JOURNAL OF MASS SPECTROMETRY

J. Mass Spectrom. 2008; 43: 903907

Published online 20 June 2008 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/jms.1455
SPECIAL FEATURE:
COMMENTARY
New and old challenges of sports drug testing
Francesco Botr ` e
1,2
1
Laboratorio Antidoping FMSI, Largo Giulio Onesti 1, 00197 Roma RM, Italy
2
Dipartimento per le Tecnologie, le Risorse e lo Sviluppo, Sapienza Universit ` a di Roma, Via del Castro Laurenziano 9, 00161 Roma RM, Italy
Received 17 April 2008; Accepted 21 May 2008
This brief note gives a general overview on the activity of the antidoping laboratories accredited by the
World Anti-Doping Agency (WADA), outlining the evolution, over the last four decades, of the analytical
methods and techniques in the detection of prohibited substances and methods.
Special emphasis is given to the future trends of the ght against doping in sports, as seen from the
perspective of a laboratory scientist, in the wider context of fair play, health protection, and perception of
the activity of the antidoping laboratories by the general public. Copyright 2008 John Wiley &Sons, Ltd.
KEYWORDS: sport doping; antidoping analysis; gas-chromatography/mass spectrometry; liquid chromatography/mass
spectrometry; isotopic ratio mass spectrometry
THE EVOLUTION OF THE PROHIBITED LIST,
AND THE ROLES OF WADA AND WAADS
The problem of drug abuse in sports in the modern age
was rst tackled by the International Olympic Committee
(IOC) in the 1960s, with a rst denition of doping given
in 1964 and the foundation of the Medical Commission in
1967. After a pilot project carried out in the summer of 1968
at the Olympic Games of Mexico City, the ofcial antidoping
tests performed on the occasion of a multisport, international
event, were rst initiated in 1972, at the Summer Olympic
Games of Munich.
Forty years ago, the only prohibited substances were
those capable of producing a signicant effect on sports
performance only if administered, in sufcient amounts,
right before or during the competition. Viewed through the
eyes of a doping analyst of today, those analytical challenges
appear now relatively easy, for the following reasons:
1. All the forbidden substances were represented by nonen-
dogenous compounds.
2. Their pharmacokinetic prole had previously been stud-
ied in sufcient detail.
3. To be effective, their intake had to take place not more
than several hours before the collection of the sample, so
that their concentration in the urine (the only biological
uid considered at that time) was still relatively high.
In the last four decades, the prohibitedlist progressively
expanded, being periodically updated, rst by the IOC

Correspondence to: Francesco Botr` e, Dipartimento per le

Tecnologie, le Risorse e lo Sviluppo, Sapienza Universit` a di Roma,
Via del Castro Laurenziano 9, 00161 Roma RM, Italy.
E-mail: francesco.botre@uniroma1.it
Medical Commission itself, and then by the World Anti-
Doping Agency (WADA), to reach its present format
(Table 1
1
). Also, the number of testing laboratories increased,
reaching the present tally of 33 laboratories (Table 2),
analyzing more than 200 000 biological samples per year.
Briey, three main periods can be identied in the race
between cheaters and testers (also outlined in Table 3):
1. anearlyage, outlinedabove, correspondingtothe abuse of
in-competition drugs: most, if not all, laboratorymethods
for the detection of doping substances of this kind were
based on gas chromatography;
2. the androgenic anabolic steroids (AAS) age (includ-
ing their endogenous prototype, testosterone), starting
in the early 1970s, in which the administration of the
performance-enhancing drugs took place mainly in peri-
ods of training and not (only) in competition; this era
also signs the development of specic analytical methods
based on gas-chromatography/mass-spectrometry and,
later on, on liquid-chromatographymass spectrometry,
also in their more advanced congurations (GC/high res-
olution mass spectrometry (HRMS), GC/MS-MS, GC/
isotope ratio mass spectrometry (IRMS), LC/MS-MS);
3. the protein chemistry and molecular biology age, which
follows the application of routine techniques in molecular
biology and genetic engineering by the pharmaceutical
companies, with the production (and abuse in sport dop-
ing) of peptide hormones: analytical techniques extended
beyond the borders of chromatography/spectrometry,
including immunological techniques and, in general, all
those analytical approaches that are generally typical of
a laboratory of protein chemistry. Chronologically, this
period also includes the recourse to blood doping, i.e.
Copyright 2008 John Wiley & Sons, Ltd.
904 F. Botr` e
Table 1. The World Anti-Doping code. The 2008 prohibited
list
Substances and methods prohibited at all times (in and out
of competition)
Prohibited substances
S1. Anabolic agents
1. Anabolic androgenic steroids (AAS)
a. Exogenous AAS (e.g. methyltestosterone, nandrolone,
stanozolol)
b. Endogenous AAS (e.g. testosterone, androstenedione,
DHT, DHEA)
2. Other anabolic agents (e.g. clenbuterol, selective
androgen receptor modulators)
S2. Hormones and related substances (e.g. EPO, hGH, IGFs,
gonadotropins, insulins)
S3. Beta-2-agonists (e.g. salbutamol, salmeterol, terbutaline,
formoterol)
S4. Hormone antagonists and modulators (e.g. antiestrogens,
myostatin inhibitors)
S5. Diuretics and other masking agents (e.g. diuretics,
epitestosterone, probenecid, alpha-reductase inhibitors,
plasma expanders)
Prohibited methods
M1. Enhancement of oxygen transfer (e.g. blood transfusions,
use of blood derivatives and analogs)
M2. Chemical and physical manipulation (e.g. tampering,
intravenous infusions)
M3. Gene doping
Substances and methods prohibited in-competition
S6. Stimulants (e.g. amphetamines, cocaine, strychnine,
ecstasy-like drugs)
S7. Narcotics (e.g. morphine, opioids)
S8. Cannabinoids (e.g. hashish, marijuana)
S9. Glucocorticosteroids
Substances prohibited in particular sports
P1. Alcohol
P2. Beta-blockers
bloodtransfusions andadministration of substances capa-
ble of increasing the capacity of oxygen transport to the
muscle.
In addition to the above, a fourth period is feared by
many as the next step in the illicit search for the better
performance-enhancingdrugs andmethods: the gene doping
age. It is expected, however, that gene doping will not
develop before gene therapy will be practically available.
Unluckily for the testers (and for the health of the ath-
letes), the distinction in these three ages did not occur as it
does in a relay competition; instead, it proceeded and contin-
ues to proceed with an overlapping use of drugs/methods
belongingto eachone of the different periods. Inother words,
the adverse analytical ndings (formerly known as positive
cases), for instance, amphetamines, did not end with the
beginning of those for AAS or peptide hormones. This is also
shown by the statistical data the WADA publishes yearly on
its website (www.wada-ama.org). It follows that a further,
and not at all minor, task for the accredited laboratories
is to ensure that the internal organization of the analytical
Table 2. The 33 antidoping laboratories accredited by the
World Anti-Doping Agency and their geographical distribution
Africa: South Africa (Bloemfontein), Tunisia
(Tunis)
Americas: Brazil (Rio de Janeiro), Canada
(Montreal), Colombia (Bogota), Cuba (La Habana),
United States (Los Angeles, Salt Lake City)
Asia: China (Beijing), Korea (Seoul), Japan (Tokyo),
Malaysia (Penang), Thailand (Bangkok)
Europe: Austria (Seibersdorf), Belgium (Ghent),
Czech Republic (Prague), Finland (Helsinki),
France (Paris), Germany (Cologne, Kreischa),
Greece (Athens), Italy (Rome), Norway (Oslo),
Poland (Warsaw), Portugal (Lisbon), Russian
Federation (Moscow), Spain (Barcelona, Madrid),
Sweden (Stockholm), Switzerland (Lausanne),
Turkey (Ankara), United Kingdom (London)
Oceania: Australia (Sydney)
methods is compatible with the expected workload, ensur-
ing, on a daily basis, the parallel screening of hundreds of
target compounds in all samples.
2
Despite the increased complexity of the list, especially
if compared to its ancestor of almost half a century ago,
the analytical challenge for the experts working in the eld
of drug testing in sports remained basically the same: to
improve the effectiveness of antidoping tests, this meaning
to reduce as much as possible the percent of false-negative
cases, avoiding at the same time the risk of any false-positive
result.
3,4
More specically, the main tasks for the accred-
ited laboratories are (1) to widen the range of detectable
substances and methods, also discriminating, whenever nec-
essary, whether a substance is produced endogenously or
administered exogenously; (2) to prolong the interval of time
after administrationduringwhichthe abuse of a drugand/or
the recourse to a prohibited method (e.g. blood transfusion)
can be detected; and (3) to increase the solidity and the inter-
laboratory reproducibility of the analytical results, thus also
allowing the worldwide application of any newly developed
method. These tasks are also achieved with the scientic
support of the World Association of Anti-Doping Scientists
(WAADS), an International scientic society, constituted in
2001, in which all WADA-accredited laboratories are rep-
resented, promoting the sharing of knowledge among the
accredited laboratories and basic and applied research in the
development of new analytical methods.
The cooperation between WADA and WAADS is a key
component in the progress of the scientic knowledge in
doping analysis, which in turn represents the main driving
force capable of increasing the efcacy of any antidoping
strategy. In the framework of the WADAWAADS interac-
tion, signicant results have already been achieved so far:
the great majority of the most recent advancements in the
eld of antidoping research has originated due to increased
cooperation among WAADS, especially in the last fewyears,
in the framework of research projects funded by the WADA.
This has proved to be a winwin strategy.
Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 903907
DOI: 10.1002/jms
New and old challenges of sports drug testing 905
Table 3. Chronological evolution of the main challenges and solutions in doping control analysis
Period Challenge Solution
Origin early 1970s Stimulants, narcotics, drugs of abuse GC/NPD
Mid 1970s Synthetic anabolic androgenic
steroids (AAS)
GC/MS
19801990 Beta-blockers, diuretics, cannabinoids,
glucocorticoids
GC/MS, immunoanalysis (for
screening only)
Early to mid-1990s Low concentration of AAS by
GC/HRMS
High sensitivity GC/MS techniques
(e.g. GC/HRMS, GC/MS-MS)
Mid- to Late 1990s Human chorionic gonadotropin Immunoanalysis (mainly ELISA)
Endogenous Testosterone and/or
precursors
GC/IRMS, also in combination with
longitudinal proling
Mid-19902000 Erythropoietin and analogs Isoelectrofocusing with double
blotting
20002005 Designer steroids LC/MS and LC/MS-MS
Hormone and hormone receptors
modulators
GC/MS and LC/MS
2005present Peptide hormones Advanced LC/MS-MS techniques,
used in combination with molecular
biology and protein chemistry
technologies
2003present Blood doping Direct tests for hemoglobin-based
oxygen carriers (HBOCs) and for
homologous blood transfusions;
indirect tests (e.g. longitudinal
proling, biological passport) for
other forms of blood doping
2008. . . Gene doping Longitudinal proling (biological
passport), advanced molecular
biology methods
THE CURRENT SITUATION: HOW THE
INVISIBLE BECOMES VISIBLE
As stated above, the current prohibited list includes
hundreds of substances, ranging from volatile stimulants
to modied polysaccharides and glycoproteins, considering
also an increasing variety of doping methods, including
blood transfusions and other forms of blood doping.
With few exception, most of the substances inserted in
the WADA prohibited list can be detected by the accred-
ited laboratories. The detection, and, when so required by
the WADA rules, the quantitative determination of low
molecular weight (i.e. <700800 Da) substances (stimu-
lants, narcotics, anabolic agents, glucocorticosteroids, beta-2-
agonists, beta-blockers, diuretics/masking agents, antiestro-
gens, cannabinoids) is nowadays possible, mainly by chro-
matographicspectrometric techniques. This means that any
known xenobiotic (and/or any known metabolite) excreted
in urine in a concentration higher than the limit of detection
of the method is not invisible: it can be detected, and the
result of the analysis (of course if carried out in compli-
ance with the WADA rules) can be reported as an adverse
analytical nding.
Chromatographymass spectrometry (with a progres-
sive shift from GC/MS to LC/MS and LC/MS-MS, also
including the most recent development in the eld of high
resolution, micro, nano- and fast chromatography) is still the
gold standardfor antidoping analysis, allowing the detection
of trace compounds in complex mixtures in concentration
ranges that can be far below1 ng/ml. It can be easily forecast
that mass spectrometry will be increasingly used also in the
eldof highmolecular weight analytes, primaryamongthem
being peptides and glycoproteins.
5
Synthetic insulins are
indeed the rst examples of high molecular weight analytes
that can be effectively detected only by LC/MS-MS-based
techniques, which allow to distinguish the synthetic forms
of the hormone both short-acting and long-acting from
the endogenous one.
6
The goal of the scientists working in
the 33 WADA-accredited laboratories is to annul or at least
to minimize the gap between the substances included in the
list and those that can practically be detected. Signicant
progress in this direction has been recorded in the last few
years.
In general, a substance can be invisible for the following
reasons:
1. The substance is unknown, or (which is the same, fromthe
point of viewof the antidoping laboratory) its metabolism
is unknown: this means that there is no known analyte at
which to aim the analysis.
2. The concentration of the substance in the considered
biological matrix is below the limit of detection of the
analytical method.
Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 903907
DOI: 10.1002/jms
906 F. Botr` e
3. The substance is very similar or even identical to
an endogenous substance, i.e. a naturally produced
substance which is normally present in all the biological
samples collected for the antidoping analysis.
4. The administration of a forbidden drug is masked by the
use of other substances that are not yet recognized as such
and therefore not yet included in the list, in the class of
masking agents.
Nonetheless, a substance cannot remain invisible forever.
Antidoping research proceeds restlessly. Several milestones
in the race between dopers and testers conrm such a trend.
Synthetic testosterone was invisible, and its abuse could
only be proved indirectly, by longitudinal tests, until a
method based on the ratio between the two stable carbon
isotopes was developed and routinely applied in antidoping
analysis.
7
Recombinant erythropoietin was invisible until a
specic test, including a specically developed step (the so
called double blotting) in an otherwise almost standardized
method based on isoelectrofocusing (IEF) combined with
immunochemiluminescence, was developed.
8
Tetrahydro-
gestrinone (THG) and other designer steroids were invisible
until they were discovered, isolated, and characterized by
the antidoping laboratories.
911
Methods for the detection of
selective androgen receptor modulators (SARM) were devel-
oped and are already available, even before the marketing
of this new class of drugs.
12
Other methods, also based on
LC/MS-MS techniques, can, in principle, ensure the detec-
tion of previously unknown doping substances.
1316
The
number of invisible peptidic and glycoproteic hormones is
also getting smaller and smaller with specic tests devel-
oped not only for new forms of erythropoietin (EPO),
17
but
also for human growth hormone, insulins, and other syn-
thetic analogs of peptide hormones.
18
Increasing attention is
also being devoted to the potential use of masking agents
and methods, whose detection should also involve extra
laboratory resources, especially if the masking strategy (e.g.
catheterization, addition of sufcient amounts of proteases
to the urine samples) is activated inside the doping control
station, that is at the moment of production/collection of the
sample.
In short, the period of invisibility depends on many
parameters, and can range from a few days to several years;
to tell the truth, new invisible substances and methods are
expected to show up in the future but as far as the scientic
and technologic development continues to progress, and as
far as the antidoping scientists continue to be involved in
research projects running parallel to their routine activity,
none of the few forms of doping will remain undetectable
forever.
Nonetheless, a list with no undetectable substances will
not represent the end of the research activity of the labs, since
the original development of the method for a new target (a
drug/metabolite either previously unknown and/or newly
added to the prohibited list) has to be made as compliant
as possible with all the other methods followed by the lab
to routinely screen up to several hundreds of samples per
week. In other words, it is not enough for a substance to be
detectable in theory: that substance has to be detectable in
practice, this meaning within the routine analytical activity of
any accredited laboratory, whose average workload includes
the screening analysis for hundreds of other substances in
each urine sample, on a large number of samples everyday,
for approximately 300 days or more per year. Optimizing
the result/resources ratio in a lab is also a fundamental
form of applied research, allowing to dedication of more
energy (manpower, instrumentation, time, money) to the
development of new methods and to the reduction of the
response times.
Finally, it has to be stressed that laboratories can con-
tribute remarkably toward the general improvement of the
effectiveness of antidoping tests but a similar effort has also
to be made by other components (stakeholders) of the anti-
doping system. Adequate planning of effective strategies
for antidoping tests is as important as the development of
effective laboratory methods. No technologically advanced,
costly, time-intensive laboratorymethodcanindeedcounter-
balance a single, but critical, pitfall in the selection, collection,
and transport of the samples. In most aspects, the situation
is very similar to that occurring in environmental analyses:
a laboratory method can be as effective as it is scientically
possible, ensuring, for instance the detection of a specic
environmental contaminant in the sub-ppt concentration
range; but there is no way that the same method can detect
a case of pollution if the sample that reaches the laboratory
is clean; and if its cleanness is due to the fact that the pro-
cedures for the selection and collection of the sample and
especially its timing were not planned in sufcient detail.
SOME CONCLUSIONS
A rst consideration that should be outlined is that the
antidoping laboratories keep pace with the evolving scenario
of doping substances and methods: the forbidden list
expanded tenfold over the last 40 years; the detection limits
of the laboratory methods improved by at least of three
orders of magnitude, from the submicrogram per milliliter
to the subnanogram per milliliter range; the response times
can be as short as few hours from the receipt of samples
on the occasion of tests carried out at the Olympic Games
and on the occasion of major international events; quality
assurance is warranted not only by ISO 17 025 accreditation
(a mandatory prerequisite for the accredited labs in this
third millennium) and by WADA accreditation (which also
includes, but it is not limited to, successful analysis of
samples from prociency tests and double blind tests),
but also by a series of inter-laboratory evaluations and
educational studies, mostly carried out within the WAADS
network.
The recent, fruitful cooperation between two relatively
young bodies, like the WADA and the WAADS, was already
mentioned. There is another aspect this cooperation should
consider, and it is to promote all the efforts necessary to
hold up the credibility of laboratory results, to raise it to
the same level of other, more traditional, sport results.
A representative example of this situation is given by
the relatively recent need of calculating, and possibly
minimizing, the measurement uncertainty (clearly relevant
to threshold substances only) a sensitive scientic issue
Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 903907
DOI: 10.1002/jms
New and old challenges of sports drug testing 907
that deserves to be dealt with in depth. Nonetheless,
researchers involved in drug testing in sports may nd it
difcult to accept that all results of the measurement of
sports performance (i.e. time in a race; distance in a disc
throw competition; weight in weightlifting) are given by
simple values, without any interval of condence; while
urinary concentration values should be expressed as mean
measurement uncertainty (including also the coverage
factor). Is the measurement of a world record really so
much more precise (and accurate) than the measurement of
the concentration of a threshold substance in urine? Or is
it rather a matter of perception, by the athletes and by the
general public, on which a fundamental role is also played by
the rapidity by which the result is calculatedandshownup?
Nonetheless, would it not be more appropriate to express
the record of a 100 m race not as, let us say, 10
00
000, but
rather as, let us say, 10
00
000 0
00
001?. This is, of course, a
provocative approach, but it shows how useless it can be to
try to increase the scientic solidity of a result whenever the
credibility of the system is not universally accepted, or, even
worse, questioned.
A nal remark deals with the health risks of doping
substances and methods: to protect the athletes means,
rst of all, to protect their health. This cannot be achieved
simply by the antidoping tests, but by promoting the culture
of a doping-free sport and circulating information on the
many potential health risks consequent to the misuse of
substances/methods included in the prohibited list.
19,20
A deeper knowledge of the actual risks of the abuse of
doping substances and methods should be promoted, going
beyond the limits imposed by the current forensic approach,
and considering the toxicological relevance not only of
markers of exposure but also of markers of effect of doping
substances and methods.
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