Drug Testing

and Analysis

Annual bannedsubstance review

Received: 23 November 2014

Accepted: 1 December 2014

Published online in Wiley Online Library

(www.drugtestinganalysis.com) DOI 10.1002/dta.1769

Annual banned-substance review: analytical

approaches in human sports drug testing
Mario Thevis,a,b* Tiia Kuuranne,c Hans Geyera and Wilhelm Schnzera
Within the mosaic display of international anti-doping efforts, analytical strategies based on up-to-date instrumentation as well as
most recent information about physiology, pharmacology, metabolism, etc., of prohibited substances and methods of doping are
indispensable. The continuous emergence of new chemical entities and the identification of arguably beneficial effects of
established or even obsolete drugs on endurance, strength, and regeneration, necessitate frequent and adequate adaptations
of sports drug testing procedures. These largely rely on exploiting new technologies, extending the substance coverage of
existing test protocols, and generating new insights into metabolism, distribution, and elimination of compounds prohibited
by the World Anti-Doping Agency (WADA). In reference of the content of the 2014 Prohibited List, literature concerning human
sports drug testing that was published between October 2013 and September 2014 is summarized and reviewed in this annual
banned-substance review, with particular emphasis on analytical approaches and their contribution to enhanced doping controls.
Copyright 2014 John Wiley & Sons, Ltd.
Keywords: gene doping; sport; mass spectrometry; xenon; cobalt

Introduction
Nowadays, doping in sport is more multifaceted than ever before
with numerous standpoints and opinions coming from all possible
conceivable perspectives.[1,2] Spearheaded by the consistently
recurring question as to whether athletes should generally be
allowed to utilize doping practices,[3] juridical,[4,5]medical,[69] and
philosophical as well as ethical aspects[1012] have been discussed
in detail in 2013/2014. In addition, viewpoints on current and future
challenges[13] and the (in)efficiency of the existing doping control
system[14,15] have been presented, underlining the complexity of
modern sports drug testing, one core element of which is the annually issued Prohibited List as established by the World Anti-Doping
Agency (WADA).[16] In order to probe for the compliance of athletes
to the anti-doping regulations, analytical methods created,
optimized, and expanded to meet the growing demands of doping
controls evolve continuously as summarized in this annual bannedsubstance review for human sports drug testing from scientific
literature published between October 2013 and September 2014.
Besides technological and methodological improvements, alternative test matrices potentially offering complementary information
and benefits to current doping control procedures have been the
subject of in-depth studies.
Identical to the 2013 Prohibited List, the 2014 issue also contains
12 classes of prohibited substances (S0S9 plus P1 and P2) and
three categories of prohibited methods (M1M3) (Table 1). Major
modifications compared to the preceding 2013 version include
the addition of vasopressin V2 antagonists (commonly referred to
as vaptans) to the subclass of diuretics and the addition of
cathinone and its analogues as well as trimetazidine to Section S6
(stimulants). Moreover, as of 1 September 2014, substances acting
as hypoxia-inducible factor (HIF) activators, such as xenon and
argon, have been listed as explicitly prohibited, necessitated by recently surfaced documents on an arguably licit and extensive use of

Drug Test. Analysis (2014)

xenon/oxygen mixtures among selected athletes.[17,18] WADA further continued the monitoring programme in order to generate information on potential patterns of abuse concerning defined
substances that are currently not (or not at all times or at any concentration) prohibited. The 2014 in-competition monitoring programme was complemented by the narcotic agent mitragynine
(Figure 1), covering now collectively the ratio of morphine over codeine, hydrocodone, tramadol, tapentadol, and mitragynine as well
as the stimulants bupropion, caffeine, phenylephrine, phenylpropanolamine, pipradrol, pseudoephedrine (< 150 g/mL), synephrine,
and nicotine. Further, as in 2013, the potential (mis)use of corticosteroids in out-of-competition periods has been monitored.[19]

Alternative test matrices

Human routine doping control samples currently include the matrices urine, serum, and whole blood for the various different test
menus allowing the conduction of targeted as well as non-targeted
analyses. Despite the broad analytical picture provided by these
specimens, complementary options such as oral fluid, sweat, and
dried blood spots (DBS) are continuously assessed to probe for
added value enabled by these alternative matrices.

* Correspondence to: Mario Thevis, Institute of Biochemistry - Center for Preventive

Doping Research, German Sport University Cologne, Am Sportpark Mngersdorf 6,
50933 Cologne, Germany. E-mail: [email protected]
a Center for Preventive Doping Research - Institute of Biochemistry, German Sport
University Cologne, Am Sportpark Mngersdorf 6, 50933 Cologne, Germany
b European Monitoring Center for Emerging Doping Agents, Cologne, Germany
c Doping Control Laboratory, United Medix Laboratories, Hylmtie 14, 00380
Helsinki, Finland

Copyright 2014 John Wiley & Sons, Ltd.

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Copyright 2014 John Wiley & Sons, Ltd.

Stimulants

S6

Narcotics
Cannabinoids
Glucocorticosteroids

Diuretics and other masking agents

S5

S7
S8
S9

Beta-2-Agonists
Hormone and metabolic modulators

Peptide hormones, growth factors

and related substancesa

S2

S3
S4

Non-approved substances
Anabolic Agents

S0
S1

Class

Diuretics

2
Non-Specified Stimulants
Specified Stimulants

Aromatase inhibitors
Selective estrogen receptor modulators (SERMs)
Other anti-estrogenic substances
Agents modifying myostatin function(s)
Metabolic modulators
Masking agents

1
2
3
4
5
1

Growth hormone (GH), Insulin-like growth factors

(e.g. IGF-1), Mechano Growth Factors (MGFs),
Platelet-Derived Growth Factor (PDGF), Fibroblast
Growth Factors (FGFs) Vascular-Endothelial Growth
Factor (VEGF), Hepatocyte Growth Factor (HGF)

Chorionic Gonadotrophin (CG)b and

Luteinizing hormone (LH)b
Corticotrophins

2
3

Erythropoiesis-Stimulating Agents

Other anabolic agents

b) endogenous

Anabolic androgenic steroids

a) exogenous

Sub-group

fenoterol, reproterol, brombuterol, bambuterol

anastrozole, letrozole, exemestane, formestane, testolactone
raloxifene, tamoxifen, toremifene
clomiphene, cyclophenil, fulvestrant
stamulumab, bimagrumab
insulins (rhInsulin, Humalog LisPro, etc.), GW1516, AICAR
diuretics, probenecid, hydroxyethyl starch, glycerol,
desmopressin
acetazolamide, bumetanide, canrenone, furosemide,
triamterene
adrafinil, amphetamine, cocaine, modafinil, benfluorex
cathine, ephedrine, etamivan, methylephedrine,
methylhexaneamine, octopamine, pseudoephedrine,
sibutramine, strychnine, tuaminoheptane
buprenorphine, fentanyl, morphine
hashish, marijuana, JWH-018, HU-210
betamethasone, dexamethasone, prednisolone,
fluocortolone

tetracosactide-hexaacetate (Synacthen),
adrenocorticotrophic hormone (ACTH)
Genotropin, Increlex

1-androstendiol, boldenone, clostebol, danazol,

methandienone, methyltestosterone, methyltrienolone,
stanozolol, tetrahydrogestrinone
androstenediol, testosterone, dehydroepiandrosterone,
19-norandrosterone
clenbuterol, selective androgen receptor modulators
(SARMs), tibolone, zeranol, zilpaterol
erythropoietin (EPO), darbepoietin (dEPO), methoxy
polyethylene glycol-epoetin beta (CERA), peginesatide, xenon

rycals (ARM036), sirtuins (SRT2104), LH receptor agonists

Examples

Table 1. Overview of prohibited substances and methods of doping according to the World Anti-Doping Agency (WADA) Prohibited List of 2014

x
x

x
x

at all times

x
x
x

in-competition
only

Prohibited

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and Analysis
M. Thevis et al.

Drug Test. Analysis (2014)

Drug Testing
and Analysis

x
x
x
x

and their releasing factors

males only
c
depending on the rules of the international sport federations

acebutolol, atenolol, bisopropol, metoprolol

Alcohol
Beta-blockers
P1
P2

Drug Test. Analysis (2014)

Gene doping
M3

1
2
1
2
Chemical and physical manipulation
M2

Enhancement of oxygen transfer

M1

Administration or reintroduction of any quantity

of blood or blood products
Artificial enhancement of uptake, transport or
delivery of oxygen
Intravascular manipulation of the blood or
blood components
Tampering
Intravenous infusion
Transfer of nucleic acids or nucleic acid sequences
Use of normal or genetically modified cells

urine substitution, proteases

Figure 1. Structures of mitragynine (1, mol wt = 398), ARM036 (2, mol

wt = 267), ACP-105 (3, mol wt = 290), and NEP28 (4, mol wt = 326).

DNA, RNA

autologous, homologous and heterologous blood, red blood

cell products
perfluorocarbons (PFCs), efaproxiral, haemoglobin-based
oxygen carriers (HBOCs)

xc
xc

Annual banned substance review

Anizan and Huestis[20] comprehensively reviewed the potential

role of oral fluid in sports drug testing, outlining the apparent
advantages (e.g. fast and non-invasive/non-intrusive sample
collection, analysis of intact drug, correlation of blood and oral fluid
concentrations particularly helpful with drugs prohibited incompetition only) and current limitations (drug stability, limited
volume, short detection windows, considerable knowledge gaps
concerning drug disposition, contamination issues, etc.). Especially
under consideration of the few controlled drug administration
studies currently available with respect to oral fluid analysis, a
potential use of this matrix for selected compounds banned incompetition only was concluded.
Similarly, the option of using sweat for doping control purposes
was reviewed, suggesting this matrix as a viable means for detecting drugs of basic pH (e.g. stimulants and narcotics) due to their accumulation in perspired liquid.[21] However, although declared as a
preferred sample for doping control by the authors, the substantial
limitations (control of sample volume, restricted analyte coverage,
limited knowledge on drug distribution, etc.) and few advantages
(non-invasiveness/intrusiveness) do not seem to promote this matrix in the focus of sports drug testing whilst its utility in clinical settings, for example in the diagnosis of cystic fibrosis, is undisputed.
The growing interest in measuring drugs and drug candidates
from whole blood and dried blood spots (DBS) in general[2225]
and particularly in doping controls[26] also continued in 2013/2014.
The combined advantages of blood testing and DBS sampling,
transport, and storage have contributed to a renaissance of the
importance of blood testing in human doping controls, certainly
supported by significant improvements in analytical instrumentation. Requiring minimal-invasive sampling devices and minimizing
the possibility of sample manipulation, frequent and cost-efficient
sample collections are enabled that allow the detection of numerous intact drugs and drug candidates prohibited according to
WADA regulations. Further, enhanced analyte stabilities are
reported, which is a central aspect of modern doping controls.
It remains to be clarified however which legitimacy applies in
case of presumptive analytical findings as the established A- and
B-sample procedure is not in place for DBS testing today. Notwithstanding, proof-of-concept studies with DBS have been conducted,
demonstrating their value to anti-doping organizations as shown in
respective sections of this review (vide infra) (Table 2).

Non-approved substances
Pharmacological substances such as, for example, non-approved
therapeutics or designer drugs that are not covered by any of the
drug classes defined in WADAs Prohibited List can be classified

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Copyright 2014 John Wiley & Sons, Ltd.

Stimulants
Narcotics
Cannabinoids
Glucocorticosteroids
Enhancement of oxygen transfer

Chemical and physical manipulation

S6
S7
S8
S9
M1

M2

Gene doping
Alcohol
Beta-blockers

Diuretics and other masking agents

S5

M3
P1
P2

Beta-2-Agonists
Hormone and metabolic modulators

Hormones and related substances

S2

S3
S4

Non-approved substances
Anabolic Agents

S0
S1

Class

1
2

1
2

1
2
3
4
5
1
2

2
1
2
4
5

Sub-group

Administration or reintroduction of blood or blood products

Artificial enhancement of uptake, transport or delivery
of oxygen
Intravascular manipulation of the blood or
blood components
Tampering
Intravenous infusion

Aromatase inhibitors
Selective estrogen receptor modulators (SERMs)
Other anti-estrogenic substances
Agents modifying myostatin function(s)
Metabolic modulators
Masking agents
Diuretics

Anabolic androgenic steroids

a) exogenous
b) endogenous
Other anabolic agents
Erythropoiesis-Stimulating Agents
Chorionic Gonadotrophin (CG) and Luteinizing hormone (LH)
Corticotrophins
Growth hormone (GH), Insulin-like growth factors (e.g. IGF-1),
Mechano Growth Factors (MGFs), etc.
95

44-46

GC/MS (/MS)

202

172-174, 177, 178

171, 179-181
183

201

189, 194-198

158-164, 168, 169

165-167

133, 138
142-145

140

134, 135, 139

146, 147

116-122

101, 201, 104-106, 109, 114

41-43, 51-59
57-62, 69
73, 74, 76, 78
83-86, 91, 92
98

complementary
methods & general

131

126

64-68

GC/C/IRMS

References

129

123-125

103, 110-113

49, 54, 55
63, 70-72
75
88
97

27, 28

LC/MS (/MS)

Table 2. References to new data and/or improved screening and confirmation methods regarding human sports drug testing published in 2013/2014

Drug Testing
and Analysis
M. Thevis et al.

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Drug Testing
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Annual banned substance review

under S0 (non-approved substances).[16] Exemplary candidates for
this category are so-called Rycals including S107 and ARM036
(Aladorian, Figure 1, 2). While test methods for S107 and its putative
metabolic products were established several years ago, the disclosure of the structure of next generation Rycal (Aladorian) suggests
to date analytical approaches by similarity only.[27] In contrast, the
class of sirtuin 1 activating compounds (STACs), which are emerging therapeutics for the treatment of age- and affluence-related
health issues (e.g. obesity and type-2 diabetes mellitus) was the subject of studies concerning comprehensive doping control detection
strategies. By means of animal models, the elimination of three
STACs including SRT1720 was investigated, and analytical approaches for urine, plasma, and DBS were evaluated.[28] Employing
liquid chromatography-(tandem) mass spectrometry (LC-MS/MS)
and isotopically labelled internal standards, the intact drug candidates and major metabolites were measured from in vivo studies,
suggesting limits of detection (LODs) of 1050 ng/mL for DBS.
The peptidic substance AOD-9604 has been recently confiscated
and observed at various occasions including customs controls[29]
and investigations concerning professional sport teams.[30] Due to
its structural similarity to the C-terminus of the human growth hormone (hGH), its potential to interfere with the frequently employed
doping control hGH isoform test was assessed.[31] Human serum
samples were fortified with AOD-9604 at different concentration
levels in the presence of defined amounts of recombinant hGH to
probe for any enhancing or suppressing effect. The isoform test
proved unaffected by AOD-9604, corroborating the specificity of
the methodology.

Anabolic agents
Anabolic-androgenic steroids
Despite the continuously growing body of evidence concerning adverse health effects of anabolic androgenic steroids (AAS), the class
of anabolic agents (in particular AAS) has been most frequently
reported with regard to adverse analytical findings in doping control samples in 2013.[32] Reported complications associated with
AAS misuse included impaired post-exercise heart rate recovery,[33]
acute hepatitis (secondary to 17-alkylated steroid abuse),[34] collagen dysplasia,[35] and general adverse cardiovascular effects.[36]
Moreover, negative effects on mental health were observed in a
retrospective study with retired elite athletes,[37] and once more,
the commonly reported organic lesions such as testicular atrophy,
testicular fibrosis, arrested spermatogenesis, and left ventricular
hypertrophy, were substantiated by the autopsy results, which
were conducted in cases of sudden or unnatural deaths where toxicology revealed the individuals AAS use.[38] All these facts become
arguably irrelevant in the light of potential benefits provided by
testosterone and its synthetic derivatives to selected athletes.
Ever since the enormous breadth of testosterones effects on
the human endocrine system has been studied,[39] the temptation has existed to particularly exploit the long-lasting anabolic
effects that have recently been shown to prevail in skeletal muscle
tissue even after cessation of the drug regimen through a cellular
memory mechanism.[40]
Initial testing procedures multi-analyte screening methods
and new mass spectrometric techniques
Utmost comprehensiveness and analytical sensitivity are vital for
efficient initial doping-control testing procedures, especially in the

Drug Test. Analysis (2014)

case of anabolic agents, and they require continuously improving

detection strategies.[41] In this context, a considerable challenge is
presented by the ever-increasing market of designer steroids for
which different strategies have been installed in sports drug testing
laboratories as recently summarized and reviewed by Abushareeda
et al. and Pozo et al.[42,43] A main pillar of doping controls
concerning AAS has been the use of gas chromatography(tandem) mass spectrometry (GC-MS/MS) with electron ionization
(EI) especially due to the robust and reproducible nature of the
provided analytical data[44] that, amongst other features, allows
for comprehensive steroid profiling (vide infra). Maintaining the advantages of the GC separation, alternative atmospheric pressure
ionization strategies have been pursued, employing either
chemical (atmospheric pressure chemical ionization, APCI)[45] or
microscale photo (atmospheric pressure photo ionization, APPI)
ionization sources.[46] Using GC-APCI-MS/MS, the mass spectrometric behaviour of 60 underivatized and trimethylsilylated steroidal
analytes was studied following nitrogen plasma-assisted protonation via water as the modifier.[45] Structure-ionization/dissociation
relationships were established to support particularly the identification of unknown steroid substances as the soft ionization strategy
largely allowed to maintain the intact (protonated) molecule. A
subset of 7 steroidal analytes with relevance to doping controls
was determined from human urine by means of enzymatic hydrolysis, liquid-liquid extraction (LLE), trimethylsilylation, and subsequent GC-APPI-MS/MS. The use of chlorobenzene as the dopant
supported the formation of predominantly radical cations of the
model substances as well as cations resulting from the loss of a
hydrogen atom ([M-H]+) or a methyl group ([M-CH3]+). Employing
multiple reaction monitoring (MRM), the approach was characterized and demonstrated competitive detection limits between 0.05
and 0.5 ng/mL for frequently observed metabolites of AAS such as
nandrolone and metandienone.[46]
The use of online turbulent flow solid-phase extraction (SPE)
for the preconcentration of four AAS from urine for subsequent
LC-MS/MS analysis was reported by Guo et al.[47] While the described sensitivity at pg/mL levels was convincing, the fact that
three out of the four studied analytes are largely metabolized and
conjugated if administered to humans was not accounted for.
Hence, the methodology as presented is not applicable to authentic doping control urine samples. A similar misconception apparently prevailed in another study employing molecular imprinted
polymer filaments for online extraction and on-coating derivatization of steroidal substances for subsequent GC-MS analysis.[48]
Urine was spiked with native testosterone, epitestosterone,
methyltestosterone, and nandrolone, extracted and determined
at limits of detection as low as 0.040.18 ng/mL; however, the
necessity of enzymatic hydrolysis and significance of phase-I
metabolism was not considered and thus also here the applicability
of the approach to sports drug testing is not demonstrated.
A facile and sensitive dilute-and-inject methodology covering
21 AAS and respective metabolites was presented by Tudela et al.[49]
targeting both free (12) and conjugated (8 glucuronidated and
one sulfated) analytes. LODs between 0.5 and 18 ng/mL were
accomplished by liquid chromatography-high resolution/high
accuracy mass spectrometry (LC-HRMS) measuring the protonated
and/or deprotonated species of the analytes as well as sodium, ammonium, or acetate adduct ions. Although the method represents a
useful and fast complement to commonly conducted GC-MS(/MS)based approaches, limitations in meeting minimum required
performance levels (MRPLs)[50] for selected model substances were
observed and discussed by the authors.

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M. Thevis et al.

Initial testing procedures metabolism studies and new

target analytes
To add to the comprehensiveness as well as specificity and sensitivity of the aforementioned initial testing procedures in doping controls, studies dedicated to the identification and characterization of
new or alternative target analytes are of utmost importance.
Exploiting human liver microsomal in vitro approaches and
a mouse model exhibiting humanized liver properties, the metabolic fate of methylstenbolone was elucidated using LC-MS/MS
and GC-MS/MS.[51] Most of the 13 observed metabolites represented monohydroxylated derivatives and resulted only from
in vitro incubations. In vivo studies in mice resulted mainly into
two bishydroxylated metabolites, which were not, however,
detected in human urine samples tested positive for the intact
drug. Instead, a metabolite of yet unclear structure was found to
provide a viable means to screen for a methylstenbolone administration. Guddat et al. elaborated on the synthesis, characterization,
and implementation of recently identified long-term metabolites
of oxandrolone into routine doping controls.[52] Utilizing the fungus
Cunninghamella elegans, the two epimeric oxandrolone metabolites
(17-hydroxymethyl-17-methyl-18-nor-2-oxa-5-androst-13en-3-one and 17-hydroxymethyl-17-methyl-18-nor-2-oxa-5androst-13-en-3-one) were produced from 18-noroxandrolone
and characterized by HRMS as well as nuclear magnetic resonance
(NMR) spectroscopy. Sensitive GC-MS/MS-based analytical strategies allowed for LODs of 1 ng/mL for these target analytes, and
an extension of detection windows up to 18 days for oxandrolone
misuse. The metabolism of danazol was studied using the fungal
strains of Beauveria bassiana and Gliocladium viride, and yielded
four main products from which 6-hydroxydanazol was a formerly
unreported product.[53] Characterized by NMR, options to produce reference material for doping control purposes are given.
Focusing on sulfoconjugated metabolites of metandienone,
Gomez et al. characterized seven products in elimination study
urine samples by means of LC-MS/MS and GC-MS/MS.[54] One of
these sulfoconjugates was identified as 18-nor-17-hydroxymethyl-17-methylandrost-1,4,13-triene-3-one sulfate, which
corresponds to the earlier reported long-term metabolite for
metandienone obtained from the glucuronide fraction. Implementing this analyte into respective sample preparation and analysis strategies, the intake of metandienone was monitored for up to
26 days. The metabolic picture of stanozolol was further
complemented in a recent study by Schnzer et al. who identified
stanozolol-N-glucuronide and 17-epistanozolol-N-glucuronide as
-glucuronidase-resistant long-term metabolites in elimination
study and doping control urine samples.[55] Employing LC-MS/MS
with high resolution/high accuracy mass spectrometry using either
native urine or SPE-purified sample extracts, LODs between 5 and
25 pg/mL were accomplished for 3-OH-stanozolol-glucuronide,
which was used as surrogate reference material in the absence
of certified synthetic N-glucuronides of stanozolol and 17epistanozolol. By means of these target analytes combined with
state-of-the-art analytical instrumentation, the number of adverse
analytical findings for stanozolol was substantially increased in the
course of 2013.
Initial testing procedures steroid profiling
Despite the fact that much is known about the absorption, distribution, metabolism, and elimination (ADME) of approved therapeutics, the human metabolism particularly concerning androgens

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has been shown to be substantially influenced by various different pharmacological interventions as well as other confounding
factors. In consideration of the significance of steroid profiling
in the anti-doping field and incorporation of steroidal module
to the athlete biological passport (ABP), continuous research is
conducted to expand the knowledge on aspects potentially or
evidently affecting urinary steroid concentrations and result
interpretation.[56]
One of the most abundant steroid hormones in humans is dehydroepiandrosterone (DHEA).[57] Due to the arguably ergogenic
properties of DHEA, it has been the subject of misuse in sport in
the past and thus it is one parameter of the commonly determined
athletes urinary steroid profile. Consequently, the dissemination of
knowledge about factors potentially affecting DHEA and DHEA
sulfate concentrations such as exercise intensity, age, gender, and
training frequency is of great importance as recently done via a
comprehensive review.[58] First data on serum levels of DHEA
sulfate, testosterone (T), androstenedione, sex hormone-binding
globulin (SHBG), and gonadotropin in elite female athletes were
generated and compiled by Bermon et al.[59] Under consideration
of sample collection time, age, type of sport discipline, ethnicity,
and use/non-use of contraceptives, a total of 849 elite female
athletes serum specimens was analyzed. As a main outcome
of the study, the 99th percentile for serum T concentrations
was found at 3.1 nmol/L, with a prevalence of disorders of sex
development (DSD) of approximately 0.7%. Focusing on urinary
steroid profiles of female athletes, the impact of hormonal contraceptives was found to reduce the excretion of epitestosterone
(EpiT) by approximately 40%, resulting in T/EpiT ratios elevated by
29% compared to females not using hormonal contraceptives.[60]
Pregnancy was shown to result in quite the contrary picture by
causing overall elevated urinary EpiT glucuronide concentrations
while most other urinary androgen levels fell below basal concentrations after a brief increase during the first trimester.[61]
As a result, especially the T/EpiT ratio (as a determinant parameter of the steroid profile) was significantly reduced in all three
study volunteers.
The influence of pharmacological interventions on the serum
and urine steroid profile was studied by Handelsman et al. who
investigated the effect of administrations of the superactive
gonadotropin analogue leuprolide to men.[62] Serum T, dihydrotestosterone (DHT), and 5-androstane-3,17-diol (Adiol) were significantly increased along with urinary T, EpiT, and androsterone (A)
concentrations upon five days of leuprolide administration,
resulting in modest (if any) changes in T/EpiT ratios. Urinary steroid
levels returned to baseline values at day 10, and a detection
strategy involving both the direct analysis of leuprolide and its
main metabolite as well as using the ratio of luteinizing hormone (LH)/T were suggested. Further to the indirect stimulation
of T secretion via releasing factors, the option of direct enhancement of serum T concentrations by transdermal applications
remains a challenging doping control analytical task. By means
of LC-MS/MS targeting 12 urinary steroid glucuro- and sulfoconjugates, Badoud et al. assessed the possibility to complement
routine steroid profiling protocols by measuring intact phase-II
metabolites.[63] Due to substantial inter-individual variabilities,
only intra-individual profiles demonstrated the capability of
uncovering topical (transdermal) T applications by targeting
specifically the ratios of the glucuronides of T and EpiT as well
as A and etiocholanolone (E). For oral T undecanoate administrations, E sulfate was found to be a promising marker to complement the currently employed steroid profile.

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Confirmatory testing procedures IRMS: new/improved
approaches
Following suspicious initial test results obtained by steroid profile
analyses, confirmatory testing procedures preferably employing
gas chromatography-combustion-isotope ratio mass spectrometry
(GC-C-IRMS) are indicated. However, since IRMS analyses are
comparably time consuming and costly, optimized screening
methods providing appropriate sensitivity and specificity for
natural/endogenous steroid abuse are desirable.
The metabolism of boldenone and boldione was revisited by de
la Torre et al. aiming at the identification of metabolic products
allowing for the improved correlation between the ingested
substances and resulting urinary metabolites as well as alternative
target analytes for IRMS confirmatory measurements.[64] A
minor but diagnostic metabolite of boldione was identified with
17-boldenone (epiboldenone). Moreover, for economic reasons
and extended detection windows, support to the confirmation of
the presence of pseudoendogenous steroids was suggested to be
generated with 5-androstane-3,17-diol (Bdiol), a common
metabolite of boldione, boldenone, testosterone, etc. Extending
the steroid profile analyses by additional metabolites can contribute to an enhanced efficiency of steroid abuse screening, but
requires substantial information on normal urinary concentrations
of these analytes. Polet et al. investigated the added value of
quantifying 6-hydroxyandrostenedione, which represents a minor
metabolite of testosterone.[65] Being considerably affected by
testosterone, androst-4-ene-3,17-dione, and androst-4-ene-3,6,17trione administrations, a total of 2128 reference population
samples was analyzed and a threshold level of 5 ng/mL of 6hydroxyandrostenedione was suggested. Samples exceeding that
threshold were classified as suspicious and analyzed by a newly
established GC-C-IRMS approach, the fitness-for-purpose of which
was demonstrated by the analyses of elimination study urine samples collected after administrations of T, androst-4-ene-3,17-dione,
and androst-4-ene-3,6,17-trione.
Confirmatory testing procedures IRMS: complementary
information
Most doping control methods employing IRMS utilize 13C
values calculated from an endogenous reference compound
(ERC) and one or more target compounds (TCs). The possibility
to pharmacologically influence the 13C value of an ERC such
as 5-pregnane-3,20-diol (PD) was studied by oral and intramuscular administrations of progesterone, the biosynthetic precursor of PD, to three female individuals.[66] Urinary PD concentrations
as well as 13C values were significantly altered, suggesting
both parameters as indicators for progesterone (mis)use in
sport. Whenever atypical urinary PD concentrations and/or 13C
values are observed, the use of 5-androst-16-en-3-ol or 11hydroxyandrosterone as ERC is recommended. Since the illicit use
of musk preparations has been shown to affect the urinary steroid
profile as well as the carbon isotope signature of doping control
TCs in 2011,[67] a systematic study of deer musk grains and their effect on doping control analytical results was pursued by He et al.[68]
Four batches of musk grains were subjected to IRMS analyses, revealing 13C values of ten steroidal agents (including amongst
others A, E, DHEA, T, and EpiT) between -18 and -30. The oral
administration of 100 mg of musk grains to two male volunteers,
however, did not result in atypical or adverse analytical findings, arguably due to insufficient amounts of steroids contained in the
100 mg of musk.

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While GC-C-IRMS is the state-of-the-art doping control analytical

approach for differentiating natural from xenobiotic analytes,
LC-IRMS has been shown to become a valid complementary tool
for selected applications. While limitations exist concerning chromatographic flexibility and sensitivity of the methodology, added
value was recognized concerning 13C value assignments of
underivatized substances such as typical steroidal TCs in sports
drug testing. Zhang et al. demonstrated the utility of LC/IRMS in
separating 19-norandrosterone, T, EpiT, A, E, and 5-pregnane3,17,20-triol using high-temperature LC and determining
respective carbon isotope ratios, which might open new possibilities of sports drug testing in the future.[69]
Alternative matrices and complementary approaches
Detection possibilities concerning AAS in alternative matrices have
been assessed with particular attention being paid to testosterone
and its esterified derivatives. Since transdermal testosterone applications have been shown to result in only modest alterations of
the urinary steroid profile, the option to use oral fluid was tested
by Thieme et al.[70] Following the transdermal administration of
approximately 37 mg of testosterone, salivary concentrations of
the target analyte increased from baseline (30142 pg/mg) to more
than 1000 pg/mg over a period of 16 h, suggesting a viable means
to efficiently screen for the misuse of transdermal testosterone
preparations. Confirmation of the finding might however still
require IRMS-based methods and conventional doping control
specimens. Due to the fact that testosterone formulations frequently contain esters of the active principle, the options to test
for the presence of testosterone esters in plasma[71] as well as
DBS[72] were elucidated as an immediate proof of doping. Forsdahl
et al. isolated and identified nine testosterone esters from 1 mL of
plasma containing two internal standards (methyltestosterone
and six-fold deuterated T acetate). The target analytes were converted to respective oxime derivatives using hydroxylamine and
determined by means of LC-MS/MS with LODs between 5 and
400 pg/mL. Complementary, Tretzel et al. employed DBS to test
for the presence or absence of eight AAS esters of nandrolone, testosterone and trenbolone. Three isotopically labelled internal standards were added prior to sample extraction and methyloxime
derivatization for LC-MS/MS analysis with a high resolution/high accuracy mass analyzer. The LODs accomplished from DBS samples
ranged between 0.1 and 0.5 ng/mL, which was shown to allow for
the detection of testosterone undecanoate in administration study
DBS samples for at least 8 h.[72]
Other anabolic agents
Among the other anabolic agents, mainly selective androgen
receptor modulators (SARMs) were the subject of anti-doping
research during the past 12 months.
In order to improve doping control detection assays, reference
material for metabolites of established arylpropionamide-derived
SARMs was prepared using a novel strategy of combined microbial
and chemical (bio)synthesis. By means of the aforementioned fungus Cunninghamella elegans, glucosides of hydroxylated phase-I
metabolites of SARMs were produced, which were subsequently
oxidized via tetramethylpiperidinyl-1-oxy (TEMPO) to yield the
corresponding glucuronides. Three representatives (S-1, S-22,
and S-24) were successfully converted into the B-ring hydroxylated and glucuronidated metabolic products and characterized
by liquid chromatography-high resolution/high accuracy mass

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spectrometry[73]; one analogue (S-4) however failed due reasons

still unknown. In case of S-1, a follow-up study including
upscaling of the (bio)synthetic protocol was conducted. Sufficient amounts were obtained for sufficient for NMR characterization, which confirmed the -conjugation of the glucuronide with
the moiety attached to C-10 of the SARM S-1.[74] The in vivo
metabolism of another, structurally different SARM referred to
as ACP-105 was investigated by means of animal administration
studies.[75] The tropanol-derived SARM yielded seven major
metabolites, mainly mono- and bishydroxylated products, which
can be used as target analytes in routine doping controls. However, it remains to be shown which of these metabolites de facto
occur(s) in humans and which might offer the longest window
of opportunity to detect the misuse of ACP-105 (Figure 1, 3).
Comparably little is known about the ADME properties of the
recently reported SARM NEP28 (Figure 1, 4).[76] Its anabolic efficiency equivalence was demonstrated in comparison studies
with dihydrotestosterone (DHT) and methyltestosterone, suggesting its utility in the treatment of sarcopenia as well as osteoporosis and, thus, the necessity to include it in doping control
analyses. Furthermore, the potential benefit concerning therapeutic means of NEP28 treatment for Alzheimers disease was
suggested due to an increase in neprilysin activity and consequently, degradation of amyloid beta peptide.
Among the other anabolic agents, clenbuterol has been a prominent representative due to its evidential misuse in sport as well as
meat contamination issues and corresponding analytical challenges. The extent of its misuse has been illustrated by a report
published by the New South Wales poisons information centre,
who summarized calls with regard to clenbuterol abuse, the majority of which necessitated hospitalization of the affected individual,
for example due to tachycardia.[77] In a doping control context,
clenbuterol remains a two-sided situation. On the one hand, excellent detection limits have been accomplished with LC-MS/MS as
well as lateral flow test strip biosensors,[78] allowing for adequate
retrospectivity for sports and food drug testing; on the other hand,
contaminated dietary products have been shown result in the
inadvertent ingestion and eventually in AAFs. This issue is still
pending and the subject of various different yet unpublished
research projects.

Peptide hormones, growth factors and related

substances
Erythropoiesis-stimulating agents (ESAs)
The most prominent erythropoiesis-stimulating agent (ESA) is
undoubtedly erythropoietin (EPO). Despite its suspected, purported, and proven misuse in elite sport, the debate as to its utility
in different clinical settings, performance enhancing properties,
and modus operandi of increasing endurance in healthy individuals
is ongoing.[79,80] Unaffected, the need to test for xenobiotic EPO, to
distinguish unambiguously between endogenous and exogenous
origin of the EPO and to improve test methods to cope with trends
in doping practices, such as repetitive microdosing is respected and
timely reviews summarize recent developments and accomplishments as well as future goals in a comprehensive manner.[81,82]
Improvements to existing analytical strategies such as isoelectric
focusing (IEF) or polyacrylamide gel electrophoresis (PAGE) with
sodium dodecyl sulfate (SDS) or sodium N-lauroylsarcosinate
(SAR) have been published, exploiting either alternative analyte

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pretreatment steps or modified analytical protocols. Desharnais

et al. reported on the beneficial effect of desialylation of EPO prior
to SDS-PAGE analysis.[83] Due to the reduced apparent molecular
mass, in-gel migration properties of human urinary EPO (huEPO)
and recombinant EPO (rEPO) developed differently, enabling an
improved separation of these analytes and, thus, a better differentiation of natural from xenobiotic EPO. Aiming at enhanced analytical turnaround times, the utility of a vacuum-assisted blotting
system was evaluated, suggesting much faster result generation
for EPO analyses.[84] This would be of paramount importance at
great sporting events, where the pertinence of rapid decisions on
athletes doping control samples is obvious.
Increasing the initial testing frequency by competitively sensitive
but potentially cheaper and faster approaches was the subject of
studies employing the so-called EPO WGA MAIIA test kit. Lnnberg
and Lundby conducted an administration study using 65 IU of
NeoRecormon/kg of bodyweight, injected subcutaneously every
other day over a period of 14 days, and plasma as well as urine were
sampled up to 21 days after the last injection.[85] In a different setting, Dehnes et al. used intravenously administered microdoses of
7.5 IU of NeoRecormon/kg of bodyweight, injected twice per week
over a period of 21 days. Also here, blood and urine was collected
for analysis.[86] Both sample types were tested for EPO by the MAIIA
lateral flow isoform test, which demonstrated its capability of providing analytical results indicating the presence or absence of rEPO
within several hours. The detection windows were approximately
24 h (in case of microdosing) and 7 days (in case of therapeutic dosing), and the assays features of rapidity and appropriate specificity
were considered as a useful complement to existing doping control
strategies.
Mass spectrometric studies on rEPO and its next-generation derivatives have been an ongoing endeavor for both in-depth characterization of glycoforms in pharmaceutical products[87] as well as
alternative detection methods in sports drug testing. Okano et al.
utilized the fact that darbepoetin alpha comprises a modified
protein backbone, enabling the generation of a peptide of unique
amino acid composition, which serves as target structure for
doping controls.[88] Following immunopurification of darbepoetin
alpha, enzymatic hydrolysis using Glu-C was conducted to yield a
28-residue spanning proteotypical peptide, which was subsequently analyzed by means of ultrahigh performance LC (UHPLC)/
high resolution/high accuracy tandem mass spectrometry. The
detection limit of the assay was 1.2 pg/mL, and the assay was found
to be suitable for high throughput with an overall time investment
of 6 h per confirmatory analysis. Complementary, though not fitfor-purpose in a doping control analytical context, methodologies
based on capillary electrophoresis (CE) and electrochemical biosensors were shown to possibly provide alternatives for future
approaches.[89] Using pre-analytical dye labeling of EPO, CE combined with laser-induced fluorescence detection was shown to be
useful for the characterization of pharmaceutical products; the
limitations concerning the methods LOD (10 ng/mL) however excludes its use in doping controls.[90] In contrast, an excellent LOD
of 0.1 fg/mL was accomplished using an electrochemical biosensor
composed of a nano-AU/ZnO-sol-gel modified glassy carbon electrode carrying EPO receptors.[91] While ultrasensitive measurements
concerning EPO were shown, a differentiation between recombinant and natural EPO is not enabled by the presented methodology; nevertheless, combining analyzers of utmost sensitivity with
devices allowing for the separation of rEPO and huEPO could possibly result in new future options. Finally, as addition or substitute for
the immunopurification, the potential utility of antisense peptides

Copyright 2014 John Wiley & Sons, Ltd.

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(paratopes) for isolating EPO from biological matrices was
presented.[92] Targeting the C-terminal binding region of EPO as
epitope, the phenomenon of protein-protein interaction was
exploited to create a high-affinity anti-EPO peptide, which could
be further developed into cost-effective extraction resins.
Besides EPO and its derivatives, hypoxia-inducible factor (HIF)
stabilizers/activators are considered as relevant to doping controls.
This class of compounds is particularly heterogeneous and includes
low molecular mass organic compounds such as FG-4592[27] as well
as the recently added noble gas xenon and, as of January 2015
explicitly named, the inorganic substance cobalt. Ionic cobalt,
more specifically Co2+ administered as CoCl2, was the method of
choice for the treatment of renal and non-renal anemia in the
pre-EPO era. Due to serious adverse effects and a undesirable
cost-benefit-ratio, CoCl2 was removed from the therapeutic arsenal; however, anecdotal evidence exists that the cobalt-option
might be exploited in sports despite all reported health risks as
summarized recently by Ebert and Jelkmann.[93] Detection strategies for cobalt from urine exist, preferably utilizing inductivelycoupled plasma (ICP) MS,[27] but due to the natural abundance of
the element, threshold levels may become necessary. The (mis)
use of xenon in sport was reported early 2014,[18] resulting in
the banning of the approved anesthetic, which arguably exhibits
HIF-activating but yet not proven erythropoietic properties.[94] The
reasonably good solubility of the gas in biofluids allows detecting
xenon in blood and plasma by means of conventional GC-MS approaches with headspace injection. Since whole blood samples
are collected in the context of the Athlete Biological Passport
(ABP) tests, frequent testing for this substance is enabled. First test
methods were reported reaching LODs of 0.5 nmol/mL, which was
found sufficient to identify xenon in post-operative patient samples
for at least 24 h.[95]
Chorionic gonadotropin (CG) and luteinizing hormone (LH)
Chorionic gonadotropin (CG) has been the most frequently determined substance in AAFs and atypical findings (ATFs) among the
class of peptide hormones, growth factors, and related substances
of WADAs Prohibited List in 2013.[32] CGs natural heterogeneity is
of enormous physiological and diagnostic relevance; at the same
time, it represents a major challenge to doping controls due to
the same reasons.[96] Consequently, confirmatory analyses
employing mass spectrometric methodologies have been pursued
and developed for nearly 10 years. Most recently, Woldemariam
and Butch presented procedure using a two-step immunoextraction sample preparation, followed by bottom-up quantification of the three most abundant isoforms of CG including intact
CG, the free -subunit, and the -subunit core fragment.[97] With
two isotopically labelled internal standard peptides being unique
to the -subunit or the respective core fragment, quantities of all
three analytes were determined, with an LOD of 0.2 IU/L. Considering the reporting level of currently 5 IU/L for intact CG, the method
proved fit-for-purpose with excellent specificity, making it the
preferred approach for CG confirmation analyses. Besides a substantial heterogeneity of glycoproteins such as CG and luteinizing
hormone (LH), issues concerning reproducible immunoreactivity
under long-term storage conditions have been reported in the
past. The performances of an immunofluorimetric (IF) and an
immunochmiluminometric (ICL) LH assay were compared using a
set of urine samples collected in the course of an LH administration
study, demonstrating that intra-assay reproducibility was given in
both cases.[98] However, a consistently lower readout was found

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for the IF kit and further reductions in measured values were recorded after a 4-year storage of the urine samples at -20 C without
the addition of stabilizing additives.
Growth hormone, Insulin-like growth factor-1 (IGF-1), and
other growth or releasing factors
Detecting growth hormone (GH) misuse in sport has required
substantial research investment over more than 15 years, and two
approaches have eventually been approved by WADA to be used
in doping controls.[99,100] The first methodology is referred to as
the GH isoform differential immunoassay that exploits the fact that
the administration of pharmaceutical GH preparations significantly
influences the distribution of naturally produced and circulating GH
isoforms. To determine whether isoform ratios are non-natural,
decision limits are required. On the basis of 21 943 serum samples
analyzed between 2009 and 2013, variations of isoform ratios
were studied and decision limits were calculated, enabling laboratories to report AAFs whenever the assay-specific values are
exceeded.[101] Since elite athletes in particular are frequently exposed to unusual conditions (e.g. extreme weather conditions combined with extreme physical stress), the potential influence of such
factors deserves in-depth consideration. Investigating the effect of
intense exercise accompanied with altered plasma volumes of
athletes recently demonstrated the robustness of the isoform
differential immunoassay and no significant changes for GH test
results were observed.[102] Further, the trend towards mass spectrometric quantifications of peptides and proteins has also expanded
to GH as shown by Pritchard et al.[103] Designed for clinically relevant serum concentrations, an isotope dilution (ID) LC-MS/MS approach was established, enabling the quantification of the 22 kDa
GH variant from 800 L of serum. Following two separate SPE steps,
GH (and the correspondingly lysine-labelled 13C6,15 N4-GH) is
trypsinized and the resulting peptides are measured by means of
LC-MS/MS. While the approach is promising and has shown good
validation data, the required serum volume as well as the reported
LOD of 10 ng/g is not yet appropriate for sports drug testing
purposes. Due to the comparably limited number of serum samples, testing GH isoforms from urine would be desirable, but lowest
urinary concentrations of the target analytes have been a limiting
factor. Bosch et al. employed hydrogel-based nanoparticles to
enrich GH isoforms from urine and to probe for the possibility to
use the differential immunoassay on this matrix.[104] In a pilot study
with spiked urine samples as well as administration study specimens, volumes of 20 mL of urine allowed for applying the isoform
test, which yielded results of comparable ratios found in timematched serum samples in case of elimination study specimens.
Whilst urine analysis might be an interesting complement, the issue of rather short detection windows for uncovering GH misuse
will likely remain here as well. Hence, research into indirect,
biomarker-based approaches has been pursued for many years,
one of which (the marker approach) represents the second option
currently available to doping control laboratories for testing for GH
misuse. Focusing on the two main parameters insulin-like growth
factor I (IGF-1) and the N-terminal propeptide of type III procollagen
(PIIINP), scores indicating the pharmacological stimulation of these
markers by GH administration were defined. Studies concerning
the intra-individual variability of these parameters and resulting
GH-2000 scores were conducted by Kniess et al., who measured
up to 8 samples of 50 male and 50 female athletes over a period
of 18 months.[105] The observed variations were found to be smaller
than those reported for inter-individual subject comparisons, and

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the possibility of individual test scores was discussed as a potential

means to increase the efficacy of the biomarker approach. An entirely different methodology was assessed by Kelly et al. who studied the effect of GH administrations on different plasma microRNA
levels.[106] Comparing a control group of healthy individuals with
patients undergoing GH replacement therapy and patients with
reported pituitary GH overproduction, the group identified four
differently-expressed microRNA candidates, which could potentially serve as indicators for GH misuse in the future.
Sports drug testing laboratories might further benefit from the
aforementioned IGF-I/PIIINP-based marker approach with regards
to the assays potential contribution to future test methods aiming
at the detection of IGF-I misuse.[107] Revealing publications on the
extent of the use and misuse of peptidic drugs in sports corroborated the tendency of a growing practice lately,[30] flanked by an
increasing number of reports indicating intentional adulteration
of products with little (if any) scientific rationale. In the context of
IGF-I, nutritional supplements based on deer antler velvet were
found to be enriched with human IGF-I;[108] however, since
the products were intended for oral application, effects on serum
IGF-I levels are not expected.
Concerning authentic IGF-I-based drug formulations, the response of serum IGF-I, PIIINP, and the corresponding GH-2000 score
to the administration of an equimolar IGF-I/IGF-I binding protein 3
(IGFBP-3) complex was investigated by Guha et al.[109] Two
placebo-controlled studies with low (ca. 6.3 mg of IGF-I/d) and high
(ca. 12.6 mg of IGF-I/d) amounts of complexed IGF-I/IGFBP-3 were
conducted with 56 participants receiving the drug over a period
of 28 days. A significant increase in serum IGF-I concentrations
accompanied by a modest response in PIIINP levels was observed,
resulting in a higher sensitivity of the test assay when utilizing
sole IGF-I as a marker rather than the GH-2000 score. Currently,
both IGF-I and PIIINP are largely measured in doping controls
and clinical context using immunological approaches. Mass
spectrometry has been shown to be a robust and accurate
alternative for quantifying particularly IGF-I, which resulted in a
variety of top-down as well as bottom-up methodologies for this
analyte recently. Using the so-called mass spectrometric immunoassay (MSIA) technology, IGF-I (plus its derivative long-R3-IGF-I
used as internal standard) was dissociated from its binding proteins and extracted from serum/plasma by anti-IGF-I antibodies
immobilized in pipette tips. Subsequently, the extract was either
enzymatically digested and subjected to LC-MS/MS analysis[110]
or forwarded to matrix-assisted laser desorption ionization (MALDI)
time-of-flight (TOF) MS measurements.[111] While the LC-MS/MS
bottom-up approach proved slightly more sensitive (1 ng/mL
vs. 1.5 ng/mL), the MALDI-TOF MS top-down methodology
allowed for high throughput analyses of more than 1000 sample measurements/day. In addition, point mutations (e.g. A67T)
were observed. Complementary, antibody-free protocols for the
top-down and bottom-up-based quantification of IGF-I were
presented by Lopes et al.[112] and Cox et al.,[113] respectively.
Dedicated specifically to doping controls, sample preparations
were conducted under the control of a fully 15 N-labelled IGF-I
internal standard in both cases. This allowed compensating
for any irregularity in preparation and/or analysis of the
obtained specimen, and the accurate quantification of IGF-I
from serum was accomplished between 50 and 1000 ng/mL.
The robustness of the bottom-up approach was demonstrated
in a comprehensive inter-laboratory comparison study, where
imprecisions between 5 participating parties were found
16%.

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IGF-I and PIIINP serum concentrations were shown to be of value

though limited use for the detection of IGF-I abuse in sport. Alternative biomarkers for the administration of IGF-I/IGFBP were sought
and found in IGFBP-2 and IGF-II, with IGFBP-2 being increased by
approximately 120% and IGF-II being depleted by ca. 50%.[114] In
women, the acid-labile subunit (ALS) could possibly serve as additional marker also. In contrast, parameters such as IGFBP-3,
osteocalcin, procollagen type I carboxy-terminal propeptide (PICP),
and type I collagen cross-linked carboxy-terminal telopeptide
(ICTP), were not shown to be significantly affected by a 28-day
treatment of the volunteers with an IGF-I/IGFBP-3 complex drug.

Beta-2-agonists
According to the 2014 Prohibited List,[16] all beta-2-agonists
(2-agonists) are prohibited with the exemption of three drugs
(salbutamol, formoterol, and salmeterol), for which allowed
dosages and routes of administration apply. The relevance of
2-agonists and their potential to increase athletic performance
(as well as promote cattle fattening) has been debated in
extenso in the past,[115] and also in 2013/2014 the ergogenic
effects of salbutamol in particular were assessed in different
experimental settings. Decorte et al. assessed the effect of
inhaled salbutamol (200 g and 800 g) on a quadriceps
fatigue test.[116] Placebo-controlled studies were conducted,
demonstrating that the drug did not affect the maximum
voluntary contraction; however, the number of repetitions of
submaximal contractions until fatigue was significantly elevated
by salbutamol applications. Whilst the mechanism of this phenomenon remains unclear, a beneficial/performance-enhancing
effect of the drug was noted. Complementary, Dickinson et al.
focused on the acute impact of inhaled salbutamol on the 5-km
time trial run performance of athletes.[117] In a placebo-controlled
study with 800 g and 1600 g of inhaled salbutamol administered 15 min prior to the exercise to non-asthmatic athletes,
no significant difference in total run times was observed. Moreover, urinary concentrations of salbutamol (samples collected
30180 min post-exercise) did not exceed the established threshold of 1000 ng/mL (or decision limit of 1200 ng/mL) at any time
point, suggesting that the allowed dosage and urinary levels for
salbutamol are justified. Conflicting results were generated by
the same group in a project investigating the influence of ethnicity, gender and dehydration on urinary levels of salbutamol following the same dosing regimen.[118] A total of 32 individuals
(18 male and 14 female, three different ethnicities) performed endurance exercise until a target weight loss (2% or 5%) indicating
dehydration was reached. While ethnicity and gender did not significantly influence the urinary elimination of the drug, dehydration in combination with the application of the maximum
allowed dose (1600 g) of salbutamol generated 20 findings
above the established WADA threshold/decision limits. In another
study, nine individuals received 8 mg of salbutamol (orally) and
were subjected to intense exercise (to exhaustion).[119] Urine
samples were collected up to 12 h post-exercise, resulting in
salbutamol findings (up to 4265 ng/mL) above the established
limits within the first 8 h, especially when the urine samples specific gravity was corrected to 1.020.
The questions whether differences in salbutamol enantiomer
tissue disposition or metabolism (due to single-nucleotide
polymorphism (SNP) in sulfotransferase SULT 1A3) might require
consideration in doping controls were pursued by Jacobson

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et al.[120,121] In a study with 25 asthmatic individuals including four
with homozygous SNP, plasma levels of salbutamol enantiomers
were measured following an inhaled dose of 400 g of salbutamol.
As no difference in pharmacokinetics of either isomer was observed, SNP in SULT 1A3 was excluded as confounding factor in
sports drug testing, although corresponding urine samples were
not analyzed. Using a rat model, the enantioselective disposition
of salbutamol in different tissues (cardiac and skeletal) was
elucidated, indicating an increased muscle partitioning of the
pharmacologically active R-salbutamol. Considering the presumed
availability of enantiomerically pure substance, cheating athletes
might exploit this option and R-enantiomer specific analysis was
suggested for doping controls.
Further to the aforementioned factors potentially affecting doping control analytical result interpretations, salbutamol stability
studies were conducted under different conditions. Storage of
urine samples at elevated temperatures for up to several weeks
and non-physiological pH (3 and 11) resulted in the degradation
of salbutamol; the composition of the degradation products, however, was not reported.[122] In contrast, three methylated
salbutamol products were identified in stock solutions of the drug
in methanol when stored for weeks at 40 C. Although these conditions are not in agreement with standard laboratory practice, the
amenability of salbutamol towards degradation is noted.
Besides salbutamol, formoterol represents a 2-agonist necessitating quantitation in sports drug testing. Using six-fold deuterated
formoterol as internal standard, enzymatic hydrolysis and subsequent LLE of urine samples followed by LC-MS/MS analysis, He
et al. established a quantitative approach for the target analyte
covering urinary concentration ranges from 0.1 500 ng/mL.[123]
Omitting the LLE but using an acetonitrile dilution/precipitation
step, Lee et al. presented a similar LC-MS/MS-based methodology
allowing to quantify formoterol from 10-100 ng/mL, covering the
required range of 50200% regarding the drugs urinary threshold
value of 40 ng/mL.[124]
Aiming at improved qualitative detection methods for 2-agonists,
three sulfoconjugated metabolites of fenoterol, a non-threshold
substance, were synthesized in vitro.[125] By means of recombinant sulfotransferases as well as S9 fractions of different human
tissues (liver, kidney, lung, and intestine), two mono- and one
bis-sulfoconjugate of fenoterol were generated and characterized
by MS-approaches. These allowed verifying the presence of
fenoterol metabolites in post-administration urine samples following inhalation and oral application and the availability of reference material enables the inclusion of new target analytes into
the emerging approaches of dilute-and-inject analytics.

Hormone and metabolic modulators

The class of hormone and metabolic modulators of WADAs
Prohibited List comprises five categories. Category 1, the aromatase
inhibitors, includes amongst others formestane, a steroidal
substance that is commonly monitored along with AAS and their
metabolites using GC-MS/MS approaches. Due to formestanes
natural occurrence in human urine, a threshold value of
150 ng/mL applies but lower concentrations could also be the result of exogenous sources of the drug. Hence, IRMS methodologies
are desirable as well as appropriately sensitive and specific action
levels that trigger confirmatory analyses on a sensible basis. Following the analysis of a reference population (n = 3031) and elimination study urine samples collected after administration of T and

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androst-4-ene-3,17-dione, a threshold at 25 ng/mL was suggested

to serve as a viable compromise between sufficient sensitivity for
drug abuse and limiting confirmatory burdens using IRMS.[126]
Tamoxifen belongs to the class of selective estrogen receptor
modulators (SERMs) and has been the most frequently observed
drug of the category S.4 in 2013.[32] Due to its clinical relevance
arguably attributable mainly to its metabolite referred to as
(Z)-endoxifen, in-depth investigations into blood-borne metabolic profiles have been conducted using either DBS[127] or
plasma samples[128] analyzed by MS methodologies. A similarly
comprehensive picture of urinary metabolites was provided by
Lu et al., providing target analytes detectable up to one week
after a single oral dose of 50 mg of tamoxifen.[129] In addition
to the existing information on relevant metabolites, seven
new products were reported as deduced from mass spectrometric information. With the knowledge that tamoxifen has
been sold as so-called dietary supplements,[130] the need to
comprehensively screen for this therapeutic is corroborated.
Agents not prohibited according to WADA can have a substantial impact on the metabolic pattern of prohibited substances
as demonstrated by Mazzarino et al. concerning the SERM
toremifene.[131] In vitro assays were employed to study the formation of phase-I metabolites and the corresponding impact of
antifungal agents, antidepressants, and H2 receptor antagonists, demonstrating that in particular antifungal substances
such as ketoconazole and related compounds considerably altered the production of hydroxylated and carboxylated metabolites of toremifene. Since these frequently serve as target
analytes in doping controls, careful assessment of analytical results is advised if drug-drug-interactions with the aforementioned therapeutics are possible.
A substantial amount of effort was invested into the detection of
insulins in clinical research projects in 2013/2014 and gained information could support future anti-doping activities. Concerning
sample preparation, the utility of molecularly imprinted polymer
(MIP) extraction cartridges coupled on-line to an LC-UV system
was reported.[132] With recoveries of 87% from urine and plasma
along with reported LODs of 0.03 and 0.2 ng/mL, the strategy appears promising also for sports drug testing applications; however,
the presented proof-of-concept data were acquired from nonphysiologically enriched specimens containing 50 ng/mL of insulin
and thus an assessment of the practical impact is difficult. The consecutive use of different nanoporous materials allowed to isolate
and enrich human insulin from artificial urine as demonstrated by
Lei et al.[133] Using 0.5 mL of the urine surrogate and two extraction
steps followed by MALDI-MS analysis, human insulin was measured
with a methods LOD of 0.05 ng/mL. The sensitivity is competitive to
established routine doping control procedures; the only aspect to
demonstrate the fitness-for-purpose of the herein assay is to provide data from authentic urine samples, which indicate specificity
and robustness of the certainly interesting protocol. An alternative
approach for measuring insulins from human plasma included
protein precipitation, mixed-mode SPE, and subsequent twodimensional LC-MS/MS analysis.[134] Requiring 250 L of sample
volume, quantification limits between 50 and 200 pg/mL were
accomplished for human insulin and five structural analogues
employing a trapping/separation step using two different stationary phases and MRM of diagnostic precursor/product ion pairs. In
contrast to the aforementioned methodologies, Peterman et al.
reported on a high-throughput MSIA for the analysis of insulins
in human serum/plasma,[135] similarly to the strategy pursued
concerning the determination of IGF-I (vide supra). Combining

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immunoaffinity purification by immobilized anti-insulin antibodies

with LC-MS/MS (using high resolution/high accuracy MS), LODs between approximately 9 and 23 pg/mL (1.53.75 pM) were accomplished, using a time-efficient semi-automated approach. Being
specifically dedicated to insulin(s), options concerning multiplexing
would be desirable. Recent literature on analytical assays for insulins further elaborated on new, yet non-approved drug candidates
such as pegylated human insulin[136] or the degludec-analogue
HS061.[137] For pegylated insulin, rat plasma underwent a simple
protein precipitation followed by reduction (i.e. cleavage of insulins
A- and B-chain) and alkylation of the solution-retained material,
followed by conventional LC-MS/MS analysis. The working range
of this approach was limited to 1001000 ng/mL and hence, it
is not an option for doping controls. HS061 presents a modified
B-chain (ProB28Asp and ThrB30Glu) and a hexadecanedioic acid
conjugated via glutamic acid to the C-terminal lysine of the B-chain.
In a study by Zhu et al. HS061 was measured from rat plasma by
LC-MS/MS in MRM mode after a simple protein precipitation
step.[137] Whilst being appropriate for the rat administration and
pharmacokinetic study, the achieved LOD of 10 ng/mL would not
be adequate for routine doping controls. Nevertheless, the reports
show that new modified insulins might become available and
should be considered in future sports drug testing programmes.
In addition to insulins, peroxisome proliferator-activated receptor
(PPAR) agonists are prohibited in sports as so-called metabolic
modulators, and MS-based test methods particularly concerning
the representatives GW1516 and GW0742 and respective metabolites have been reported in the past. A more generic and comprehensive approach towards detecting the intact compounds
(mainly in human and animal dietary products) was reported by
Bovee et al. in 2014.[138] The combined use of bioactivity screening,
which allows covering known as well as unknown PPAR agonists,
with targeted LC-MS/MS confirmation presents a fast and sensitive
approach for testing active/intact drugs. Another frequently
discussed metabolic modulator with regards to abuse in sport is
5-aminoimidazole-4-carboxamide ribofuranoside (AICAR). Its quantitative analysis in human urine was found to be comparably facile;
however, its natural abundance complicates the identification of
AICAR administration. Hence, the viability to test for AICAR-ribotide,
the corresponding 5-monophosphate of AICAR, in erythrocytes
was assessed.[139] Since increasing amounts of bioavailable AICAR
lead to an increased formation of AICAR-ribotide in red blood cells
(RBCs), the intra-erythrocytic concentration of this analyte could
provide indications towards abuse of AICAR. Using IDMS, a methodology necessitating 20 L of RBCs was established, enabling the
quantification of AICAR-ribotide between 10 and 1000 ng/mL. A
reference population of 99 individuals provided information on
normal physiological values (10500 ng/mL), and in vitro incubation
tests demonstrated a rapid increase of intracellular AICAR-ribotide
levels up to 12 000 ng/mL. As the phosphoconjugate of AICAR is
trapped in the erythrocyte, the elevated concentration prevails for
a prolonged period of time and might thus serve as a complement
to the ABP concerning AICAR abuse. Confirmation of the
exogenous origin of AICAR was accomplished by urine analysis utilizing IRMS.[140] Due to substantially different carbon isotope ratios
of synthetic AICAR products and naturally produced AICAR, the selectively trimethylsilylated derivative was subjectable to GC/C/IRMS
and significant differences in isotopic signatures of ERCs (e.g. A, E,
PD) and AICAR were observed in an elimination study for more than
40 h post-administration.
Due to the assumed performance enhancing effects of PPAR
agonists and AICAR, PPAR agonists such as pioglitazone were

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the subject of studies concerning their ability to stimulate mitochondrial biogenesis. As presented by Sanchis-Gomar et al. no such
effect was observed in an animal model study.[141]

Diuretics and other masking agents and

stimulants
Among masking agents, glycerol especially has been the subject of
interest in doping-control-related studies and its relevance as a
plasma-volume expanding substance has been questioned. From
a meta-analysis it was concluded that the plasma volume is affected
and increased when fluid administration is accompanied by
glycerol; however, the impact on parameters relevant for the interpretation of the hematological module of ABP such as hemoglobin
or hematocrit were found to be modest.[142] Following this metaanalysis, a placebo-controlled administration study with glycerol
(prior to exercise) was conducted and blood parameters as well as
urinary glycerol concentrations were determined.[143] The results
obtained confirmed the meta-analysis outcome with a significant
difference in plasma volume between the glycerol administration
and the control group. Moreover, urinary concentrations of the
substance were measured and the obtained values supported the
currently enforced threshold level of 4.3 mg/mL.[144] While athletes
of the control group stayed well below the established threshold,
individuals having received glycerol (1 g/kg of bodyweight) considerably exceeded the limit. In order to account for potential differences in glycerol elimination originating from endogenous
sources, e.g. due to ethnicity, gender, etc., 959 athletes samples
from North America were quantified concerning urinary glycerol
concentrations.[145] As a result of the findings presented therein,
the above mentioned conservative threshold of 4.3 mg/mL was
suggested and adopted since urinary glycerol values observed in
post-administration samples reached up to more than 60 mg/mL.
Most commonly, glycerol has been measured from urine by means
of GC-MS-based approaches. Alternatively, the option to analyze
the compound by LC-MS/MS following derivatization was
reported.[146] Using 100 L of urine, the contained glycerol is
derivatized under alkaline conditions with benzoyl chloride for
4 h, before the analyte is partitioned between an aliquot of water
and n-hexane. The method proved sufficiently sensitive
(LOQ = 1 g/mL) and accurate to quantify glycerol in urine between
1 and 1000 g/mL.
Elimination studies concerning the plasma volume expanders
hydroxyethyl starch (HES) and dextran were published by Esposito
et al.[147] Comparison between full scan MS and in-source collisioninduced dissociation (CID) data showed the substantially better
LODs of in-source CID analyses, allowing detecting HES and dextran
abuse for 72 and 12 h, respectively.

Stimulants
As in previous years, WADAs Prohibited List accounts for two
groups of stimulants, namely non-specified and specified
compounds.[16] If a stimulant is not explicitly mentioned as being
non-specified, it is automatically considered as specified. While
traditional stimulants have been well covered in the past, the continuous emergence of designer (psycho)stimulants has become a
major multidisciplinary challenge in legal, forensic, toxicological,
and doping control contexts, with hundreds of new entities registered since 2012.[148157] Due to the fact that this Annual Banned

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Substance Review is dedicated to sports drug testing, the topic of
stimulants is covered with particular focus on anti-doping-related
work and should not be considered exhaustive concerning
designer stimulants in general.
Dietary supplements can be a source for stimulants reportedly
causing AAFs in sport. Song et al. screened 17 arguably herbal
weight loss supplements for the presence of sibutramine and 11
of these products contained the synthetic stimulant between 0.3
and 20 mg per application unit (e.g. capsule, bag).[158] In 2013,
health issues associated with the intake of a dietary supplement advertised with energizing and weight-loss supporting properties
were reported in the Netherlands. The analysis of the retained products revealed the presence of several different stimulating agents,
amongst which the prohibited compound oxilofrine (1-5 mg/capsule) and two phenylethylamine derivatives were detected.[159]
Similarly, the analysis of another nutritional supplement revealed
the presence of N,-diethyl-phenethylamine at 6 mg/g.[160] Besides
the fact that its acute and long-term effects on humans is unknown,
its ingestion via the dietary supplement would likely result in an
AAF in doping controls. The still most frequently detected stimulant
in human doping controls is 1,3-dimethylamylamine (DMAA,
methylhexanamine), which has also been an ingredient of nutritional supplements in the past. DMAAs natural occurrence in
Geraniaceae has been debated in extenso, and results of a recent
study concerning DMAA in relevant plant species, Geranium and
Pelargonium oils, and nutritional supplements supported the hypothesis that DMAA is not a natural component of Geraniaceae.[161]
Consequently, the amounts of DMAA detected in dietary products
were not considered as of plant origin but synthetically derived. A
rapid and quantitative means to determine DMAA in nutritional
supplements was reported by Monakhova, utilizing 1H NMR
spectroscopy.[162] In total, 16 products were tested yielding findings
between 3 and 415 mg/g of DMAA in 9 cases. Due to DMAAs considerable prevalence in dietary supplements, studies concerning
physiological effects and safety profile of the drug were conducted
with either a single oral dose of 25 mg[163] or a 12-week intervention including the daily intake of 50 mg of DMAA (alone and in
combination with caffeine).[164] In both studies, the selected scenario did not result in significant changes of the measured variables
including, for example, body mass/composition, blood pressure,
and heart rate.
Due to the aforementioned growing number of stimulants potentially relevant for doping controls, comprehensive test methods,
knowledge about metabolism, physico-chemical properties and analytical behaviour of the substances are required. Using positive
electrospray ionization (ESI)-MS/MS, Fornal investigated the dissociation pathways of 39 protonated cathinones.[165] Based on common and individual collisionally activated dissociation patterns,
the identification of known as well as structurally related substances is facilitated and initial test methods of broad coverage
are supported. The detection of -methylphenethylamine in eight
doping control urine samples was accomplished by LC-MS/MS as
published by Cholbinski et al.[166] Using acidic hydrolysis followed
by LLE or, alternatively, dilute-and-inject approaches, the methods
LOD was determined as 10 ng/mL which readily demonstrated
its fitness-for-purpose. Also by means of dilute-and-inject but
employing a stable-isotope labelled internal standard, the possibility to test for the presence of lisdexamfetamine, a prodrug of amphetamine, was assessed.[167] Using high resolution/high accuracy
mass spectrometry, lisdexamfetamine was quantified down to
0.15 ng/mL, which allowed for detecting the analyte in postadministration samples up to 11 h. In case of AAFs, measurements

Drug Test. Analysis (2014)

targeting also the prodrug support identification of the original

source of urinary amfetamine.
Test methods complementary to conventional GC- or LC-MS(/MS)
methodologies were presented employing direct analysis in real
time (DART) MS. Both dietary supplements as well as urine samples collected after ingestion of DMAA-containing products were
analyzed by means of DART MS with (LLE or SEP) and without
sample preparation.[168] With this method DMAA was identified
in neat post-administration urine samples collected up to 48 h,
but in the absence of any method characterization, the detection
limit, robustness, etc. the performance is difficult to estimate. Similarly, following thin-film solid-phase microextraction (SPME),
DART MS was used to determine also cocaine from human
urine.[169] LOD of 0.5 ng/mL, which was determined by employing
a deuterated analogue to cocaine, is well below the substance
class MRPL. For urine testing, targeting the metabolite(s) would
have been an alternative due to higher abundances but the
proof-of-concept is nevertheless given.

Cannabinoids
The primordial matter of all cannabinoids, 9-tetrahydrocannabinol
(THC), has been studied in great detail in the context of many different scenarios in the past. Concerning sports drug testing, the recent
use (here: shortly before competition) of THC has been of particular
interest for many years to allow doping control authorities to
appropriately interpret urinary concentrations of AAFs. This is especially relevant as THC use is prohibited in-competition only, and
THCs metabolic fate has been shown to be influenced by numerous factors, even exercise.[170] Desrosiers et al. investigated the potential utility of THC-glucuronide as a urinary marker for the recent
inhalation of THC.[171] In a study comprising a total of 24 frequent
and occasional smokers, urinary THC-glucuronide concentrations
were quantified by LC-MS/MS showing peak concentrations between 0.6 and 7.4 h post-administration. Collecting and analyzing
two consecutive urine samples was suggested, which predicted
recent cannabis smoking amongst the occasional users at an
efficiency of 77%. The option to screen for indicative biomarkers
by means of metabonomic strategies was assessed by Kiss et al.,
who demonstrated the presence of up- as well as down-regulated
parameters in a proof-of-concept study with 15 THC-using athletes,
5 control samples, and 9 doping control samples tested negative
for the use of THC.[172] How these can contribute to future sports
drug testing programs remains to be shown and is the subject
of ongoing projects. Alternative matrices might provide desirable
information and are more and more the subject of anti-doping
research. The possibility to sensitively test for one of the main metabolites 11-nor-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)
in hair was presented by Thieme et al.[173] By means of selective
esterification of the analyte and LC-MS3 measurements, a
substantial improvement in limit of quantification down to
100 fg/mg was accomplished. While hair analysis allows for excellent retrospectivity albeit of limited temporal resolution, oral fluid
analysis might be a viable alternative in the future.[20,174]
Similar to the class of stimulants, a continuously growing supply
of synthetic cannabinoids has been recorded in the recent past, being of concern for public health and sports drug testing.[175,176] In
order to comprehensively cover these substances also in routine
doping controls, adequate target analytes are required, which
necessitates metabolism studies and/or drug-class-specific analytical strategies. For instance, employing human hepatocytes, the

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metabolism of newly identified synthetic cannabinoids such as

AKB-48[177] and PB-22 (as well as its 5-fluorinated analogue)[178]
was studied, corroborating the frequently observed intense metabolism of synthetic cannabinoids revealing mostly hydroxylation(s)
and glucuronidation. A UHPLC-TOF MS-based approach covering
55 cannabimimetic agents (or respective metabolites) was
presented by Sundstrm et al.[179] LODs down to 0.2 ng/mL were
accomplished following enzymatic hydrolysis of 1 mL of urine and
subsequent two-step SPE. Since a variety of the analytes were intact
synthetic cannabinoids, the method might benefit from including
further metabolites due to the aforementioned extent of metabolic
conversion of most cannabimimetics as shown in a similar LC-MS/
MS approach by Scheidweiler and Huestis.[180] Using enzymatic
hydrolysis and supported liquid extraction (SLE), 20 synthetic
cannabinoids and 21 metabolites were identified from human urine
down to 0.1 ng/mL using MRM data recording. Exploiting the generation of diagnostic product ions of distinct pharmacophores of, for
example, indole-derived synthetic cannabinoids was shown to be a
viable alternative for comprehensive analyte coverage too.[181] By
means of precursor ion scanning using three ions (m/z 121, m/z
144, and m/z 155) typically generated from indole-structured
compounds, saliva, blood, and urine was successfully tested for
19 model substances, and LODs between 0.1 and 0.5 ng/mL
were achieved.

Glucocorticosteroids
Glucocorticosteroids are prohibited in sports when systemic application by oral, intravenous, intramuscular or rectal administration
routes is conducted. Analytically, the differentiation of the drug administration route is challenging and local injections such as intraarticular or intratendinuous treatments of triamcinolone acetonide
have been shown to create urinary drug concentrations of up to
200 ng/mL.[182] To support the identification of drug application
regimens, the metabolic pattern of corticosteroids following topical
and oral administration might be useful as shown by Matabosch
et al. concerning methylprednisolone.[183] Comparing the urinary
metabolite profiles following oral (once 4 or 40 mg) and topical
(5 10 mg/day), discrimination of both applications was best accomplished by considering 16,17,21-trihydroxy-6-methylpregna1,4-diene-3,11,20-trione and 17,20,21-trihydroxy-6-methylpregna1,4-diene-3,11-dione, which were significantly lower in case of topical
administrations.

Manipulation of blood and blood components

The most frequently addressed questions relating to this class of
prohibited methods have been autologous and homologous blood
transfusion, which in particular have shown to represent complex
tasks to doping control laboratories. Principles of underlying doping procedures, physiological reactions and potential health risks
associated with these clandestine practices as well as current and
potential future detection strategies have been revisited by several
authors,[184188] with specific focus on various different approaches
using blood and urine as doping control matrices.
Homologous (allogeneic) blood transfusion has been shown to
be detectable by means of measuring arrays of minor blood group
antigens, which allows illustrating the presence or absence of more
than one population of erythrocytes. Complementary, the use of
high resolution quantitative polymerase chain reaction (qPCR)
targeting eight polymorphic markers (seven for the determination

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of ins/del polymorphisms and one specific for the Y-chromosome)

was assessed concerning its utility to determine doping-derived
microchimerism using leukocytes.[189] In proof-of-concept studies
the potential of the methodology to identify donor-DNA in a recipients sample was demonstrated, and the limit of detection was
estimated to require 10% of donor blood. The authors comprehensively discussed the advantages (low cost, speed, complementary
information) but also the caveats (limited sensitivity, limited specificity in case of first-degree relatives, a need for sufficient amounts
of leukocytes) of the assay, which overall can be a valuable addition
to the anti-doping arsenal.
Indirect markers suggesting blood transfusions have been
so-called plasticizers (e.g. di(2-ethylhexyl)phthalate, DEHP)
and respective metabolites in human urine. Whether the intracellular content of DEHP of ex vivo stored erythrocytes
would be another complement was tested by Varlet-Marie
et al. who measured DEHP in RBCs prior to and after infusion
by means of GC-MS.[190] While the content of DEHP before
transfusion was significantly elevated, the rapid decline of the
analytes concentration in vivo did not support using this
marker for doping control purposes.
Concerning the practice of blood doping in general, the ABP
with its hematological module is most likely the most universal tool
of sports drug testing. It can provide information for subsequent
target testing (e.g. regarding recombinant EPO or homologous
blood transfusion) and independently indicate anti-doping rule
violations.[191193] However, longitudinal monitoring of blood parameters, modeling, and interpretation of measurement results necessitates a robust approach in which potentially confounding
factors are accounted for. In that context, the influence of varying atmospheric pressure and vibration as occurring during air
freight on ABP parameters were tested and no effect was recorded within 72 h.[194] Further, plasma osmolality is known to
change during exercise, and depending on the chosen analytical
approach (e.g. manual vs. automated measurements) significant
differences in plasma volume-depending parameters such as
hematocrit can occur.[195] Since ABP measurements are harmonized internationally, these observations should not apply to
routine doping controls. Additionally, suggestions concerning
the improved accounting for real and/or simulated altitude in
the ABP algorithm were made, especially in the light of the different scenarios athletes might exploit altitude exposure or the
assumed effects thereof.[196]
In addition to conventional blood markers such as hematocrit,
hemoglobin content, and percentage of reticulocytes, the utility
and variability of alternative serum markers was assessed. Voss
et al. monitored the exercise-induced changes in plasma volume,
total protein, albumin, ferritin, and soluble transferrin receptor during a 6-day cycling intervention.[197] The obtained data showed that
physical stress as applied to the cyclist by six stage races significantly influences the aforementioned parameters by plasmavolume associated processes and acute phase/inflammatory
reactions. If new parameters are to be implemented into the ABP,
effects as those reported will necessitate systematic recording
during testing and careful consideration.
Probing for alternative test methods concerning autologous
blood doping, the utility of capillary electrophoresis (CE) in separating ex vivo stored erythrocytes from native RBCs was demonstrated
by Harrison et al.[198] Owing to vesiculation, stored erythrocytes exhibit distinct relative size distributions, which can be visualized by
CE after cross-linking of erythrocytic proteins (accomplished by glutaraldehyde) to stabilize the RBCs for CE analytical conditions. Using

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in vitro mixing models, 5% of stored RBCs were detectable using the
proof-of-concept study. If the approach is applicable to authentic
post-transfusion blood samples remains to be shown.

Gene doping

Acknowledgements

Advances in therapeutic benefit and safety of gene therapies inevitably increase the risk of their misuse in sport.[199] Proactively
established detection methods for gene doping practices have
largely focused on the direct detection of the employed transgene
(s) and vector control elements using PCR-based methodologies as
recently reviewed by Perez et al.[200] New additions to this armamentarium have resulted from vector distribution and clearance studies
as reported by Baoutina et al., who developed and validated different real-time PCR assays targeting transgenic human erythropoietin
cDNA.[201] Five assays spanning four different splicing sites were
assessed and ultimate sensitivity was accomplished after linearization of the plasmid, enabling the detection of five copies of the
transgene in the presence of substantial genomic DNA background.
Further to gene therapy methods, the abuse of drug candidates
operating via RNA interference such as small interfering RNA
(siRNA) has necessitated consideration by doping control laboratories. Due to the fact that siRNA-based drugs predominantly require
stabilizing modifications, for example 2-O-methylation or the use
of locked nucleic acids, non-natural targets are provided for sports
drug testing purposes. To probe for the metabolic fate of intravenously administered siRNA and the possibility to detect the intact
substance as well as metabolites in urine, animal studies were conducted with model siRNA designed to interfere with myostatin.[202]
Using conventional RNA extraction kits, LC-HRMS top-down as well
as bottom-up, and alternatively SDS-PAGE analyses, the model
substances and metabolites were detected in rat urine for up to
24 h following a single dose of siRNA administered intravenously
at 1 mg/kg.

Conclusion
With the continuously growing complexity of the options presumably enhancing athletic performance, research in anti-doping science is becoming extremely multifaceted. Besides the efforts
made to expand test methods and to include more/new prohibited
substances and methods of doping into routine sports drug testing
programs, an increasing number of studies have been dedicated to
complementary goals. These include predominantly the identification of alternative doping control matrices as well as the fine-tuning
of existing approaches to reduce the burdens imposed on both the
testing and the tested party and to improve the sensitivity for
traditional doping agents. As a result of the latter, findings for
stanozolol and dehydrochloromethyltestosterone for instance
increased considerably from approximately 290 in 2012 to over
540 in 2013. To accommodate the enormous number of legal highs,
designer stimulants, and synthetic cannabinoids, a substantial
number of publications has been recorded also in an anti-doping
context although the larger misuse of these substances by elite athletes has not been observed in laboratory analyses or obtained by
other means of anti-doping investigations. In contrast, the issue of
(adulterated) nutritional supplements has seemingly affected antidoping statistics, where the drug with arguably modest stimulating
properties methylhexaneamine has resulted in 169 findings in
2013. Overall, the scientific efforts undertaken by numerous international research groups have allowed for significant

Drug Test. Analysis (2014)

accomplishments in the international fight against doping; much

information has been generated on doping agents and their detection as well as traps that athletes might fall into inadvertently. For
both scenarios, scientific data are invaluable and indispensable.

The authors thank the Federal Ministry of the Interior of the Federal
Republic of Germany and Manfred-Donike-Institute for Doping
Analysis, Cologne, for supporting the presented work.

References
[1] J.R. Delanghe, T.M. Maenhout, M.M. Speeckaert, M.L. De Buyzere.
Detecting doping use: More than an analytical problem. Acta Clin.
Belg. 2014, 69, 25.
[2] J. Dvorak, N. Baume, F. Botre, J. Broseus, R. Budgett, W.O. Frey,
H. Geyer, P.R. Harcourt, D. Ho, D. Howman, V. Isola, C. Lundby,
F. Marclay, A. Peytavin, A. Pipe, Y.P. Pitsiladis, C. Reichel, N. Robinson,
G. Rodchenkov, M. Saugy, S. Sayegh, J. Segura, M. Thevis, A. Vernec,
M. Viret, M. Vouillamoz, M. Zorzoli. Time for change: A roadmap to
guide the implementation of the World Anti-Doping Code 2015.
Brit. J. Sport Med. 2014, 48, 801.
[3] J. Savulescu, L. Creaney, A. Vondy. Should athletes be allowed to use
performance enhancing drugs? Brit. Med. J. 2013, 347, f6150.
[4] B.R. Alexander. War on drugs redux: Welcome to the war on doping in
sports. Subst. Use Misuse 2014, 49, 1190.
[5] D. Valkenburg, O. de Hon, I. van Hilvoorde. Doping control, providing
whereabouts and the importance of privacy for elite athletes. Int. J.
Drug Policy 2014, 25, 212.
[6] H.G. Pope Jr., R.I. Wood, A. Rogol, F. Nyberg, L. Bowers, S. Bhasin.
Adverse health consequences of performance-enhancing drugs: An
Endocrine Society scientific statement. Endocr. Rev. 2014, 35, 341.
[7] A. Ljungqvist. The fight against doping is a fight for the protection of
the clean athlete, the health of the athlete and the integrity of sport.
Brit. J. Sport Med. 2014, 48, 799.
[8] M.A. Klein. Protecting athletes and ensuring sports--free of doping.
Subst. Use Misuse 2014, 49, 1198.
[9] C. Johnson, R.J. Peters Jr. Stereotypes of athletes use of performance
enhancing products. J. Sport Sci. Med. 2014, 13, 456.
[10] P. Bonte, S. Sterckx, G. Pennings. May the blessed man win: A critique
of the categorical preference for natural talent over doping as proper
origins of athletic ability. J. Med. Philos. 2014, 39, 368.
[11] J. Hoberman. Physicians and the sports doping epidemic. Virtual
Mentor 2014, 16, 570.
[12] S. Camporesi, M.J. McNamee. Performance enhancement, elite
athletes and anti doping governance: Comparing human guinea
pigs in pharmaceutical research and professional sports. Philos.
Ethics Humanit. Med. 2014, 9, 4.
[13] J. Dvorak, M. Saugy, Y.P. Pitsiladis. Challenges and threats to
implementing the fight against doping in sport. Brit. J. Sport Med.
2014, 48, 807.
[14] W. Maennig. Inefficiency of the anti-doping system: Cost reduction
proposals. Subst. Use Misuse 2014, 49, 1201.
[15] F. Botre, X. de la Torre, F. Donati, M. Mazzarino. Narrowing the gap
between the number of athletes who dope and the number of
athletes who are caught: Scientific advances that increase the
efficacy of antidoping tests. Brit. J. Sport Med. 2014, 48, 833.
[16] World Anti-Doping Agency. The 2014 Prohibited List. Available at:
https://wada-main-prod.s3.amazonaws.com/resources/files/WADARevised-2014-Prohibited-List-EN.PDF [4 November 2014].
[17] J. Weinreich. Dokumente zum russischen "Sauerstoff-Cocktail" mit
Xenon im Hochleistungssport, in Jens Weinreich - sport and politics.
Available at: https://www.jensweinreich.de/2014/02/25/dokumentezum-russischen-sauerstoff-cocktail-mit-xenon-im-hochleistungs
sport-doping-sochi2014/ [30 September 2014].
[18] Economist. Breathe it in, in The Economist. Available at: http://www.
economist.com/news/science-and-technology/21595890-obscuregas-improves-athletes-performance-breathe-it [30 September 2014].
[19] World Anti-Doping Agency. The 2014 Monitoring Program. Available
at: https://www.wada-ama.org/en/files/wada-monitoring-program2014-enpdf [26 September 2014].

Copyright 2014 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/dta

Drug Testing
and Analysis

M. Thevis et al.

[20] S. Anizan, M.A. Huestis. The potential role of oral fluid in antidoping
testing. Clin. Chem. 2014, 60, 307.
[21] A. Mena-Bravo, M.D. Luque de Castro. Sweat: A sample with limited
present applications and promising future in metabolomics.
J. Pharmaceut. Biomed. 2014, 90, 139.
[22] T.W. McDade. Development and validation of assay protocols for use
with dried blood spot samples. Am. J. Hum. Biol. 2014, 26, 1.
[23] A. Sharma, S. Jaiswal, M. Shukla, J. Lal. Dried blood spots: Concepts,
present status, and future perspectives in bioanalysis. Drug Test.
Anal. 2014, 6, 399.
[24] R.N. Rao. Emerging liquid chromatography-mass spectrometry
technologies improving dried blood spot analysis. Expert Rev.
Proteomics 2014, 11, 425.
[25] R.V. Oliveira, J. Henion, E. Wickremsinhe. Fully-automated approach
for online dried blood spot extraction and bioanalysis by twodimensional-liquid chromatography coupled with high-resolution
quadrupole time-of-flight mass spectrometry. Anal. Chem. 2014, 86,
1246.
[26] M. Thevis, A. Thomas, W. Schnzer. Targeting prohibited
substances in doping control blood samples by means of
chromatographic-mass spectrometric methods. Anal. Bioanal.
Chem. 2013, 405, 9655.
[27] M. Thevis, W. Schnzer. Analytical approaches for the detection of
emerging therapeutics and non-approved drugs in human doping
controls. J. Pharm. Biomed. Anal. 2014, 101, 66.
[28] S. Hppner, P. Delahaut, W. Schnzer, M. Thevis. Mass
spectrometric studies on the in vivo metabolism and excretion of
SIRT1 activating drugs in rat urine, dried blood spots, and plasma
samples for doping control purposes. J. Pharm. Biomed. Anal.
2014, 88, 649.
[29] C. Vanhee, G. Moens, E. Deconinck, J.O. De Beer. Identification and
characterization of peptide drugs in unknown pharmaceutical
preparations seized by the Belgian authorities: Case report on
AOD9604. Drug Test. Anal. 2014, 6, 964.
[30] P.R. Harcourt, F. Marclay, B. Clothier. A forensic perspective of the AFL
investigation into peptides: An antidoping investigation case study.
Brit. J. Sport Med. 2014, 48, 810.
[31] A.K. Orlovius, A. Thomas, W. Schnzer, M. Thevis. AOD-9604 does not
influence the WADA hGH isoform immunoassay. Drug Test. Anal.
2013, 5, 850.
[32] World Anti-Doping Agency. 2013 Anti-Doping Testing Figures Laboratory Report. Available at: https://wada-main-prod.s3.amazonaws.
com/resources/files/WADA-2013-Anti-Doping-Testing-Figures-LABORATORY-REPORT.pdf [9 May 2014].
[33] M.R. dos Santos, R.G. Dias, M.C. Laterza, M.U. Rondon, A.M. Braga, R.
L. de Moraes Moreau, C.E. Negrao, M.J. Alves. Impaired post exercise
heart rate recovery in anabolic steroid users. Int. J. Sport Med. 2013,
34, 931.
[34] C.M. Girgis, I. Kuo, D.J. Handelsman. Acute hepatitis and fevers in an
amateur body-builder: A new complication of synthetic androgen
abuse? Endocr. Pract. 2014, 20, e130.
[35] S. Tsitsilonis, C.E. Panayiotis, M.S. Athanasios, K.K. Stavros, V.S. Ioannis,
A. George, F. Konstantinos, P.N. Despina, Z.B. Aristides. Anabolic
androgenic steroids reverse the beneficial effect of exercise on
tendon biomechanics: An experimental study. Foot Ankle Surg.
2014, 20, 94.
[36] M.A. Santos, C.V. Oliveira, A.S. Silva. Adverse cardiovascular effects
from the use of anabolic-androgenic steroids as ergogenic
resources. Subst. Use Misuse 2014, 49, 1132.
[37] A.S. Lindqvist, T. Moberg, B.O. Eriksson, C. Ehrnborg, T. Rosen,
C. Fahlke. A retrospective 30-year follow-up study of former
Swedish-elite male athletes in power sports with a past anabolic
androgenic steroids use: A focus on mental health. Brit. J. Sport Med.
2013, 47, 965.
[38] S. Darke, M. Torok, J. Duflou. Sudden or unnatural deaths involving
anabolic-androgenic steroids. J. Forensic Sci. 2014, 59, 1025.
[39] E. Nieschlag, S. Nieschlag. Testosterone deficiency: A historical
perspective. Asian J. Androl. 2014, 16, 161.
[40] I.M. Egner, J.C. Bruusgaard, E. Eftestol, K. Gundersen. A cellular
memory mechanism aids overload hypertrophy in muscle long
after an episodic exposure to anabolic steroids. J. Physiol. 2013,
591, 6221.
[41] H. Geyer, W. Schnzer, M. Thevis. Anabolic agents: Recent strategies
for their detection and protection from inadvertent doping. Brit. J.
Sport Med. 2014, 48, 820.

wileyonlinelibrary.com/journal/dta

[42] W. Abushareeda, A. Fragkaki, A. Vonaparti, Y. Angelis, M. Tsivou,

K. Saad, S. Kraiem, E. Lyris, M. Alsayrafi, C. Georgakopoulos.
Advances in the detection of designer steroids in anti-doping.
Bioanalysis 2014, 6, 881.
[43] O.J. Pozo, N. De Brabanter, A. Fabregat, J. Segura, R. Ventura,
P. Van Eenoo, K. Deventer. Current status and bioanalytical
challenges in the detection of unknown anabolic androgenic
steroids in doping control analysis. Bioanalysis 2013, 5, 2661.
[44] D. Cuervo, P. Diaz-Rodriguez, J. Munoz-Guerra. An automated sample
preparation for detection of 72 doping-related substances. Drug Test.
Anal. 2014, 6, 516.
[45] M. Raro, T. Portoles, J.V. Sancho, E. Pitarch, F. Hernandez, J. Marcos,
R. Ventura, C. Gomez, J. Segura, O.J. Pozo. Mass spectrometric
behavior of anabolic androgenic steroids using gas chromatography
coupled to atmospheric pressure chemical ionization source. Part I:
Ionization. J. Mass Spectrom. 2014, 49, 509.
[46] L. Hintikka, M. Haapala, T. Kuuranne, A. Leinonen, R. Kostiainen.
Analysis of anabolic steroids in urine by gas chromatographymicrochip atmospheric pressure photoionization-mass spectrometry
with chlorobenzene as dopant. J. Chromatogr. A 2013, 1312, 111.
[47] F. Guo, J. Shao, Q. Liu, J.B. Shi, G.B. Jiang. Automated and sensitive
determination of four anabolic androgenic steroids in urine by
online turbulent flow solid-phase extraction coupled with liquid
chromatography-tandem mass spectrometry: A novel approach for
clinical monitoring and doping control. Talanta 2014, 125, 432.
[48] Q. Zhong, Y. Hu, G. Li. A novel protocol for molecularly imprinted
polymer filaments online coupled to GC-MS for the determination
of androgenic steroids in urine. J. Sep. Sci. 2013, 36, 3903.
[49] E. Tudela, K. Deventer, L. Geldof, P. Van Eenoo. Urinary detection of
conjugated and unconjugated anabolic steroids by dilute-and-shoot
liquid chromatography-high resolution mass spectrometry. Drug
Test. Anal. 2014, in press. DOI: 10.1002/dta.1650
[50] World Anti-Doping Agency. Minimum Required Performance Levels
for Detection and Identification of Non-Threshold Substances. Available at: http://www.wada-ama.org/Documents/World_Anti-Doping_
Program/WADP-IS-Laboratories/Technical_Documents/WADATD2013MRPL-Minimum-Required-Performance-Levels-v1-2012-EN.
pdf [20 September 2013].
[51] L. Geldof, L. Lootens, M. Polet, D. Eichner, T. Campbell, V. Nair, F. Botre,
P. Meuleman, G. Leroux-Roels, K. Deventer, P.V. Eenoo. Metabolism of
methylstenbolone studied with human liver microsomes and the
uPA(+)/(+)-SCID chimeric mouse model. Biomed. Chromatogr.
2014, 28, 974.
[52] S. Guddat, G. Fusshller, S. Beuck, A. Thomas, H. Geyer, A. Rydevik,
U. Bondesson, M. Hedeland, A. Lagojda, W. Schnzer, M. Thevis.
Synthesis, characterization, and detection of new oxandrolone
metabolites as long-term markers in sports drug testing. Anal.
Bioanal. Chem. 2013, 405, 8285.
[53] A.M. Marzouk, G.T. Maatooq. Microbial metabolism of danazol: A
contribution to doping analysis. Z. Naturforsch. 2014, 69, 245.
[54] C. Gomez, O.J. Pozo, L. Garrostas, J. Segura, R. Ventura. A new sulphate
metabolite as a long-term marker of metandienone misuse. Steroids
2013, 78, 1245.
[55] W. Schnzer, S. Guddat, A. Thomas, G. Opfermann, H. Geyer, M. Thevis.
Expanding analytical possibilities concerning the detection of
stanozolol misuse by means of high resolution/high accuracy mass
spectrometric detection of stanozolol glucuronides in human sports
drug testing. Drug Test. Anal. 2013, 5, 810.
[56] T. Kuuranne, M. Saugy, N. Baume. Confounding factors and genetic
polymorphism in the evaluation of individual steroid profiling. Brit. J.
Sport Med. 2014, 48, 848.
[57] K. Rutkowski, P. Sowa, J. Rutkowska-Talipska, A. Kuryliszyn-Moskal,
R. Rutkowski. Dehydroepiandrosterone (DHEA): Hypes and hopes.
Drugs 2014, 74, 1195.
[58] K. Collomp, C. Buisson, F. Lasne, R. Collomp. DHEA, physical exercise
and doping. J. Steroid Biochem Mol. Biol. 2015, 145C, 206.
[59] S. Bermon, P.Y. Garnier, A. Linden Hirschberg, N. Robinson, S. Giraud,
R. Nicoli, N. Baume, M. Saugy, P. Fenichel, S.J. Bruce, H. Henry,
G. Dolle, M. Ritzen. Serum androgen levels in elite female athletes.
J. Clin. Endocrinol. Metab. 2014, 99, 4328.
[60] J.J. Schulze, J.E. Mullen, E. Berglund Lindgren, M. Ericsson, L. Ekstrom,
A.L. Hirschberg. The impact of genetics and hormonal contraceptives
on the steroid profile in female athletes. Front. Endocrinol. 2014, 5, 50.
[61] A. Fabregat, J. Marcos, L. Garrostas, J. Segura, O.J. Pozo, R. Ventura.
Evaluation of urinary excretion of androgens conjugated to cysteine

Copyright 2014 John Wiley & Sons, Ltd.

Drug Test. Analysis (2014)

Drug Testing
and Analysis

Annual banned substance review

[62]

[63]

[64]

[65]

[66]
[67]

[68]
[69]

[70]

[71]
[72]
[73]

[74]

[75]

[76]

[77]
[78]

[79]
[80]

[81]

in human pregnancy by mass spectrometry. J. Steroid Biochem. 2014,

139, 192.
D.J. Handelsman, A. Idan, J. Grainger, C. Goebel, L. Turner, A.
J. Conway. Detection and effects on serum and urine steroid and
LH of repeated GnRH analog (leuprolide) stimulation. J. Steroid
Biochem. 2014, 141, 113.
F. Badoud, J. Boccard, C. Schweizer, F. Pralong, M. Saugy, N. Baume.
Profiling of steroid metabolites after transdermal and oral
administration of testosterone by ultra-high pressure liquid
chromatography coupled to quadrupole time-of-flight mass
spectrometry. J. Steroid Biochem. 2013, 138C, 222.
X. de la Torre, D. Curcio, C. Colamonici, F. Molaioni, F. Botre.
Metabolism of boldione in humans by mass spectrometric
techniques: detection of pseudoendogenous metabolites. Drug Test.
Anal. 2013, 5, 834.
M. Polet, P. Van Renterghem, W. Van Gansbeke, P. Van Eenoo. Studies
on the minor metabolite 6a-hydroxy-androstenedione for doping
control purposes and its contribution to the steroid profile. Drug
Test. Anal. 2014, 6, 978.
J. Wang, R. Yang, W. Yang, X. Liu, Y. Xing. Impact of progesterone
administration on doping test of endogenous steroids. Anal.
Bioanal. Chem. 2014, 406, 6061.
M. Thevis, W. Schnzer, H. Geyer, D. Thieme, J. Grosse, C. Rautenberg,
U. Flenker, S. Beuck, A. Thomas, R. Holland, J. Dvorak. Traditional
Chinese medicine and sports drug testing: Identification of natural
steroid administration in doping control urine samples resulting
from musk (pod) extracts. Brit. J. Sport Med. 2013, 47, 109.
Y. He, J. Wang, X. Liu, Y. Xu, Z. He. Influences of musk administration
on the doping test. Steroids 2013, 78, 1047.
L. Zhang, M. Thevis, T. Piper, M.A. Jochmann, J.B. Wolbert, D.
M. Kujawinski, S. Wiese, T. Teutenberg, T.C. Schmidt. Carbon isotope
ratio analysis of steroids by high-temperature liquid
chromatography-isotope ratio mass spectrometry. Anal. Chem.
2014, 86, 2297.
D. Thieme, C. Rautenberg, J. Grosse, M. Schoenfelder. Significant
increase of salivary testosterone levels after single therapeutic
transdermal administration of testosterone: Suitability as a
potential screening parameter in doping control. Drug Test. Anal.
2013, 5, 819.
G. Forsdahl, H.K. Vatne, T. Geisendorfer, G. Gmeiner. Screening of
testosterone esters in human plasma. Drug Test. Anal. 2013, 5, 826.
L. Tretzel, A. Thomas, H. Geyer, G. Gmeiner, G. Forsdahl, V. Pop,
W. Schanzer, M. Thevis. Use of dried blood spots in doping control
analysis of anabolic steroid esters. J. Pharmaceut. Biomed. 2014, 96, 21.
A. Rydevik, U. Bondesson, M. Thevis, M. Hedeland. Mass spectrometric
characterization of glucuronides formed by a new concept,
combining Cunninghamella elegans with TEMPO. J. Pharmaceut.
Biomed. 2013, 84, 278.
A. Rydevik, A. Lagojda, M. Thevis, U. Bondesson, M. Hedeland. Isolation
and characterization of a beta-glucuronide of hydroxylated SARM S1
produced using a combination of biotransformation and chemical
oxidation. J. Pharmaceut. Biomed. 2014, 98, 36.
M. Thevis, A. Thomas, T. Piper, O. Krug, P. Delahaut, W. Schnzer.
Liquid chromatography-high resolution/high accuracy (tandem)
mass spectrometry-based identification of in vivo generated
metabolites of the selective androgen receptor modulator ACP-105
for doping control purposes. Eur. J. Mass. Spectrom. 2013, 20, 73.
K. Akita, K. Harada, J. Ichihara, N. Takata, Y. Takahashi, K. Saito. A novel
selective androgen receptor modulator, NEP28, is efficacious in
muscle and brain without serious side effects on prostate. Eur. J.
Pharmacol. 2013, 720, 107.
J. Brett, A.H. Dawson, J.A. Brown. Clenbuterol toxicity: A NSW poisons
information centre experience. Med. J. Aust. 2014, 200, 219.
C. Song, A. Zhi, Q. Liu, J. Yang, G. Jia, J. Shervin, L. Tang, X. Hu, R. Deng,
C. Xu, G. Zhang. Rapid and sensitive detection of beta-agonists using a
portable fluorescence biosensor based on fluorescent nanosilica and
a lateral flow test strip. Biosens. Bioelectron. 2013, 50, 62.
V. Cernaro, A. Lacquaniti, A. Buemi, R. Lupica, M. Buemi. Does
erythropoietin always win? Curr. Med. Chem. 2014, 21, 849.
M. Hardeman, T. Alexy, B. Brouwer, P. Connes, F. Jung, H. Kuipers, O.
K. Baskurt. EPO or PlacEPO? Science versus practical experience:
Panel discussion on efficacy of erythropoetin in improving
performance. Biorheology 2014, 51, 83.
C. Reichel. Detection of peptidic erythropoiesis-stimulating agents in
sport. Brit. J. Sport Med. 2014, 48, 842.

Drug Test. Analysis (2014)

[82] M. Citartan, S.C. Gopinath, Y. Chen, T. Lakshmipriya, T.H. Tang.

Monitoring recombinant human erythropoietin abuse among
athletes. Biosens. Bioelectron. 2015, 63, 86.
[83] P. Desharnais, J.F. Naud, C. Ayotte. Desialylation improves the
detection of recombinant erythropoietins in urine samples analyzed
by SDS-PAGE. Drug Test. Anal. 2013, 5, 870.
[84] F. Garribba, S. Turi, G. Corpetti, A. Khiabani, X. De la Torre, F. Botre. A
modified procedure based on a vacuum-driven blotting system for
the detection of erythropoietin and its analogs. Bioanalysis 2014, 6,
1605.
[85] M. Lnnberg, C. Lundby. Detection of EPO injections using a rapid
lateral flow isoform test. Anal. Bioanal. Chem. 2013, 405, 9685.
[86] Y. Dehnes, A. Shalina, L. Myrvold. Detection of recombinant EPO in
blood and urine samples with EPO WGA MAIIA, IEF and SAR-PAGE
after microdose injections. Drug Test. Anal. 2013, 5, 861.
[87] J. Jiang, F. Tian, Y. Cai, X. Qian, C.E. Costello, W. Ying. Site-specific
qualitative and quantitative analysis of the N- and O-glycoforms in
recombinant human erythropoietin. Anal. Bioanal. Chem. 2014, 406,
6265.
[88] M. Okano, M. Sato, S. Kageyama. Identification of the long-acting
erythropoiesis-stimulating agent darbepoetin alfa in human urine
by liquid chromatography-tandem mass spectrometry. Anal.
Bioanal. Chem. 2014, 406, 1317.
[89] F. Mazzei, R. Antiochia, F. Botre, G. Favero, C. Tortolini. Affinity-based
biosensors in sport medicine and doping control analysis.
Bioanalysis 2014, 6, 225.
[90] N. Pang, Y. Bai, Y. Zhou, X. Yang, Z. Zhang, H. Nie, X. Fu, H. Liu. Rapid
and subnanomolar assay of recombinant human erythropoietin by
capillary electrophoresis using NanoOrange precolumn labeling and
laser-induced fluorescence detection. J. Sep. Sci. 2014, 37, 2233.
[91] L. Zhang, Y. Wang, J. Wang, J. Shi, K. Deng, W. Fu. rhEPO/EPO
discrimination with ultrasensitive electrochemical biosensor
based on sandwich-type nano-Au/ZnO sol-gel/nano-Au signal
amplification. Biosens. Bioelectron. 2013, 50, 217.
[92] N. Stambuk, Z. Manojlovic, P. Turcic, R. Martinic, P. Konjevoda,
T. Weitner, P. Wardega, M. Gabricevic. A simple three-step method
for design and affinity testing of new antisense peptides: An
example of erythropoietin. Int. J. Mol. Sci. 2014, 15, 9209.
[93] B. Ebert, W. Jelkmann. Intolerability of cobalt salt as erythropoietic
agent. Drug Test. Anal. 2014, 6, 185.
[94] W. Jelkmann. Xenon misuse in sports Increase of hypoxia-inducible
factors and erythropoietin, or nothing but hot air? Dtsch. Z.
Sportmed. 2014, 65, 267.
[95] M. Thevis, T. Piper, H. Geyer, A. Thomas, M.S. Schaefer, P. Kienbaum,
W. Schnzer. Measuring xenon in human plasma and blood by
gas chromatography/mass spectrometry. Rapid Commun. Mass
Spectrom. 2014, 28, 1501.
[96] U.H. Stenman, H. Alfthan. Determination of human chorionic
gonadotropin. Best Pract. Res. Clin. Endocrinol. Metab. 2013, 27, 783.
[97] G.A. Woldemariam, A.W. Butch. Immunoextraction-tandem mass
spectrometry method for measuring intact human chorionic
gonadotropin, free beta-subunit, and beta-subunit core fragment in
urine. Clin. Chem. 2014, 60, 1089.
[98] G.K. Singh, M. Jimenez, R. Newman, D.J. Handelsman. Immunoreactive
LH in long-term frozen human urine samples. Drug Test. Anal.
2014, 6, 336.
[99] R.I. Holt. Detecting growth hormone misuse in athletes. Indian J.
Endocrinol. Metab. 2013, 17, S18.
[100] G.A. Green. Drug testing in sport: hGH (human growth hormone).
Virtual Mentor 2014, 16, 547.
[101] J.A. Hanley, O. Saarela, D.A. Stephens, J.C. Thalabard. hGH isoform
differential immunoassays applied to blood samples from athletes:
Decision limits for anti-doping testing. Growth Horm. IGF Res. 2014,
24, 205.
[102] S.C. Voss, S. Giraud, M. Alsayrafi, P.C. Bourdon, Y.O. Schumacher,
M. Saugy, N. Robinson. The effect of a period of intensive exercise
on the isoform test to detect growth hormone doping in sports.
Growth Horm. IGF Res. 2013, 23, 105.
[103] C. Pritchard, K.J. Groves, S. Biesenbruch, G. OConnor, A.E. Ashcroft,
C. Arsene, D. Schulze, M. Quaglia. Quantification of human growth
hormone in serum with a labeled protein as an internal standard:
Essential considerations. Anal. Chem. 2014, 86, 6525.
[104] J. Bosch, A. Luchini, S. Pichini, D. Tamburro, C. Fredolini, L. Liotta,
E. Petricoin, R. Pacifici, F. Facchiano, J. Segura, E. Garaci,
R. Gutierrez-Gallego. Analysis of urinary human growth hormone

Copyright 2014 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/dta

Drug Testing
and Analysis

[105]

[106]
[107]
[108]
[109]

[110]

[111]

[112]

[113]

[114]

[115]
[116]
[117]
[118]

[119]

[120]
[121]

[122]

M. Thevis et al.

(hGH) using hydrogel nanoparticles and isoform differential

immunoassays after short recombinant hGH treatment: Preliminary
results. J. Pharmaceut. Biomed. 2013, 85, 194.
A. Kniess, E. Ziegler, D. Thieme, R.K. Mller. Intra-individual variation of
GH-dependent markers in athletes: Comparison of population based
and individual thresholds for detection of GH abuse in sports.
J. Pharmaceut. Biomed. 2013, 84, 201.
B.N. Kelly, D.M. Haverstick, J.K. Lee, M.O. Thorner, M.L. Vance, W. Xin, D.
E. Bruns. Circulating microRNA as a biomarker of human growth
hormone administration to patients. Drug Test. Anal. 2014, 6, 234.
N. Guha, D.A. Cowan, P.H. Sonksen, R.I. Holt. Insulin-like growth factor-I
(IGF-I) misuse in athletes and potential methods for detection. Anal.
Bioanal. Chem. 2013, 405, 9669.
H.D. Cox, D. Eichner. Detection of human insulin-like growth factor-1
in deer antler velvet supplements. Rapid Commun. Mass Spectrom.
2013, 27, 2170.
N. Guha, I. Erotokritou-Mulligan, C. Bartlett, S.P. Nevitt, M. Francis, E.
E. Bassett, D.A. Cowan, P.H. Sonksen, R.I. Holt. Biochemical markers
of insulin-like growth factor-I misuse in athletes: The response of
serum IGF-I, procollagen type III amino-terminal propeptide, and the
GH-2000 score to the administration of rhIGF-I/rhIGF binding
protein-3 complex. J. Clin. Endocrinol. Metab. 2014, 99, 2259.
E.E. Niederkofler, D.A. Phillips, B. Krastins, V. Kulasingam, U.A. Kiernan,
K.A. Tubbs, S.M. Peterman, A. Prakash, E.P. Diamandis, M.F. Lopez,
D. Nedelkov. Targeted selected reaction monitoring mass
spectrometric immunoassay for insulin-like growth factor 1. PLoS
One 2013, 8, e81125.
P.E. Oran, O. Trenchevska, D. Nedelkov, C.R. Borges, M.
R. Schaab, D.S. Rehder, J.W. Jarvis, N.D. Sherma, L. Shen,
B. Krastins, D.C. Schwenke, P.D. Reaven, R.W. Nelson. Parallel
workflow for high-throughput (>1,000 samples/day) quantitative
analysis of human insulin-like growth factor 1 using mass
spectrometric immunoassay. PLoS One 2014, 9, e92801.
F. Lopes, D.A. Cowan, M. Thevis, A. Thomas, M.C. Parkin. Quantification
of intact human insulin-like growth factor-I in serum by nanoultrahigh-performance liquid chromatography/tandem mass
spectrometry. Rapid Commun. Mass Spectrom. 2014, 28, 1426.
H.D. Cox, F. Lopes, G.A. Woldemariam, J.O. Becker, M.C. Parkin,
A. Thomas, A.W. Butch, D.A. Cowan, M. Thevis, L.D. Bowers, A.
N. Hoofnagle. Interlaboratory agreement of insulin-like growth
factor 1 concentrations measured by mass spectrometry. Clin. Chem.
2014, 60, 541.
N. Guha, I. Erotokritou-Mulligan, S.P. Nevitt, M. Francis, C. Bartlett,
D.A. Cowan, E.E. Bassett, P.H. Sonksen, R.I. Holt. Biochemical markers
of recombinant human insulin-like growth factor-I (rhIGF-I)/rhIGF
binding protein-3 (rhIGFBP-3) misuse in athletes. Drug Test. Anal.
2013, 5, 843.
A.G. Fragkaki, C. Georgakopoulos, S. Sterk, M.W. Nielen. Sports doping:
Emerging designer and therapeutic beta-agonists. Clin. Chim. Acta
2013, 425C, 242.
N. Decorte, D. Bachasson, M. Guinot, P. Flore, P. Levy, S. Verges,
B. Wuyam. Effect of salbutamol on neuromuscular function in
endurance athletes. Med. Sci. Sports Exerc. 2013, 45, 1925.
J. Dickinson, J. Hu, N. Chester, M. Loosemore, G. Whyte. Acute impact
of inhaled short acting b2-agonists on 5 km running performance.
J. Sport Sci. Med. 2014, 13, 271.
J. Dickinson, J. Hu, N. Chester, M. Loosemore, G. Whyte. Impact of
ethnicity, gender, and dehydration on the urinary excretion of
inhaled salbutamol with respect to doping control. Clin. J. Sport
Med. 2014, 24, 482.
M. Hostrup, A. Kalsen, M. Auchenberg, S. Rzeppa, P. Hemmersbach,
J. Bangsbo, V. Backer. Urine concentrations of oral salbutamol in
samples collected after intense exercise in endurance athletes. Drug
Test. Anal. 2014, 6, 528.
G.A. Jacobson, K.C. Yee, D. Premilovac, S. Rattigan. Enantioselective
disposition of (R/S)-albuterol in skeletal and cardiac muscle. Drug
Test. Anal. 2014, 6, 563.
G.A. Jacobson, K.C. Yee, R. Wood-Baker, E.H. Walters. SULT 1A3 singlenucleotide polymorphism and the single dose pharmacokinetics of
inhaled salbutamol enantiomers: Are some athletes at risk of higher
urine levels? Drug Test. Anal. 2014, in press. DOI: 10.1002/dta.1645
B.C. Garrido, M.L. Silva, R.M. Borges, M.C. Padilha, F.R. de Aquino Neto.
Salbutamol extraction from urine and its stability in different
solutions: Identification of methylation products and validation of a
quantitative analytical method. Biomed. Chromatogr. 2013, 27, 1630.

wileyonlinelibrary.com/journal/dta

[123] G. He, J. Lu, X. Wang, Y. Xu, Y. Wu, Y. Dong, L. Shen, Z. He, J. Zhao,
H. Yuan. An improved liquid chromatography-tandem mass
spectrometric method to quantify formoterol in human urine.
J. Chrom. Sci. 2014, 52, 848.
[124] K.M. Lee, H.J. Kim, J. Son, J.H. Park, O.S. Kwon, J. Lee. Simple quantitation
of formoterol and 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic
acid in human urine by liquid chromatography-tandem mass
spectrometry in doping control. J. Chromatogr. B 2014, 967, 8.
[125] A.K. Orlovius, S. Guddat, M. Gutschow, M. Thevis, W. Schnzer. In vitro
synthesis and characterisation of three fenoterol sulfoconjugates
detected in fenoterol post-administration urine samples. Anal.
Bioanal. Chem. 2013, 405, 9477.
[126] M. Polet, P. Van Renterghem, W. Van Gansbeke, P. Van Eenoo. Profiling
of urinary formestane and confirmation by isotope ratio mass
spectrometry. Steroids 2013, 78, 1103.
[127] N.G. Jager, H. Rosing, J.H. Schellens, J.H. Beijnen, S.C. Linn. Use of dried
blood spots for the determination of serum concentrations of
tamoxifen and endoxifen. Breast Cancer Res. Treat. 2014, 146, 137.
[128] E. Dahmane, J. Boccard, C. Csajka, S. Rudaz, L. Decosterd,
E. Genin, B. Duretz, M. Bromirski, K. Zaman, B. Testa, B. Rochat.
Quantitative monitoring of tamoxifen in human plasma
extended to 40 metabolites using liquid-chromatography highresolution mass spectrometry: new investigation capabilities for
clinical pharmacology. Anal. Bioanal. Chem. 2014, 406, 2627.
[129] J. Lu, C. He, G. He, X. Wang, Y. Xu, Y. Wu, Y. Dong, G. Ouyang. Structural
elucidation of new urinary tamoxifen metabolites by liquid
chromatography quadrupole time-of-flight mass spectrometry.
J. Mass Spectrom. 2014, 49, 570.
[130] M. Evans-Brown, A. Kimergard, J. McVeigh, M. Chandler, S.D. Brandt. Is
the breast cancer drug tamoxifen being sold as a bodybuilding
dietary supplement? Brit. Med. J. 2014, 348, g1476.
[131] M. Mazzarino, X. de la Torre, I. Fiacco, A. Palermo, F. Botre. Drug-drug
interaction and doping, part 1: An in vitro study on the effect of nonprohibited drugs on the phase I metabolic profile of toremifene. Drug
Test. Anal. 2014, 6, 482.
[132] M.M. Moein, M. Javanbakht, B. Akbari-adergani. Molecularly
imprinted polymer cartridges coupled on-line with high
performance liquid chromatography for simple and rapid analysis
of human insulin in plasma and pharmaceutical formulations.
Talanta 2014, 121, 30.
[133] C. Lei, O. Noonan, S. Jambhrunkar, K. Qian, C. Xu, J. Zhang,
A. Nouwens, C. Yu. Sensitive detection of human insulin using a
designed combined pore approach. Small 2014, 10, 2413.
[134] E.E. Chambers, K.J. Fountain, N. Smith, L. Ashraf, J. Karalliedde,
D. Cowan, C. Legido-Quigley. Multidimensional LC-MS/MS enables
simultaneous quantification of intact human insulin and five
recombinant analogs in human plasma. Anal. Chem. 2014, 86, 694.
[135] S. Peterman, E.E. Niederkofler, D.A. Phillips, B. Krastins, U.A. Kiernan, K.
A. Tubbs, D. Nedelkov, A. Prakash, M.S. Vogelsang, T. Schoeder,
L. Couchman, D.R. Taylor, C.F. Moniz, G. Vadali, G. Byram, M.F. Lopez.
An automated, high-throughput method for targeted quantification
of intact insulin and its therapeutic analogs in human serum or
plasma coupling mass spectrometric immunoassay with high
resolution and accurate mass detection (MSIA-HR/AM). Proteomics
2014, 14, 1445.
[136] K. de Dios, A. Manibusan, R. Marsden, J. Pinkstaff. Comparison of
bioanalytical methods for the quantitation of PEGylated human
insulin. J. Immunol. Method 2013, 396, 1.
[137] X. Zhu, J. Liu, J. Wu, R. Cao, T. Li. Pharmacokinetic study of HS061, a
new human insulin, in non-diabetic rat using ultra performance
liquid chromatography-tandem mass spectrometry. J. Chromatogr. B
2014, 967, 50.
[138] T.F. Bovee, M. Blokland, S. Kersten, A.R. Hamers, H.H. Heskamp, M.
L. Essers, M.W. Nielen, L.A. van Ginkel. Bioactivity screening and
mass spectrometric confirmation for the detection of PPARdelta
agonists that increase type 1 muscle fibres. Anal. Bioanal. Chem.
2014, 406, 705.
[139] A. Thomas, M. Vogel, T. Piper, O. Krug, S. Beuck, W. Schnzer, M. Thevis.
Quantification of AICAR-ribotide concentrations in red blood cells by
means of LC-MS/MS. Anal. Bioanal. Chem. 2013, 405, 9703.
[140] T. Piper, A. Thomas, N. Baume, T. Sobolevsky, M. Saugy,
G. Rodchenkov, W. Schnzer, M. Thevis. Determination of (13) C/(12)
C ratios of endogenous urinary 5-amino-imidazole-4-carboxamide
1beta-D-ribofuranoside (AICAR). Rapid Commun. Mass Spectrom.
2014, 28, 1194.

Copyright 2014 John Wiley & Sons, Ltd.

Drug Test. Analysis (2014)

Drug Testing
and Analysis

Annual banned substance review

[141] F. Sanchis-Gomar, H. Pareja-Galeano, V.E. Martinez-Bello. PPARgamma
agonist pioglitazone does not enhance performance in mice. Drug
Test. Anal. 2014, 6, 922.
[142] K. Koehler, M. Thevis, W. Schaenzer. Meta-analysis: Effects of glycerol
administration on plasma volume, haemoglobin, and haematocrit.
Drug Test. Anal. 2013, 5, 896.
[143] K. Koehler, H. Braun, M. de Marees, H. Geyer, M. Thevis, J. Mester,
W. Schaenzer. Glycerol administration before endurance exercise:
Metabolism, urinary glycerol excretion and effects on dopingrelevant blood parameters. Drug Test. Anal. 2014, 6, 202.
[144] World Anti-Doping Agency. DECISION LIMITS FOR THE CONFIRMATORY QUANTIFICATION OF THRESHOLD SUBSTANCES. Available at:
https://wada-main-prod.s3.amazonaws.com/resources/files/WADATD2014DL-v1-Decision-Limits-for-the-Quantification-of-ThresholdSubstances-EN.pdf [14 November 2014].
[145] B.N. Kelly, M. Madsen, K. Sharpe, V. Nair, D. Eichner. A population study
of urine glycerol concentrations in elite athletes competing in North
America. Drug Test. Anal. 2013, 5, 890.
[146] Y. Dong, Y. Ma, K. Yan, L. Shen, X. Wang, Y. Xu, G. He, Y. Wu, J. Lu,
Z. Yang, F. Feng. Quantitative analysis of glycerol levels in human
urine by liquid chromatography-tandem mass spectrometry.
J. Chromatogr. B 2014, 957, 30.
[147] S. Esposito, K. Deventer, A.J. Giron, K. Roels, L. Herregods, A. Verstraete,
P. Van Eenoo. Investigation of urinary excretion of hydroxyethyl starch
and dextran by uhplc-hrms in different acquisition modes. Biol. Sport
2014, 31, 95.
[148] L.A. King. New phenethylamines in Europe. Drug Test. Anal. 2014, 6,
808.
[149] E. Smolianitski, E. Wolf, J. Almog. Proactive forensic science: A novel
class of cathinone precursors. Forensic Sci. Int. 2014, 242, 219.
[150] C.L. German, A.E. Fleckenstein, G.R. Hanson. Bath salts and synthetic
cathinones: An emerging designer drug phenomenon. Life Sci.
2014, 97, 2.
[151] L.J. De Felice, R.A. Glennon, S.S. Negus. Synthetic cathinones: Chemical
phylogeny, physiology, and neuropharmacology. Life Sci. 2014, 97, 20.
[152] A. Peterfi, A. Tarjan, G.C. Horvath, T. Csesztregi, A. Nyirady. Changes in
patterns of injecting drug use in Hungary: a shift to synthetic
cathinones. Drug Test. Anal. 2014, 6, 825.
[153] V. Uralets, S. Rana, S. Morgan, W. Ross. Testing for designer stimulants:
Metabolic profiles of 16 synthetic cathinones excreted free in human
urine. J. Anal. Toxicol. 2014, 38, 233.
[154] J.D. Power, P. Kavanagh, J. OBrien, M. Barry, B. Twamley, B. Talbot,
G. Dowling, S.D. Brandt. Test purchase, identification and synthesis
of 2-amino-1-(4-bromo-2, 5-dimethoxyphenyl)ethan-1-one (bk-2C-B).
Drug Test. Anal. 2014, in press. DOI: 10.1002/dta.1699
[155] N. Uchiyama, S. Matsuda, M. Kawamura, Y. Shimokawa,
R. Kikura-Hanajiri, K. Aritake, Y. Urade, Y. Goda. Characterization of
four new designer drugs, 5-chloro-NNEI, NNEI indazole analog,
alpha-PHPP and alpha-POP, with 11 newly distributed designer
drugs in illegal products. Forensic Sci. Int. 2014, 243, 1.
[156] S.D. Brandt, M.H. Baumann, J.S. Partilla, P.V. Kavanagh, J.D. Power,
B. Talbot, B. Twamley, O. Mahony, J. OBrien, S.P. Elliott, R.P. Archer,
J. Patrick, K. Singh, N.M. Dempster, S.H. Cosbey. Characterization of a
novel and potentially lethal designer drug (+/-)-cis-para-methyl-4methylaminorex (4,4-DMAR, or Serotoni). Drug Test. Anal. 2014, 6,
684.
[157] R.A.
Glennon.
Bath
salts,
mephedrone,
and
methylenedioxypyrovalerone as emerging illicit drugs that will need
targeted therapeutic intervention. Adv. Pharmacol. 2014, 69, 581.
[158] F. Song, D. Monroe, A. El-Demerdash, C. Palmer. Screening for multiple
weight loss and related drugs in dietary supplement materials by flow
injection tandem mass spectrometry and their confirmation by liquid
chromatography tandem mass spectrometry. J. Pharmaceut. Biomed.
2014, 88, 136.
[159] B. Venhuis, P. Keizers, A. van Riel, D. de Kaste. A cocktail of synthetic
stimulants found in a dietary supplement associated with serious
adverse events. Drug Test. Anal. 2014, 6, 578.
[160] P.A. Cohen, J.C. Travis, B.J. Venhuis. A methamphetamine analog
(N,alpha-diethyl-phenylethylamine) identified in a mainstream
dietary supplement. Drug Test. Anal. 2014, 6, 805.
[161] K.G. Austin, J. Travis, G. Pace, H.R. Lieberman. Analysis of 1,3
dimethylamylamine concentrations in Geraniaceae, geranium oil
and dietary supplements. Drug Test. Anal. 2014, 6, 797.
[162] Y.B. Monakhova, M. Ilse, J. Hengen, O. El-Atma, T. Kuballa,
M. Kohl-Himmelseher, D.W. Lachenmeier. Rapid assessment of the

Drug Test. Analysis (2014)

[163]

[164]
[165]
[166]

[167]

[168]
[169]

[170]

[171]

[172]

[173]

[174]
[175]

[176]

[177]

[178]

[179]

[180]

illegal presence of 1,3-dimethylamylamine (DMAA) in sports

nutrition and dietary supplements using 1H NMR spectroscopy.
Drug Test. Anal. 2014, 6, 944.
B.K. Schilling, K.G. Hammond, R.J. Bloomer, C.S. Presley, C.R. Yates.
Physiological and pharmacokinetic effects of oral 1,3dimethylamylamine administration in men. BMC Pharmacol.
Toxicol. 2013, 14, 52.
R.J. Bloomer, T.M. Farney, I.C. Harvey, R.J. Alleman. Safety profile of
caffeine and 1,3-dimethylamylamine supplementation in healthy
men. Hum. Exp. Toxicol. 2013, 32, 1126.
E. Fornal. Study of collision-induced dissociation of electrospraygenerated protonated cathinones. Drug Test. Anal. 2014, 6, 705.
P. Cholbinski, M. Wicka, K. Kowalczyk, A. Jarek, P. Kaliszewski,
A. Pokrywka, E. Bulska, D. Kwiatkowska. Detection of betamethylphenethylamine, a novel doping substance, by means of
UPLC/MS/MS. Anal. Bioanal. Chem. 2014, 406, 3681.
M. Thevis, G. Sigmund, A. Thomas, M. Vogel, K. Walpurgis,
D. Kwiatkowska, H. Geyer, W. Schnzer. Isotope-dilution mass
spectrometric quantification of the prodrug lisdexamfetamine in
human urine in doping control analysis. Rapid Commun. Mass
Spectrom. 2014, 28, 781.
A.D. Lesiak, K.J. Adams, M.A. Domin, C. Henck, J.R. Shepard. DART-MS
for rapid, preliminary screening of urine for DMAA. Drug Test. Anal.
2014, 6, 788.
A. Rodriguez-Lafuente, F.S. Mirnaghi, J. Pawliszyn. Determination of
cocaine and methadone in urine samples by thin-film solid-phase
microextraction and direct analysis in real time (DART) coupled
with tandem mass spectrometry. Anal. Bioanal. Chem. 2013, 405,
9723.
A. Wong, M.E. Montebello, M.M. Norberg, K. Rooney, N. Lintzeris,
R. Bruno, J. Booth, J.C. Arnold, I.S. McGregor. Exercise increases
plasma THC concentrations in regular cannabis users. Drug Alcohol
Depen. 2013, 133, 763.
N.A. Desrosiers, D. Lee, M. Concheiro-Guisan, K.B. Scheidweiler, D.
A. Gorelick, M.A. Huestis. Urinary cannabinoid disposition in
occasional and frequent smokers: Is THC-glucuronide in sequential
urine samples a marker of recent use in frequent smokers? Clin.
Chem. 2014, 60, 361.
A. Kiss, C. Bordes, C. Buisson, F. Lasne, P. Lanteri, C. Cren-Olive. Datahandling strategies for metabonomic studies: Example of the
UHPLC-ESI/ToF urinary signature of tetrahydrocannabinol in
humans. Anal. Bioanal. Chem. 2014, 406, 1209.
D. Thieme, H. Sachs, M. Uhl. Proof of cannabis administration by
sensitive detection of 11-nor-Delta(9)-tetrahydrocannabinol-9carboxylic acid in hair using selective methylation and application of
liquid chromatography- tandem and multistage mass spectrometry.
Drug Test. Anal. 2014, 6, 112.
D. Lee, M.A. Huestis. Current knowledge on cannabinoids in oral fluid.
Drug Test. Anal. 2014, 6, 88.
O. Corazza, P. Simonato, J. Corkery, G. Trincas, F. Schifano. "Legal
highs": Safe and legal "heavens"? A study on the diffusion,
knowledge and risk awareness of novel psychoactive drugs among
students in the UK. Riv. Psichiatr. 2014, 49, 89.
S. Dugan, J. Roesler, A. Westbrook, E. Bilden, D. Anderson,
N. Van Deelen, S. Wolff, M. Kinde, R. Lynfield. The high cost of bath
salts: a study of the health care burden of illicit synthetic drug use
in Duluth, Minnesota. Minn. Med. 2014, 97, 34.
A.S. Gandhi, M. Zhu, S. Pang, A. Wohlfarth, K.B. Scheidweiler, H.F. Liu,
M.A. Huestis. First characterization of AKB-48 metabolism, a novel
synthetic cannabinoid, using human hepatocytes and highresolution mass spectrometry. AAPS J. 2013, 15, 1091.
A. Wohlfarth, A.S. Gandhi, S. Pang, M. Zhu, K.B. Scheidweiler,
M.A. Huestis. Metabolism of synthetic cannabinoids PB-22 and
its 5-fluoro analog, 5 F-PB-22, by human hepatocyte incubation
and high-resolution mass spectrometry. Anal. Bioanal. Chem.
2014, 406, 1763.
M. Sundstrm, A. Pelander, V. Angerer, M. Hutter, S. Kneisel,
I. Ojanper. A high-sensitivity ultra-high performance liquid
chromatography/high-resolution time-of-flight mass spectrometry
(UHPLC-HR-TOFMS) method for screening synthetic cannabinoids
and other drugs of abuse in urine. Anal. Bioanal. Chem. 2013,
405, 8463.
K.B. Scheidweiler, M.A. Huestis. Simultaneous quantification of 20
synthetic cannabinoids and 21 metabolites, and semi-quantification
of 12 alkyl hydroxy metabolites in human urine by liquid

Copyright 2014 John Wiley & Sons, Ltd.

wileyonlinelibrary.com/journal/dta

Drug Testing
and Analysis

[181]

[182]
[183]

[184]
[185]
[186]
[187]
[188]
[189]
[190]
[191]

M. Thevis et al.

chromatography-tandem mass spectrometry. J. Chromatogr. A 2014,

1327, 105.
M. Mazzarino, X. de la Torre, F. Botre. A liquid chromatography-mass
spectrometry method based on class characteristic fragmentation
pathways to detect the class of indole-derivative synthetic
cannabinoids in biological samples. Anal. Chim. Acta 2014, 837, 70.
C.W. Chang, T.Y. Huang, Y.C. Tseng, G.P. Chang-Chien, S.F. Lin, M.
C. Hsu. Positive doping results caused by the single-dose local
injection of triamcinolone acetonide. Forensic Sci. Int. 2014, 244C, 1.
X. Matabosch, O.J. Pozo, N. Monfort, C. Perez-Mana, M. Farre, J. Marcos,
J. Segura, R. Ventura. Urinary profile of methylprednisolone and its
metabolites after oral and topical administrations. J. Steroid Biochem.
Mol. Biol. 2013, 138C, 214.
T. Pottgiesser, Y.O. Schumacher. Current strategies of blood doping
detection. Anal. Bioanal. Chem. 2013, 405, 9625.
C.D. Oliveira, A.V. Bairros, M. Yonamine. Blood doping: Risks to
athletes health and strategies for detection. Subst. Use Misuse 2014,
49, 1168.
J. Segura, C. Lundby. Blood doping: Potential of blood and urine
sampling to detect autologous transfusion. Brit. J. Sport Med. 2014,
48, 837.
Y.P. Pitsiladis, J. Durussel, O. Rabin. An integrative omics solution to
the detection of recombinant human erythropoietin and blood
doping. Brit. J. Sport Med. 2014, 48, 856.
N. Leuenberger, N. Robinson, M. Saugy. Circulating miRNAs: A new
generation of anti-doping biomarkers. Anal. Bioanal. Chem. 2013,
405, 9617.
I. Manokhina, J.L. Rupert. A DNA-based method for detecting
homologous blood doping. Anal. Bioanal. Chem. 2013, 405, 9693.
E. Varlet-Marie, J. Ashenden, M.J. Moerkeberg, C. Vela, M. Audran.
Relevance of di(2-ethylhexyl)phthalate blood levels for blood
transfusion detection. Bioanalysis 2014, 6, 1591.
A.R. Vernec. The athlete biological passport: An integral element
of innovative strategies in antidoping. Brit. J. Sport Med. 2014,
48, 817.

wileyonlinelibrary.com/journal/dta

[192] M. Saugy, C. Lundby, N. Robinson. Monitoring of biological markers

indicative of doping: the athlete biological passport. Brit. J. Sport
Med. 2014, 48, 827.
[193] M. Zorzoli, A. Pipe, P.Y. Garnier, M. Vouillamoz, J. Dvorak. Practical
experience with the implementation of an athletes biological
profile in athletics, cycling, football and swimming. Brit. J. Sport Med.
2014, 48, 862.
[194] M. Ashenden, K. Sharpe, J. Plowman, G. Allbon, L. Lobigs, A. Baron,
C.J. Gore. Stability of athlete blood passport parameters during air
freight. Int. J. Lab. Hematol. 2014, 36, 505.
[195] P. Watson, R.J. Maughan. Artifacts in plasma volume changes due to
hematology analyzer-derived hematocrit. Med. Sci. Sport Exer. 2014,
46, 52.
[196] F. Sanchis-Gomar, H. Pareja-Galeano, T. Brioche, V. Martinez-Bello,
G. Lippi. Altitude exposure in sports: The athlete biological passport
standpoint. Drug Test. Anal. 2014, 6, 190.
[197] S.C. Voss, M. Alsayrafi, P.C. Bourdon, F. Klodt, D. Nonis, W.G. Hopkins,
Y.O. Schumacher. Variability of serum markers of erythropoiesis
during 6 days of racing in highly trained cyclists. Int. J. Sport Med.
2014, 35, 89.
[198] C.R. Harrison, J.C. Fang, K.J. Walthall, C.C. Green, V. Porobic. Towards
the identification of autologous blood transfusions through capillary
electrophoresis. Anal. Bioanal. Chem. 2014, 406, 679.
[199] G. Fischetto, S. Bermon. From gene engineering to gene modulation
and manipulation: Can we prevent or detect gene doping in sports?
Sports Med. 2013, 43, 965.
[200] I.C. Perez, C. Le Guiner, W. Ni, J. Lyles, P. Moullier, R.O. Snyder. PCRbased detection of gene transfer vectors: Application to gene
doping surveillance. Anal. Bioanal. Chem. 2013, 405, 9641.
[201] A. Baoutina, T. Coldham, B. Fuller, K.R. Emslie. Improved detection of
transgene and nonviral vectors in blood. Hum. Gene Ther. Methods.
2013, 24, 345.
[202] A. Thomas, K. Walpurgis, P. Delahaut, M. Kohler, W. Schanzer, M. Thevis.
Detection of small interfering RNA (siRNA) by mass spectrometry
procedures in doping controls. Drug Test. Anal. 2013, 5, 853.

Copyright 2014 John Wiley & Sons, Ltd.

Drug Test. Analysis (2014)