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R eview
S pecial Focus: A nti- doping
analysis & the
O lympics
Analytical progresses of the International
Olympic Committee and World Anti-Doping
Agency Olympic laboratories
The Summer Olympic Games constitute the biggest concentration of human sports and activities in a particular
place and time since 776 BCE, when the written history of the Olympic Games in Olympia began. Summer and
Winter Olympic anti-doping laboratories, accredited by the International Olympic Committee in the past and the
World Anti-Doping Agency in the present times, acquire worldwide interest to apply all new analytical advancements
in the fight against doping in sports, hoping that this major human event will not become dirty by association with
this negative phenomenon. This article summarizes the new analytical progresses, technologies and knowledge
used by the Olympic laboratories, which for the vast majority of them are, eventually, incorporated into routine
anti-doping analysis.
The Olympic Games (OG) played a very important role in the ethics, the philosophy and the
mentality of the ancient Greeks. Their impact
on the divided – in ancient times – Greek citystates was continuous. To name only a few:
the Olympic Truce was stopping wars among
Greek cities; the calendar was adjusted to the
every 4-year Olympic event; and Greeks were
concentrating from the mainland and the
colonies (Figure 1) [1] , creating the concept of
a mega event with religious, sports and political dimensions. The OG today remain a mega
event as the sociology science defines [2] with
respective dimensions: financial instead of religious, sports and political. In that framework,
the anti-doping system has a very important
role: to keep the OG impact and dimensions
as much as possible not influenced and clean
from doping. Consequently, the Olympic antidoping laboratories, responsible for performing
the anti-doping analysis of the OG, accredited
by the International Olympic Committee (IOC)
up to 2004 and the World Anti-Doping Agency
(WADA) afterwards, have a worldwide interest in applying and utilizing all the analytical
advancements. International experts in the field
are contributing to this direction.
IOC analytical specifications for the Olympic
laboratories comprise the reporting time of negative samples in 24 h and of positives in 48 h.
WADA publishes the catalogue of the accredited laboratories for doping control analysis [101] .
None of the accredited laboratories, in each routine analytical capacity, is able to cope with this
sort of turnaround time for the hundreds of OG
anti-doping samples daily. The number of the
in- and out-of-competition anti-doping samples
has been constantly increasing since the 1996
Atlanta OG from around 1500 [3] to a planned
number of 6250 for the forthcoming 2012
London OG [102] . The Olympic laboratories
are supported not only to enhance their qualitative–technological capacities, and to comply
with the mega event needs, but they must also
have infrastructure that multiplies their routine
analytical capacity.
The present article is a review of the qualitative–technological progress of the Olympic
laboratories starting from the 1972 Munich
OG, since at the 1968 Mexico OG only limited
testing was introduced by IOC. This progress
was not always an easy and overnight task for
many reasons: it adds to existing pre-Olympic
rush status where budget, space, staff training
and instrumentation should be ensured; the
laboratory has to implement the new method
without experience in quite a demanding environment, which comprises 24 to 48-h reporting
time, consecutive shifts of staff and presence
of independent observers; and, any laboratory
error can influence the entire Olympic investment of the country, since media can now give
full worldwide publicity in seconds.
10.4155/BIO.12.148 © 2012 Future Science Ltd
Bioanalysis (2012) 4(13), 1549–1563
Costas Georgakopoulos*1,
Martial Saugy2 , Sylvain
Giraud2 , Neil Robinson2
& Mohammed Alsayrafi1
1
Anti-Doping Laboratory Qatar,
PO Box 27775, Doha, State of Qatar
2
Laboratoire Suisse d’Analyse du
Dopage, Switzerland
*Author for correspondence:
Tel.: +974 4413 2991
Fax: +974 4413 2997
E-mail: [email protected]
1972 Munich OG
The anti-doping control of the 1972 Munich
OG was the beginning of the modern antidoping system, which eventually progressed to
ISSN 1757-6180
1549
R eview | Georgakopoulos, Saugy, Giraud, Robinson & Alsayrafi
Figure 1. Map showing routes from Greek cities and colonies to the Olympic Games in Olympia later than the
5th century BCE.
Reproduced with permission from The British Museum Company Ltd [1] .
the WADA Anti-Doping Code. The IOC and
Donike organized a large daily number of antidoping tests, to be conducted according to the
List of Prohibited Substances and Methods,
using GC screening, MS confirmatory methods,
24-h sample reporting times, A and B sample
analysis and double-blind QC samples (control samples introduced by the sports authority
among the routine samples, which are are not
identifiable as control samples, to check laboratory operations) [3] . With the use of chromatographic systems equipped with nitrogen–phosphorus detectors (NPD) and autosamplers, it
was possible to analyze up to 220 samples per
day [4] . Chromatographic identification criteria were based on chromatographic retention
parameters and in 1970 Donike published a
study for the reproducibility of the retention
times [5] . In this preparative for the OG study,
Donike suggested the use of autosamplers and
identical equilibration times between each chromatographic run, in order to improve retention
reproducibility. As it was published after the end
of the 1972 OG [6] , the retention time reproducibility was better than 0.2%. The NPD-based
stimulant screen developed by Donike is still in
use in some WADA laboratories, today 40 years
1550
Bioanalysis (2012) 4(13)
later. The 1972 IOC Prohibited List is presented
in Box 1 [4] . The 1972 Munich OG anti-doping
organization resulted in 11 positive cases and the
reported prohibited substances were: ephedrine
(three cases), methamphetamine, dimethylamphetamine, nikethamide (two cases), amphepramone, amphetamine, phenmetrazine and
norephedrine [4] .
1976 Montreal OG
The organizational progress of the Olympic
anti-doping system was continued with the
1976 Montreal OG: IOC Medical Commission
acquired a daily working experts committee; a
training program for the anti-doping sample collection officers; and, a coordination department
for the logistics of the tests and the samples chain
of custody – practices that are all followed today.
But the most important progress was the inclusion in the analytical repertoire of the anabolic–
androgenic steroids (AAS) [7] , probably one of
the most powerful performance-enhancing
pharmacological classes of compounds. In 1975,
the St Thomas’s Hospital Medical School team
of Brooks published a method for the detection
of AAS by radioimmunoassay (RIA) [8] , which
was applied to the analysis of 275 out of 1800
future science group
Analytical progresses of the IOC & WADA Olympic laboratories
Montreal OG samples. The method was detecting the 17a-methyl and 17a-ethyl AAS. Despite
the fact of its 2–3 day duration for completion, it
was followed because of its higher sensitivity and
lower cost compared with the GC–MS method.
Obviously, the contribution of the instrumental and the manpower cost to the total cost of
analysis were different in the 1970s than today.
The antiserum sensitivity for the 17a-methyl
AAS was 5 pg. Among the detected AAS were
norethandrolone, ethylestrenol, methyltestosterone, methandienone, stanozolol, fluoxymesterone, oxandrolone, clostebol, mestanolone,
methandriol and oxymetholone. Analysis
was performed in urine in the unconjugated
or hydrolyzed conjugated fraction. An unacceptable crossreactivity to testosterone higher
than 5% resulted in an additional preparative
step, which was the acetylation of testosterone
with acetic anhydrite. The 17a-alkylated AAS
remained underivatized due to presence of the
bulky methyl or ethyl group. The testosterone
acetate had no crossreactivity with the antisera. Another antiserum was produced for the
detection of 19-nortestosterone and the method
was run without acetylation [9] . RIA suspicious
samples underwent GC–MS confirmatory ana
lysis [10] , which resulted in eight positive samples.
The GC–MS reported AAS concentrations of
higher than 50 ng/ml in the seven of them.
The GC–MS method was based on Amberlite
XAD-2 column cleanup, enzymatic hydrolysis
by Helix Pomatia juice, another one XAD-2
extraction, Sephadex LH-20 fractionation and
O-trimethylsilyl (TMS) derivatization analyzed
in MAT 112 GC–MS [3] .
1980 Moscow OG
The 1980 Moscow OG anti-doping analysis suffered from the technological embargo of the US
government to the instruments to be sold to the
USSR. The RIA method of Brooks [8] combined
with two HP5840/5985 GC–MS machines were
used for the anti-doping analysis.
1984 Los Angeles OG
The University of California, Los Angeles
(UCLA), Olympic laboratory was equipped for
the first time in OG anti-doping analysis with
a full set of GC and GC–MS machines [11] .
The repertoire of analysis comprised screening
for the ratio of testosterone to epitestosterone and the synthetic AAS clostebol, dehydrochloromethyltestosterone, mesterolone,
oxandrolone, oxymetholone, methenolone,
| R eview
Box 1. The International Olympic Committee Prohibited List for the
1972 Munich Olympic Games.
Psychomotor stimulants

Ethylamphetamine, amphetamine, benzphetamine, cocaine,
diethylaminopropiophenone, dimethylaphetamine, fencamphetamine,
methylamphetamine, methylphenidate, norpseudoephedrine, phendimetrazine,
phenmetrazine, prolintane and related substances.
Sympathomimetic amines

Ephedrine, methylephedrine, methoxyphenamine and related substances.
Various stimulants of the CNS

Amiphenazol, bemegrid, leptazol, nikethamide, strychnine and related substances.
Narcotics & analgesics

Dextromoramide, dipipanone, heroin, methadone, morphine, pethidine and
related substances.
Data taken from [4].
nandrolone, methandienone, methyltestosterone, methandriol, mestanolone, norethandrolone, fluoxymesterone and stanozolol.
Here, it is important to mention that the TMS
derivatization protocol developed by Donike
and Zimmermann for the conversion of the
keto-steroids to pure isomerically enol O-TMS
steroids using the trimethyliodosilane catalyst
[12] was used. This development allowed the
improvement of the GC behavior of the ketosteroids, reducing their polarity and improving
chromatographic stability. UCLA developed
the method for the analysis of b-blockers, but
at the last minute IOC decided not to include
them in the repertoire of the OG analyses. It is
worthy to mention that the routine anti-doping
analysis, as described in [11] , was used by all the
IOC-accredited laboratories for more than the
next 10 years, with only the addition of diuretics
analysis incorporated.
1988 Seoul OG
In the 1988 Seoul OG, new elements were added
to the routine anti-doping analysis: screening for
diuretics in HPLC and diode-array UV detection [13,14] , a Laboratory Automation System [13] ,
QC regulations of the GLP specifications [15] and
screening for corticosteroids in thermospray
LC–MS [16] . The mass spectra of the corticosteroids comprised the m/z (MH)+, (MH)+ -18,
(MH)+ -60, (MH)+ -30 and ammonium adducts
(MNH4)+. However, despite all those substantial progresses of the anti-doping OG analysis,
what attracted attention was the announcement
that the Olympic winner of the athletics 100-m
distance run, Johnson, was found to be positive
for use of the AAS stanozolol. The detection of
stanozolol metabolites was based on the work
www.future-science.com
Key Terms
Trimethylsilyl
derivatization:
Derivatization for the GC
analysis that converts polar
molecular groups, such as
hydroxyl, to nonpolar groups by
the substitution of the hydrogen
atom with a Si-(CH3)3 group.
This way, the chromatographic
behavior is improved.
Ratio of testosterone to
epitestosterone: Indirect
marker of exogenous
administration of testosterone,
based on the quantitation of
testosterone and its epimer
epitestosterone. Ratio mean
population value is one. World
Anti-Doping Agency Code sets
the ratio threshold to four.
1551
R eview |
Georgakopoulos, Saugy, Giraud, Robinson & Alsayrafi
conducted by the Cologne laboratory [17] and
the relevant study of the Seoul laboratory [18] .
Before the Cologne study, stanozolol detection
in human urine was based on the detection
of the parent compound, whose detectability
is quite short compared with the well-known
hydroxylated metabolites (WADA-accredited
laboratories today do not include the parent
stanozolol in screening procedures, since it
is useless compared with the detection of the
hydroxylated metabolites).
1992 Barcelona OG
The 1992 Barcelona OG anti-doping control
was directly related to the detection of the first
of a series of Soviet stimulants – the mesocarb.
This detection was accompanied by another
progress; the mesocarb confirmation analysis
was based on the use of LC–MS technology
for first time, using a particle-beam interface
to generate electron ionization mass spectra [19] .
Screening analysis of mesocarb was performed
by HPLC–UV, in the same analytical procedure
as for the diuretic class. GC–MS confirmation
analysis of mesocarb artifact was also performed.
Similar to the mesocarb, the LC–MS confirmation analysis was also developed for diuretics
[20] . Another interesting substance reported in
positive samples during the 1992 OG was clenbuterol, the veterinary b2-agonist with increasing popularity for its anabolic action in cheating
athletes, but also with epidemiological records in
Catalonia in 1992 [21] . The following literature
references constitute a thorough presentation of
the 1992 Barcelona Olympic laboratory [22,23] .
In those references, information is related to the
1992 Barcelona OG laboratory concerning the
new building, the staff and its organizational
structure, instrumentation related to the analytical methodologies, computer network system,
reagents, quality assurance aspects, a summary
of the analytical methodologies and the results.
Especially in the results section [22,23] , the wealth
of the analytical parameters per sample recorded
during the OG can be concluded. The organizational structure of the laboratory was completed
by the custom-made Laboratory Information
Management System [24,25] . The Laboratory
Information Management System was able to
organize the thousands of samples, aliquots,
tubes and autosampler vials involved in the daily
workload of the Olympic laboratory, to generate
reports/working forms, to prepare instrumental sequences of sample analyses and to follow
all cases requiring repeats, investigations of
1552
suspicious analytical findings, confirmations
and almost every kind of laboratory activity.
Numerical reading was performed through the
barcode system. This system was so successful
that its descendant is still in use in the Barcelona
laboratory today.
1994 Lillehammer winter OG
Two important advancements were applied by
the Oslo laboratory for the 1994 Winter OG in
Lillehammer: blood analysis for the detection of
the nonautologous blood transfusions and highresolution MS (HRMS) coupled with GC [3] for
the detection of synthetic AAS in low concentrations in urine. Blood collection for detection of
nonautologous blood transfusions was based on
the detection of the blood groups of the allogenic
transfused erythrocytes [26] . The method was
new and used microtubes filled with gel containing agglutinating antibodies to the erythrocyte’s antigens: the erythrocytes with negative
antigens could pass through the microtube with
the respective antibody, while erythrocytes with
expressing antigens remained at the top of the
gel. The method could detect AB0, Rh, K and Jk
blood groups, while for other blood groups, such
as Fy and MNSs and for confirmatory analyses,
the classic microscopic approach was followed.
The microtube method could detect transfused
blood of the afore-mentioned blood groups at a
range of 1–5%. GC–HRMS was introduced in
order to increase the detection window of AAS
abuse in periods chronologically far from the
expected in-competition testing [27] . The GC–
HRMS was the result of progress made in the
instrumental electronics and the MS geometry
itself. GC–HRMS constitute a magnetic analyzer coupled with one or two electrostatic analyzers. The accelerating voltages can reach up
to 10 kV and the mass resolution in 5% peak
valley in GC–MS measurements can reach up
to 40,000. The mass resolution is defined as the
ratio of the mass m/z to the difference between
that mass m/z and the next mass m/z, when
these two consecutive masses m/z can be separated at an in-between valley of 5% (or 10%)
of their respective peak profiles [27] . In a wellcalibrated GC–HRMS, the drop of the signal
of the background noise is multiple to the drop
of the signal of the compound of interest, such
a phenomenon results in a significant increase in
the S/N and, consequently, the limit of detection. The GC–HRMS instrument of the Oslo
laboratory was a magnetic sector Finnigan MAT
95 coupled with a HP5890 GC, with routine
resolution capability of 3000–5000 in 10%
peak valley definition of the MS resolution, also
described in [27] . The instrument could easily
analyze entire batches of samples for AAS at a
concentration of less than 10 ng/ml of urine.
Based on this technology, the IOC Medical
Commission decided some years later (1996)
to introduce specifications for the detection of
certain AAS metabolites to the Olympic movement Anti-Doping Code, such as the long-term
stanozolol metabolite 3´-hydroxy-stanozolol,
at a concentration of 2 ng/ml of urine for all
anti-doping samples.
1996 Atlanta OG
The Atlanta OG laboratory was programmed to
implement the carbon isotope 13C/12C ratio MS
analysis (GC–C–isotope-ratio MS [IRMS]) to
identify origins of naturally occurring AAS, such
as testosterone and metabolites, but eventually
the method, developed by Becchi and collaborators [28] , was not used. However, the 1996 OG
anti-doping reports created another surprise –
detection of another new Soviet stimulant:
bromantane [29] .
1998 Nagano Winter OG
For the Nagano OG, GC–C–IRMS was
introduced for the first time. The Tokyo IOCaccredited laboratory developed the method to
detect dihydroepiandrosterone (DHEA) abuse
among others [30,31] . An interesting collection
of data presented in [31] concerns the d values of
AAS originating from pharmaceutical preparations, food supplements and reference material
providers. Those data proved the validity of the
GC–C–IRMS technology. During the OG, all
samples were analyzed by GC–C–IRMS methodology, an approach that has not been repeated
since. Another interesting progress the Tokyo
laboratory implemented, in collaboration with
the Barcelona IOC-accredited laboratory, was
the development of the method for the discrimination of oral versus inhaled salbutamol in
human urine [32] . This method tried to solve the
problem of the extensive use of b2-agonists in the
winter sports for medical reasons. Eventually, the
method was not officially approved because of
the complexity of the analysis and the statistical
evaluation of the data.
2000 Sydney OG
Recombinant EPO became available in the
middle of 1980s and was popular with cheating
athletes for its capacity to induce erythropoiesis
and increase the transfer of oxygen to the muscles, while becoming undetectable relatively
quickly compared wtih the length of time over
which it enhanced endurance performance.
EPO was placed on the IOC Prohibited List in
1990, but until its detection became a reality,
10 years passed: “The Olympic movement yesterday announced measures to trace the use of
the previously undetectable drug EPO,” wrote
the UK newspaper The Telegraph on 2 August
2000 [103] . Another article of the same newspaper, published on 29 August 2000, 2 weeks
before the 2000 OG Opening Ceremony,
referred to: “Selected competitors will be asked
to give a sample of blood and if they refuse, even
on religious grounds, they will be excluded from
the Games” [104] . This announcement was one
of the most important issues of the 2000 OG:
the athletes that did not agree to the blood collections had to leave the OG. This new test for
EPO was one of the most important elements
of the atmosphere of the 2000 Sydney OG. The
test of EPO implemented from the Sydney IOCaccredited laboratory was the combination of
two individual methods that were both applied
for the first time: the blood test for the calculation of the blood ON/OFF models and the urine
test for the measurement of the distribution of
the EPO isoforms.
The blood test was a funding of the Australian
government and IOC to Australian government
analytical laboratories, the parent organization
of the Sydney IOC-accredited laboratory, to
introduce new technologies and tests for the
OG. The studies were conducted with volunteer athletes who were divided in groups with
or without administration of EPO and/or iron
supplementation and had blood samples taken.
Several blood parameters were measured and
statistically processed. These studies led to the
validation of the first-generation of the ON- and
OFF-score models, using data from the athletes.
The ON model, when applied to data from an
athlete, could indicate that they were still in the
phase of application of EPO. The OFF model
provided data that indicated that the athlete had
stopped using EPO [33] . The ON model was
based on the blood parameters of EPO, hematocrit (Hct), reticuloc yte Hct (which is a function of % reticulocytes, red blood cells count and
mean cell volume of reticulocytes), the soluble
transferrin receptor (sTfr) and percent Macro
(percentage of cells with mean cell volume
higher than 120 fl), while the OFF model was
based on the first three of the above parameters.
| R eview
Key Term
Blood ON/OFF models:
Indirect markers of EPO abuse,
constituted by functions of
hematological parameters,
indicating EPO abuse close to
the testing (on) or late abuse
(off) developed initially for the
2000 Sydney Olympic Games.
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R eview |
Key Terms
Novel erythropoiesisstimulating protein
(NESP): Also known as
darbepoetin a, is produced in
Chinese hamster ovary cells and
is marketed under the names of
Aranesp ® and Nespo ®. NESP is
a hyperglycosylated analogue of
EPO that contains two novel
N-linked sialic acid-containing
carbohydrate side chains.
Homologous blood
transfusion: Allogeneic or
homologous refers to the
transfusion of blood of another
ABO-compatible person and
Rhesus D blood groups in order
to increase red blood cells and
oxygen transfer to muscles, in
particular in endurance sports.
hGH: Growth hormone that is
secreted throughout life mainly
by the pituitary gland and acts
through the specific growth
hormone receptor. The growth
hormone receptor is expressed
in almost all tissues of the
human body. It is synthesized
and abused by athletes for
anabolic action.
1554
The main achievement of this study was that
when the multiple indirect markers of the altered
erythropoiesis are used simultaneously, they can
identify current or recent EPO use.
Another big heritage of the 2000 Sydney OG,
which also opened the anti-doping analysis to
a new technology and made EPO a detectable
drug, was the isoelectric focusing (IEF) method
of the direct detection of recombinant EPO
isoforms in urine, developed by Lasne and de
Ceaurriz in the Paris IOC-accredited laboratory [34] . An isoform of EPO is a subset of the
EPO molecules that has a defined charge mainly
due to the differences in the carbohydrate side
chains. The method was based on that developed by Lasne for the double-blotting protocol
[35] and allowed IEF separation and discrimination of synthetic to endogenous EPO isoforms.
Recombinant EPO (rEPO) was synthesized in
Chinese hamster ovary cells and has modifications in the isoforms compared with human
endogenous EPO that is synthesized in the
kidney. The modifications affect the electrical
charge allowing the IEF separation.
Before the Sydney OG, in a meeting held in
Lausanne early in August 2000 with the participation of the Australian and French researchers,
the IOC decided that neither process was going
to be sanctioned on its own and a procedure
that required both the blood data of the ON
model as well as the IEF to be undertaken was
required before a sample could be reported as
positive. If both met the parameters required to
declare EPO use, then a positive result could be
reported. The OFF model was not accepted as an
indicator of EPO use, which, on its own, could
result in a positive finding. No adverse results
were reported from the OG, but a number of
blood results indicating ‘outliers’ in scores for
both the ON and OFF models were reported
to the IOC and subsequently the International
Federations for future follow-up. In the 2 years
following the Sydney 2000 OG, further work
by the Australian Institute of Sports using data
from the previous studies allowed modification
and refinement of the models with the development of the second-generation ON- and
OFF-score models [36] . It is interesting that in
the following years, WADA reassessed the IEF
procedure and declared that it could be used
independently of the blood test and that it was
a standalone test, and subsequently produced the
Technical Document to support it [105] . The ON
model has not had further acceptance, but the
new-generation OFF model is now part of the
Athlete’s Biological Passport [37] . 11 years after
the models development for the 2000 Sydney
OG, in March 2011, the Court of Arbitration
for Sport (CAS) ruled against the cyclists
Pellizotti and Caucchioli, not because they
failed drug tests or were nabbed in a criminal
investigation – the conventional ways athletes
are caught for doping – but because, indirectly,
several changes in their blood signaled that they
had illicitly manipulated their blood to improve
performance [106] .
2002 Salt Lake City Winter OG
The 2002 Salt Lake City Winter OG presented another challenge for the IEF method of
Lasne [34] , because the new long-lasting rEPO
Aranesp® (novel erythropoiesis-stimulating
protein [NESP] ) had just come out in the pharmaceutical market. Following is an extract from
an article on ABC News on 8 February 2002:
“Anti-doping agencies hope to have a reliable test
for Aranesp ready by the 2004 Summer OG in
Athens” [107] . Obviously, some participant athletes of the 2002 OG believed these articles. The
case of one athlete, the 50-km cross-country
skier Muehlegg, ended in the CAS in Lausanne
on 24 January 2003 as follows:
“In their decisions, the three arbitrators considered that:
Darbepoetin (Aranesp) is a substance analogue and mimetic of rEPO and is therefore a
prohibited substance;
n
The direct urine test is valid for the detection
of Aranesp as it is valid for the detection of
rEPO;
n
The presence of Aranesp in the human body
can be established with certainty by the urine
test, considering that darbepoetin cannot be
produced naturally; accordingly, there is no
risk of confusion between Aranesp and natural
EPO;
n
The EPO test method used by the Salt Lake
City laboratory is in accordance with the prevailing standards of the scientific community
and the particular urine test performed on
Muehlegg’s sample was properly conducted by
the laboratory” [108] .
n
The IEF method in the period between
August 2000, when it was not considered a
standalone method of detection of EPO, and
February 2002 had acquired the fair scientific
respect to be applied to a synthetic EPO, such
as NESP, without being named in the IOC
Prohibited List of 2002, which was unfortunate
for the cheating athletes.
The UCLA IOC-accredited laboratory, which
ran the temporary IOC-accredited laboratory
at Salt Lake City for the 2002 OG period, had
developed methods for the IRMS analysis of
endogenous steroids, such as epitestosterone
[38] . Epitestosterone is prohibited not because of
its anabolic action – it is inactive – but because
of its use by cheating athletes to hide testosterone abuse through the distortion of the ratio of
testosterone to epitestosterone. This particular
method is one of first where a HPLC cleanup
step was introduced in the IRMS preparative
steps to remove interferences to the measured
d values of steroids [28] .
2004 Athens OG
The first implementation of the WADA
Anti-Doping Code (International Standard
for Laboratories, version 3.0, published in
June 2003) in a major event was during the
2004 Athens OG. The WADA Code became
the worldwide anti-doping specification on
1 January 2004, and replaced the IOC Olympic
movement Anti-Doping Code for the everyday/
everywhere anti-doping application. An overview of the analytical methods and results of the
2004 Athens anti-doping analysis is presented
in [39] . The structure and organization of the
Athens Olympic laboratory were presented in
[40] . The hematological profile parameters ana
lysis and validation had been included in the
scope of the ISO/IEC 17025 accreditation of
the Athens laboratory. The validation report of
the analysis for reticulocytes has been presented
in [41] . The validation parameters comprised
linearity, precision, uncertainty, intermediate
and long-term precision, comparability with
other analyzers, effect of drift, carryover, stability and accuracy. LC–MS/MS technology (ion
trap) was used for the first time at an OG for
all samples. For the first time also, direct urine
injection methods were used for quantification
of the ephedrines, morphine and salbutamol [42] .
The chromatographic systems used for the direct
urine injection quantitation were the same as
for the LC–MS/MS screening method in order
to avoid loss of time in conditioning from the
one chromatographic system to another. After
the norbolethone [43] and the THG [44] stories,
the UCLA laboratory revealed the circulation
of another designer steroid, MADOL [45] . Some
days before the OG period, the Athens laboratory
| R eview
was provided by the WADA-accredited UCLA
laboratory with the information and the reference standard to screen for this steroid. However,
no positive sample was reported based on the
steroid.
Apart the new Anti-Doping Code, in the
2004 Athens OG three new analytical technologies were used to test for prohibited pharmacological classes in blood (except for homologous
blood transfusions that were tested for at the
1994 Winter OG by gel chromatography). One
of the new methods used by the 2004 Athens
OG laboratory was the detection of hemoglobin-based oxygen carriers (HBOCs) [46–48] . A
quick screening of blood samples for HBOCs
is based on colorimetric detection, since use of
HBOCs causes discoloration of the plasma. For
the suspect samples, electrophoretic analysis or
size exclusion HPLC followed, and for bovine
an additional LC–MS confirmatory analysis is
completed [46–48] . During the implementation of
the colorimetric methodology, it became apparent that there was a need to increase the specificity to avoid unnecessary activation of both the
chromatographic methods for hemolyzed plasma
samples. An additional ultrafiltration step was
added in the colorimetric, electrophoretic and
HPLC methods in order to remove any free
hemoglobin. Due to HBOCs chemistry, which
is based on intra- or inter-molecular crosslinked
hemoglobin with molecular weight higher than
200 kDa, the ultrafi ltration step can visually
distinguish hemolyzed against positive HBOC
plasma samples [49] , saving valuable laboratory
resources, since no one sample underwent confirmatory analysis in the OG period. Moreover, the
LC ion-trap analysis provided full-scan MS/MS
capabilities of the bovine-based HBOC peptides
in order to improve the specificity.
The second new method used by the 2004
Athens OG laboratory concerned the detection of the synthetic recombinant hGH (rhGH)
in serum samples, a method developed by
Strasburger and coworkers [50] . The so-called isoform method is based on the detection of rhGH
isoforms. Endogenous pituitary GH occurs in
multiple isoforms; approximately 70% of circulating GH is in the form of a 22-kDa polypeptide, 5–10% as a 20-kDa isoform and the rest
as dimers, oligomers and acidic, desaminated,
acylated and fragmented forms of GH, whereas
the rhGH comprises exclusively the 22-kDa isoform. When rhGH is administered, endogenous
pituitary secretion is inhibited through negative
feedback and an increase to the 22-kDa isoform
1555
R eview |
relative to other non-22-kDa isoforms occurs.
Statistically, an increased proportion of 22-kDa
GH is attributed to GH administration. The
isoform method relies on measurement of GH
isoforms by two pairs of antibodies specific to
either 22-kDa GH or pituitary-derived hGH.
The current isoform method is described in the
WADA guidelines [109] . The 2004 method was
based on the same pairs of antibodies 1B3/5D7
and 8B11/8A9 as the contemporary method,
but the basis of measurement was fluorescence
immunoassays. In Figure 2 , the negative and
positive controls for one of the pair assays, the
8B11/8A9, are presented. In Figure 3, distribution of OG 2004 samples ratios according to the
gender of sample and the assays used are presented. The isoform GH assays throughout the
2004 OG period run in an excellent way and the
blood samples were clearly negative.
The third new method used by the 2004
Athens OG laboratory concerned the detection
of homologous blood transfusion (blood transfused from a human donor with ABO blood
group compatibility) in whole blood samples.
The method was developed by Nelson and
16
14
ng/ml or ratio
12
n = 26
CV% = 13.9
10
8
6
n = 26
CV% = 12.1
4
2
0
ve
iti
s
Po
ve
iti
s
Po
O
IO
AT
R
T
PI
EC
R
e l
tiv
si g/m
Po n
I
AT
R
ive
at
eg
N
T
PI
ive l
at /m
eg ng
N
EC
R
ive l
at /m
eg ng
N
Figure 2. Distribution of positive and negative controls of the 8B11/8A9
assays through the 2004 Olympic Games period. The horizontal lines in the
box denote the 25th, 50th and 75th percentile values. The error bars denote the
5th and 95th percentile values. The two symbols below the 5th percentile error
bar denote the 0th and 1st percentile values. The two symbols above the 95th
percentile denote the 99th and 100th percentiles. The square symbol in the box
denotes the mean of the column of data. The definitions of the terms REC, PIT
and RATIO can be found in [109] .
1556
co-workers [51,52] . The method was transferred
and developed in the Athens OG laboratory
in collaboration with the colleague WADAaccredited laboratory of Lausanne and the
Genimatas Hospital of Athens [42] . One of the
first amendments to the original Australian
method [52] , which was decided in Athens, was
the implementation of the method as qualitative and not quantitative, in order for the
validation and accreditation procedures to be
facilitated. The same approach for the method is
followed today (detection of homologous blood
transfusion [53,54]).
The three new technologies applied at the
2004 OG completed their anti-doping validation shortly before the opening of the OG,
therefore creating a complex logistical situation.
In the plan of the blood collection containers,
serum and whole-blood tubes were planned to be
sealed in the same sample container at the collection site. It is well known that serum and whole
blood have different storage conditions: freezing and refrigerating, respectively. However, in
order to facilitate the logistical implementation
of the new methods so close to the opening of the
OG, taking into account this conflicting storage
conditions, the decision was taken to give priority to store the whole blood first (refrigerating)
and then the serum sample (freezing), to protect
hGH. Retrospectively, several practical issues
would have been avoided if, simply, another
200–300 containers would have been used to
store whole blood and serum in separate tubes,
an additional marginal expense in comparison
with the entire OG anti-doping program. The
blood transfusion method was developed in the
Athens laboratory during July 2004, very close
to the OG period. The Athens laboratory was
participating in proficiency testing schemes
organized by the laboratory loop of Athens–
Sydney–Lausanne. Unfortunately, on the day
of the Opening Ceremony (13 August 2004),
some contradictory discussions on the interpretation of the last proficiency test took place.
Even though within hours such discussions were
found to be based on a misunderstanding by one
of the experts involved, they had already created
a situation in which a positive case was observed
by the Athens laboratory on a blood sample,
but could not be reported as such. The sample
was collected on the 19 August 2004 from the
US cyclist and Olympic winner Hamilton, but
because the Athens laboratory had no time
to have the blood-transfusion method fully
accredited under the ISO accreditation process
0.7
5D7/1B3
8B11/8A9
0.8
0.6
Ratio assay 1:2
| R eview
0.5
0.6
0.4
0.4
0.3
0.2
0.2
0.0
0.1
Female
average: 0.36
SD: 0.11
n = 91
Male
average: 0.39
SD 0.13
n = 123
Female
average: 0.46
SD: 0.18
n = 61
Male
average: 0.48
SD: 0.23
n = 108
Figure 3. Olympic Games 2004 samples ratios distribution according to gender of sample
and the assays used. The horizontal lines in the box denote the 25th, 50th and 75th percentile
values. The error bars denote the 5th and 95th percentile values. The two symbols below the 5th
percentile error bar denote the 0th and 1st percentile values. The two symbols above the 95th
percentile denote the 99th and 100th percentiles. The square symbol in the box denotes the mean
of the column of data. All Olympic Games 2004 samples were clearly negatives.
(a situation that would have created a legally
weak position in case of an appeal in court),
it was decided that the case be reported to the
IOC as a negative, but with suspicions of blood
transfusion, rather than a positive. Then, in
line with the established procedure, to store the
sealed containers with the serum and frozen
whole blood; the A and B samples were moved
to the freezer some days after reporting, to preserve the serum for retrospective hGH analysis.
When the IOC asked on 15 September 2004
for Hamilton’s B sample to be analyzed in the
Lausanne Laboratory, the red blood cells in
the whole-blood sample were destroyed, which
prevented proper reanalysis of the sample and
Hamilton kept the 2004 OG gold medal. In the
meantime, in September 2004, Hamilton was
already targeted by WADA and the International
Cycling Union, based on the previous suspicious
sample in Athens when he participated in the
2004 Tour of Spain competition. Due to the
erythrocytes’ long life in the blood, homologous blood transfusion can be detected over a
similarly long period of time. In the 2004 Tour
of Spain competition, Hamilton was also tested
in blood and reported positive for blood transfusions by the Lausanne Laboratory. For this
positive case, Hamilton was given a 2-year ban
from the US Anti-Doping Agency. Hamilton
appealed this verdict to CAS, but on the basis
of the Lausanne results and with the support
of the Athens data, the appeal was rejected on
10 February 2006 [110] . It is worthy to mention that later, on 20 May 2011, after a life ban
from sports due to a second positive finding on
the steroid dehydroepiandrosterone, Hamilton
admitted his doping case in Athens and surrendered the 2004 Athens OG medal [111] , and this
first blood transfusion positive case was closed
definitely.
Besides the Hamilton case, in the Athens
2004 OG five athletes, two Greeks and three
Hungarians, were excluded from the OG and
banned from competing, without a positive antidoping sample, due to irregularities in the collection procedure or manipulating the samples.
These five cases, the Hamilton case and the 23
reported positives from the Athens 2004 OG
laboratory made a total of 29 doping cases, which
set a record of anti-doping cases for the OG.
Among the 23 reported positive samples were the
first two positives for endogenous steroids based
on IRMS. Another one of the reported positive
cases was based on the stimulant heptaminol,
but it was an eventually proven that the heptaminol substance was artifact of the stimulant
isometheptene, used originally by the athlete [55] .
A number of the Athens 2004 OG positive
results were based on synthetic AAS detected
in concentrations 10- to 100-times below the
WADA Minimum Required Performance Limits
[112] , analyzed with GC–HRMS operating
1557
R eview |
routinely in resolutions of 10–20 K (in 5% peak
valley MS resolution definition in the Waters
Autospec Ultima [Manchester, UK]). It is well
justified in historical books that women were
not competing in the ancient OG, they could
not even enter the stadium in Olympia as spectators [1] . Women competed in the stadium in
Olympia for the first time in 2004 during the
Athens OG, taking part in the shot-put. The
anti-doping control proved that the first winner
of the first women’s competition in the Olympia
stadium in history, was positive for the steroid
stanozolol (among the low concentration positive samples as for above). In Figure 4, the screening chromatogram for this sample is presented.
In 2004, for the first time the IOC implemented
an 8-year storage of Olympic samples for future
reanalysis.
Intensity (0–100%)
2006 Torino Winter OG
Continuing the 1998 Tour de France practice
of the French police, Italian police raided the
Austrian athletes’ quarter in search of evidence
of doping during the 2006 OG period. The raid
was conducted due to suspicions over the presence of the biathlon coach Mayer, who had been
banned from all Olympic events due to previous doping convictions. Around the time of the
raid, Mayer and two Austrian biathletes tried to
escape and flee back to Austria. The samples that
were collected from the rest of the athletes in the
team after that event were all reported as negative. Even in the period during the preparation
of this article, this case has not been finalized in
the status of an official publishable report. In the
analytical part of the 2006 Torino Winter OG,
the Torino OG laboratory was a satellite laboratory of the Rome WADA-accredited laboratory.
The Rome laboratory developed an LC–MS to
detect a large number of prohibited substances
from different classes, proving the analytical
capacity and the multitrace character of this
technology [56] .
Especially for the homologous blood transfusion analysis and, due to insufficient time for its
implementation, it was decided that a satellite
Lausanne WADA-accredited laboratory unit be
set within the 2006 Winter OG laboratory. The
relocation of the homologous blood transfusion
activity required a lot of organization. Some of
the personnel, reagents and instruments were
transferred for nearly 1 month to Torino. This
was the minimum time necessary to settle
and crossvalidate the method. Then the Swiss
Accreditation Service came to audit the laboratory and activities to give the official approval.
After accreditation, a total of 153 blood samples
were analyzed.
Time (min)
Time (min)
Time (min)
Time (min)
Time (min)
Figure 4. The Olympia stadium, for first time in history, opened to a women’s competition – the Athens 2004 Olympic
Games athletics shot-put – and the gold medalist reported positive for the steroid stanozolol. Screening chromatograms
are shown. (A) Methyltestosterone (internal standard; m/z 446.3036), (B) 16β-OH stanozolol (anabolic androgenic steroid stanozolol
metabolite; upper m/z 560.3650, below m/z 545.3415) and (C) 3’-OH stanozolol (anabolic androgenic steroid stanozolol metabolite;
upper m/z 560.3650, below m/z 545.3415).
1558
2008 Beijing OG
The partnership between WADA and pharmaceutical and biotechnology companies was
desired to reduce the gap between the marketing of a drug with potential doping properties
and its detection by anti-doping laboratories.
WADA, Roche and the Lausanne laboratory
decided together the right strategy to have a test
implemented as soon as possible. Well before
the drug was available on the market, an ELISA
test was designed for anti-doping requirements.
Finally, a fully validated and accredited test was
implemented in the Lausanne laboratory in summer 2008, approximately 6 months after the
release of the continuos EPO receptor activator
(CERA) on the European market (drug name:
| R eview
Key Term
Continuos EPO receptor
activator (CERA):
A PEGylated form of epoetin b
(NeoRecormon ®), a methoxy
polyethylene glycol-epoetin b.
This synthetic EPO has a
prolonged life in the human
body.
Table 1. Summary of the progress of the Olympic laboratories.
Olympic Games (year) Summary of progress
Munich (1972)
Montreal (1976)
Los Angeles (1984)
Seoul (1988)
Barcelona (1992)
Lillehammer (1994)
Atlanta (1996)
Nagano (1998)
Sydney (2000)
Salt Lake (2002)
Athens (2004)
Torino (2006)
Beijing (2008)
The beginning of the contemporary anti-doping system
GC–NPD screening of stimulant and narcotics, method still in use in several WADA-accredited laboratories
Use of autosamplers in GC–NPDs
Studies for GC retention parameters reproducibility
RIA steroids screening
GC–MS steroids confirmation
All samples analyzed for steroids including ratio of testosterone to epitestosterone
GC–MS steroids analysis with O-TMS, enol-TMS derivatives
GC–MS benchtop as modern instrumentation
b-blockers screening (but not eventually applied to Olympic Games)
LIMS
LC–MS for corticosteroids
Stanozolol metabolites
Mesocarb: the first of a series of Soviet stimulants
LC–MS confirmations of mesocarb and diuretics
Homemade LIMS
Clenbuterol detection
Blood collections
Homologous blood transfusion detection using immunoaffinity columns
GC–HRMS for steroids
Bromantane: the next in the Soviet stimulants series
GC–C–IRMS analysis
Salbutamol: oral or inhaled application discrimination
EPO indirect detection with ON/OFF models on hematological parameters
EPO direct detection of isoforms on IEF bands
NESP positives despite the rumors of undetectable EPO
GC–C–IRMS on epitestosterone
WADA Anti-Doping Code
Homologous blood transfusion using flow cytometry
Recombinant hGH using the isoforms detection in fluorescence immunoassays
HBOCs detection using filtered serum colorimetric, electroforetic, HPLC and LC–MS/MS analysis
LC–MS/MS screening analysis of all samples
LC–MS/MS direct urine injection for salbutamol, ephedrines and morphine
MADOL: another one of the US series of designer steroids
Positive steroid cases on IRMS
ISO17025 validation/accreditation of hematological analyzers
First ever women’s competition in Olympia stadium
Olympic record in doping cases: 29, 23 laboratory positives. five disqualifications and one admission (after
7 years)
IOC stores the Olympic samples for 8 years
Multitrace LC–MS/MS analysis for almost all class of prohibited small molecules
CERA detection at the end of Olympic Games in Lausanne – Paris WADA laboratories
Continuous reference in the analysis of prohibited substances with LC–MS does not mean that the GC–MS analysis is outdated. This reference is originated by the fact
that the LC–MS analysis is a newer technology compared with GC–MS and relatively more progress has been made.
C–IRMS: Combustion–isotope ratio MS; CERA: Continuos EPO receptor activator; HBOC: Hemoglobin-based oxygen carrier; HRMS: High-resolution MS;
IEF: Isoelectric focusing; IOC: International Olympic Committee; IRMS: Isotope-ratio MS; LIMS: Laboratory Information Management System; NESP: Novel
erythropoiesis-stimulating protein; NPD: Nitrogen–phosphorus detector; RIA: Radioimmunoassay; TMS: Trimethylsilyl; WADA: World Anti-Doping Agency.
1559
R eview |
MIRCER A®). This period corresponded to
the Summer OG in Beijing. Unfortunately, the
test could not be put in place for the 2008 OG
because of a lack of time. At that moment, it was
decided to ship all serum samples to Lausanne
at the end of the OG and have them reanalyzed
just for the detection of CERA. In the meantime,
a confirmation test was developed by the Paris
anti-doping laboratory [57–59] . The collaboration
between the Lausanne and Paris laboratories
and the reanalysis of the 1190 OG blood samples resulted in eight positive cases. This work
was possible thanks to an agreement between
WADA, Roche (MIRCERA producers) and the
Lausanne laboratory. This collaboration was
officially initiated on the 23 June 2006 and the
method was fully validated at the end of 2008.
Conclusion
In Table 1, a summary of the analytical progresses
performed by the OG laboratories since 1972 is
presented. This progress, to be implemented,
required the collaboration of the laboratory, IOC
and WADA scientists to overcome the stressful
environment of the periods before the OG.
In a few months, the 2012 London OG will be
held. The heritage of the methods of the indirect
doping detection of the ratio of testosterone to
epitestosterone of Donike [60] and the ON and
OFF models of the 2000 Sydney OG [33] is in
place. The science is preparing to apply a new indirect detection method of GH abuse through the
markers approach [61] , which is complementary
to the existing isoform method [54] applied in the
2004 Athens OG. The legal support for the GH
indirect method has already been prepared [62] and
introduced to the WADA Code [113] : “Presence
of a Prohibited Substance or its Metabolites or
Markers in an Athlete’s Sample.”
For 2788 years (776 + 2012) of written OG
history, the OG remain a human mega event and
the anti-doping system is ready to protect this
heritage by making continuous progress.
Future perspective
OG currently are and will continue to be a sports
mega event. The fight against doping is a continuous need, which will not end in the coming future. The threat of the degradation of the
sports impact maximizes during the OG due
to the publicity of the event and the concentration of the entire family of elite athletes. In
this framework, the Olympic laboratories shall
apply all the latest technological progresses to
cover the gap between doping and anti-doping
and protect the sports from degradation. As the
doping practices are shifting from ‘exogenous’ to
the ‘endogenous’ human organism, more indirect methods will become routine tools for the
WADA-accredited laboratories.
Acknowledgements
C Georgakopoulos wishes to thank P Simitsek and ME
Spyridaki for the hGH data presentations and friends from
the World Anti-Doping Agency-accredited laboratories who
assisted in writing this article.
Executive summary

The Olympic Games (OG) is a mega sports event, which brings the biggest family of elite athletes together and huge publicity, but there is
also a great threat to sport because of doping.

Olympic laboratories face the challenges of the introduction of new technologies into their analytical repertoire and of the multiplication of
their analytical capacity in volume and in time.

The anti-doping organization of the Munich OG of 1972 was the beginning of the modern anti-doping system. The
GC–nitrogen–phosphorus detector method developed by Donike is still in use in World Anti-Doping Agency-accredited laboratories.

In the 1976 Montreal OG, the most challenging class of prohibited substances, anabolic steroids, started to be detected.

International Olympic Committee-accredited laboratories in Los Angeles 1984, Seoul 1988 and Barcelona 1992 created the tradition of the
highest analytical quality work in the most demanding environment of the OG.

The application of the isoelectric focusing electrophoretic method for EPO ended the story of the undetected substance in Sydney 2000.

In Sydney 2000, ON/OFF models were developed that were the basis for indirect blood detection methods and the Athlete’s Biological
Passport.

Athens 2004 had an OG record in doping with 29 positive cases, and in regard to new methods to be applied, three new technologies were
setup.

Retesting of negative samples to apply new technologies became a reality with the detection of the continuos EPO receptor activator in
samples at Beijing 2008.

The London 2012 Olympic laboratory faces the new challenge of indirect methods in the fight against doping – indirect markers to detect
abuse of growth hormone.
1560
Financial & competing interests
disclosure
The authors have no relevant affiliations or financial
involvement with any organization or entity with a
financial interest in or financial conflict with the
subject matter or materials discussed in the manuscript. This includes employment, consultancies,
honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or
royalties.
No writing assistance was utilized in the
production of this manuscript.
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