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An Overview on the Testing An Overview on the Testing Technology in Doping Control for Technology in Doping Control for
Human and Horse SportsHuman and Horse Sports
Dr Terence S. M. WanHead of Racing Laboratory
The Hong Kong Jockey Club
Seminar on Doping Control in Sports, 5 Dec 2010, Hong Kong
22
OutlineOutlineBACKGROUND• What is doping in sports and means of doping control• Comparing forensic drug testing for equine vs human athletes • Interpretation of analytical findingsTESTING TECHNOLOGYTESTING TECHNOLOGY FOR DOPING CONTROL•• Laboratories for human and equine doping controlLaboratories for human and equine doping control• General screening and confirmatory analyses• EPO and Growth Hormone detection in horses and humans• Longitudinal monitoring (athlete biological passports)• Retrospective testingDRUG CASE EXAMPLES• 2008 Beijing Olympic equestrian events in Hong Kong• 2009 EPO analogue in samples from Australian racehorses• 1999 Investigation of mephenesin in the winner 'WhyTellYou'
33
What is Doping in Sports?What is Doping in Sports?• An attempt to change the inherent performance of an athlete by
introducing pharmacodynamic or physiological changes
• IOC: administration or use of a banned substance or banned method (WADA has a much broader definition)
• Majority of doping through the use of banned substances
• To many, winning is everything (prize, pride, reputation, sponsorship)
• Surveys conducted on human athletes:
• 30% firmly believed that over half of athletes have used drugs!
• >50% of elite athletes would commit to doping IF guaranteed not being caught and win but have to die within 5 years!!
• With betting, can also dope an (animal) athlete to lose!
44
Means of Doping ControlMeans of Doping Control• Surveillance, investigation, & confession – not very effective
• Direct testing for performance-altering effects – costly & unreliable
• Indirect testing (testing for banned substances or their markers)
• “biological” problem with a “chemical” solution!
• evidence (banned substance) => performance-altering effect?
• negative test results => absence of doping?
• undetectable substances => OK to use?
• ENFORCEMENT: strict liability principle (a practical necessity)
• preventing collective injustice (a high objective) Vs depriving one’s natural justice & presumption of innocence
• participants voluntarily abide by the relevant rules
• private law character
55
Difficulties with Difficulties with Equine DrugEquine Drug TestingTesting1.1. Difficulties in Difficulties in urine collectionurine collection (blood is limited in volume and (blood is limited in volume and
not preferred for comprehensive drug screening)not preferred for comprehensive drug screening)
2.2. Horse urine Horse urine –– large matrix variations, and large matrix variations, and highly complexhighly complex
3.3. OpenOpen--ended scope (drug coverage) ended scope (drug coverage) –– all kinds of past and all kinds of past and present drugs, present drugs, bothboth performance enhancing performance enhancing andand impairingimpairing; ; veterinary medications; other substances prohibited by the veterinary medications; other substances prohibited by the rules rules => no confined targets.=> no confined targets.
4.4. Metabolic fateMetabolic fate of drugs in horses not as well studied as in of drugs in horses not as well studied as in humans (drugs may be given in unconventional ways)humans (drugs may be given in unconventional ways)
5.5. Higher sensitivityHigher sensitivity generally required (more stringent generally required (more stringent requirements for equine athletes, generally more extensive requirements for equine athletes, generally more extensive distribution and metabolism in horses)distribution and metabolism in horses)
=> technologically more demanding and takes longer.
66Human UrineHuman Urine Horse UrineHorse Urine
sediment
Difficulties with Difficulties with Equine DrugEquine Drug TestingTesting
complex matrix
77
Interpretation of Analytical FindingsInterpretation of Analytical Findings1. When was the athlete exposed?
2. How much has the athlete been exposed to?
3. How (by which route) was the athlete exposed?
With high individual variability of substance clearance from the body, and only a single concentration in a sample => most difficult if not impossible to give an answer to the above.
Normally not much help from the athlete accused of doping!
Difficult to predict the extent of systemic or localised, intended or side effect at the time of competition, if any, from just the concentration in urine voided (or blood collected) some time after the competition.
Also cannot distinguish between deliberate abuse and inadvertent misuse!
88
Laboratories for Human and Equine Laboratories for Human and Equine Doping ControlDoping Control
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Doping Control TestingDoping Control TestingHuman:
WADA and ISO/IEC 17025 accreditation (General Requirements for the Competence of Testing and Calibration Laboratories).
Conform to the Conform to the requirements of the World requirements of the World AntiAnti--Doping Agency Doping Agency (WADA) code (WADA) code –– and the five and the five International StandardsInternational Standards. .
Meet the Meet the Minimum Minimum Required Performance Required Performance LevelsLevels (MRPL) for (MRPL) for detecting prohibited detecting prohibited substances.substances.
Equine (jurisdiction-dependent):Most require ISO/IEC 17025 accreditation
Many conform to the requirements of Many conform to the requirements of ILACILAC--G7G7 ((Accreditation Requirements Accreditation Requirements and Operating Criteria for Horseracing and Operating Criteria for Horseracing LaboratoriesLaboratories). ).
Many are signatories to Many are signatories to Article SixArticle Six ((Prohibited SubstancesProhibited Substances) of the ) of the International Agreement on Breeding, International Agreement on Breeding, Racing and WageringRacing and Wagering of the of the IFHAIFHA (International Federation of Horseracing (International Federation of Horseracing Authorities).Authorities).Many meet the Many meet the Performance Performance SpecificationSpecification for doping control for doping control laboratories required by the IFHA.laboratories required by the IFHA.
1010
Criteria for IdentificationCriteria for IdentificationIn human doping control:
WADA Technical Document – TD2010IDCR, Identification Criteria for Qualitative Assays Incorporating Column Chromatography and Mass Spectrometry
In equine doping control:
Association of Official Racing Chemists (AORC) Guidelines for the Minimum Criteria for Identification by Chromatography and Mass Spectrometry, April 2003
htthttp://www.aorcp://www.aorc--online.org/AORConline.org/AORC MS MS Criteria.pdfCriteria.pdf
1111
““AA”” and and ““BB”” Sample AnalysisSample AnalysisHuman: “A” and “B” sample analysis are analysed in the same laboratory.
Equine: In many cases, “A” and “B” sample analysis are analysed in different laboratories.
Advantages of both samples tested in the same laboratory:– Detection capability assured.– Sample degradation or loss during shipment minimised.
Advantages of both samples tested in different laboratories:– Independent re-analysis as a reliable countercheck (mistakes
spotted readily with different procedures, reagents, standards, instruments, etc)
– Observer Effect (unintentional bias) minimised.– Robustness and acceptability of evidence enhanced.
1212
Conventional Conventional Drug TestingDrug Testing
1313
General ApproachGeneral Approach• Extraction (isolate and enrich)
• Screening (efficient and simple, wide drug coverage, sensitive)
• Confirmation (qualitative, definitive, specific and sensitive)
• Quantification (where relevant; accurate, need to estimate uncertainty of measurement)
1414
Sample Extraction ProcessesSample Extraction Processes
Examples of Common Techniques:
Liquid-Liquid Extraction (LLE)
Partition between an aqueous phase (urine or blood) and an organic phase (solvent).
Solid-Phase Extraction (SPE)
Retention of target drug(s) on bonded sorbents, e.g., C18 sorbents or ion-exchange sorbents; followed by elution with a solvent.
1515
Liquid-Liquid Extraction
1616
Solid-Phase Extraction Cartridges
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Automated SPE Equipment
1818
Common Analysis TechniquesCommon Analysis Techniques
Immunoassays (e.g. ELISA, RIA)Immunoassays (e.g. ELISA, RIA)Competition between the target Competition between the target drug(sdrug(s) in the sample ) in the sample
and a labelled drug for specific antibodies; good for and a labelled drug for specific antibodies; good for screeningscreening but not considered a definitive technique in but not considered a definitive technique in doping control testingdoping control testing
GC/MSSeparation of components by Gas Chromatography, and
detection of target drug(s) by a Mass Spectrometer for screening, confirmation or quantification.
LC/MSSeparation of components by Liquid Chromatography,
and similarly detection by Mass Spectrometry
1919
Immunoassay Workstation
2020
Gas Chromatograph / Mass Spectrometer
2121
Liquid Chromatograph / Tandem Mass Spectrometer(Ion Trap)
2222
Liquid Chromatograph/Tandem Mass Spectrometer(Triple Quadrupole)
2323
Liquid Chromatograph / Tandem Mass Spectrometer(Quadrupole Linear Trap)
2424
High Resolution Accurate Mass Liquid Chromatograph / Tandem Mass Spectrometer (LTQ Orbitrap Velos)
2525
Inductively-Coupled PlasmaMass Spectrometer
RT: 4.00 - 12.00 SM: 7G
4 5 6 7 8 9 10 11 12Time (min)
0
50
1000
50
1000
50
1000
50
1000
50
100RT: 6.71
RT: 5.09
RT: 5.42
RT: 5.45
RT: 5.61
RT: 4.00 - 12.00 SM: 7G
4 5 6 7 8 9 10 11 12Time (min)
0
50
1000
50
1000
50
1000
50
1000
50
100RT: 6.79
RT: 7.05
RT: 7.02
RT: 7.07
RT: 7.14
RT: 4.00 - 12.00 SM: 7G
4 5 6 7 8 9 10 11 12Time (min)
0
50
1000
50
1000
50
1000
50
1000
50
100RT: 8.94
RT: 8.95
RT: 9.14
RT: 9.19
RT: 9.28
RT: 4.00 - 12.00 SM: 3G
4 5 6 7 8 9 10 11 12Time (min)
0
50
1000
50
1000
50
1000
50
1000
50
100RT: 5.68
5.97
RT: 5.73
RT: 5.68
RT: 5.09
RT: 6.64
RT: 4.00 - 12.00 SM: 7G
4 5 6 7 8 9 10 11 12Time (min)
0
50
1000
50
1000
50
100 RT: 8.42
7.97
RT: 8.68
RT: 8.75
RT: 4.00 - 12.00 SM: 7G
4 6 8 10 12Time (min)
0
1000
1000
1000
1000
1000
100RT: 10.08
RT: 10.36
RT: 10.50
RT: 10.60
RT: 10.93
RT: 11.07
RT: 4.00 - 12.00 SM: 7G
4 5 6 7 8 9 10 11 12Time (min)
0
50
1000
50
1000
50
100RT: 7.89
RT: 8.02
RT: 9.73
RT: 4.00 - 12.00 SM: 7G
4 5 6 7 8 9 10 11 12Time (min)
0
50
1000
50
1000
50
1000
50
1000
50
100RT: 5.72
RT: 5.95
5.69
RT: 6.43
RT: 8.28
RT: 8.67
RT: 4.00 - 12.00 SM: 7G
4 5 6 7 8 9 10 11 12Time (min)
0
50
1000
50
1000
50
1000
50
1000
50
100RT: 8.70
RT: 9.07
RT: 9.09
RT: 10.13
RT: 10.63
Nadolol m/z 310 m/z 254
Morphine m/z 286 m/z 201
Heptaminol m/z 146 m/z 128
Oxymorphone m/z 302 m/z 284
Famotidine m/z 338 m/z 259
Oxycodone m/z 316 m/z 298
Butorphanol m/z 328 m/z 310
Guanabenz m/z 231 m/z 214
Benzoylecgonine m/z 290 m/z 168
Etafedrine m/z 194 m/z 176
Nalbuphine m/z 358 m/z 340
Pindolol m/z 249 m/z 116
Buspirone m/z 386 m/z 222
Nylidrin m/z 300 m/z 282
Sildenafil m/z 475 m/z 311
Hydroxyalprazolam m/z 325 m/z 307
Anileridine m/z 353 m/z 120
Cocaine m/z 304 m/z 182
Atenolol m/z 267 m/z 225
Cimetidine m/z 253 m/z 159
Nizatidine m/z 332 m/z 232
Ranitidine m/z 286 m/z 185
Hydroxydetomidine m/z 293 m/z 237
Haloperidol m/z 376 m/z 165
Desipramine m/z 267 m/z 236
N-Norpropoxyphene m/z 326 m/z 252
Nortriptyline m/z 264 m/z233
Perphenazine m/z 404 m/z 171
Methadone m/z 310 m/z 265
Prazosin m/z 384 m/z 366
Clenbuterol m/z 277 m/z 259
Buprenorphinem/z 468 m/z 414
Sotalol m/z 273 m/z 255
Practolol m/z 267 m/z 225
Romifidine m/z 258
Clonidine m/z 296 m/z 219
Labetalol m/z 329 m/z 311
Bisoprolol m/z 326 m/z 116
Benperidol m/z 382 m/z 165
Droperidol m/z 380 m/z 194
Carvedilol m/z 407 m/z 283
Trifluperidol m/z 410 m/z 165
Screening: 42 basic drugs or metabolites in horse urine at ca. 5 ng/mL each by LC/SRM
RT: 0.00 - 9.99 SM: 9G
0 1 2 3 4 5 6 7 8 9Time (min)
10
20
30
40
50
60
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80
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100
Rel
ativ
e Ab
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20
30
40
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Rel
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bund
ance
5.95
5.95
NL: 5.36E4m/z= 131.50-132.50 F: + c APCI Full ms4 [email protected] [email protected] [email protected] [ 100.00-220.00] MS nr112222_02
NL: 4.81E4m/z= 166.50-167.50 F: + c APCI Full ms4 [email protected] [email protected] [email protected] [ 100.00-220.00] MS nr112222_02
nr112222_02 #141-146 RT: 5.86-6.07 AV: 6 SB: 16 5.32-5.61, 6.49-6.78 NL: 4.39E4F: + c APCI Full ms4 [email protected] [email protected] [email protected] [ 100.00-220.00]
100 110 120 130 140 150 160 170 180 190 200 210 220m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
132
167
131
168 203
186
RT: 0.00 - 9.97 SM: 9G
0 1 2 3 4 5 6 7 8 9Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
5.95
5.95
NL: 3.27E5m/z= 131.50-132.50 F: + c APCI Full ms4 [email protected] [email protected] [email protected] [ 100.00-220.00] MS clen_04
NL: 3.87E5m/z= 166.50-167.50 F: + c APCI Full ms4 [email protected] [email protected] [email protected] [ 100.00-220.00] MS clen_04
clen_04 #141-146 RT: 5.83-6.02 AV: 6 SB: 16 5.29-5.58, 6.44-6.73 NL: 3.06E5F: + c APCI Full ms4 [email protected] [email protected] [email protected] [ 100.00-220.00]
100 110 120 130 140 150 160 170 180 190 200 210 220m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
167
132
131168
203
186
5.95'
5.95'
132
132
167
167
186
186
203
203
Confirmation: Clenbuterol in urine by LC-MS4
Extracted-ion Chromatogramsof m/z 132 and 167
clenbuterol standard
Referee urine sample
Product-ion scan (m/z 277 m/z 259 m/z 203 m/z 100 - 220)
2828
7.55 7.60 7.65 7.70 7.75 7.80 7.85 7.900
5000
10000
15000
20000
25000
30000
Time-->
Abundance
Ion 580.00 (579.70 to 580.70): SAM_1B.D\data.ms
7.55 7.60 7.65 7.70 7.75 7.80 7.85 7.900
5000
10000
15000
20000
25000
30000
Time-->
Abundance
Ion 583.00 (582.70 to 583.70): SAM_1B.D\data.ms
PFP-Testosterone m/z 580
PFP-d3 -Testosterone m/z 583
Quantification of Testosterone by GC/SIM
y = 0.043x + 0.025R2 = 0.999
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
4.000
0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0
Conc. of Testosterone (ng/mL)
Are
a R
atio
(T
esto
ster
one/
d3- T
esto
ster
one)
Quantification: Testosterone in Horse Urine by GC/SIM
2929
Mass Spectrometry: Fragmentation
Art work courtesy of Jessica Wan
3030
Mass Spectrometry: Acceleration and Separation
Art work courtesy of Jessica Wan
3131
Mass Spectrometry: Detection
Art work courtesy of Jessica Wan
3232
Mass Spectrometry:Identification (and Quantification)
Art work courtesy of Jessica Wan
3333
Gas Chromatography-Combustion-Carbon Isotope Ratio Mass Spectrometry
Measurement of the relative 13C/12C ratio (δ13C) for confirming the exogenous administration of endogenous steroids, as synthetic anabolic agents are generally made from soy sterols with defined and low 13C content.
In human, the range of δ13C value for endogenous steroids is quite narrow and significantly higher than that obtained after exogenous administrations.
In equine, differentiation of the δ13C value before and after exogenous administrations is not as clear cut, perhaps due to its plant-based diet (i.e. similar to synthetic steroids). Moreover, comparing with baseline (reference) values in racehorses is based on not more than 1/10,000 risk.
Not feasible to use IRMS.
3434
EPO and Growth Hormone EPO and Growth Hormone Detection in HorsesDetection in Horses
and Humansand Humans
3535EPO
Detection of EPODetection of EPOErythropoietin (EPO) Erythropoietin (EPO) –– the major glycoprotein the major glycoprotein regulator for regulator for erythropoiesiserythropoiesis..
Exogenous EPO can be identified in blood or urine Exogenous EPO can be identified in blood or urine and differentiated clearly from endogenous EPO. and differentiated clearly from endogenous EPO.
In In human, human, generally by generally by IsoelectricIsoelectric Focussing Focussing –– Double Double Blotting (IEFBlotting (IEF--DB)DB)..
In In equine,equine, also by IEFalso by IEF--DB, but mass spectrometryDB, but mass spectrometry-- based techniques preferred.based techniques preferred.
3636
IsoelectricIsoelectric FocussingFocussing –– Double BlottingDouble BlottingIn In humanhuman, the confirmation of recombinant , the confirmation of recombinant erythropoietinserythropoietins and and analogues are carried out using IEF, followed by Double Blottinganalogues are carried out using IEF, followed by Double Blotting with with chemiluminescentchemiluminescent detection.detection.
WADA Technical Document TD2009EPO Ver. 2.0WADA Technical Document TD2009EPO Ver. 2.0
3737
Mass SpectrometryMass Spectrometry--basedbasedIn In equineequine, the confirmation of recombinant , the confirmation of recombinant erythropoietinserythropoietins and and analogues can be achieved by analogues can be achieved by immunoaffinityimmunoaffinity purification, followed purification, followed by by trypsintrypsin digestion, and then detection of the unique peptide digestion, and then detection of the unique peptide fragment VNFYAWK by LC/MS (Selective Reaction Monitoring).fragment VNFYAWK by LC/MS (Selective Reaction Monitoring).
Yu NH, Yu NH, et al. et al. Anal. Anal. BioanalBioanal. Chem. Chem., ., 396396 (2010), 2513(2010), 2513--2521.2521.
Negative plasma freshly spiked with rhEPO at 0.1 ng/mL (3 fmol/mL)
m/z 464.5 214.4
m/z 464.5 714.8
m/z 464.5 811.9
m/z 464.5 828.7
R.A. = 91%
R.A. = 100%
R.A. = 27%
R.A. = 95%
Peptide VNFYAWK standard
m/z 464.5 214.4
m/z 464.5 714.8
m/z 464.5 811.9
m/z 464.5 828.7
R.A. = 99%
R.A. = 94%
R.A. = 28%
R.A. = 100%
3838
Detection of Growth HormoneDetection of Growth HormoneIn In human: human: detection of specific detection of specific isoformsisoforms using sandwichusing sandwich--type type immunoassays. Recombinant human growth hormone (immunoassays. Recombinant human growth hormone (rhGHrhGH) is ) is monomericmonomeric 2222--kDa kDa hGHhGH isoformisoform only. The ratio of the 22only. The ratio of the 22--kDa kDa hGHhGH to other endogenous to other endogenous hHGhHG isoformsisoforms increasesincreases after after rhGHrhGH administration, and these changes can be detected for more than administration, and these changes can be detected for more than 36 hrs post36 hrs post--administration administration [1] [1] ..In In equine:equine: (i) detection of endogenous(i) detection of endogenous insulininsulin--like growth factorlike growth factor--1 1 (IGF(IGF--1)1) exceeding a threshold. However, this has proved difficult exceeding a threshold. However, this has proved difficult due to lack of a due to lack of a metrologicallymetrologically--traceable reference standard.traceable reference standard.In In equine:equine: (ii) identification of recombinant equine growth hormone (ii) identification of recombinant equine growth hormone ((reGHreGH) by LC/SRM detection) by LC/SRM detection of a unique peptide after of a unique peptide after immunoimmuno affinity purification and affinity purification and trypsintrypsin digestiondigestion. The specific peptide . The specific peptide detected, detected, MMFPAMPLSSLFANAVLRFPAMPLSSLFANAVLR, differs from endogenous , differs from endogenous eGHeGH by an additional by an additional methioninemethionine at the Nat the N--terminal. This method can terminal. This method can detect detect reGHreGH up to 48 hrs postup to 48 hrs post--administration administration [2][2]..
[1] [1] BidlingmaierBidlingmaier et alet al, , ClinClin. . ChemChem, , 20092009, , 5555, 445, 445--453.453.[2] [2] LudovicLudovic et alet al, Anal. , Anal. ChemChem, , 20082008, , 8080, 8340, 8340--8347.8347.
3939
Longitudinal Monitoring Longitudinal Monitoring (Athlete Biological Passport)(Athlete Biological Passport)
4040
Athlete Biological PassportA means to detect a doping violation whereby a substance, its metabolite or marker, cannot be detected either due to its low dose or intermittent use.The biological passport, along with longitudinal monitoring, detects changes in the body from one’s own “normal” profile due to the effects of doping. In human, a doping violation can be pursued based on conclusion drawn from longitudinal profiling according to Article 2.2 of the World Anti-Doping Code, “Use or attempted use by an Athlete of a Prohibited Substance or a Prohibited Method.”At present, a procedure is in place for the establishment of biological passports in human blood (haematological variables), but not yet in human urine (steroid profiles).In equine, biological passports have yet to be implemented (whereabouts of horses problematic!).
4141
Retrospective TestingRetrospective Testing
4242
Why Retrospective TestingN
o. o
f dru
gs o
r pro
hibi
ted
subs
tanc
es
Time
No. of drugs detectable
No. of drugs available
Testing in futureCurrent Testing in future
Capabilitygap
4343
Retrospective TestingAn obvious need for longAn obvious need for long--term storage and term storage and retrospective testing of official samples toretrospective testing of official samples to identify identify doping agents that should never be presentdoping agents that should never be present;;
A powerful deterrent A powerful deterrent –– usingusing future technologyfuture technology to to test current samples for doping agentstest current samples for doping agents
Part of the deterrent effect is Part of the deterrent effect is psychologicalpsychological
In human, samples may be stored up to 8 years for testing.
In equine, samples are stored in some authorities for 5 years or more.
4444
The HKJC Racing LaboratoryThe HKJC Racing Laboratory Positive Drug CasesPositive Drug Cases
4545
Case 1
First 5 cases ofCapsaicin and Nonivamidein equestrian competitions
2008 Olympics
4646
Case 1 Case 1 –– CapsaicinCapsaicin
NH
CH3H3CO
HO
O
CH3
Active component of chilli peppers (Capsicum) - 16,000,000 Scoville units (Tabasco sauce: 2,500 – 5,000 Scoville units).
Severe lachrymator (active ingredient in pepper spray)Severe lachrymator (active ingredient in pepper spray)
Capsaicin has both Capsaicin has both hypersensitisinghypersensitisingand and pain relievingpain relieving properties. At the properties. At the time the FEI classified it as bothtime the FEI classified it as botha Medication Class A and a Dopinga Medication Class A and a DopingSubstance.Substance.
4747
Case 1 Case 1 –– Confirmation MethodConfirmation Method
Urine sampleUrine sample
Blood sampleBlood sample
Enzyme hydrolysisEnzyme hydrolysis
SolidSolid--Phase ExtractionPhase ExtractionOROR
Evaporate and reconstituteEvaporate and reconstitutewith bufferwith buffer
LC/SRM LC/SRM (Positive ESI)(Positive ESI)Urine sampleUrine sample Blood sampleBlood sample
4848
Case 1 Case 1 –– LC/SRM DataLC/SRM Data
C:\Xcalibur\...\nr081902_07 8/20/2008 5:20:09
RT: 3.36 - 7.36 SM: 9G
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)
50
100
50
100
50
100
50
100
50
100
RT: 5.35AA: 108276
RT: 5.35AA: 150708 5.90
RT: 5.35AA: 716391
5.91
RT: 5.35AA: 44647
4.53
RT: 5.35AA: 90236
5.17
C:\Xcalibur\...\capsaicin_std_10 8/20/2008 5:52:4
RT: 3.36 - 7.36 SM: 9G
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)
50
100
50
100
50
100
50
100
50
100
RT: 5.36AA: 2237361
RT: 5.36AA: 2878520
RT: 5.36AA: 13275074
RT: 5.35AA: 881738
RT: 5.36AA: 1676407
m/z 306 → 94
Relative abundance = 15.1 %
Positive Sample
m/z 306 → 122
m/z 306 → 137
m/z 306 → 170
m/z 306 → 182
Relative abundance = 21.0 %
Relative abundance = 100 %
Relative abundance = 6.23 %
Relative abundance = 12.6 %
m/z 306 → 94
Relative abundance = 16.9 %
m/z 306 → 122
m/z 306 → 137
m/z 306 → 170
m/z 306 → 182
Relative abundance = 21.7 %
Relative abundance = 100 %
Relative abundance = 6.64 %
Relative abundance = 12.6 %
Capsaicin Standard
Presence of capsaicin confirmed by LC/SRM.
4949
Case 1 Case 1 –– The positive casesThe positive cases
4 horses in the 2008 Olympics with either urine or blood or 4 horses in the 2008 Olympics with either urine or blood or both found positive for capsaicin.both found positive for capsaicin.
First reported cases in equestrian samples.First reported cases in equestrian samples.
All horses were from All horses were from jumpingjumping..
Capsaicin can be smeared on the horse's front legs. The Capsaicin can be smeared on the horse's front legs. The horse would jump much higher to avoid its horse would jump much higher to avoid its hypersensitisedhypersensitised legs touching the fence. Perhaps due to its potency, volatilitylegs touching the fence. Perhaps due to its potency, volatility and relatively poor absorption, it is very hard to detect (and relatively poor absorption, it is very hard to detect (low low pg/pg/mLmL levelslevels).).
5050
Case 1 Case 1 –– NonivamideNonivamide
NH
H3CO
HO
OCH3
Synthetic analogue of capsaicin, but also found in chilli Synthetic analogue of capsaicin, but also found in chilli peppers in a much smaller amount. peppers in a much smaller amount.
Has the same Has the same hypersensitisinghypersensitising and pain relieving properties and pain relieving properties as capsaicin.as capsaicin.
Structurally different to capsaicin, Structurally different to capsaicin, nonivamidenonivamide is an attractive is an attractive alternative to alternative to delude doping control laboratoriesdelude doping control laboratories..
One horse in the 2008 Olympics was found positive for One horse in the 2008 Olympics was found positive for nonivamidenonivamide in its urine sample, again in jumping (in its urine sample, again in jumping (worldworld’’s s first report in horsesfirst report in horses).).
PPelargonic elargonic aacid cid vvanillylanillylaamide (PAVA)mide (PAVA)
5151
Case 1 Case 1 –– The legal battlesThe legal battlesCountry /
Horse nameSample involved
Banned substance
Analysis completed
End of proceedings
FEI Tribunal decision besides horse & rider disqualification
Brazil /Chupa Chup
Blood(no urine)
Capsaicin Aug 2008 Oct 2008 105 days + CHF 1750 fine + CHF 2500 costs
Brazil /Rufus
Urine Nonivamide Sep 2008(B sample w/ expert witness)
Oct 2008 135 days + CHF 2000 fine + CHF 1750 costs
Germany /Cöster
Urine + Blood
Capsaicin Aug 2008 Oct 2008 (Apr 2009
CAS)
120 days + CHF 2000 fine + CHF 1500 costs (increased to 8 months + CHF 5000 costs)
Ireland /Lantinus
Urine + Blood
Capsaicin Aug 2008 Oct 2008 90 days + CHF 1750 fine + CHF 2000 costs
Norway /Camiro(Team Bronze Medal)
Urine Capsaicin Aug 2008 Dec 2008 (Dec 2009 CAS) (Jul
2010 Swiss Supreme
Court)
135 days + CHF 3000 fine + CHF 8000 plus costs (rejected appeal + CHF 6000 costs) (rejected appeal + CHF 11000 costs & fees)
5252
''CCamiroamiro'' case filescase files
5353
Case 2
Australia’s first EPO detection in harness & thoroughbred
racehorses(World’s first in thoroughbreds)
Lab work in Jul - Aug 2009
5454
Case 2 Case 2 –– The findingsThe findingsEPOsEPOs best known for its widespread abuse in cycling.best known for its widespread abuse in cycling.
EPOsEPOs have been found in samples from have been found in samples from standardbredstandardbred (harness) (harness) racehorses in N. America since 2006. This time racehorses in N. America since 2006. This time 8 positive 8 positive blood samplesblood samples from Melbourne.from Melbourne.
Including the worldIncluding the world’’s first report in thoroughbred racehorses s first report in thoroughbred racehorses (3 samples):(3 samples):
A Samples A Samples –– screened suspicious by ELISA in Australiascreened suspicious by ELISA in Australia
A Samples (remaining portions) A Samples (remaining portions) –– confirmed by confirmed by immunoaffinityimmunoaffinity purification, purification, trypsintrypsin digestion and 2Ddigestion and 2D--nano nano LC/SRM of the unique peptide VNFYAWK in Hong KongLC/SRM of the unique peptide VNFYAWK in Hong Kong..
B Samples B Samples –– confirmed by IEFconfirmed by IEF--Double Blotting in UK.Double Blotting in UK.
After about 15 months since the samples were reported After about 15 months since the samples were reported positive, one of the trainers of the training partnership positive, one of the trainers of the training partnership pleaded guilty and was disqualified for pleaded guilty and was disqualified for THREE yearsTHREE years..
5555
Plasma (5 mL)Plasma (5 mL)IncubateIncubate
overnight overnight atat 37 37 ººCC
Immuno-affinity purification
NR
H
O SO
O
NH
HR N
H
HR
HO SO
OHO S
O
O
antibody
Tosylactivated magnetic beads
Analysis of rhEPO/DPO/PEGAnalysis of rhEPO/DPO/PEG--EPOEPO
Wash & eluteWash & elute
2D-Nano-LC/MS
Denaturation
80 ºC for 10 min37 ºC for 3 hours
Trypsin digestion
ExtractionExtraction
AnalysisAnalysis
5656
Case 2 Case 2 –– The findings (contThe findings (cont’’d)d)One case in harness racing involving 3 blood samples One case in harness racing involving 3 blood samples collected from a pacer 'collected from a pacer 'EmEm MaguaneMaguane':':
with the licensed with the licensed stablehandstablehand / part owner disqualified for / part owner disqualified for 13 years (and fined AUD 30,000)13 years (and fined AUD 30,000) and the trainer for and the trainer for 6 6 years (and fined AUD 500).years (and fined AUD 500). TrainerTrainer’’s disqualification s disqualification reduced to reduced to 4 years4 years on appeal. on appeal.
TWO other harness racing cases involving 2 TWO other harness racing cases involving 2 standardbredstandardbred horses horses yet to be heardyet to be heard..
One outOne out--ofof--competition sample on 19 May 2009 competition sample on 19 May 2009
One prior to running third on 22 May 2009One prior to running third on 22 May 2009
One prior to running fourth on 5 June 2009One prior to running fourth on 5 June 2009
5757
Case 3
MephenesinHKJC post-race sample
from the winner 'Whytellyou'in a HK$ 1.9 m Class 1 cup race
(an extensively investigated case)April 1999
5858
Post-race Sample of 'Whytellyou'
Sample type: urine
Drug: MEPHENESIN (old muscle relaxant, 10-20 mg/kg)(not an approved veterinary medication in Hong Kong)
Concentration in urine: 3 g/mL (after enzyme hydrolysis)
CH3
O OHOH
(~ 3000 ng/mL)
5959
Enzyme-hydrolysed Urine
liquid-liquid extraction
GC/MS
BSTFA derivatisation
Case 3 Case 3 –– AnalysisAnalysis
6060
7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.400
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
Time-->
Abundance
Ion 180.00 (179.70 to 180.70): SPK1UG.D 8.48
7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.400
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
Time-->
Abundance
Ion 326.00 (325.70 to 326.70): SPK1UG.D
8.49
7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.400
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Time-->
Abundance
Ion 180.00 (179.70 to 180.70): QU041022NT.D 8.47
7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.400
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Time-->
Abundance
Ion 326.00 (325.70 to 326.70): QU041022NT.D
8.48
Mass Spectra Extracted-ion Chromatogramsof m/z 180 and 326
Sample
Mephenesin standard
GC/MS Confirmation of Mephenesin in Urine Sample
8.47 min
8.47 min
8.48 min
8.48 min
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000
220000
240000
m/z-->
Abundance
Scan 274 (8.473 min): QU041022NT.D (-)73
147
103
180
129 205326
165 2214723688 259 294 343310279 394364
326205180
147103
73
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 3800
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
600000
650000
m/z-->
Abundance
Scan 275 (8.482 min): SPK1UG.D (-)73
147
103
180
205129
326
16522147
23688 281253 311296 364342 397
326205180
147103
73
6161
Results from samples of 'Whytellyou'(race time at 20:15 hrs on 10.4.1999 in HV Racecourse)
Post-race urine (sampled ~ 2030 hrs)Urinary mephenesin after enzyme hydrolysis 3 g/mLUrinary free mephenesin (without enzyme hydrolysis) <3 ng/mL
(more free mephenesin formed upon storage)
Post-race blood (sampled ~ 2030 hrs) Plasma mephenesin 0 ng/mL
Pre-race urine (sampled ~ 0630 hrs, or 14 hrs before)Urinary mephenesin after enzyme hydrolysis 0 ng/mLUrinary free mephenesin (without enzyme hydrolysis) 0 ng/mL
=> exposure within 14 hours of post=> exposure within 14 hours of post--race sampling.race sampling.
6262
Administration experiments with mephenesin
6363
>10 ng/mL for more than 40 hours!
Topical administration, urine free mephenesin
0
100
200
300
400
500
600
0 10 20 30 40 50 60 70 80Time after administration (hour)
Urin
ary
free
mep
hene
sin
(ng/
mL)
N211 (4.7 g)
K080 (4.7 g)S289 (3.3 g)
K126 (3.3 g)P295 (1.7 g)
S045 (1.7 g)
6464
Detected for more than 30 hours!
Topical administration, plasma mephenesin
0
20
40
60
80
100
120
140
0 10 20 30 40 50 60Time after administration (hour)
Pla
sma
mep
hene
sin
(ng/
mL)
N211 (4.7 g)
K080 (4.7 g)
K126 (3.3 g)S289 (3.3 g)
P295 (1.7 g)
S045 (1.7 g)
6565
Conclusion from Topical Administration Experiments
Topical Administrations Whytellyou
Plasma mephenesin
Detected for more than 30 hours
Not detected
Urinary free mephenesin
>10 ng/mL for more than 40 hours
<3 ng/mL
Conjugated mephenesin
3 g/mL before 17 hours or after 32 hours
3 g/mL within 14 hours (0630 – 2030 hrs)
Results were therefore inconsistent with a topical administration of mephenesin to 'Whytellyou'.
6666
Oral administration, urinary mephenesin after enzyme hydrolysis
0.1
1
10
100
1000
10000
0 5 10 15 20 25 30 35 40 45 50
Time after administration (hour)
Urin
ary
mep
hene
sin
(g/
mL)
P192 (2.5 g)S125 (2.5 g)S305 (1.0 g)P183 (1.0 g)P184 (0.25 g)N166 (0.05 g)P291 (0.05 g)
2.2 h2.8 h 8 h
9.4 h 14 h 16 h 28 h
3 g/mL
Time matchedwithin 14 hrs !
6767
Oral administration, plasma free mephenesin
0.0
50.0
100.0
150.0
200.0
0 2 4 6 8 10 12 14 16
Time after administration (hour)
Pla
sma
mep
hene
sin
(ng/
mL)
P183 (1.0 g)
S305 (1.0 g)P184 (0.25 g)
N166 (0.05 g)P291 (0.05 g)
6868
Oral administration, plasma free mephenesin
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0 2 4 6 8 10 12 14 16
Time after administration (hour)
Pla
sma
mep
hene
sin
(ng/
mL)
P183 (1.0 g)
S305 (1.0 g)
P184 (0.25 g)
N166 (0.05 g)
P291 (0.05 g)
2.2 h2.8 h 8 h 9.4 h 14 h
Conc. = 0 ng/mL
6969
Oral administration, urinary free mephenesin
0.1
1.0
10.0
100.0
1000.0
0 2 4 6 8 10 12 14 16 18 20
Time after administration (hour)
Urin
ary
free
mep
hene
sin
(ng/
mL) P183 (1.0 g)
S305 (1.0 g)
P184 (0.25 g)
14 h8 h
3 ng/mL
9.4 h
Conc. < 3 ng/mL;concentration
matched !
7070
Summary of Oral Administration ExperimentsDose 2.5 g 1.0 g 0.25 g 0.05 g
Horse 'Whytellyou' P192 S125 P183 S305 P184 N166 P291
Urinary free mephenesinConc. in ng/mL (when the conjugated form is ~3 g/mL) <3 ng/mL 50 28 1.5 1.7 2.9 8.5 6
% Free / Conjugated <0.1% 1.7 0.9 <0.1 <0.1 <0.1 0.3 0.2
Urinary mephenesin after enzyme hydrolysis
Time in hours (at 3 g/mL) < 14 h 16 28 9.4 14 8 2.2 2.8
??
0 ng/mL 00 0
9 12< 14 h 3 3
1.8 0.6
6
Plasma free mephenesinConc. in ng/mL (when the conjugated form in urine is ~3 g/mL)
0 0
Time to clear in hours 1513
7171
Three horses (ages 3-5) given mephenesin orally were able to generate the conditions found in the post-race samples from the 4-year-old 'Whytellyou'.
Results were consistent with an oral administration of about 0.25 to 1 gram (a sub-therapeutic dose) of mephenesin to 'Whytellyou' between 8 - 14 hours prior to the collection of post-race samples, namely, between 0630 and 1230 hrs on 10 Apr 1999.
Further investigations suggested that powder mephenesin was mixed with the fodder and given to 'Whytellyou' in HV.
Formal enquiry: the trainer was also held responsible for boldenone (an anabolic steroid not approved for use on Hong Kong racehorses) detected in post-race urine from another horse in his stable:
=> horses disqualified and trainer’s licence suspended.
Case 3: Investigation results
7272
The ultimate aim of a doping control systemin sports is not high incidents of positive
findings and sanctions, but rather, minimum violations resulting from the deterrent
of a highly effective and well respected doping control and drug testing program.
CONCLUSION
Our quest continues...
7373
Staff of the HKJC Racing LaboratoryStaff of the HKJC Racing Laboratory
November 2009, HKJC 125November 2009, HKJC 125thth AnniversaryAnniversary
