l ded in eal life testing n ret el et al i otine s onitoring smo ers sn s sers n rogram ontrol ret el et al ormone and dministration st dy n eldoni m meta oli mod lators healthy ol nteers or hine entanyl ar oti s i ed sam les er laetse and y odone dminsitration st dy enion ydro odone n odeine ar oti s in l ded in healthy ol nteer s onitoring ydro odone rogram 2 1.7.2 Detectability of testosterone esters and clenbuterol in DBS/DPS try hnine n the ne t ste it is of o io s im ortan e to fo s on fre ently o rring s stan es s h as ethylhe aneamine testosterone esters and len terol romising methods to dete t len terol and inta t ana oli ethyl henidate o aine steroid esters from s i ed sam les ha e een ro osed ith limit of dete tion of i ethamide ng m for len terol and ng m for the testosterone esters testosterone henyl ro ionate eso ar testosterone iso a roate and testosterone de anoate o e er oth methods anna inoids e amethasone sho ed in om lete e tra tion of the analytes or len terol the e tra tion re o ery as hile l o orti oids desonide for the testosterone esters and only to ere e tra ted artly d e to the man ins lin adsor tion effe t of the esters to the sam le material n reased sensiti ity thro gh im ro ed sam le ns lin lis ro ns lin as art re aration and analyti al ro ed res is desira le for rolonged indo s of dete tion as ell as for ns lin gl lisine i ed sam les the ossi ility to dete t lo administration doses his might e a hie ed sing alternati e e tra tion homas et al ormone and dministration st dy ns lin glargine meta oli mod lators ins lin as art n onditions and deri ati ation agents i e signal enhan ement as ell as sensiti e instr mentation ns lin detemir dia eti atient ns lin tresi a s h as nano s ale ano oreo er in om lete el tion of the analytes o ine ins lin from the sam ling ard might e ir m ented y alternati e olle tion materials ne romising or ine ins lin a roa h is to se ater sol le materials s h as ar o ymethyl ell lose hi h an e estosterone nde anoate almost om letely dissol ed in a an a eo s ffer sol tion ma ing the entire sam le a aila le for estosterone de anoate f rther ro essing estosterone henyl ro ionate i ed sam les estosterone a etate dministration st dy ret el et al na oli agents estosterone ollo ing method de elo ment and alidation only the mentioned testosterone ester assay’s estosterone y ionate nde anoate n estosterone iso a rioate healthy ol nteer sensiti ity as e al ated in a roof of on e t st dy for do ing ontrol r oses en e a st dy androlone on h man administration of len terol has yet to e ond ted he testosterone ester assay as henyl ro ionate ren olone enanthate roofed relia le for dete tion of orally administered testosterone nde anoate mg ndriol androlone esto a s in a healthy s e t to eight ho rs ost administration des ite limited analyte e tide hormones ret el et al dministration st dy n yna then gro th fa tors related healthy ol nteer re o ery and the lo ioa aila ility of oral testosterone nde anoate s a roof of on e t a s stan es and mimeti s tano olol sam le si e of one n is often sed in the field of anti do ing to demonstrate that a method has dministration st dy n na oli agents ret el et al ehydro hlormethyl healthy ol nteers a li a le sensiti ity ota ly d e to the large onse en es of san tioning an athlete ased on a testosterone dministration st dy n false ad erse analyti al finding high s e ifi ity is of great im ortan e his is arti larly im ortant se doe hedrine tim lants healthy ol nteers tim lant in l ded in eal life testing n for dire t dete tion methods s h as dire t dete tion of testosterone esters and len terol in ret el et al i otine s onitoring smo ers sn s sers n herefore and d e to the et een s e t aria ility in adsor tion elimination rates it rogram ontrol ret el et al ormone and dministration st dy n eldoni m is re ired to olle t sam les from more than one healthy s e t ndergoing administration meta oli mod lators healthy ol nteers or hine resem ling the s s e ted ra ti e y heating athletes lean aseline sam les in a lini al trial an e entanyl ar oti s i ed sam les sed to e al ate the s e ifi ity ho e er randomi ed la e o ontrolled st dies ith sam les from er laetse and y odone dminsitration st dy enion ydro odone n a la e o gro ndergoing the same inter ention as the dr g administration gro is desira le to odeine ar oti s in l ded in healthy ol nteer s onitoring provide extensive information of the assay’s specificity for a period of time. These points should be ydro odone rogram 2 2 ta en into consideration in the further development and validation of the and methodolo ies for detection of fre uently occurrin dopin substances and methods.
efore the and methodolo ies can be implemented in a dopin control settin the transport and stora e procedures must be optimi ed and standardi ed amon dopin control a encies and laboratories. n this re ard it is re uired to evaluate the stability of analytes on cards under varyin stora e conditions. de radation as observed for anabolic steroid esters on after ee s stora e at room temperature hile clenbuterol as found to be stable on card for at least one ee hen stored at cooled conditions . ince the lon term stability of clenbuterol and testosterone esters on cards at cooled or fro en conditions has yet to be reported studies assessin analyte stability at varyin stora e temperatures are arranted.
1.8 Applicability to anti-doping: objectives
mportant elements of an efficient anti dopin system are tar eted testin and fre uent and unpredictable sample collection to maximi e detection and deterrence. o ever as hi hli hted in the previous sections the system is limited by challen es includin time consumin and expensive collection and shipment limited analyte stability labour intensive analysis and in some cases insufficient sensitivity and specificity of the existin state of the art analytical methods to detect some dopin substances. Thus continued research to improve the time and cost efficiency and increase the li elihood of catchin cheatin athletes and recreationally active is arranted.
The minimally invasive easy and cost reduced and techni ues have the potential to improve the time and cost efficiency compared to traditional collection shipment and analysis. s complementary matrices and can increase deterrence and the capacity to better reveal dopin practices by allo in more fre uent out of competition testin and lar e scale testin also in eo raphically remote areas. Therefore the present h pro ect aimed to compare the specificity sensitivity and applicability of and materials for analysis of fre uently occurrin dopin substances to find the best performin method for routine dopin analysis. dditionally the pro ect aimed to evaluate the lon term stability of four different testosterone esters on cards after stora e at cooled and fro en conditions to achieve a robust matrix suitable for a dopin control settin . oreover e aimed to evaluate hether the administration of the four testosterone esters T T T and testosterone propionate T ustanon or clenbuterol to healthy male volunteers could be detected in samples.
ta en into consideration in the further development and validation of the and no led e about the pharmaco inetics and pharmacodynamics of a dopin substance is important methodolo ies for detection of fre uently occurrin dopin substances and methods. for tar eted testin . Therefore the present pro ect aimed to evaluate detection indo s for testosterone esters and clenbuterol in and or urine samples collected from humans sub ected to efore the and methodolo ies can be implemented in a dopin control settin the transport administration resemblin the suspected practice by cheatin athletes. oreover e aimed to examine and stora e procedures must be optimi ed and standardi ed amon dopin control a encies and the acute er o enic effects of a mixed testosterone ester in ection m ustanon on human laboratories. n this re ard it is re uired to evaluate the stability of analytes on cards under physical performance and hether the basal serum testosterone concentration influences the varyin stora e conditions. de radation as observed for anabolic steroid esters on performance in stren th and po er exercises to help anti dopin authorities determine the most cost after ee s stora e at room temperature hile clenbuterol as found to be stable on card efficient testin pro rams. for at least one ee hen stored at cooled conditions . ince the lon term stability of clenbuterol and testosterone esters on cards at cooled or fro en conditions has yet to be reported To further develop already existin detection methods is a cost efficient ay to improve the li elihood studies assessin analyte stability at varyin stora e temperatures are arranted. of revealin dopin practices. ased on the erythropoietic stimulatin effect of testosterone the pro ect aimed to evaluate the potential of the adaptive model in the hematolo ical module to infer testosterone dopin and as a screenin tool to identify suspicious samples for subse uent 1.8 Applicability to anti-doping: objectives analysis. mportant elements of an efficient anti dopin system are tar eted testin and fre uent and unpredictable sample collection to maximi e detection and deterrence. o ever as hi hli hted in the previous sections the system is limited by challen es includin time consumin and expensive The follo in hypotheses ere investi ated collection and shipment limited analyte stability labour intensive analysis and in some cases . in le and repeated intramuscular in ections of m testosterone esters ustanon insufficient sensitivity and specificity of the existin state of the art analytical methods to detect some affect biomar ers in the hematolo ical module of the thlete iolo ical assport. dopin substances. Thus continued research to improve the time and cost efficiency and increase . mix of testosterone esters administered to healthy participants is detectable by the li elihood of catchin cheatin athletes and recreationally active is arranted. application of a based analytical method ith sufficient specificity and sensitivity to complement existin analytical strate ies hile increasin time cost efficiency. urther The minimally invasive easy and cost reduced and techni ues have the potential to improve lon term stability of four different testosterone esters on cards can be achieved by the time and cost efficiency compared to traditional collection shipment and analysis. s stora e at fro en conditions. complementary matrices and can increase deterrence and the capacity to better reveal . ne intramuscular in ection of mixed testosterone esters enhances physical dopin practices by allo in more fre uent out of competition testin and lar e scale testin also in performance in stren th and po er exercises acutely h after in ection. dditionally the eo raphically remote areas. Therefore the present h pro ect aimed to compare the specificity basal serum testosterone concentration influences the performance in countermovement sensitivity and applicability of and materials for analysis of fre uently occurrin dopin ump s all out cycle sprint and one arm isometric elbo flexion. substances to find the best performin method for routine dopin analysis. dditionally the pro ect . lenbuterol can be detected in and urine samples follo in sin le dose in estion of aimed to evaluate the lon term stability of four different testosterone esters on cards after oral clenbuterol. stora e at cooled and fro en conditions to achieve a robust matrix suitable for a dopin control settin . oreover e aimed to evaluate hether the administration of the four testosterone esters The present h pro ect as a part of the nnovation und enmark’s Industrial Researcher T T T and testosterone propionate T ustanon or clenbuterol to healthy male ro ram. The ndustrial h pro ect as a collaboration bet een nti opin enmar and the volunteers could be detected in samples. niversity of openha en ith the accredited or e ian opin ontrol aboratory as a
third art he resent Industrial h thesis is based on two of the Industrial PhD’s studies, from hich ur a ers ha e een r ed –three su itted and ne dra t he a ers are re erred t as Paper I, II, III and IV, corresponding to those listed in ‘List of apers’. Data collection took place in enha en en ark under the uidance ss ciate r ess r ik lai aastru rds r at the ni ersit enha en and r ak rke er the eni r cience ana er at nti in en ark he anal tical rk r Paper I-III as er r ed at the r e ian in ntr l a rat r in sl r a the h student and la rat r ers nnel under uidance the a rat r irect r r ette ehnes he anal tical rk r Paper IV as er r ed at the accredited la rat r in l ne er an
2 third art he resent Industrial h thesis is based on two of the Industrial PhD’s studies, from 2. Results and discussion hich ur a ers ha e een r ed –three su itted and ne dra t he a ers are re erred t as Paper I, II, III and IV, corresponding to those listed in ‘List of apers’. Data collection took place in he main results from Paper I-IV are described and discussed in the following subchapters. enha en en ark under the uidance ss ciate r ess r ik lai aastru rds r at the ni ersit enha en and r ak rke er the eni r cience ana er at nti in en ark he anal tical rk r Paper I-III as er r ed at the r e ian in ntr l 2.1 Acute performance-enhancing effects of testosterone in humans
a rat r in sl r a the h student and la rat r ers nnel under uidance the s outlined in the introduction, findings indicate that testosterone administration rapidl can affect a rat r irect r r ette ehnes he anal tical rk r Paper IV as er r ed at the skeletal muscle contractions, – energ metabolism and reco er , as well as increase the accredited la rat r in l ne er an cardiorespirator capacit , and aggressi e beha ior in some indi iduals. hese ph siological and ps chological effects could potentiall pro ide testosterone dopers with an ad antage if administering testosterone right before or during a competition, but studies on the acute performance enhancing effect of testosterone administration in humans are lacking. he following sections address potential mechanisms b which acute testosterone administration can enhance muscular strength, sprint performance and ertical ump performance, and relate the current knowledge to the results of Paper III.
2.1.1 Acute ergogenic effects of testosterone administration on muscular strength It is possible that acute testosterone administration can enhance muscular strength. Intracellular calcium concentration has been reported to increase within seconds to minutes in cultured rat m otubes after testosterone stimulation. In skeletal muscles, intracellular calcium regulates contraction ia binding to troponin . urther, stimulation b the testosterone metabolite D has been found to rapidl increase force production ia phosphor lation of the regulator m osin light chain kinase in fast twitch mouse muscle fibres. In skeletal muscles, phosphor lated m osin light chain kinase can increase the sensiti it of the contractile proteins to calcium. hus, acute testosterone administration can potentiall stimulate increased ma imal force production e.g. measured as in humans b both augmented intracellular calcium release and calcium sensiti it for cross bridge formation.
oteworth , the abilit to deduce human effects from rodent studies and in vitro studies is unclear. or e ample, human skeletal muscles appear to ha e low acti it of α reductase, the en me that con erts testosterone to D . he con ersion of testosterone to D , and thereb the potential D mediated effect of testosterone on human skeletal muscle contractilit , might thus be limited. In Paper III, we were the first to e aluate the acute effects of testosterone administration on human ph sical performance. e found that the in a one arm isometric elbow fle ion test remained 2 Paper III in vitro
m. interosseus dorsalis I
2.1.2 Acute ergogenic effects of testosterone administration on sprint performance – in vitro
Paper III Paper III in vitro Paper III Paper III m. interosseus dorsalis I 2.1.3 Acute ergogenic effects of testosterone administration on vertical jump performance
2.1.2 Acute ergogenic effects of testosterone administration on sprint performance – in vitro Paper III
2.1.4 Serum testosterone and performance capacities Paper III subjects’ serum testosterone levels and performance outcomes in Paper III Paper III Paper III
2.1.5 Implications for anti-doping – Paper III
anti dopin aut orities s ould focus on out of competition testin f testin in competition it is note ort t at t ere seem to be no performance en ancin effects of ustanon at a time point ere it is detectable in Paper II plasma and urine Paper I
2.1.4 Serum testosterone and performance capacities
2.1.6 Study limitations Paper III ne can ar ue t at t e applied dose in Paper III as too lo to induce mar ed p siolo ical effects because findin s indicate t at testosterone induced effects on muscle stren t le po er and subjects’ serum testosterone levels and performance outcomes in muscle si e in ealt oun men are dependent on dose o ever e observed a t reefold Paper III increase in mean serum testosterone level from pre to post injection in t e testosterone roup ile it remained unc an ed in t e placebo roup Paper III oreover ma imal circulator concentrations of t e testosterone esters measured in occurred durin t e first one to t ree da s post injection Paper III Paper II ese findin s indicate t at t e performance measurements ere carried out in t e period ere acute effects can be e pected and t at t e applied dose as sufficient to elevate serum testosterone to suprap siolo ical levels ort mentionin is t e et ical concerns en conductin testosterone administration studies e c ose m ustanon because t is as previousl been safel administered to males in controlled studies is dose is in t e lo er ran e of t e dopin practice reported b at letes and recreationall active in uestionnaires and intervie s m to more t an m per ee Paper III et serum concentrations of ere reduced post injection in t e testosterone ester roup Paper III ic illustrates t at t e testosterone administration caused a feedbac in ibition of endo enous testosterone production s an important note it re ards to t e safet of participants one subject e perienced suppressed testosterone levels compared to baseline at t e medical follo up t ree ee s after t e second injection but t e levels ad returned to baseline at t e additional medical follo up t ree mont s later o additional severe side effects ere reported Paper III
2.1.5 Implications for anti-doping dditionall it can be speculated t at a performance en ancement ould ave been observed if ot er e ercise modalities ere applied erefore furt er researc it t e use of ot er e ercise modalitiesare needed –
Paper III
2.2 Indirect detection of testosterone esters
2.2.1 The steroid profile s i li ted in t e introduction t e implementation of t e individual based steroidal module of t e as improved t e sensitivit and specificit for screenin for testosterone dopin n Paper I a total of clean urine samples includin all placebo samples and t e baseline samples for t e testosterone ester roup ere anal ed en usin population based cut off values t e specificit as i e incorrect identifications of suspicious samples ile t e adaptive model of t e steroidal module on t e ot er and ad a specificit of i e incorrect identifications of suspicious samples n t e da follo in t e first and second testosterone ester injection t e ratio as suspicious in and subjects respectivel en usin population based reference ran es en usin t e adaptive model t e ratio identified all subjects in t e ustanon roup on t e first da after bot injections is confirms t e superiorit of usin individuali ed t res olds
imilar to t e application of transdermal and intranasal testosterone el in ealt oun volunteers and 5α diol ere t e most sensitive steroidal mar ers for intramuscular ustanon injections Paper I ese mar ers remained increased compared it bot baseline levels and levels in t e placebo roup for ten and fourteen da s after t e first and second testosterone ester injection respectivel o ever t e steroidal module as man confoundin factors t at c allen e t e passport evaluation or e ample alco ol consumption evident b t e presence of et l lucuronide t in t e urine sample li el caused t e at pical ratio in one placebo sample as t is sample ad t e i est measured t concentration m imilarl alco ol consumption mi t ave contributed to t e at pical ratios in t e ten ustanon samples it t concentrations above n m but since t e samples simultaneousl s o ed at pical α diol ratios t e ere still evaluated as suspicious
n t e presence of confoundin factors t e t lete assport ana ement nit evaluates t e sample validit and possible s in t e conte t of t e remainin urinar mar ers n t is re ard it could be speculated t at combinin information from t e ematolo ical profile it t e steroid profile could be elpful in t e passport evaluation as previousl su ested b ot ers
2.2.2 The hematological profile ased on t e proven er t ropoietic stimulatin effect of testosterone e evaluated t e potential of t e adaptive model in t e ematolo ical module to infer testosterone dopin e to identif suspicious samples for subse uent anal sis Paper I ecent studies ave establis ed
2.2 Indirect detection of testosterone esters t at m testosterone el can elevate b percenta e point and b it in ours and t at an intramuscular injection of m testosterone enant ate can increase t e 2.2.1 The steroid profile b it in four da s n contrast to Paper I t ese studies did not appl a placebo controlled s i li ted in t e introduction t e implementation of t e individual based steroidal module of desi n uc desi n is of importance to e clude t e possibilit t at ot er factors t an t e testosterone t e as improved t e sensitivit and specificit for screenin for testosterone dopin n Paper I administration caused t e observed fluctuations n Paper I e demonstrated t at bot sin le and a total of clean urine samples includin all placebo samples and t e baseline samples for t e repeated intramuscular injections of m mi ed testosterone esters elevated t e and testosterone ester roup ere anal ed en usin population based cut off values t e specificit and decreased t e score t ree to ten da s after t e first and or second injections ontrar to as i e incorrect identifications of suspicious samples ile t e adaptive model of t e t e findin of a increase in four da s post injection of m testosterone enant ate in steroidal module on t e ot er and ad a specificit of i e incorrect identifications of ealt males remained unc an ed t rou out t e intervention in Paper I is mi t be suspicious samples n t e da follo in t e first and second testosterone ester injection t e e plained b a lo er dose in Paper I, because t e testosterone induced er t ropoiesis as been ratio as suspicious in and subjects respectivel en usin population based reference reported to be dose dependent us t e relativel lo response percenta e point ran es en usin t e adaptive model t e ratio identified all subjects in t e ustanon roup mi t ave been too small to induce a measurable c an e in econdl t e subjects did not et on t e first da after bot injections is confirms t e superiorit of usin individuali ed t res olds iron supplementation t us insufficient iron availabilit for s nt esis cannot be e cluded
imilar to t e application of transdermal and intranasal testosterone el in ealt oun Paper I was the first study to use WADA’s ABP software and a placebo controlled desi n to evaluate volunteers and 5α diol ere t e most sensitive steroidal mar ers for intramuscular ustanon t e sensitivit and specificit of t e ematolo ical module to infer sin le and repeated injections Paper I ese mar ers remained increased compared it bot baseline levels and levels testosterone ester injections n a reement it t e pot esis e s o ed t at intramuscular in t e placebo roup for ten and fourteen da s after t e first and second testosterone ester injection testosterone ester injections affect not onl t e steroidal module but also some of t e mar ers in t e respectivel o ever t e steroidal module as man confoundin factors t at c allen e t e passport ematolo ical module verall t e ematolo ical module identified suspect samples in evaluation or e ample alco ol consumption evident b t e presence of et l lucuronide t ustanon treated subjects e and identified a lar er proportion of doped subjects in t e urine sample li el caused t e at pical ratio in one placebo sample as t is sample ad and respectivel t an t e and core ic bot identified ustanon t e i est measured t concentration m imilarl alco ol consumption mi t ave treated subjects ote ort in t e placebo roup as at pical in of t e subjects and as contributed to t e at pical ratios in t e ten ustanon samples it t concentrations above elevated from baseline ten da s after t e second injection n increase in t e as previousl n m but since t e samples simultaneousl s o ed at pical α diol ratios t e ere still evaluated been reported as indication of blood dopin ence our results s o t at t e lac s as suspicious specificit in some cases is is possibl because t e score contains blood variables t at are influenced
b pre anal tical conditions suc as prolon ed stora e alt ou t e stable in t e placebo n t e presence of confoundin factors t e t lete assport ana ement nit evaluates t e sample roup indicates t at t e pre anal tical conditions in Paper I ere not sub optimal n summar t e validit and possible s in t e conte t of t e remainin urinar mar ers n t is re ard it could seemed to be t e best mar er in t e ematolo ical module for indicatin testosterone dopin be speculated t at combinin information from t e ematolo ical profile it t e steroid profile could be elpful in t e passport evaluation as previousl su ested b ot ers s a proof of concept e conducted anal ses based on at pical ematolo ical profiles in Paper
2.2.2 The hematological profile I. e found t at t e and core could in addition to t e ratio elp identif in ased on t e proven er t ropoietic stimulatin effect of testosterone e evaluated t e suspicious samples for more tar eted testin ndeed t e steroidal profile is more sensitive to potential of t e adaptive model in t e ematolo ical module to infer testosterone dopin e to testosterone dopin t an t e ematolo ical module onet eless t e use of t ese mar ers could be identif suspicious samples for subse uent anal sis Paper I ecent studies ave establis ed beneficial in cases ere confoundin factors affect t e steroid profile urt ermore sometimes no
urine sa ples but only ABP blood sa ples are collected to sa e the costs related to urinary analysis and or to sa e ti e when testin any athletes n cases of suspicious blood profiles with ele ated alues but stable B alues and where subse uent A analysis ha e shown ne ati e results the ABP he atolo ical odule could be used to tar et follow up urine sa ple collection and analysis his is supported by Paper I where out of ustanon treated sub ects had an atypical ratio fourteen days after the second in ection i e. four days after the was ele ated co pared with baseline and placebo his i plies that the ratio could still be ele ated if a follow up urine sa ple was collected days after an ele ated
2.3 Direct detection of testosterone esters
oday dopin with testosterone esters is directly detected in urine by analysis or in seru plas a by steroid ester analysis owe er as addressed in the introduction these test ethods ha e so e li itations or e a ple low ad inistration doses or the use of testosterone
preparations with δ PDB close to or within the ran e of those for endo enous urinary steroids i ht be difficult to detect by Additionally direct steroid ester analysis is li ited by rapid te perature dependent de radation of esters in seru plas a especially in blood collection tubes not stabili ed with the esterase inhibitor sodiu fluoride urther collection and transportation of urine and enous blood sa ples are sub ect to strict criteria as described in WADA’s standards and can be ti e consu in lo istically challen in and re uire substantial resources
2.3.1 Further development of DBS analysis of testosterone esters DB and DP are alternati e low cost sa ple atrices which i ht be applicable for detection of dopin with testosterone esters especially because inacti ation of the hydrolase en y es occurs when the blood is dried on the sa plin aterial n support of this a pro isin ethod to detect intact anabolic steroid esters fro spi ed DB sa ples with a D of l n has been proposed e ertheless the ethod showed inco plete e traction of the analytes and resulted in double chro ato raphic pea s hus we ai ed to further de elop the ethod for analysis of the testosterone esters D PP and P by i pro in sa ple preparation and analytical procedures Paper II
arious alternati e e traction conditions were tested includin different sol ents te peratures and incubation periods in sonification and sha in in order to find the ost efficient urine sa ples but only ABP blood sa ples are collected to sa e the costs related to urinary analysis tra t n r d r s r D and s str n r an s nts as n ssar and or to sa e ti e when testin any athletes n cases of suspicious blood profiles with ele ated a s t ads r t n t s st rs t t s as d D ard n t s r s t d n alues but stable B alues and where subse uent A analysis ha e shown ne ati e art s r t sa n at r a n t s rnatant a t r n st as add d t r n ant d results the ABP he atolo ical odule could be used to tar et follow up urine sa ple collection and art s and r t t t nstr nt t r a t n tr and t an r d d analysis his is supported by Paper I where out of ustanon treated sub ects had an atypical t st r r s t t d ss n t s n t rs Paper II ratio fourteen days after the second in ection i e. four days after the was ele ated co pared with baseline and placebo his i plies that the ratio could still be ele ated if a follow na t d tra t n nd t ns r s t d n r r s t n and Paper II up urine sa ple was collected days after an ele ated s ara t t s r s r rt d s s sts t at s as d D at r a s ar n t t a r D ana s s t st st r n st rs r r t st d n t t n d r nt d t s a at r s at r a A a dr r n a D s d as s n r at t nt a r t r ana t s Add t na a D ard 2.3 Direct detection of testosterone esters D t rt W st r A as a at d d t t t nt a n n oday dopin with testosterone esters is directly detected in urine by analysis or in at r t n tra t n n at ast at at r t s r r s D seru plas a by steroid ester analysis owe er as addressed in the introduction these test and r t r s at r a r ara t t s r and t D ethods ha e so e li itations or e a ple low ad inistration doses or the use of testosterone ard d s t t s an a s r s t n t d r n t D ard a s
preparations with δ PDB close to or within the ran e of those for endo enous urinary steroids r t n t s ts and s r a a a a as a r d t s t d r n a tr a s and i ht be difficult to detect by Additionally direct steroid ester analysis is li ited by rapid d n ntr nt r ard t t s at r a te perature dependent de radation of esters in seru plas a especially in blood collection tubes not stabili ed with the esterase inhibitor sodiu fluoride urther collection and transportation of r s r rt d t d r d t t n t st st r n st rs n D s d t urine and enous blood sa ples are sub ect to strict criteria as described in WADA’s standards and d r at at n as a an t n an n at n and t r r at ra s na and assa can be ti e consu in lo istically challen in and re uire substantial resources s ns t t r t d r at at n an ad t t r at n t r at ra a s r s t st st r n st rs r d s t s na str n t ar d t a s n a n r at ra a s r ta n d d r at at n dr a n n 2.3.1 Further development of DBS analysis of testosterone esters r d n n t t d r t st st r n st r ana s s n s r as a t t s d r at at n DB and DP are alternati e low cost sa ple atrices which i ht be applicable for detection of r a nt an rr s and ar t nstr nt and t s r r s sa an n n dopin with testosterone esters especially because inacti ation of the hydrolase en y es occurs when s dra n r d n r d t s na str n t and r s t d n s n a s r t the blood is dried on the sa plin aterial n support of this a pro isin ethod to detect t st st r n st rs d r t n n t r s nd n s r st r d r n s and as t r r intact anabolic steroid esters fro spi ed DB sa ples with a D of l n has been proposed s t d as d r at at n r a nt n t na t d r t Paper II e ertheless the ethod showed inco plete e traction of the analytes and resulted in double chro ato raphic pea s hus we ai ed to further de elop the ethod for analysis of the n t a ana s s as r r d n nt na tra r ss r s st testosterone esters D PP and P by i pro in sa ple preparation and analytical procedures nt r a d t a a t ass s tr t r s r s t d n Ds n r n Paper II r and n r and D t rt t s nan s st t r d d
as and nn r n d a t r n d t a a t s ass s tr t r arious alternati e e traction conditions were tested includin different sol ents te peratures n an d t method’s sensitivity markedly and resulted in LODs of n r and and incubation periods in sonification and sha in in order to find the ost efficient n mL for n mL D Paper II his as onsidered suffi ient ased on the hi her lasma on entrations of inta t esters follo in an intramus ular in e tion of m ustanon hi h is an administration dose ell ithin the sus e ted and ur orted doses used y do ers
urther the method validation sho ed that the final method Paper II as s e ifi ith a ro riate re ision for ualitative analysis and no nota le arry over effe t hus the develo ed method a eared suita le for analysis of testosterone esters
2.3.3 Detection of testosterone ester administration in DBS he reviously re orted D method as roven relia le for dete tion of orally administered testosterone unde anoate m ndriol esto a s et een t o and ei ht hours ost administration in a n trial n this res e t it must e stressed that the harma okineti s seem to differ et een su e ts after testosterone ester administration hus iven the im ortan e of hi h s e ifi ity in a dire t dete tion method it is ne essary to evaluate the DBS assay’s sensitivity and s e ifi ity in an administration study ith more than one su e t randomi ed la e o ontrolled desi n ith a setu mimi kin a do in ontrol settin ould rovide the most a li a le information of the DBS method’s specificity and sensitivity herefore e analy ed D sam les from nine men re eivin m ustanon and ten men re eivin la e o in a randomi ed dou le linded la e o ontrolled desi n usin the develo ed nanoL ased method Paper II o mimi a do in ontrol settin the sam les ere trans orted from the sam le olle tion site in Denmark to the la oratory in Oslo ithin hours
m ortantly the method sho ed s e ifi ity i e no false adverse analyti al findin s urther D and ould e dete ted ith sensitivity for five days after in e tion and for u to days in one su e t u e t he shortest hained ester sho ed the shortest indo s of dete tion ma five days and as only dete ta le in all su e ts for one day after in e tion oreover the individuals ith the hi hest o served relative a undan es ere also amon the individuals ith the lon est dete tion times en e the varyin dete tion indo s a eared to e due to oth the len th of the ester side hain and inter individual variation in a sor tion elimination rates ote orthy a shorter dete tion eriod hours as re orted for the lon hained testosterone unde anoate in D after a sin le oral in estion of m of the dru o ever the lo er dete ta ility as ro a ly a result of the e tensive first ass meta olism and onse uently lo ioavaila ility of orally administered testosterone unde anoate
2 n mL for n mL D Paper II his as onsidered suffi ient ased on the hi her lasma r findin s are in a reement ith the detection indo s reported for D and in h man on entrations of inta t esters follo in an intramus ular in e tion of m ustanon hi h is ser m p asma after an in ection of S stanon his is of interest since the samp e vo me in an administration dose ell ithin the sus e ted and ur orted doses used y do ers DBS is on y of that sed in p asma ana ysis he e p anation is i e y the eve of en ymatic activity and th s de radation of testosterone esters in p asma compared ith dried ood evident urther the method validation sho ed that the final method Paper II as s e ifi ith a ro riate y increased sta i ity of testosterone esters hen stored on DBS cards as reported previo s y and re ision for ualitative analysis and no nota le arry over effe t hus the develo ed method in Paper II. n this re ard it is of note that the increased ana yte sta i ity in DBS i e y ma es coo ed a eared suita le for analysis of testosterone esters transportation red ndant hich i red ce the costs compared ith shippin of standard ood o ective y these findin s s est that DBS co d e a favora e samp e matri for testosterone ester ana ysis in oth e ite and recreationa sports 2.3.3 Detection of testosterone ester administration in DBS he reviously re orted D method as roven relia le for dete tion of orally administered nterestin y the detection indo s in DBS Paper II are compara e to the indirect detection periods testosterone unde anoate m ndriol esto a s et een t o and ei ht hours ost o served in the B steroida mod e after sin e and repeated in ections of m S stanon Paper administration in a n trial n this res e t it must e stressed that the harma okineti s seem I n Paper I e o served that the rinary and α dio ratios co d indicate testosterone to differ et een su e ts after testosterone ester administration hus iven the im ortan e of hi h dopin ith sensitivity for at east five days and in some individ a s p to days after the s e ifi ity in a dire t dete tion method it is ne essary to evaluate the DBS assay’s sensitivity and in ections his s ests that DBS ana ysis potentia y co d e sed for nam i o s confirmation s e ifi ity in an administration study ith more than one su e t randomi ed la e o ontrolled of dopin ith testosterone esters ased on atypica steroid profi es desi n ith a setu mimi kin a do in ontrol settin ould rovide the most a li a le information of the DBS method’s specificity and sensitivity herefore e analy ed D sam les from nine men re eivin m ustanon and ten men re eivin la e o in a randomi ed dou le 2.4 Detection of clenbuterol linded la e o ontrolled desi n usin the develo ed nanoL ased method Paper II o en tero is a ide y sed dopin s stance y ody i ders and ath etes see in to increase ean mimi a do in ontrol settin the sam les ere trans orted from the sam le olle tion site in ody mass ts ana o ic and ipo ytic effects have een o served in severa mamma ian species – t Denmark to the la oratory in Oslo ithin hours nti no it is n no n hether c en tero has hypertrophic and meta o ic effects in hea thy h mans n a para e investi ation to Paper IV e ere the first to eva ate the effects of ac te m ortantly the method sho ed s e ifi ity i e no false adverse analyti al findin s urther c en tero administration on restin meta o ism and s e eta m sc e si na in in hea thy yo n men D and ould e dete ted ith sensitivity for five days after in e tion and for u to e o served increased ener y e pendit re fat and car ohydrate o idation as e as s e eta m sc e days in one su e t u e t he shortest hained ester sho ed the shortest indo s of si na in of m and aro nd ho rs after the in estion of of c en tero in si hea thy dete tion ma five days and as only dete ta le in all su e ts for one day after in e tion oreover men essen et a np ished data Decem er he o served effects can potentia y trans ate the individuals ith the hi hest o served relative a undan es ere also amon the individuals ith into increased fat free mass he st dy as imited y a sma samp e si e and the ac of a p ace o the lon est dete tion times en e the varyin dete tion indo s a eared to e due to oth the ro p ot ithstandin the o served chan es ere of re ative y reat ma nit de ith o inter len th of the ester side hain and inter individual variation in a sor tion elimination rates individ a variation and are th s n i e y to e res ts of random variation herefore this st dy ote orthy a shorter dete tion eriod hours as re orted for the lon hained testosterone indicates that the detection of dopin ith c en tero even a sin e dose is of re evance rther unde anoate in D after a sin le oral in estion of m of the dru o ever the lo er ased on the potentia hea th ris associated ith c en tero mis se detection of c en tero dopin dete ta ility as ro a ly a result of the e tensive first ass meta olism and onse uently lo is a so important for the protection of the hea th of ath etes and recreationa y active ioavaila ility of orally administered testosterone unde anoate
2 2.4.1 Detection of clenbuterol in urine Paper IV
Paper IV
2.4.1 Detection of clenbuterol in urine 2.4.2 Detection of clenbuterol in DBS evaluated. Such clinical trials are needed to demonstrate the method’s applicability in doping analysis. Paper IV Paper IV
Paper IV Paper IV Paper IV
Paper IV Paper IV Paper IV
ollectively our indings imply that S is a suitable matri or long term storage and routine doping analysis o clenbuterol even a ter a single dose administration. o ever studies evaluating di erent collection and transportation procedures ould be valuable to optimi e the procedures or use in doping control and analysis. iven the longer detection indo s or clenbuterol in urine urine is e pected to remain as the pre erred sample matri or clenbuterol analysis hile S sampling could be use ul e.g. hen testing a large number o athletes in a short time.
2.5 Implementation of DBS in doping control
or S to be implemented by in routine doping analysis standardi ed and optimi ed protocols or collection transportation and storage procedures are re uired. n the ollo ing sections possible procedures and bene its o S collection are discussed.
2.5.1 Sample collection procedures ne o the bene its ith S is the minimally invasive collection o capillary blood e.g. rom a inger pric . n this respect it must be noted that hile inger pric blood sampling is a ast and minimally invasive sample collection procedure it appears to have some limitations. or e ample in the or ith Paper II and Paper IV e e perienced di iculties collecting S samples rom individuals ith poor blood circulations in the hands. cessive s uee ing or ‘milking’ of the site to orce blood out o the inger may cause hemolysis and or s uee e tissue luid into the blood drop and a ect the content o the collected sample. hus su icient training o the doping control o icers and ritten guidelines including pre sampling techni ues to stimulate blood lo e.g. hand rubbing commercial hand armers or running ater might improve S collection. urther athletes ho use their ingers in per orming their sport such as in s imming handball or archery may be reluctant to provide a inger pric blood samples. lternatively collection devices and collection o capillary blood rom other anatomical sites may be used to facilitate the DBS collection. One example is the TAP™ push button blood collection device SeventhSense iosystems ed ord S hich uses microneedles and vacuum to collect capillary blood e.g. rom the upper arm. his circumvents the potential problem o poor blood lo in the hands. Subse uently the blood can be deposited on the S card. n a recent pilot study ith ten healthy volunteers a strong correlation as observed bet een the e pression levels o S a speci ic biomar er or heme biosynthesis in S collected by the system and in S collected by inger pric . his illustrates the potential o in S sampling. evertheless other possible collection devices should be evaluated in uture studies. ollectively our indings imply that S is a suitable matri or long term storage and routine doping n gene al analyte concent ations measu ed in DBS co elate ell ith those measu ed in enous analysis o clenbuterol even a ter a single dose administration. o ever studies evaluating di erent blood. ence it appea s that DBS can be accu ately used fo uantitati e pu poses. n this collection and transportation procedures ould be valuable to optimi e the procedures or use in ega d it must be noted that hen a pa tial punch out f om a DBS is analy ed diffe ent hematoc it doping control and analysis. iven the longer detection indo s or clenbuterol in urine urine is le els and the eby the dist ibution of the spot onto the ca d can lead to inco ect uantification. e pected to remain as the pre erred sample matri or clenbuterol analysis hile S sampling could u the inconsistent DBS olumes pa ticula ly hen spotting di ectly f om the finge tip complicates be use ul e.g. hen testing a large number o athletes in a short time. the estimation of analyte concent ations Paper IV o elati e abundances Paper II hen analy ing the enti e spot. Accu ate dete mination of analyte concent ations is pa ticula ly impo tant in uantitati e analysis of th eshold substances. As illust ated by T et el and co o ke s these challenges can be 2.5 Implementation of DBS in doping control o e come by hole spot analysis of kno n spot olumes. The efo e it appea s that fixed olume DBS or S to be implemented by in routine doping analysis standardi ed and optimi ed protocols sampling should be the p ocedu e of choice in doping cont ol hen analy ing fo th eshold substances. or collection transportation and storage procedures are re uired. n the ollo ing sections possible hen hole blood is not needed fo analysis an alte nati e is the use of d ied plasma i.e. DPS. Du ing procedures and bene its o S collection are discussed. the method de elopment p eceding Paper II e obse ed no diffe ences in the pe fo mance of the tested mate ials i.e. comme cial DBS and DPS and continued ith the p oduct best suited fo an 2.5.1 Sample collection procedures administ ation study and doping cont ol. o e e DPS might be the best pe fo ming sampling mate ial ne o the bene its ith S is the minimally invasive collection o capillary blood e.g. rom a inger fo othe compounds. The efo e studies compa ing diffe ent collection mate ials fo d ied mat ix pric . n this respect it must be noted that hile inger pric blood sampling is a ast and minimally analysis of othe substances a e needed. invasive sample collection procedure it appears to have some limitations. or e ample in the or ith Paper II and Paper IV e e perienced di iculties collecting S samples rom individuals ith n this thesis e collected t o DBS ca ds ith fou spots each i.e. A B samples and the collected DBS poor blood circulations in the hands. cessive s uee ing or ‘milking’ of the site to orce blood out samples d ied fo a minimum of t o hou s at oom tempe atu e befo e being packed in indi idual o the inger may cause hemolysis and or s uee e tissue luid into the blood drop and a ect the ate tight sealable plastic bags containing t o desiccant packets Paper II and Paper IV . This appea s content o the collected sample. hus su icient training o the doping control o icers and ritten p efe able in many aspects. The collection of se e al spots makes it possible to combine spots fo guidelines including pre sampling techni ues to stimulate blood lo e.g. hand rubbing commercial one analysis if sensiti ity e ui es o the possibility to de elop substance specific p e analytical hand armers or running ater might improve S collection. urther athletes ho use their ingers pu ification p otocols if necessa y and use one spot fo each designated analysis. u the A B sample in per orming their sport such as in s imming handball or archery may be reluctant to provide a collection makes B confi mation analysis possible hile indi idual sealable bags ith humidity inger pric blood samples. lternatively collection devices and collection o capillary blood rom abso bents likely enhances analyte stability and educes the likelihood of sample manipulation. ont a y other anatomical sites may be used to facilitate the DBS collection. One example is the TAP™ push t o hou s of d ying is unsuitable in a doping cont ol setting hen t ying to imp o e the time and cost button blood collection device SeventhSense iosystems ed ord S hich uses efficiency. Thus studies e aluating sho te d ying as ell as alte nati e p ocedu es fo packing and microneedles and vacuum to collect capillary blood e.g. rom the upper arm. his sealing of DBS samples a e needed. circumvents the potential problem o poor blood lo in the hands. Subse uently the blood can be deposited on the S card. n a recent pilot study ith ten healthy volunteers a strong correlation as observed bet een the e pression levels o S a speci ic biomar er or heme 2.5.2 Transportation and storage procedures biosynthesis in S collected by the system and in S collected by inger pric . his nfo mation about analyte stability at a ying tempe atu es is impo tant hen optimi ing the illustrates the potential o in S sampling. evertheless other possible collection devices t anspo tation and sto age p ocedu es to be used in outine doping analysis. hen sto ed at oom should be evaluated in uture studies. tempe atu e fo fou eeks a deg adation of as obse ed fo anabolic ste oid este s on DBS hile e obse ed less deg adation follo ing sto age at Paper II . once ning clenbute ol the analyte has been epo ted to be stable fo at least one eek hen sto ed ef ige ated . nte estingly e found that testoste one este s and clenbute ol a e highly stable i.e. and months especti ely hen sto ed at Paper II and Paper IV . Based on these obse ations it appea s that DBS samples fo analysis of ste oid este s and clenbute ol can be t anspo ted at oom tempe atu e but should be sto ed f o en if subse uent e analysis is conside ed.
2.5.3 Possibilities and benefits with DBS Based on the small sample olume in DBS hich may limit the assay sensiti ity and the fact that the p od ugs a e only momenta ily in the systemic ci culation u ine ill likely continue to be the backbone fo doping analysis. ot ithstanding the e a e many possibilities ith DBS sampling in anti doping testing. As highlighted in Paper II Paper IV and by othe s the fast collection and simplified logistics ith DBS sampling make mo e f e uent and o la ge scale testing possible hich may imp o e detection and ende dete ence f om doping. u the the imp o ed analyte stability in DBS compa ed ith li uid mat ices as epo ted by othe s – and in Paper II as ell as the possibility fo long te m sto age Paper II and Paper IV could allo fo testing in mo e emote a eas and sto age of samples fo futu e analyses. o eo e anothe benefit ith DBS sampling is the gende aspect of sample collection. Opposed to u ine sample collection he e the doping cont ol office must be of the same gende as the athlete DBS sampling can be pe fo med independent of gende . n addition to the easy setup i.e. no toilet needed this ill likely make doping cont ols mo e feasible.
n a pilot project, we tested the use of DBS collection by a TAP™ device (SeventhSense Biosystems, edfo d A SA in eal life doping cont ol ith Anti Doping Denma k. ineteen athletes e e selected fo in competition testing and p o ided a pai of A B DBS samples each as ell as esponded to a uestionnai e conce ning thei expe iences of DBS sample collection. Of the tested athletes had been tested befo e. n total ould p efe the DBS sampling p ocedu e compa ed ith u ine sampling. Simila ly ould p efe this sampling p ocedu e compa ed ith collection of enous blood. The a e age pain sco e hen g ading pain sensation f om to i.e. is most painful as . . . Based on these p elimina y data it appea s that DBS sampling is e y ell accepted by athletes. Because DBS sampling seems mo e athlete f iendly than con entional sampling methods DBS could potentially be the p efe ed sample mat ix in anti doping education p og ams e.g. hen young athletes a e int oduced to the doping cont ol p ocess.
DBS hile e obse ed less deg adation follo ing sto age at Paper II . once ning f interest for anti dopin is also the possibility of automatin the entire analytical wor flow from sample clenbute ol the analyte has been epo ted to be stable fo at least one eek hen sto ed ef ige ated e traction to S S analysis , This can reduce the manual laboratory wor load, analyte loss . nte estingly e found that testoste one este s and clenbute ol a e highly stable i.e. durin sample transfer and ris of contamination, as well as increase the sample throu hput in routine and months especti ely hen sto ed at Paper II and Paper IV . Based on these analysis obse ations it appea s that DBS samples fo analysis of ste oid este s and clenbute ol can be t anspo ted at oom tempe atu e but should be sto ed f o en if subse uent e analysis is conside ed. 3. Conclusion and perspectives 2.5.3 Possibilities and benefits with DBS Based on the small sample olume in DBS hich may limit the assay sensiti ity and the fact that the This thesis demonstrated that DBS analysis allows for detection up to days after an intramuscular p od ugs a e only momenta ily in the systemic ci culation u ine ill likely continue to be the backbone injection of m Sustanon , and for at least three days after an oral in estion of clenbuterol, fo doping analysis. ot ithstanding the e a e many possibilities ith DBS sampling in anti doping with no false adverse analytical findin s Additionally, preliminary data indicate that DBS samplin is testing. As highlighted in Paper II Paper IV and by othe s the fast collection and simplified logistics very well accepted by athletes and re arded as more athlete friendly than e istin matrices Thus, DBS ith DBS sampling make mo e f e uent and o la ge scale testing possible hich may imp o e based analytical methods for detection of dopin with testosterone esters and clenbuterol seem to detection and ende dete ence f om doping. u the the imp o ed analyte stability in DBS compa ed have sufficient specificity and sensitivity to complement e istin analytical strate ies while improvin ith li uid mat ices as epo ted by othe s – and in Paper II as ell as the possibility fo long te m time and cost efficiency and reducin intrusiveness This can potentially allow for more fre uent sto age Paper II and Paper IV could allo fo testing in mo e emote a eas and sto age of samples fo and or lar e scale testin to increase detection and deterrence in both elite and recreational athletes futu e analyses. o eo e anothe benefit ith DBS sampling is the gende aspect of sample collection. urther, the hi h analyte stability on DBS li ely ma es cooled shippin redundant and ma es DBS a Opposed to u ine sample collection he e the doping cont ol office must be of the same gende as the suitable sample matri for lon term stora e for future analysis owever, future studies should athlete DBS sampling can be pe fo med independent of gende . n addition to the easy setup i.e. no evaluate different collection procedures and shorter dryin and pac in procedures suited for use in toilet needed this ill likely make doping cont ols mo e feasible. dopin control
n the present thesis, it was shown that sin le and repeated intramuscular injections of m mi ed n a pilot project, we tested the use of DBS collection by a TAP™ device (SeventhSense Biosystems, testosterone esters affect mar ers in the hematolo ical module of the ABP, with the lar est detectable edfo d A SA in eal life doping cont ol ith Anti Doping Denma k. ineteen athletes e e effects three to ten days after an injection The T seemed to be the most selective mar er Thus, selected fo in competition testing and p o ided a pai of A B DBS samples each as ell as esponded the hematolo ical module could help tar etin follow up sample collection and confirmatory analysis to a uestionnai e conce ning thei expe iences of DBS sample collection. Of the tested athletes had by S if only ABP blood samples have been collected, or initiate the analysis for testosterone ester been tested befo e. n total ould p efe the DBS sampling p ocedu e compa ed ith u ine sampling. in DBS, if such samples have been collected urther, the evaluation of mar ers in the steroid module Simila ly ould p efe this sampling p ocedu e compa ed ith collection of enous blood. The and hematolo ical module to ether mi ht improve detectability of testosterone dopin , especially in a e age pain sco e hen g ading pain sensation f om to i.e. is most painful as . . . Based cases when steroid mar ers are affected by confoundin factors on these p elimina y data it appea s that DBS sampling is e y ell accepted by athletes. Because DBS sampling seems mo e athlete f iendly than con entional sampling methods DBS could potentially be the n contrast to the hypothesis, an intramuscular injection of m mi ed testosterone esters did not p efe ed sample mat ix in anti doping education p og ams e.g. hen young athletes a e int oduced to enhance performance acutely in a countermovement jump test, a one arm isometric elbow fle ion test the doping cont ol p ocess. nor a sec cycle sprint test urther, the serum testosterone levels appeared not to influence performance in stren th and power e ercises at baseline Thus, when determinin the most cost efficient testin pro rams in power and stren th sports, it appears that anti dopin authorities should focus on collectin samples out of competition when testin for testosterone esters
The present thesis demonstrated that a sin le oral in estion of clenbuterol can be detected for at least days in urine The lon er detection windows in urine compared to in DBS su est that urine is still the preferred sample matri for clenbuterol analysis, while DBS samplin could be useful e when testin a many athletes in a short time urther, low ( n m urinary concentrations of clenbuterol can occur rapidly (≤24 h) after ingestion of 80 µg of drug in some individuals. Thus, an atypical findin for clenbuterol necessitates careful investi ation to determine the li elihood of food contamination
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2 PAPER I
2
PAPER II
Figure 4. Hematological and steroidal passports for Subject A’s samples selected for IRMS analysis. The red lines mark the individual reference range and the blue the individual test result. A shows the Day 5 blood sample (sample no. 5) that was flagged atypical for ABPS, and B shows the steroidal profile of the matched urine sample. C shows the Day 10 blood sample (sample no. 6) that was flagged atypical for high RET%, and D shows the steroidal profile of the matched urine sample.
Figure 5. Relative abundance of testosterone propionate in DBS (n = 9). The first dose of Sustanon® was administered on day 0 and the second on day 21. Samples were collected at baseline and on day 1 (22), 3 (24), 5 (26), 10 (31), 14 (35) and 21 (42) after administration. PAPER III
TABLES
Table 1. Subject characteristics
PLA TE Total n 10 9 19
Age (years) 25 ± 2 23 ± 1 24 ± 2
Height (cm) 186 ± 6 183 ± 7 185 ± 7
Body weight (kg) 82 ± 14 83 ± 10 82 ± 12
PLA = placebo group, TE = testosterone ester group
Table 2. Sprint performance
Variable Treatment Pre Post
PLA 1124 ± 186 1115 ± 197 Peak power (W) PAPER IV TE 1110 ± 186 1119 ± 182
PLA 815 ± 109 816 ± 104 Mean power (W) TE 811 ± 109 811 ± 106
PLA 52 ± 5 52 ± 6 Fatigue index (%) TE 50 ± 6 51 ± 6
Performance outcomes (mean ± SD) for the 30-second all-out cycle sprint test before (pre) and after (post) placebo (PLA) and testosterone ester (TE) administration.
ISBN 978-87-7209-334-5 Sara Amalie Solheim Improving time- and cost-efficiency in doping analysis
Improving time- and cost-efficiency in doping analysis
PhD Thesis Sara Amalie Solheim
December 2019