Sports drugs testing - what are the odds?

D Brynn HibbertSchool of Chemistry

University of New South WalesSydney, Australia

2

Where we are – Sydney, Australia

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

5

The perils of bad measurement!

Map based on Abel Tasman’s voyage of 1644 and subsequent discoveries.

Note : Error in longitude ~ 2800 km

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

DEVELOPMENTS IN ANALYSIS

Isotope ratio mass spectrometry

1000

CC

CC

CC

C

PDBV

12

13

PDBV

12

13

sample

12

13

PDBVsample,13

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’

GC-IRMS

12

Testosterone abuse

HO O

O

HO

O

HO

H

OH

H

O

O

OH

HO

O

OH

HO

H

OH

OH

O

OH

HO

O

H

HO

O

H

HO

O

H

HO

HO

O

H

O

O

HO

OH

O

OH

Pregnanediol

Cholesterol

PregnanetriolTestosterone

Androsterone Etiocholanolone 11-ketoEtiocholanolone

11b-hydroxyAndrosterone

Testosterone & Metabolites

Endogenous Reference Compounds

Testosterone – adverse finding

1. If one of the following holds1. 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 d > 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” ...

14

Administration Study

Effect of multiple DHEA administration on metabolites

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-34

-32

-30

-28

-26

-24

-22

-20

-18

0 24 48 72 96 120 144 168 192

Time (h)

13C

(‰)

A Et11-ketoEt DHEASeries5

d13C trends of Et, A and 11-ketoEt of subject B with multiple DHEA administrations (each marked by on the lower axis)

Endogenous reference compound

Testosterone Metabolites

Dd13C

DHEA = dehydroepiandrosterone, testosterone precursor

Gra

ph: A

dam

Caw

ley

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

DEVELOPMENTS IN METROLOGY

Measurement uncertainty, metrological traceability Bayes theorem

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)

D B. Hibbert

Ancient History 3000 BCThe Royal Cubit … ± 0.05 %

Calibration hierarchy and metrological traceability

chain

samplevalueuc

Measurement Result

Stated reference

Met

rolo

gica

l tra

ceab

ility

Calibration hierarchy

Traceability chain

samplevalueuc

Measurement Result

samplevalueuc samplevalueuc

Measurement Result

Stated reference

Met

rolo

gica

l tra

ceab

ility

Met

rolo

gica

l tra

ceab

ility

Calibration hierarchy

Calibration hierarchy

Traceability chain

Traceability chain

Traceability of the certified values to the international standard VPDB

Metrological traceability chain

International standard

VPDBIRMS at

NISTNatural Gas Reference Material

IRMS atBrenna LaboratorySteroid Reference

Materials

IRMS atNMIInternal Standard

IRMS atNMISteroids in CRM

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)

26The EURACHEM Guide http://www.eurachem.bam.de/index.htm

The EURACHEM Guide http://www.eurachem.bam.de/index.htm

Guide To The Expression Of Uncertainty In Measurement. JCGM:100, Geneva (2008).

www.bipm.org/en/publications/guides/gum.html

Guide To The Expression Of Uncertainty In Measurement. JCGM:100, Geneva (2008).

www.bipm.org/en/publications/guides/gum.html

Uncertainty (GUM) concept

combined uncertainty (uc)

expandeduncertainty (U)

coverage interval

measured value corrected for systematic effects= best estimate of true value

estimated pdf

Possible values of measurand

PD

F

Total CO2 measurements in horses - where to draw

the lineD. Brynn Hibbert

Nicholas ArmstrongJohn H. Vine

29

Expanded standard uncertainty

• uc = 0.22 mM

• veff = 400

• U = 0.44 mM (95% coverage)

• odds of concluding a horse at 36 mM is over 37 mM

• = 1 in 190,000

Rounding

Calibrator purity

Calibration

Reproducibil-ity

Bias

Combined

0 0.05 0.1 0.15 0.2 0.25

u /mM

Advantages of the GUM approach

1. Statistically sound2. Give probability of the true value

being in a range3. Understanding of contributions to

uncertainty4. 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)

32

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

33

codeinemorphineheroin

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GC-MS: TIC of acid/neutral fraction

1. = O6,N-Diacetylnorcodeine2. = O6,N-Diacetylnormorphine3. = 4-Acetoxy3,6-dimethoxy-5-[2-N-Methylacetamido]ethylphenanthrene4. = O6,O3,N-Triacetylnormorphine5. = Noscapine6. = N-Acetylnornoscapine7. = 1-Acetoxy-N-Acetyl-1,9-dihydroanhydronornarceine

35

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 …

UN for Codeine/Morphine

C/M

0 4 8 12 16 20 24 28 32 36 40 44 48 52M

ore

ratio

Fre

qu

en

cy

SA

SWA

SEA

MEX

Heroin database

37

Around 2500 entries

A Bayesian interpretation

• Determine the distributions of ratios by origin

38

C/M for SEA

0

100

200

300

400

500

600

700

800

900

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

mor

e

Ratio

Nu

mb

er o

f en

trie

s in

dat

abas

e

Pr(25>C/M>30 | SEA) =199/2004

Probability

39

MXSASWASEAiii XX

.,,

)Pr()|28C/MPr(

)SEAPr()SEA|28C/MPr()28C/M|SEAPr(

Hence we can redefine the ratios for different regions in terms ofprobabilities

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 …

Thank you!

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