Sports drugs testing - what
are the odds?
D Brynn Hibbert
School of Chemistry
University of New South Wales
Sydney, Australia
Where we are – Sydney, Australia
2
Acknowledgements
• Ellaine Munton, Michael Collins, Helen
Salouros, John Murby (NMIA)
• Diako Ebrahimi (UNSW)
• Nicholas Armstrong (UNSW)
• Steve Sidney and the NLA
Outline
• Catch up football
• Developments in analysis
– isotope ratio mass spectrometry
• Developments in metrology and statistics
– metrological traceability
– measurement uncertainty
– Bayes theorem and chemometrics
The perils of bad measurement!
Map based on
Abel Tasman’s
voyage of
1644 and
subsequent
discoveries.
Note : Error in longitude ~ 2800 km
5
Catch up football
• Are the drug cheats one step ahead?
• In 2009 there were 277,928 samples
analysed in the world
• 5610 Atypical or adverse findings (2.02
%), up from 2008 (1.84 %)
Who was caught in 2009?
… by country
Isotope ratio mass spectrometry
DEVELOPMENTS IN ANALYSIS
Developments in analysis
• Stable isotope analysis by gas chromatography
– isotope ratio mass spectrometry (GC-IRMS)
• Look at 2H/1H, 13C/12C, 15N/14N, 18O/16O
• Measure ratio as a ‘delta’ scale in ‰ with
reference to standards e.g. 13C ‘Vienna Pee Dee
Belemnite’
  13 C 

 13 C 
  12 

  12 
  C  sample  C  V  PDB 
13
 Csample,V PDB  
 1000
13
 C


 12 C 




V

PDB


GC-IRMS
Testosterone abuse
Testosterone & Metabolites
O
O
HO
Cholesterol
Endogenous Reference
Compounds
O
HO
OH
O
OH
H
HO
O
O
OH
OH
OH
O
O
HO
H
HO
Pregnanediol
OH
OH
OH
O
Testosterone
O
HO
Pregnanetriol
H
O
O
O
O
HO
HO
HO
H
Androsterone
HO
H
Etiocholanolone
12
HO
H
11-keto
Etiocholanolone
H
11b-hydroxy
Androsterone
Testosterone – adverse finding
1. If one of the following holds
1. Testosterone/Epitestosterone value equal or greater than 4;
2. concentration of testosterone or epitestosterone (equivalent to
the glucuronide) greater than 200 ng/mL;
3. concentration of androsterone or etiocholanolone (equivalent to
the glucuronide) greater than 10,000 ng/mL;
4. concentration of =dehydroepiandrosterone (equivalent to the
glucuronide) greater than 100 ng/mL
2. and then if  > 3 ‰ different from endogenous reference
The results of the IRMS analysis … shall be used to draw conclusions
as to whether a doping violation may have been committed.
If the IRMS study does not readily indicate exogenous administration,
the result should be reported as “inconclusive” ...
Administration Study
Endogenous reference compound
Graph: Adam Cawley
13 C(‰)
Effect of multiple DHEA administration on metabolites
DHEA = dehydroepiandrosterone, testosterone precursor
-18
-20
-22
-24
-26
-28
-30
-32
-34
-36
D13C
0
24
48
Testosterone Metabolites
72
96
Time (h)
120
144
A
11-ketoEt
Series5
168
192
Et
DHEA
13C trends of Et, A and 11-ketoEt of subject B with multiple DHEA
administrations (each marked by  on the lower axis)
14
Landis case
• “The World Anti-Doping Agency (WADA) is satisfied with
the decision announced today by the Court of Arbitration
for Sport (CAS). …
• … Today’s ruling further confirms that Mr Landis
committed an anti-doping rule violation and that there is
no escaping the fact that synthetic testosterone was
found in his sample.”
UNSW + NMI Australia
• We are developing well-characterised
reference methods and materials for IRMS
• There is a bias in the way that IRMS
instruments corrects for isotopes of
oxygen
• With proper bias correction we find
improved results
Bias
Results after correction
Measurement uncertainty, metrological traceability
Bayes theorem
DEVELOPMENTS IN
METROLOGY
Metrological comparability
comparability of measurement results, for
quantities of a given kind, that are
metrologically traceable to the same
reference
(VIM 2.46)
Comparisons
SI
Metrological traceability
property of a measurement result whereby
the result can be related to a reference
through a documented unbroken chain of
calibrations, each contributing to the
measurement uncertainty
(VIM 2.41)
Ancient History 3000 BC
The Royal Cubit … ± 0.05 %
D B. Hibbert
Calibration hierarchy and
metrological traceability chain
Stated reference
Calibration hierarchy
Metrological traceability
Traceability chain
uc
value
sample
Measurement Result
Metrological traceability chain
Traceability of the certified values to the international standard VPDB
International
standard
VPDB
Natural Gas
Reference Material
Steroid Reference
Materials
IRMS at
NIST
IRMS at
Brenna Laboratory
Internal Standard
IRMS at
NMI
Steroids in CRM
IRMS at
NMI
Measurement uncertainty
non-negative parameter characterizing the
dispersion of the quantity values being
attributed to a measurand, based on the
information used
(VIM 2.26)
Guide To The Expression Of Uncertainty In
Measurement. JCGM:100, Geneva (2008).
www.bipm.org/en/publications/guides/gum.html
The EURACHEM Guide
26
http://www.eurachem.bam.de/index.htm
Uncertainty (GUM) concept
coverage interval
PDF
expanded
uncertainty (U)
estimated pdf
combined
uncertainty
(uc)
Possible values of measurand
measured value corrected for systematic effects
= best estimate of true value
Total CO2 measurements in
horses - where to draw the line
D. Brynn Hibbert
Nicholas Armstrong
John H. Vine
Expanded standard uncertainty
• uc = 0.22 mM
• veff = 400
• U = 0.44 mM (95%
coverage)
Combined
Bias
Reproducibility
Calibration
Calibrator purity
• odds of concluding a
horse at 36 mM is
over 37 mM
• = 1 in 190,000
29
Rounding
0
0.05
0.1
0.15
u /mM
0.2
0.25
Advantages of the GUM
approach
1. Statistically sound
2. Give probability of the true value being in
a range
3. Understanding of contributions to
uncertainty
4. Supported by ISO, IEC, ILAC, OIML,
BIPM, IUPAC, IUPAP, IFCC
Bayes theorem
• Basis of the GUM
• Basis of all DNA evidence
p(H|E) = p(E|H)
x p(H)
H = hypothesis (e.g. cocaine present in sample)
E = evidence (e.g. mass spectrum, measurement data)
Thomas Bayes
• Essay towards solving a
problem in the doctrine of
chances
– Philosophical Transactions
of the Royal Society of
London, 1764
Thomas
Bayes
1702 – 1761
32
codeine
33
heroin
morphine
GC-MS: TIC of acid/neutral fraction
1.
2.
3.
4.
5.
6.
7.
=
=
=
=
=
=
=
O6,N-Diacetylnorcodeine
O6,N-Diacetylnormorphine
4-Acetoxy3,6-dimethoxy-5-[2-N-Methylacetamido]ethylphenanthrene
O6,O3,N-Triacetylnormorphine
Noscapine
N-Acetylnornoscapine
34
1-Acetoxy-N-Acetyl-1,9-dihydroanhydronornarceine
The heroin signature program
• Look for series of marker compounds to
identify the origin of heroin seizures
• UN signature- ratios of
– codeine/morphine,
– noscapine/morphine,
– 6MAM/morphine,
– papaverine/morphine
• US DEA – more ratios …
35
UN for Codeine/Morphine
C/M
SWA
MEX
e
52
48
44
40
M
or
ratio
36
32
28
24
20
16
8
12
SA
4
0
Frequency
SEA
Heroin database
Around 2500 entries
37
A Bayesian interpretation
• Determine the distributions of ratios by
origin
900
800
Pr(25>C/M>30 | SEA) =199/2004
700
600
500
400
300
200
100
Ratio
38
more
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
0
Number of entries in database
C/M for SEA
Probability
Pr(SEA | C/M  28) 
Pr(C/M  28 | SEA ) Pr(SEA )
 Pr(C/M  28 | X ) Pr( X )
i  SEA, SW A, SA. MX
i
Hence we can redefine the ratios for different regions in terms of
probabilities
39
i
Conclusions
• In the face of legal scrutiny we must be
exact in our conclusions
• Inferences must be supported by evidence
(traceability, uncertainty)
• Need good knowledge of underlying
chemistry and statistics
• We are unlikely to be out of a job for a few
years …
© 1999 Hua Hue Xie – finalist in the Doug Moran Portrait prize
Thank you!