Journal of Analytical Sciences, Methods and Instrumentation, 2011, 1, 19-24
doi:10.4236/jasmi.2011.12003 Published Online December 2011 (http://www.SciRP.org/journal/jasmi)
19
Substance Identification in Anti-Doping Control
by Means of Mass Spectrometry. Data Reduction
and Decision Criteria
Mats Larsson
Department of Physics, AlbaNova University Center, Stockholm University, Stockholm, Sweden.
Received September 23rd, 2011; revised October 19th, 2011; accepted November 11th, 2011.
ABSTRACT
A real doping case for which the national-level reviewing body deemed it probable that a misidentification of the na-
tional-level athlete’s sample occurred at the WADA accredited laboratory, thus making the athlete in this case strictly
anonymous, is used to discuss criteria for data reduction and tolerance windows in GC-MS and LC-MS/MS. Stricter
criteria for data reduction would remedy the present ambigu ities.
Keywords: Substance Identification, Anti-Doping, Mass S p ectrometry, Data Reduction
1. Introduction
A high-profile Swed ish female athlete, winner of an Olym-
pic gold medal and several World and European cham-
pionship s, expressed a few years ago in a TV -prog ramme
how the fear of the presence of a prohibited substance in
her sample during a doping control had made her almost
paranoid; leaving a water bottle out of sight for only a few
seconds could be enough for its being replaced with a con-
taminated bottle. She has never tested positive, but the strict
liability imposed on athletes by the World Anti-Doping
Code (WADC) leaves an extremely narrow margin of
mistakes for athletes. The bar for an athlete to bear no fault
or negligence for the presence of a prohibited substance
in an athlete’s sample is set extremely high; even to bear
no significant fault or negligence require extraordinary
circumstances.
When an athlete is convicted for an anti-doping rule
violation according to WADC 2.1 §, usually the only evi-
dence available to the anti-doping organisation is the pre-
sence of a prohibited substance, metabolite or marker in
the athlete’s sample, as determined by a laboratory accre-
dited by the World Anti-Doping Agency (WADA). Pro-
vided that the analysis has been performed according to
WADA’s International Standard for Laboratories (ISL),
the analysis cannot be challenged by the athlete. Under
the current WADC, the athlete must not only show that
the laboratory made a departure from the ISL but also that
this departure reasonably could have caused the adverse
analytical finding; this makes the laboratory result in
practise unchallengeable for an athlete.
Given the enormous consequences for an athlete to be
found guilty of an anti-dop ing rule violation, and the sin-
gular importance attributed to the laboratory results, it is
of course imperative that the science underpinning sub-
stance identification is impeccable. There are views ex-
pressed in the scientific literature that the underpinning
scienc e and the app lication of g uidelines in substance iden-
tification have shortco mings, and I will briefly review this
literature. However, in the present paper the data reduce-
tion and confirmation criteria will be in focus. In particular,
I will use a concrete case where the n ational-level r eview-
ing body determined that there was a significant probabil-
ity that the laboratory had mixed up the samples. Thus, in
the example I will use, the athlete is strictly anonymous.
2. Brief Overview of the Doping Literature
The literature on doping with a focus on analytical chemis-
try by means of chromatography and mass spectrometry
fa lls broadly into three categories. In the first category are
the original research articles and review articles concern-
ing detection of prohibited substances by various mass
spectrometric techniques. The reviews by Thevis and
Schänzer [1,2] give a good overview of this literature and
the implementation of new techniques. The WADA In-
ternational Standard for Laboratories (ISL) [3] encour-
ages the accredited laboratories to publish the results of

Substance Identification in Anti-Doping Control by Means of Mass Spectrometry. Data Reduction and Decision Criteria
20
their research in peer-reviewed journals, something which
accounts for part of the papers referenced in [1]. The con-
ventional gas chromatography-mass spectrometric tech-
niques (GC-MS) have been the work horses in the labo-
ratory anti-doping fight, but the trend is that liquid chro-
matography-(tandem) mass spectrometry (LC-MS/MS) is
emerging as the most powerful technique in doping con-
trol analysis [2]. One can note, however, that according
to the WADA International Standard for Laboratories [3],
the GC-MS or LC-MS technique is “··· the analytical
technique of choice for confirmation of Prohibited Sub-
stances, Metabolite(s) of a Prohibited Substance, or Marker
(s) of the Use of a Prohibited Substance or Prohibited
Method.” (5.2.4.3.1.2 in ISL).
The second category concerns criteria in chromatog-
raphy and mass spectrometry which should ensure that
the unambiguous presence of a substance in a biological
specimen has been established. The classic paper in this
category is the one by Sphon [4], and it is surprising how
little the confirmation criteria has changed since this pio-
neering work . The presen t methodology as used by WADA
and o ther organisations such as the Europe an Council (EC),
Food and Drug Administration (FDA, USA), etc., which
is not uniform, has been reviewed by River [5] and Van
Eenoo and Delbeke [6 ].
To the third category belongs papers that critically point
out weaknesses in substance identification [7,8] by mass
spectrometry, or other possible weaknesses in doping con-
trol such as calculation of decision limits (with papers also
appearing rebutting the criticism) [9-16], the application
of decision criteria [17-19], validation of specificity [20],
and flawed laboratory data in a specific doping case (and
rebuttal) [21-23].
The most severe critique of the doping control system,
however, was levelled in a Nature Editorial [24]:
“Nature believes that accepting ‘legal limits’ of spe-
cific metabolites without such rigorous verification goes
against the foundational standards of modern science, and
results in an arbitrary test for which the rate of false posi-
tive and false negatives can never be known”.
The same issue of Nature contained a commentary by
Berry [25], which used the Floyd Landis doping case (see
also [21-23]) as starting poin t for a critical scru tiny of the
science of doping, in particular from the point of v iew of
statistics and logic. Naturally these critical views [24,25]
in one of the leading scientific journals in the natural
sciences did not go unchallenged. Scientists associated
with doping laboratories [26] and WADA [27] delivered
rebuttals, but Nature also opened for a few further criti-
cal remarks [28,29].
3. Experiment and Data Reduction
The data to be discussed here have the advantage that de-
spite coming from an actual doping case, they have never
been scrutinized by an anti-doping reviewing body for the
reason that the anti-doping agency and the WADA accre-
dited laboratory failed to convince the national-level re-
viewing body to their comfortable satisfaction that there
had not been a misidentificatio n of samples, and the case
was dismissed on these grounds only. According to the
first page of the Laboratory Documentation Package
(LDP) from the Doping Control Laboratory, Karolinska
University Hospital, signed by the laboratory director in
accordance with [30], a sample number different from the
one of the accused athlete, but belonging to the same batch,
was assigned to the internal code used by the laboratory
during the analysis. This means that the athlete whose ana-
lyzed sample will be discussed is strictly anonymous des-
pite deriving from a real case. Thus, we completely avoid
the discussions whether a decision by a disciplinary court
can be taken as “proof” of the scientific correctness of the
statements brought forward by expert witnesses [10,13],
and the strong emotions stirred in the Floyd Landis case.
The sample of the anonymous athlete was claimed to
containing 4.9 ng/mL of 3’-OH-Stanozolol, a metabolite
of the anabolic androgenic steroid Stanozolol. The level
is about a factor of two higher than the so-called Mini-
mum Required Performance Level (MRPL) [31] of 2
ng/mL. This level is sometimes misunderstood to mean a
level below which the presence of 3’-OH-Stanozolol does
not con stitute an adverse analyti cal finding. This is wrong ,
and the level has been introduced to ensure that all WADA
accredited laboratories have the capacity to report the pre-
sence of forbidden substances and metabolites in a uni-
form way. Some laboratories are able to report lower con-
centrations than others, and concerning the specific me-
tabolite 3’-OH-Stanozolol, it is not a threshold substance.
The identification criteria for substances in urine sam-
ples combine chromatographic separation and mass spec-
trometry. The chromatographic step is of no concern here
and will not be discussed further. Mass spectrometry uses
the sequence ionization, fragmentation, and detection.
Electron impact ionization at 70 eV, where the ionization
cross section typically peaks for many small molecules,
is a “hard” ionization method, and the molecular ion will
undergo unimolecular decompos ition on the s timescale.
The fragmentation pattern reveals information about the
parent ion, but the pattern can differ depending on what
system is used for mass separation of the ionized frag-
ments, and a reference standard is required for compare-
son. When “soft” ionization is used, such as electrospray
ionization, leading to less unimolecular decay, collision
induced dissociation (CID) is used (MS/MS), and also in
this case it is well known that the CID fragmentation
pattern can depend significantly on the employed MS/MS
technique [32]. The criteria for substance identification

Substance Identification in Anti-Doping Control by Means of Mass Spectrometry. Data Reduction and Decision Criteria21
are discussed in [5,6] for different organisations (include-
ing WADA), and th e implemen tation of the sp ecif ic WADA
criteria are outlined in technical documents TD2003IDCR
[33] (prior to September 1, 2010) and TD2010IDCR [34]
(effective date September 1, 2010).
Table 1 shows the results for 3’-OH-Stanozolol from
the anonymous sample and a compari son wi t h the standard,
both taken from the LDP. The parent ion [M] is 3’-OH-
Stanozolol-3TMS+ with mass 560 Da. One can make se-
veral observations using Table 1. The WADA Technical
Document TD2003IDCR [33] (which was the document
in force when the analysis was made) requires three di-
agnostic ions to be used, yet the standard operating pro-
cedure of the WADA accredited laboratory relaxes this
requirement to only two ions; the peak at 254.1 Da hardly
qualifies as a diagnostic ion since it is normalized to 100%
for both the standard and the sample. The mass difference
between the two diagnostic ions is 15 Da, and it is very
likely that the peak at 545.3 Da corresponds to [M-CH3]+.
de Zeeuw [7] specifically points out th at this ion does not
provide much ad dition al d iagn os tic informatio n, ho weve r,
there is nothing in WADA’s technical documentations
preventing the analyst from using it. It is common, de
Zeeuw [7] notes, to not paying attention to the d iagnostic
value of the fragments. Most striking with the table is
that the two diagnostic ions only marginally fall within the
required acceptance range [33,34]. This requires the peak
at 545.3 Da to be further scrutinized. Figure 1 shows a se -
lected ion m onitoring chroma togram taken from t he LDP.
Table 1. Relative ion abundances in a GC-MS analysis of
3’-OH-Stanozolol at the Doping Control Laboratory, Karo-
linska University Hospital.
Standard
(m/z in Da) Relative abundance (%) Acceptance range (%) Sample (%)
254.1 100.0 - 100.0
545.3 60 50 - 70 51
560.3 58 48 - 68 49
Figure 1. Chromatogram from selected ion monitoring at
retention times 7.60 - 8.00 min recorded at the Doping Con-
trol Laboratory, Karolinska University Hospital (repro-
duced from the LDP) by means of GC-MS.
The relative abundance of the 545.3 peak “[···] shall
preferably be determined from the peak area or height of
integrated selected ion chromatograms” [33]. In this par-
ticular case, the standard operating procedure required
the peak area to b e integrated from “valley to v alley”, i.e.
from retention time 7.77 min to 7.83 min. It is obvious
from a simple visual inspection that the broad peak at
retention time 7.862 min will make a larger contribution
to the area in the interval 7.77 - 7.83 min than the frac-
tion of peak 7.804 min (545.3 Da) that falls outside of
this region. Taking this into account, the relative abun-
dance of ion 545.3 Da is estimated to be 46% and not
51%. The relative abundance thus falls outside of the
acceptance range, and the sample does not fulfil the WADA
criteria to constitute an adverse analytical finding.
There is a caveat to this reasoning, and this is the fol-
lowing. The technical document TD2003IDCR [33] and
the standard operating procedure do not require the ana-
lyst to proceed as just outlin ed, i.e. using the correct peak
area, and it is only in TD2010IDCR [34] the analyst is
permitted (but not required) to use computer-assisted peak-
resolving software. Thus, a procedure which would not
be tolerated in my department’s course in experimental
physics for freshman physics students is part of the stan-
dard operating procedure of a WADA accredited labora-
tory and accepted by the most recent WADA technical
document for mass spectrometry [34].
WADA accredited laboratories use more than one me-
thod to determine whether a forbidden substance or me-
tabolite is present in a sample, and in the present case th e
laboratory also used LC-MS/MS, see Figure 2. Table 2
shows the results. Four d iagnostic ions w ith a r elative abun-
dance different from 100% are now within the acceptance
range. Does this mean that the presence of 3’-OH-Stano-
zolol in the sample now has been unambiguously estab-
lished? No, it does not, on the contrary. In Table 1 in ref.
[34] WADA has tightened the maximum tolerance win-
dows for relative ion intensities. It is not exp lained in de-
tail the rationale for this, however, it is clear from TD
2010IDCR [34] that a paper by Stein and Heller [35] has
been deemed important.
We now apply the new accep tance ranges in force since
September 1, 2010 [34] to the present case, see Table 3.
Now only one out of four diag nostic ions falls within the
acceptance range and the results do not fulfil WADA’s
requirements for being reported as an adverse analytical
finding.
The technique of LC-MS/MS is widely used by WADA
accredited laboratories in the anti-doping fight. It is known
only to WADA how many athletes that have been con-
victed based on the old rules [33], which with the new
rules would have been acquitted. Since many thousand
doping controls are performed all over the world ea ch year,

Substance Identification in Anti-Doping Control by Means of Mass Spectrometry. Data Reduction and Decision Criteria
22
Figure 2. Chromatogram from selected ion monitoring at
retention times 1.50 - 2.50 min recorded at the Doping Con-
trol Laboratory, Karolinska University Hospital by means
of LC-MS/MS (reproduced from the LDP). The arrows
show the peaks given in the left columns of Tables 2 and 3.
Table 2. Relative ion abundances in an LC-MS/MS analy sis
of 3’-OH-Stanozolol at the Doping Control Laboratory,
Karolinska University Hospital.
Standard
(m/z in Da) Relative abundance (%)
Acceptance range
(%) according to
TD2003IDCR [33] Sample (%)
97.2 100.0 100.0 100.0
93.3 26.8 20.0 - 33.5 31.5
107 25.9 19.4 - 32.4 31.6
91.3 17.6 7.6 - 27.6 11.7
95.1 18.4 8.4 - 28.4 24.1
Table 3. Relative ion abundances in an LC-MS/MS analy sis
of 3’-OH-Stanozolol at the Doping Control Laboratory,
Karolinska University Hospital.
Standard
(m/z in Da) Relative abundance (%)
Acceptance range
(%) according to
TD2010IDCR [34] Sample (%)
97.2 100.0 100.0 100.0
93.3 26.8 21.4 - 32.2 31.5
107 25.9 20.6 - 31.1 31.6
91.3 17.6 14.1 - 21.1 11.7
95.1 18.4 14.7 - 22.1 24.1
it would seem unlikely that the author has happened to
come across the one and only case.
4. Conclusions
In this paper I have demonstrated that the method of data
reduction in a doping control case can make the differ-
ence between an adverse analytical finding and acquit-
tance. This is highly undesirable, in particular since the
more rigorous data reduction is the scientifically sound
one and leads to a non-fulfilment of the WADA criteria
for substance identification in a particular case. The new
technical document TD2010IDCR represents a step for-
ward [34], but as long as the use of peak-resolving soft-
ware is only permitted, not mandatory, ambiguities will
prevail. As long as the text in the technical document is
permissive, an athlete can never challenge the laboratory
result at the Court of Arbitration fo r Sport (CAS).
The importance of the exact phrasing in WADA’s
technical documents is made apparent in a recent award
delivered by CAS [36] in which the technical documents
are referred to no less than 37 times, and every “t” crossed
and “i” dotted in CAS reading of the documents. It is
beyond the scope of the present article, and beyond the
competence of the author, to review all technical docu-
ments of WADA, however, leaving open to the analyst
how to derive an ion’s relative intens ity is a flagrant loop
hole in anti-doping science.
The new, sharpened tolerance windows imposed by
WADA [34 ] are welcome but leaves open the uneasy ques-
tion concerning how many adverse analytical findings that
have been reported using the old criteria, which would
appear as negatives using the new criteria. Or to rephrase
the questio n: how many false-positives were reported prior
to September 1, 2010?
5. Acknowledgements
The author would like to thank the anonymous athlete for
access to the Laboratory Documentation Package and the
referees f or valuable comments.

Substance Identification in Anti-Doping Control by Means of Mass Spectrometry. Data Reduction and Decision Criteria23
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