The Vis 2014

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PBA-9583; No. of Pages 18
Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxxxxx
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba
Review
Analytical approaches for the detection of emerging therapeutics and

non-approved drugs in human doping controls
Mario Thevis a, , Wilhelm Schnzer b
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/Bonn, Germany
a r t i c l e
i n f o
Article history:
Received 3 March 2014
Received in revised form 5 May 2014
Accepted 6 May 2014
Available online xxx
Keywords:
Doping
Sport
Mass spectrometry
Emerging drugs
Stamulumab
Anti-myostatin antibody
a b s t r a c t
The number and diversity of potentially performance-enhancing substances is continuously growing,
fueled by new pharmaceutical developments but also by the inventiveness and, at the same time,
unscrupulousness of black-market (designer) drug producers and providers. In terms of sports drug testing, this situation necessitates reactive as well as proactive research and expansion of the analytical
armamentarium to ensure timely, adequate, and comprehensive doping controls. This review summarizes literature published over the past 5 years on new drug entities, discontinued therapeutics, and
tailored compounds classied as doping agents according to the regulations of the World Anti-Doping
Agency, with particular attention to analytical strategies enabling their detection in human blood or urine.
Among these compounds, low- and high-molecular mass substances of peptidic (e.g. modied insulin-like
growth factor-1, TB-500, hematide/peginesatide, growth hormone releasing peptides, AOD-9604, etc.)
and non-peptidic (selective androgen receptor modulators, hypoxia-inducible factor stabilizers, siRNA, S107 and ARM036/aladorian, etc.) as well as inorganic (cobalt) nature are considered and discussed in terms
of specic requirements originating from physicochemical properties, concentration levels, metabolism,
and their amenability for chromatographic-mass spectrometric or alternative detection methods.
2014 Elsevier B.V. All rights reserved.
1. Introduction
With the constantly increasing knowledge about biochemical mechanisms at cellular and molecular levels, more and more
options for pharmacological interventions have been identied that
suggest new paths to desired therapies potentially allowing cure for
severe if not fatal diseases. The ipside of such research is the misuse potential offered by a subset of new drug candidates, especially
those that promote muscle growth, stimulate erythrocyte production, or enhance physical stamina and athletic performance via
other routes [1]. Such drug candidates have been offered and sold
via Internet-based providers for years, despite the lack of clinical
approval and, in some cases, discontinuation of their development
due to severe side effects. The targeted clientele of such offerings is
composed of recreational as well as professional athletes, with the
latter ones being at risk of violating regulations established by the
Corresponding author at: Institute of Biochemistry Center for Preventive

Doping Research, German Sport University Cologne, Am Sportpark Mngersdorf 6,
50933 Cologne, Germany. Tel.: +49 221 4982 7070; fax: +49 221 4982 7071.
E-mail address: thevis@dshs-koeln.de (M. Thevis).
World Anti-Doping Agency (WADA) [2] These regulations as presented in WADAs Prohibited List include a category of substances
dedicated to particularly such compounds, i.e. non-approved for
human use/discontinued drug candidates, referred to as S0. In
order to enable comprehensive doping controls, accredited laboratories update, expand, and improve their portfolio of analytical
assays, most of which rely on chromatographic-mass spectrometric
approaches [3,4]; however, the implementation of new compounds
into sports drug testing protocols requires a substantial amount
of information including therapeutic dosage, pharmacokinetics,
metabolism, and elimination. Moreover, specic physicochemical properties might necessitate dedicated sample collection and
transport conditions, sample preparation or analytical procedures
to ensure the required sensitivity and specicity to detect the target analyte with appropriate limits of detection (LODs) [5] With
the constraints in budget, time, sample volume(s), laboratory staff
and instrumentation, sports drug testing laboratories however are
urged to combine as many detection assays as possible without
compromising the necessary analytical requirements, preferably
by using and expanding existing analytical approaches. Hence, test
menus need to be rationally arranged and their tness-for-purpose
as appropriate initial testing procedure has to be demonstrated.
http://dx.doi.org/10.1016/j.jpba.2014.05.020
0731-7085/ 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: M. Thevis, W. Schnzer, Analytical approaches for the detection of emerging therapeutics and nonapproved drugs in human doping controls, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.05.020
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M. Thevis, W. Schnzer / Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxxxxx
While formerly drug classes dictated the composition of analytical assays, nowadays the available analytical equipment commonly
governs the employed test strategies [3]. To date, routine doping
control matrices are urine, serum and blood, occasionally complemented by alternative matrices such as hair potentially providing
supporting evidence. The collection protocols follow stringent regulations and require trained doping control ofcers/phlebotomists;
transport times and conditions have to be controlled and documented especially in case of blood samples for the Athlete Biological
Passport (ABP), where also time limits for transport and analysis apply. In addition, sample storage (urine and serum) has to be
ensured for up to 10 years to allow for re-testing if requested.
In the present review, literature published between 2009 and
2013 concerning emerging, designer, and discontinued drugs is
discussed in the context of human doping controls. Challenges
arising from structural feature of substances are presented and
metabolite identication and detection strategies are outlined for
a representative selection of compounds covering low- and high
molecular mass analytes of non-peptidic, peptidic, and ribonucleic
acid composition.
2. Compounds affecting skeletal muscle performance
Due to the substantial number of compounds with evident or
presumed impact on skeletal muscle physiology and/or performance, the substances considered in the following are divided into
the categories of low and high molecular mass products.
2.1. Low molecular mass substances
2.1.1. Ryanodine receptor-calstabin-complex stabilizers (Rycals)
Studies on cardiac arrhythmia as well as sarcopenia (as dened
as the age-related loss of muscle mass, force, and exercise capacity) and muscular dystrophy have revealed the relevance of
the ryanodine receptor 1 (RyR1) and its Ca2+ -channel complex
building partner molecule calstabin-1 (FK506 binding protein 12,
FKBP12) with regard to normal skeletal and cardiac muscle function. Substantial research on mechanisms of post-translational
modications has been conducted in animal models and, more
recently, also in humans indicating particularly S-nitrosylation and
(hyper)phosphorylation of RyR1 as main factors of the aging-,
disease-, or exercise-induced functional impairment of myocytes
[68]. A potential therapy is based on benzothiazepine-derived
drug candidates such as the rst- and second-generation therapeutics JTV-519 and S107 (Fig. 1a, 1 and 2) [9], which have been
shown to reduce muscle fatigue and improve exercise capacity in
laboratory rodents by restoring the RyR1-FKBP12 complex. Consequently, the relevance of such compounds for sports drug testing
was recognized and detection assays for the intact drugs and/or in
vitro generated metabolites in blood and urine were established.
The mass spectrometric behavior of JTV-519 and S-107 was
studied in extenso using electrospray ionization (ESI) and collisioninduced dissociation (CID) [10] as well as electron ionization (EI)
[11] employing high resolution/high accuracy mass spectrometry, stable isotope labeling and, in case of ESI-CID, H/D-exchange
experiments. By means of the obtained information, test methods for urine [10,11] and plasma [12] were developed enabling
the detection of the intact molecules at LODs of 0.16 ng/ml. In
case of blood plasma, peak concentrations of the drug candidates
after therapeutic dosing were expected at approximately 40 ng/ml,
which was well within the detection window of the developed test
method. In the absence of data on the metabolism and (renal) elimination of the benzothiazepines, urine samples were subjected to
enzymatic hydrolysis followed by liquidliquid extraction (LLE) of
the target analytes and subsequent detection by means of liquid
chromatography(tandem) mass spectrometry (LCMS/MS) or gas

chromatographymass spectrometry (GCMS). In order to further
complement the analytical approach with putative metabolites,
phase-I and phase-II metabolic reactions were simulated for S107 in vitro, yielding predominantly N- and S-oxygenated species
as well as N- and O-demethylated metabolites. Moreover, glucuronic acid conjugates of the intact drug and its O-demethylated
phase-I metabolic product were identied representing viable targets for future doping controls [13]. Moreover, the development of
next-generation benzothiazepine-derived compounds needs further consideration, e.g. with regard to the phase-II clinical trial drug
candidate referred to as ARM036 (Aladorian, Fig. 1a, 3) [14], the
product ion mass spectrum of which is presented in Fig. 1b.
2.1.2. Selective androgen receptor modulators (SARMs)
Non-steroidal selective androgen receptor modulators (SARMs)
have been subject of extensive preclinical and clinical trials since
the rst-in-class compounds were identied in 1998, predominantly aiming at the treatment and prevention of sarcopenia,
osteoporosis, and disease-related losses of skeletal muscle mass,
strength, and function [15,16]. Moreover, the potential utility of
SARMs in cardiology has been discussed [17], and the substantial interest in new drug entities with SARM-like properties is
still on the incline according to recent reviews [16,18] and publications on advances in SARM-related research [1921]. With
the increasing amount of possible non-steroidal and steroidal
SARM drug candidates, examples of which are illustrated in Fig. 2
(413), the portfolio of compounds potentially misused in sports
is expanded accordingly [22,23] and detection assays plus ample
information on metabolism and elimination are vital for appropriate doping controls. Consequently, studies focusing on the
metabolism of SARMs and possibilities to detect intact as well as
metabolized SARMs have been initiated and continued, and the
relevance and necessity of adequate test methods was demonstrated with the rst adverse analytical ndings (AAFs) for SARMs
in 2010 and the following years [24,25]. The analytical assays
for SARMs have been established for plasma [12,26], dried blood
spots (DBS) [27], and urine targeting either the intact substances
(plasma and DBS) or main metabolites (urine) as identied and
characterized in in vitro [28] and in vivo studies [2931]. Despite
modest structural similarities between some SARMs comprising
e.g. a 4-substituted aniline moiety, a substantial heterogeneity of
pharmacophores is present in currently investigated SARMs including (amongst others) arylpropionamide, quinolinone, tropanol,
tetrahydroquinoline, hydantoin, thiophene, phenyl-oxadiazol, and
steroid derivatives (Fig. 2, Table 1). Hence, various projects have
been required providing insights into main metabolic pathways
and the mass spectrometric behavior of identied and characterized target compounds.
All SARMs recently studied in a doping control context demonstrated good or excellent ionization properties using electrospray,
thus supporting the sensitive detection of these compounds and
related metabolic products in sports drug test samples employing LCMS/MS-based strategies [3,32]. Arylpropionamide-derived
SARMs were among the rst category of emerging anabolic
agents investigated with ESI-MS/MS, EI-MS(/MS), and under
in vitro and in vivo metabolism conditions. Substantial agreement
between results of in vitro and in vivo studies was observed, and
post-administration study urine samples of the arylpropionamidederived SARMs S-4 and S-22 (Fig. 2, 4 and 5, respectively)
predominantly yielded the glucuronic acid conjugates of the intact
drugs and corresponding mono-hydroxylated metabolites as viable
analytes for routine doping controls [29,30] with LODs for the intact
drug candidates found below 1 ng/ml. Complementary, LODs of
0.0520 ng/ml [27,33] and 10 ng/ml [26] were determined in DBS
and plasma, respectively, for the intact therapeutics. Substance
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Fig. 1. (a) Structures of JTV-519 (1), S-107 (2), and Aladorian (ARM036, 3); (b) product ion mass spectrum of the protonated molecule [M+H]+ at m/z 268 of Aladorian,
recorded at a collision energy of 25 eV.
characterization by mass spectrometric techniques, particularly

LCMS/MS employing high resolution/high accuracy mass spectrometry, was further conducted for the related arylpropionamides
S-1, S-9, S-23, and S-24 [34], all of which were also subjected
to systems simulating metabolic reactions such as human liver
microsomal [28] or fungal [35] preparations to provide reference
material for (provisional) targets for sports drug testing. Similarly, investigations into the mass spectrometry of SARMs and
their detection in human urine were conducted for quinolinone(e.g. LGD-2226, Fig. 2, 6), tetrahydroquinoline- (e.g. S-40503, Fig. 2,
8), and hydantoin-derived substances (e.g. BMS564929, Fig. 2, 10)
[34], complemented by more recent studies on phenyl-oxadiazol(RAD140, Fig. 2, 11) and tropanol-based SARMs (ACP-105, Fig. 2,
12) [36]. The elimination of ACP-105 was further studied in
a rat model, demonstrating the production of various different
mono- and bishydroxylated metabolites serving as preferred target
Fig. 2. Structures of S-4 (Andarine, 4), S-22 (Enobosarm, 5), LGD-2226 (6), LG 121071 (7), S-40503 (8), S-101479 (9), BMS-564929 (10), RAD140 (11), ACP-105 (12), and
LGD-4033 (13).
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Table 1
Structure characteristics of selected SARMs.
No. (Fig. 2)
4
5
7
8
9
10
11
12
13
SARM
Pharmacophore
Elemental composition
Molecular mass (Da)
Refs.
S-1
S-4 (Andarine)
S-9
S-22 (Ostarine)
S-23
S-24
LGD-2226
LGD-2941
LGD-3303
LG-121071
S-40503
S-49288
S-101479
JNJ-28330835
BMS-564929
JNJ-37654032
RAD140
ACP-105
AC-262536
LGD-4033
RAD35010
Ly2452473
FTBU-1
2-FPA
GLPG0634
MK-3984
NEP28
MK-0773
Cl-4-AS-1
TFM-4AS-1
YK11
S-42
Arylpropionamide
Arylpropionamide
Arylpropionamide
Arylpropionamide
Arylpropionamide
Arylpropionamide
Quinolinone
Quinolinone
Quinolinone
Quinolinone
Tetrahydroquinoline
Tetrahydroquinoline
Tetrahydroquinoline
Phenyl-pyrazol-carboxyamide
Hydantoin
Benzoimidazole
Phenyl-oxadiazole
Tropanol
Tropanol
Pyrrolidinyl-benzonitrilea
Indole
Indole
Benzoimidazole
Pyridinylmethanamide
Diarylimidazolidinedione
Phenylmethanamide
Thiophene
Steroidal
Steroidal
Steroidal
Steroidal
Steroidal
C17 H14 F4 N2 O5
C19 H18 F3 N3 O6
C17 H14 ClF3 N2 O5
C19 H14 F3 N3 O3
C18 H13 ClF4 N2 O3
C18 H14 F4 N2 O3
C14 H9 F9 N2 O
C17 H16 F6 N2 O2
C16 H14 ClF3 N2 O
C15 H15 F3 N2 O
C15 H23 N3 O3
C25 H26 N4 O
C26 H24 F2 N4 O3
C14 H10 F6 N4 O
C14 H12 ClN3 O3
C11 H7 Cl2 F3 N2 O
C20 H16 ClN5 O2
C16 H19 ClN2 O
C18 H18 N2 O
C14 H12 F6 N2 O
C13 H11 ClF3 NO
C23 H23 N3 O2
C19 H16 FN5 OS
C17 H19 FN2 O
C19 H14 F3 N3 O3
C17 H12 F7 NO2
C10 H10 BrF3 N2 S
C27 H34 FN5 O2
C26 H33 ClN2 O2
C27 H33 F3 N2 O2
C25 H34 O6
C21 H28 O
402.0839
441.1148
418.0543
389.0987
416.0551
382.0941
392.0571
394.1116
342.0747
296.1136
293.1739
398.2107
478.1816
364.0759
305.0567
309.9888
393.0993
290.1186
278.1419
338.0854
289.0481
373.1790
381.1060
286.1481
389.0987
395.0756
325.9700
479.2697
440.2231
474.2494
430.2355
296.2140
[165]
[18]
[165]
[18]
[165]
[165]
[18]
[18]
[18]
[166]
[18]
[20]
[16]
[18]
[18]
[18]
[18]
[18]
[18]
[18]
[16]
[18]
[18]
[16]
[16]
[21]
[18]
[18]
[18]
[18]
[18]
Unconrmed.
analytes as the administered compound was detected intact only

up to 24 h [31]. In contrast, human in vitro and in vivo studies
with RAD140 suggested the use of the administered SARM as urinary target compound for doping control purposes as only modest
metabolic reactions to a monohydroxylated analog were observed
and RAD140 was detected in post-administration study urine samples up to 8 days [37]. While recent reviews on SARMs in clinical
development have referred to the structure of the drug candidate
LGD-4033 as undisclosed [16], Internet-based suppliers of SARMs
have assigned 4-(2-((S)-2,2,2-triuoro-1-hydroxyethyl)pyrrolidin1-yl)-2-(triuoromethyl)-benzonitrile (Fig. 2, 13) to it. Despite the

absence of conrmation, the drug entity is part of LIGAND PHARMACEUTICALs patents on compounds with SARM-like properties
[38] and thus also a candidate for doping controls. Hence, detection assays are required particular in the light of its arguably illicit
availability and information on the mass spectrometric properties
of the substance and its metabolite(s) are needed to complement
routine sports drug testing (Fig. 3).
Fig. 3. Product ion mass spectrum of the deprotonated molecule [MH] at m/z 337 of LGD-4033, recorded at a collision energy of 15 eV.
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2.1.3. Sirtuin-1 activators

Sirtuins (17) represent a family of NAD+ -dependent histone
deacetylase enzymes, with sirtuin-1 (SIRT1) being a key regulator
in the deacetylation of metabolism-modulating protein substrates
such as forkhead box proteins (FOXO) and peroxisome proliferatoractivated receptor coactivator 1 (PGC-1) [39]. Since the
discovery of resveratrols SIRT1-activating effect with signicant
impact on skeletal muscle mitochondrial content, extended life
span and anti-aging as well as antidiabetogenic properties in animal models, much effort was invested into research concerning
synthetic SIRT1-activating drug entities (STACs) [40,41]. Despite an
extensive debate concerning the underlying mechanism(s) responsible for the observed benecial effects [4246], a series of STACs
lead drug candidates such as SRT1720, SRT1460, and SRT2104
(Fig. 4, 14 and 15) was produced and has been subject of advanced
clinical trials since. The arguably comparable outcome of STAC
administration with other metabolic modulators such as e.g. AICAR
(vide infra) in terms of pharmacologically enhanced endurance has
triggered concerns as to the misuse potential of these substances.
Consequently, analytical assays have been established for rstgeneration STACs and additional model compounds in blood and
urine. Moreover, in vitro and in vivo biotransformation studies were
conducted to provide information on viable targets for extended
detection windows in routine sports drug testing.
Commercially available and synthesized STAC reference substances including SRT1720 and 4 structurally related compounds
were studied concerning their ESI-MS and MS/MS behavior and
measured from human plasma [47]. A total of 100 l of specimen
was enriched with an eight-fold deuterated analog of SRT1720 and
deuterated resveratrol (internal standards), and plasma proteins
were precipitated by means of acetonitrile. The supernatant was
analyzed by LCESI-MS/MS in multiple reaction monitoring (MRM)
mode, allowing for detection limits between 0.1 and 1 ng/ml.
Considering the reported plasma concentrations of STACs in clinical
trials reaching up to 390 ng/ml, the accomplished LODs demonstrate the tness-for-purpose of the presented approach. In order
to complement the approach with analyses conducted from urine,
target analytes were generated using in vitro biotransformation
systems [48]. Main metabolic reactions included N-oxidation and
hydroxylation, and the resulting products were further observed in
subsequent rat administration studies [49]. By means of the intact
STACs, the test method was characterized indicating detection limits for the target analytes in urinary matrix of 0.5 ng/ml. It remains
to be claried whether the intact drugs or selected metabolites
will be the most appropriate compounds for routine doping control
analyses; however, the principle option to detect these compounds
in blood/plasma and urine is established and serves the purpose of
proactive and preventive anti-doping research.
2.1.4. AICAR (5-amino-4-imidazolecarboxamide ribonucleoside)
Adenosine monophosphate (AMP)-activated protein kinase
(AMPK) is an essential factor of mitochondrial biogenesis and function [50]. Due to the key role of skeletal muscle mitochondria
in health and disease as well as in exercise, numerous studies
concerning the at least partially unclear mechanisms of mitochondrial biogenesis have been conducted with the goal of identifying
potential pharmacological means to cure genetically caused (e.g.
Duchenne muscular dystrophy, DMD, or amyotrophic lateral sclerosis, ALS) and non-genetic (e.g. obesity or metabolic syndrome)
disorders [51]. The so-called master regulator of mitochondrial
numbers is the earlier mentioned PGC-1, controlling the cells
powerhouse, which is affected by stressors such as caloric restriction and exercise [52]. The activation of PGC-1 can be triggered
through gene expression as well as post-translational modications
including phosphorylation, which (among other factors) depends
on the activity of AMPK, since only the phosphorylated PGC-1 is
believed to be subsequently primed by SIRT1 via deacetylation and

capable of inducing gene expression required for mitochondriogenesis. This interconnection, the relevance of AMPK and its activation
through potent agonists such as 5-amino-4-imidazolecarboxamide
ribonucleoside (AICAR, Fig. 4, 16) [53] have been the rationale to
include this compound and related substances to WADAs Prohibited List in 2009, rst categorized as gene doping substance
and later as metabolic modulator [2]. The administration of the
natural and cell-permeable AMPK activator AICAR to laboratory
rodents at 500 mg/kg/day was shown to effectively activate the
AMPK signaling pathway resulting in an improved endurance of
untrained mice by 2344% [54]. This supported the necessity of the
compounds inclusion in doping control programs as well as the
fact that the substance has been readily available as chemical and
via Internet-based suppliers [23] and has supposedly found its way
into the world of sport meanwhile [55,56].
Due to the natural occurrence of AICAR in human urine, providing evidence for the illicit use of the non-approved drug
candidate has required sophisticated analytical strategies similar
to approaches employed for the detection of natural/endogenous
steroid misuse. By means of liquid chromatography/isotopedilution tandem mass spectrometry (LC-IDMS/MS), naturally
occurring urinary concentrations of AICAR were quantied in a population of 499 athletes, demonstrating a signicant correlation of
urinary AICAR levels and gender, type of sport (e.g. endurance and
strength sport) and time point of sample collection (i.e. in/out of
competition) [57]. A mean value of approximately 2200 ng/ml was
reported, and under consideration of another set of 500 urine samples collected from recreational athletes, a tentative threshold level
of 3500 ng/ml was suggested (unpublished results). However, the
mere exceeding of urinary AICAR concentrations above reference
values will not allow proving the abuse of synthetic AICAR, particularly due to substantial intra- and inter-individual variations;
such information will nevertheless enable and trigger further analyses supporting or disproving a possible AICAR administration and
eventually revealing the source of the compound (endogenous or
exogenous).
An alternative matrix presumably conserving administered
AICAR for a prolonged period of time is the erythrocyte. Upon
introduction into the blood stream, AICAR was shown to cross
the red blood cell (RBC) membrane followed by its conversion
into the 5 -monophosphate derivative, which does not allow its
efux from the RBC and causes an accumulation of the produced
AICAR-ribotide, arguably for the lifespan of the erythrocyte. Hence,
measuring intra-erythrocytic AICAR-ribotide concentrations could
provide complementary information as to whether unnaturally
high levels prevail or not. A quantitative analytical assay also based
on LC-IDMS/MS was established for AICAR-ribotide in RBCs, and
99 recreational athletes samples were measured to obtain ranges
of normal physiological values (10500 ng/ml) of the analyte [58].
By means of in vitro incubation reactions, the administration of
pharmacological doses of AICAR were simulated, demonstrating
an increase of intra-erythrocytic AICAR-ribotide concentrations of
110 g/ml providing proof-of-concept for this approach. More
data for reference ranges and authentic administration study
samples will be required to assess the signicance of these numbers; however, the intra-individual prole of AICAR concentrations
might be a sensible contribution to the Athlete Biological Passport
(ABP) to allow agging an unusual blood parameter nding.
Conclusive test results concerning the endogenous or exogenous origin of a naturally occurring substance in an athletes doping
control sample is commonly obtained by isotope-ratio mass spectrometry (IRMS) [59]. This technical approach has been successfully
employed in steroid analyses for over a decade in doping controls, and its utility for tackling the AICAR issue was suggested
as early as 2010. Two major obstacles had to be managed for the
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Fig. 4. Structures of SRT1720 (14), SRT1460 (15), AICAR (16), Rolipram (17), Roumilast (18), Cilomilast (19), L-739943 (20), MK-0677 (Ibutamoren, 21), CP-424391
(Capromorelin, 22), and SM-130686 (23).
successful IRMS analysis of AICAR, namely the low volatility of

the analyte, which necessitates derivatization prior to introducing the substance into the gas chromatography-combustion-IRMS
(GC/C/IRMS) system, and the isolation of AICAR from urine without
isotopic fractionation. Both challenges have recently been solved,
providing a validated assay allowing to differentiate synthetic and
natural AICAR by means of their carbon isotope signature [60].
The test method requires a volume of 3 ml of urine and currently
urine samples with AICAR levels higher than 1500 ng/ml are recommended to be subjected to GC/C/IRMS. Due to the considerable
amounts of AICAR in urine, also LC-IRMS, which is commonly
inferior to GC/C/IRMS in terms of analytical sensitivity, has been
considered as a viable means of measuring the carbon isotope signature; to date however, no methodology has been established or
reported.
2.1.5. Phosphodiesterase-4 (PDE4) inhibitors
In continuation of the quest for therapeutics supporting
the therapy of mitochondria-related disorders, the impact of
phosphodiesterase-4 (PDE4) inhibitors has recently been revisited. Phosphodiesterases comprise a family of 11 currently known
members with specic characteristics and stimulating or inhibiting agents. PDE4, a major target in chronic obstructive pulmonary
disease (COPD) treatment [6163], catalyzes the conversion of
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3 -5 -cyclic adenosine monophosphate (cAMP) to 5 -AMP. Its inhibition was shown to result in a cascade of consequences allowing
to explain the benecial effects of the unspecic PDE inhibitor
resveratrol in animal test models [50,64] including, amongst others, increased mitochondrial biogenesis and function associated
with improved fat utilization and, last but not least, enhanced exercise performance. The administration of the archetypical synthetic
PDE4-inhibitor rolipram (Fig. 4, 17) to laboratory rodents yielded
similar results, indicating that another master regulator of skeletal
muscle mitochondriogenesis upstream of the above reported key
factors (e.g. AMPK and PGC-1) has been identied. Consequently,
approved drugs and drug candidates of this category inevitably
move into the focus of sports drug testing and preventive doping
research organizations, revealing a considerable number of at least
50 candidates that have been mentioned as potential therapeutic agents with PDE4-inhibiting properties [65]; however, to date
only one (roumilast, Fig. 4, 18) has received clinical approval for
the treatment of COPD [66,67]. Cilomilast (Fig. 4, 19) has advanced
to phase-III clinical trials [68], and numerous additional new drug
entities in pre-clinical or early clinical development have recently
been summarized in a comprehensive review [65].
Due to these arguments, the necessity for detection assays
capable of screening for PDE4-inhibitors such as resveratrol,
rolipram, roumilast, and cilomilast was noticed and rst analytical approaches were recently presented [69]. Based on published
DMPK data and in vitro incubation reactions, target analytes were
selected and mass spectrometrically characterized, including the
intact drugs as well as major metabolites. While roumilast and
cilomilast each yielded mainly one reasonably abundant metabolite
in vitro (roumilast-N-oxide and hydroxylated cilomilast), rolipram
was converted into six intense metabolic products resulting from
hydroxylation and dealkylation reactions. Employing an established sample preparation protocol for urine specimens consisting
of enzymatic hydrolysis followed by LLE and subsequent LCMS/MS
analysis, LODs of 15 ng/ml for the compounds of interest were
accomplished, and proof-of-concept data were achieved with a
patients urine sample collected after therapeutic use of roumilast. Due to the substantial increase in performance as observed
with rodents on a treadmill test (time-to-exhaustion) and the evidently elevated number of mitochondria in skeletal muscle of the
test animals resulting from PDE4-inhibitor administrations, misuse of these substances cannot be excluded and at least inclusion
in monitoring programs seems advisable.
2.1.6. Peptidic and non-peptidyl growth hormone secretagogues
(GHS)
The multifaceted (and arguably performance-enhancing) effects
of human growth hormone (hGH), primarily mediated via insulinlike growth factor-1 (IGF-1) to the skeletal muscle, have triggered
substantial interest and comprehensive research programs with
medicinal and therapeutic intention in the past. One of many
remarkable outcomes has been the discovery of orally active
growth hormone secretagogues (GHS) based on various different
pharmacophores including e.g. benzolactame, 4-spiropiperidine,
capromorelin, and oxindole derivatives such as L-739943, MK0677, CP-424391, and SM-130686, respectively, which are depicted
in Fig. 4 as selected examples of an enormous variety of potential
drug candidates [70,71]. Since GHS stimulate the hGH secretion
via an alternative route from the growth hormone releasing hormone (GHRH, vide infra) and arguably provide synergistic effects
[72], their clinical development has been pursued complementary
to conventional therapies indicated in hGH/IGF-1 axis-related disorders and age-related decline. Sustained increases in plasma IGF-1
were reported in GHS administration studies; however, none of the
lead drug candidates has yet received clinical approval, possibly due
to limited benets compared to hGH replacement therapies and
reported weight gain due to appetite-stimulating effects of GHS.

Nevertheless, compounds such as MK-0677 have been available
through Internet-based suppliers, and detection methods for the
heterogeneous class of non-peptidyl GHS are desirable in routine
doping controls, preferably using commonly available methodologies such as mass spectrometry (Fig. 5a). In addition to these orally
available GHS, a considerable variety of growth hormone releasing peptides (GHRPs) acting through the same pathways on hGH
secretion has been developed since the seminal works of Bowers
and co-workers demonstrated the capability of short peptides to
regulate hGH release in the early 1980s [7375]. One representative (Pralmorelin, GHRP-2) received clinical approval 2004 in Japan
as a diagnostic tool for GH deciency in adults [76]. Whilst the
majority of GHRPs, a selection of which is summarized in Table 2,
does currently not hold approval by any health authority for human
therapeutic use, numerous black-market sources have been identied as suppliers of such agents [77,78], which underlines the
importance of doping control assays allowing the sensitive and
comprehensive analysis of GHS.
A generic approach toward this issue was presented in 2012 by
Pinyot et al., who employed a competitive receptor-binding assay
[79,80]. Since all GHS share the commonality to bind to the GHS
receptor 1a, the membrane-bound receptor is preincubated with a
radiolabeled ligand (125 I-ghrelin), which is displaced by (urinary)
GHS in a dose-dependent manner as demonstrated with 7 synthetic
GHS in a proof-of-concept study. The responses or minimal positive
concentrations for GHS in urine varied from 1.5E10 M to 1.0E06
with MK-0677 being most sensitively detected. The analysis of the
growth hormone releasing peptide GHRP-2 in post-administration
study urine samples was further presented, suggesting a detection
window of approximately 4.5 h for this particular drug. Conrmation of the presence of a prohibited substance belonging to the class
of GHS was recommended and conducted via dedicated LCMS/MS
assays.
Such a mass spectrometry-based methodology focusing particularly on the detection of GHRP-2 and its main metabolite
(d-Ala-d(-naphthyl)-Ala-Ala-OH) in human urine was published
in 2010 by Okano et al. [81]. Here, an isotope-labeled GHRP-2 internal standard was used to ensure appropriate sample preparation
and analysis conditions before the specimen was subjected to solidphase extraction (SPE) and subsequent LCMS/MS analysis. The
assay enabled LODs of 2050 pg/ml and was applied to an excretion study with ten volunteers who received an intravenous bolus
of 100 g of GHRP-2 dihydrochloride. While the intact GHRP-2 was
found up to 13 h, the aforementioned metabolite was detected up to
24 h using the established approach. Similarly, a screening method
was established in 2011 targeting a family of 8 GHRPs (GHRP-1,
-2, -4, -5, -6, alexamorelin, hexarelin, and ipamorelin) plus the
earlier mentioned GHRP-2 metabolite using an isotope-dilution
LCMS approach [82]. Here, two deuterated internal standards (d4 GHRP-4 and a d3 -GHRP-2 metabolite) were added and SPE was
conducted with a weak cation exchange resin prior to microbore
reversed-phase LC separation and full scan high resolution/high
accuracy mass spectrometry, which enabled LODs of 0.20.5 ng/ml.
The applicability of the developed method to authentic urine samples was tested with an elimination study with 10 mg of GHRP-2
orally administered to one male volunteer. The analyses revealed
the absence of intact GHRP-2 and the traceability of the GHRP-2
metabolite up to 20 h post-administration. In a follow-up study, the
instrumental setup was modied to consist of a nanoUHPLC system
interfaced to a quadrupole-orbitrap mass analyzer [83]. As a result,
the LODs were lowered approximately 20-fold to allow for the
detection of 210 pg/ml of each substance. Since DMPK data of most
GHRPs are not available but of great importance in terms of doping
controls, animal in vivo studies and in vitro simulations of metabolic
reactions were conducted to identify and characterize viable target
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Fig. 5. (a) Product ion mass spectrum of the protonated molecule [M+H]+ at m/z 528 of MK-0677, recorded at a collision energy of 25 eV; (b) product ion mass spectrum of
the protonated molecule [M+H]+ at m/z 655 of GHRP-3, recorded at a collision energy of 20 eV; (c) full scan MS spectrum of the intact anti-myostatin antibody Stamulumab,
recorded at a resolution of 35,000 (FWHM).
compounds for sports drug testing approaches [84]. Focusing on

currently non-approved GHRPs (except for GHRP-2), seven compounds were administered to rats via oral and intravenous routes.
These compounds (GHRP-1, -2, -4, -5, -6, alexamorelin, hexarelin,
and ipamorelin) yielded at least 3 urinary metabolites each after

i.v. application, which were conrmed by human in vitro simulations and will extend the initial testing options for GHRPs in routine
doping controls. A typical product ion mass spectrum of an intact
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Table 2
Primary structures of selected peptidic drug candidates. Peptides with 10 or less
amino acid residues are presented in 3-letter code, larger peptides in 1-letter code.
Compound
Amino acid sequence
GHRP-1
Ala-His-d-Nal-Ala-Trp-dPhe-Lys-NH2
d-Ala-d-Nal-Ala-Trp-d-PheLys-NH2
Aib-d-Trp-d-Pro-d-Ile-ArgNH2
d-Trp-Ala-Trp-d-Phe-NH2
Tyr-d-Trp-Ala-Trp-d-Phe-NH2
His-d-Trp-Ala-Trp-d-Phe-LysNH2
Ala-His-d-Mrp-Ala-Trp-d-PheLys-NH2
His-d-Mrp-Ala-Trp-d-Phe-LysNH2
Aib-His-d-2-Nal-d-Phe-LysNH2
YADAIFTNSY RKVLGQLSAR
KLLQDIMSRQ QGESNQERGA
RARL
KLLQDIMSR-NH2
KLLQDIMSRK*-NH2
YdADAIFTNSY RKVLGQLSAR
KLLQDIMSRK*-NH2
YdADAIFTQSY RKVAGQLSAR
KLLQDILSRK*-NH2
GPETLCGAEL VDALQFVCGD
RGFYFNKPTG YGSSSRRAPQ
TGIVDECCFR SCDLRRLEMY
CAPLKPAKSA
TLCGAELVDA LQFVCGDRGF
YFNKPTGYGS SSRRAPQTGI
VDECCFRSCD LRRLEMYCAP
LKPAKSA
GPRTLCGAEL VDALQFVCGD
CAPLKPAKSA
MFPAMPLSSL FVNGPRTLCG
AELVDALQFV CGDRGFYFNK
PTGYGSSSRR APQTGIVDEC
CFRSCDLRRL EMYCAPLKPA KSA
YQPPSTNKNT KSQRRKGSTF
EERK-NH2
GPETLCGAEL VDALQFVCGD
CAPLKPAKSA RSVRAQRHTD
MPKTQKYQPP STNKNTKSQR
RKGSTFEEH
GHRP-2
GHRP-3
GHRP-4
GHRP-5
GHRP-6
Alexamorelin
Hexarelin
Ipamorelin
GHRH
Sermorelin
CJC-1288
CJC-1293
CJC-1295
IGF-1
des(1-3)-IGF-1
R3 -IGF-1
long-R3 -IGF-1
MGF
full length MGF
Molecular mass
monoisotopic
[Da]
954.5
817.4
654.4
607.3
770.4
872.4
957.5
886.5
711.4
5037.6
3355.8
3634.9
3634.9
3437.9
7643.6
7360.5
7670.6
9105.4
2865.5
12264.9
Non-standard abbreviations:
Aib = aminoisobutyric acid, Nal = naphthylalanine, Mrp 2-methyltryptophane
*maleimido-propionic acid (MPA) tag for bioconjugation, accounting for a mass of
151 Da.
GHRP is depicted in Fig. 5b representing GHRP-3. Human pharmacokinetic data for GHRP-6 were presented by Cabrales et al. in
2012/2013 [85,86]. Employing an isotope-dilution mass spectrometric approach, GHRP-6 was determined from plasma with an
LOQ of 5 ng/ml. Samples were enriched with 13 C-labeled GHRP-6,
plasma proteins were depleted by acetone-facilitated precipitation,
and the target analyte was quantied from the concentrated supernatant by nanoLC-Q/TOF MS using a monolithic nano LC column.
Following an i.v. bolus dose of 100, 200, or 400 g/kg of bodyweight, plasma samples were collected up to 72 h, and GHRP-6
concentrations were found to fall below the LOQ (5 ng/ml) after
12 h post-administration.
2.2. High(er) molecular mass substances

2.2.1. Growth hormone releasing hormones (GHRHs)
In addition to GHS, growth hormone releasing hormone (GHRH)
and its modied synthetic analogs received an enormous stimulus
for illicit and mostly Internet-based sales [77,87,88]. In contrast to
GHS, GHRHs act via receptors at the anterior pituitary, and clinically
approved representatives of GHRHs are tesamorelin and sermorelin. In addition, research concerning new potent analogs of GHRH as
therapeutic agents has been continued uninterruptedly for decades
[89]. This has resulted in a series of potential drug candidates, one
of the most frequently mentioned compound of which has been
CJC-1295 (Table 2). Bearing sequence modications at 5 positions
as well as a 3-maleimido-propionic acid for in vivo bioconjugation to albumin [90], its efcacy in rats with extended plasma
half-life was reported. Hence, complementary to GHS test methods, screening protocols for GHRHs such as CJC-1288, CJC-1293,
and CJC-1295 have been desirable albeit, similar to most GHRPs,
DMPK data in humans and information on the renal elimination
of the intact drug and relevant metabolite(s) are largely missing. A
methodology employing immunoafnity purication followed by
nanoLCMS/MS analysis was presented in 2012 [91]. By means of
preconcentration of 5 ml of urine using SPE and subsequent extraction of the target analytes GHRH (129, sermorelin) and CJC-1295
(without the maleimidopropionic acid moiety), the highly sensitive
and specic analysis of the compounds was accomplished allowing for detection limits of 5 and 1 pg/ml, respectively. It remains to
be claried, however, whether the intact peptides or corresponding metabolites will be most efcient as target analytes in routine
doping controls when using urine as primary doping control matrix.
Alternatively, blood/serum might be a viable option to test for the
intact drugs.
2.2.2. Mechano growth factors (MGFs)
Peptide hormones categorized as mechano growth factors
(MGFs) have been explicitly mentioned as prohibited substances
in relevant WADA regulations since 2005. The use of the term MGF
has been ambiguous in the literature, i.e. referring to IGF-1Eb (or Ec
in humans) mRNA, pro-IGF-1Eb, as well as the synthetic MGF peptide, and it is hence recommended to use the name MGF solely
for synthetic mechano growth factor peptides [92]. While IGF-1Ec
mRNA is derived by alternative splicing from the IGF-1 gene, the
formation and in vivo existence of MGF in humans comprising 24
amino acid residues (Table 2), as well as the effects attributed to
MGF itself are subject of substantial controversy. Earlier reports
described MGFs capability to stimulate muscle (stem) cell proliferation and thus to increase muscle strength and regeneration,
suggesting that its abuse in sport is cannot be excluded, despite
missing clinical approval [93]. However, comprehensive tests with
synthetic MGF failed to reproduce the anabolic and regenerationpromoting effects of MGF and a dedicated MGF receptor has yet to
be identied to support and corroborate the commonly accepted
MGF hypothesis [94].
To date, no dedicated detection assay for doping control purposes has been reported but ndings of MGF in black-market vials
were reported in 2012 [95], stressing the potential abuse of such
compounds in amateur and/or elite sport despite the above mentioned questionable efcacy. The black-market product was shown
to be composed of the appropriate 24 amino acid residues as listed
in Table 2 but comprised a C-terminal amidation, which has not
been postulated for the potentially naturally occurring human MGF.
2.2.3. Anti-myostatin antibody MYO-029
Myostatin, also known as growth and differentiation factor8 (GFD-8), is a highly conserved member of the transforming
growth factor beta (TGF-) superfamily which acts as a negative
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regulator of skeletal muscle mass [96,97]. In myostatin knockout mice (Mstn/ ), a substantial increase in muscle mass was
observed resulting from both an elevated volume (hypertrophy)
and number (hyperplasia) of skeletal muscle bers [9799]. Hence,
selective blocking of the myostatin signaling pathway has been
considered as a therapeutic means to counteract or cure severe diseases such as muscular dystrophies but also other arenas such as
improving livestock production were mentioned as desirable goals
[98,100102]. Approaches specically targeting myostatin include
injectable myostatin-binding proteins such as the GDF-8 propeptide [103,104] as well as recombinant antibodies [104106].
In a mouse model of muscular dystrophy, the inhibition of endogenous myostatin by specic antibodies considerably improved the
dystrophic phenotype of the animals as their muscle mass, muscle size, muscle strength and body weight were signicantly
increased [105]. Consequently, antibody-based drug candidates
for humans were developed with MYO-029, also referred to as
stamulumab, being rst in class in 2005 [107109]. Despite discontinuation from clinical development, a considerable misuse
potential is eminent and test methods on immunological or mass
spectrometric platforms are desirable. With the improving capability of high resolution/high accuracy mass spectrometry also the
direct analysis of the (puried) antibody could be a future option
(Fig. 5c).
2.2.4. RNA interference
Considering the number of research articles, ribonucleic acid
(RNA) interference (RNAi) has continued to be one of the
most dynamic arenas of biotechnological research (along with
proteomics and epigenetics) [110]. The enormous therapeutic
potential of posttranscriptional gene silencing has been recognized in numerous elds of medicinal treatment, particularly
where the temporary knock-down of negative regulators is desirable. Here, a frequently referenced example is the inhibition of
myostatin, the prominent negative regulator of myogenesis, the
elimination of which has resulted in signicant increases of muscle mass and strength in animal models and, thus, suggesting
a viable means to cure myopathies such as Duchenne muscular dystrophy (DMD) [111113]. Strategies employing antisense
oligonucleotides or small interfering RNA (siRNA) were described
as successful means but a main prerequisite of RNAi has been the
stability of the drug candidate. Due to the rapid degradation of
RNA in general, modied ribonucleic acids were introduced such as
2 -uoronucleotides, 2 -O-methylated nucleotides, phosphorothioate nucleotides, or locked nucleic acids, effectively protecting the
siRNA from hydrolysis and early elimination from the system [114].
Due to the availability of software allowing to predict viable RNAi
motifs and the rapid and the option to purchase fully automated
synthesized tailored siRNA, detection strategies for this new therapeutic approach bearing the risk abuse in sport have been initiated
[115,116].
A major step toward demonstrating the tness-for-purpose of
developed anti-doping methods was provided in 2013, when animal studies were conducted with model siRNA [117]. Following
the intravenous administration of therapeutic amounts of siRNA,
urine samples were collected and subjected to two analytical
platforms consisting of biochemical and LC-HRMS methodologies.
Sample preparation included the specic enrichment of urinary
siRNA using solid-phase extraction spin columns, the extract of
which was applied to gel electrophoresis, LC-HRMS of intact
target analytes (i.e. intact siRNA or truncated metabolic products), or chemical hydrolysis and LC-HRMS(/MS) determination
of synthetically mono- and oligonucleotides. Further to these, a
combination of gel electrophoresis followed by mass spectrometric analysis in a bottom-up approach was demonstrated to provide
the desired information whether chemically modied RNA was
present in a urine sample or not. Overall, screening for siRNA by gel

electrophoresis followed by conrmatory measurements employing LC-HRMS was found to be suitable for doping control purposes
offering detection limits of 25 pmol/ml of urine and detection windows of 24 h when single dose administration to laboratory rodents
was conducted.
3. Compounds affecting the oxygen transfer capacity
Since oxygen transfer capacity is one of the key parameters
of athletic performance, its manipulation has been attempted
by numerous means, and doping control laboratories and antidoping authorities have been urged to expand their analytical
scope beyond approved erythropoiesis-stimulating agents (ESAs)
such as erythropoietin (EPO). These have been successfully covered in the past by continuously improved and updated isoelectric
focusing (IEF) and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods [118121]; however, recent drug
developments have necessitated further considerations as shown
below.
3.1. Hypoxia-inducible factor (HIF) stabilizers
One of the main targets of pharmacological stimulation of erythropoiesis have been hypoxia-inducible factors (HIFs) since their
role in oxygen sensing and production of EPO was recognized and
demonstrated in 1992 [122]. Prolylhydroxylase-catalyzed hydroxylation of HIFs and their subsequent ubiquitination followed by
proteasomal degradation was shown to be reduced/eliminated by
various categories of small molecules, acting as prolylhydroxylase
inhibitors (PHIs), resulting in an increased expression of EPO and,
consequently, an elevated erythropoiesis [123]. The main advantage of these compounds over approved ESAs such as EPO and its
second- and third-generation successors is their oral availability
and lack of concerns about immunogenicity [124]. A multitude of
drug candidates has been presented in the past as compiled in
recent reviews [125,126], and a selection of disclosed substances
is summarized in Table 3 as well as Fig. 6.
The plethora of emerging HIF stabilizing therapeutics and the
concomitant potential for abuse as doping agents has necessitated
proactive detection method developments in doping controls. First
attempts were conducted in 2008 with the characterization of
FG-2216 (Fig. 6, 24) and its implementation into routine doping
controls followed by investigations into structurally related compounds such as FG-4592 (Fig. 7a), in vitro metabolism elucidations,
and the development of comprehensive analytical approaches
[127131]. Since most of the potential HIF stabilizers comprise
structures that suggest physico-chemical properties suitable for
chromatographic and mass spectrometric analyses, LCMS/MS has
been the method of choice in most of the published analytical approaches. By means of targeted MRM-based measurements,
detection limits for FG-2216 and related (model) compounds
between 1 and 10 ng/ml were accomplished [130], which is sufciently sensitive considering the expected therapeutic amounts and
resulting urinary concentrations of the drugs metabolites. Moreover, in order to account for the yet largely unknown structures of
drug candidates and respective metabolic products, a mass spectrometric peculiarity of isoquinoline-derived HIF stabilizers was
exploited to allow for a specic initial testing approach. In several studies, the combined elimination of methylenamine (29 Da)
or carbon monoxide (28 Da) accompanied by the addition of a water
molecule (18 Da) was observed, resulting in a nominal loss of 11 or
10 Da, respectively. Hence, a neutral loss scan for this rather specic mass difference can support and broaden the screening for
current and future drug candidates with similar structural features
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11
Table 3
Structure characteristics of selected HIF stabilizers.
No. (Fig. 6)
HIF stabilizer/company
Pharmacophore
Elemental composition
Molecular mass (Da)
24
25
26
FG-2216
FG-4592
Fibrogen
Fibrogen
Fibrogen
GSK360A
GSK
GSK
GSK
GSK
Amgen
Amgen
Bayer
Bayer
Janssen
Merck
Merck
Isoquinoline-glycineamide
Isoquinoline-glycineamide
Thienopyridine-glycineamide
Thiazolopyridine-glycineamide
isothiazolopyridine-glycineamide
Quinoline-glycineamide
Tetrahydropyrimidine-glycineamide
Dihydropyrimidine-glycineamide
Dihydropyrazolopyrimidine-glycineamide
Pyridopyrimidine-glycineamide
Naphthyridine-glycineamide
Dihydropyridopyrimidine-glycineamide
Pyrazol-picolinonitrile
Pyrazol-nicotinic acid
Benzoimidazol
Naphthyridine-glycineamide
Tetrahydropyrrolo-glycineamide
C12 H9 ClN2 O4
C19 H16 N2 O5
C10 H7 ClN2 O4 S
C9 H6 ClN3 O4 S
C24 H18 N4 O4 S
C17 H17 FN2 O5
C19 H27 N3 O6
C21 H19 N3 O5
C16 H14 N4 O5
C11 H8 BrN3 O5
C12 H11 N3 O5
C17 H13 FN4 O5
C15 H11 N5 O
C14 H9 BrN4 O3
C13 H11 ClN4 O2 S
C18 H13 F3 N4 O5
C22 H17 F3 N4 O6 S
280.0251
352.1059
285.9815
286.9738
458.1049
348.1121
393.1900
393.1325
342.0964
340.9647
277.0699
372.0870
277.0964
359.9858
322.0291
422.0838
522.0821
27
28
29
30
31
32
33
34
35
although this mass spectrometric scan mode is not particularly

sensitive.
3.2. Cobaltous chloride
An arguably outdated and old-fashioned option to stimulate erythropoiesis is the administration of cobaltous (II) chloride [132].
Having been used since the early 1950s, it later served as reference to assess the activity of one international unit of EPO [133].
Technically, cobaltous chloride can be categorized among the group
of HIF stabilizers as its erythropoetic effect is, at least partially,
attributed to an HIF stabilization via displacement of iron in the catalytically active site of relevant prolylhydroxylases. In addition or
alternatively, its mechanism of action was hypothesized to include
a reduction of ascorbate biosynthesis or a direct binding to HIF
[125]. At any rate, its erythropoietic effect was therapeutically
exploited prior to the EPO era (until its considerable health risks
necessitated the search for alternative therapeutics) and its abuse
in sport for the very same reasons cannot be excluded [134]. In the
contrary, particularly in animal sport, the abuse of cobaltous chloride has been raised very recently [135] and analytical approaches
such as LCMS/MS following complexation of cobalt (Fig. 7b) [136]
as well as commonly employed inductively coupled plasma (ICP)
MS are viable options to quantify the naturally occurring trace element in doping control specimens [137139]. However, threshold
levels might have to be established similarly to guidelines of horse
racing authorities, if the quantity of cobalt is considered relevant
for human sports drug testing in the future. This is of particular
importance in the light of dietary supplementation with cobaltous
chloride, for which amounts of up to 600 and 1400 g/day have
been reported to be acceptably safe for humans [140142].
3.3. Erythropoietin fusion protein EPO-Fc
Despite the fact that EPO has been clinically approved, extensively used in therapeutic settings, and further developed for 25
years, alternatives with either improved pharmacokinetics, proles of undesirable effects, and/or easier routes of administration
have been explored in the past [123,143146]. Among these, the
fusion protein composed of EPO and the fragment crystallizable
(Fc) part of immunoglobulin G (IgG), commonly referred to as EPOFc, has been pursued in pre-clinical and clinical studies [147]. Here,
a longer half-life of the drug candidate as compared to EPO itself
was observed and, more importantly, the inhalative application of
the substance was enabled as supported by the presence of airwayepithelial Fc receptors.
Despite missing clinical approval, doping control test methods

had to be assessed in terms of their capability to detect a potential
abuse of the experimental drug by athletes. A rst and comprehensive study concerning the traceability of EPO-Fc in human
serum was done in 2012 [148]. Both screening and conrmatory
approaches were suggested allowing the detection of EPO-Fc at
concentrations as low as 5 pg/ml of serum employing ELISA and
gel electrophoretic/immunological approaches. The ELISA-based
methodology exploited the fact that the Fc-part of the drug candidate can be captured by protein A-coated beads, which will not
allow for retaining natural EPO. Subsequently, the analysis of the
beads eluate with a commercial EPO ELISA kit is conducted, which
would trigger a conrmatory analyses if a measurable signal indicating the presence of EPO is obtained [148]. The conrmation
of an EPO-Fc nding is ideally included in routine doping control
protocols such as IEF or SDS-PAGE, both followed by Western blotting. The IEF properties of EPO-Fc under routine doping control
conditions were found to be unfavorable; however, the apparent
molecular mass of 60 kDa of EPO-Fc as determined by SDS-PAGE
yielded a distinct image enabling the unequivocal differentiation
of EPO-Fc from EPO and other related ESAs.
3.4. Peginesatide
In addition to EPO-based ESAs and HIF stabilizers, EPO mimetic
peptides (EMPs) have received much attention ever since rst
medicinal reports were published in 1996 on the capability of
EMPs to bind to and stimulate the EPO receptor [149]. A rst-inclass approved EMP-based drug referred to as peginesatide was
launched in 2012, comprising a homodimeric EMP structure linked
to a 2 20 kDa polyethylene glycol (PEG) support (Fig. 8). However,
in early 2013, the manufacturer voluntarily recalled the therapeutic agent as safety endpoint data of cardiovascular events and death
were worse for peginesatide than for the comparator EPO product, and until now it has not been re-introduced to the market.
Nevertheless, the availability of this ESA and, thus, its abuse cannot be excluded requiring appropriate test methods as established
between 2011 and 2012 [150153].
In order to allow for comprehensive doping controls, viable
matrices including serum/plasma, urine, and dried blood spots
(DBS) were evaluated, demonstrating that the drug can be detected
if therapeutic amounts have been administered. When using MSbased methodologies, precipitation of high abundant proteins from
serum (or plasma) was followed by enzymatic hydrolysis of the
peptidic moiety using subtilizin, yielding a diagnostic pentapeptide [GPIT(1-nal)] for LCMS/MS analysis [151]. The assay allowed
for detection limits of 1 ng/ml, which was found appropriate
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Fig. 6. Structures of selected HIF stabilizers including FG-2216 (24), FG-4592 (25), and GSK360A (27). Further details are given in Table 3.
considering therapeutic plasma concentrations of up to 1000 ng/ml.

Due to the considerably high blood concentrations, DBS analysis
was pursued offering a faster and cheaper sample collection strategy [152]. Here, essentially the same sample preparation strategy
was applied including extraction of the DBS, subtilizin digestion,
and subsequent LCMS/MS measurement. However, as a result of
the limited sample volume (approximately 20 l of DBS), detection limits were determined at 10 ng/ml, which nevertheless will
serve the purpose of sports drug testing. As urine is the most frequently collected doping control specimen, animal in vivo study
urine samples were used to probe for the renal elimination of either
the intact peginesatide or metabolite(s) that could be utilized as
target analytes [150]. Since the intact drug was observed up to 4
days post-administration of a single therapeutic dose of the ESA,
the methodology as adapted and optimized for urine was validated,

enabling for the analysis of 0.5 ng of peginesatide per ml of urine.
Complementary, immunological (ELISA) and gel electrophoretic
methods for the detection of peginesatide in serum were reported,
allowing for detection limits of 0.5 ng/ml [153]. The ELISA consisted
of a sandwich-like test method employing an immobilized monoclonal anti-PEG antibody and a monoclonal biotinylated antibody
directed against the peptidic moiety of peginesatide. Using serum
samples from an administration study with 50 g/kg bodyweight,
the drug was detectable up to 10 days post-administration using
the ELISA-based initial test method. Conrmatory analyses without
mass spectrometry were then suggested by means of gel electrophoretic determination of immunoprecipitated peginesatide.
Here, a different monoclonal antibody targeting a different epitope
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13
Fig. 7. (a) Product ion mass spectrum of the protonated molecule [M+H]+ at m/z 353 of FG-4592, recorded at a collision energy of 30 eV; (b) product ion mass spectrum of
M+ at m/z 355 of the cobalt-diethyldithiocarbamate (DDC) complex as analyzed with ESI-MS/MS at a collision energy of 40 eV.
of the peptide residue was used to capture and extract the ESA from
500 l of serum. Subsequently, conventional SDS-PAGE with double Western blotting was conducted, enabling the visualization of
0.5 ng/ml of the target analyte.
4. Other compounds
As the quest for faster recovery, better athletic performance, and
optimized body composition continues, new substances advertised
with such properties have frequently been observed with Internetbased suppliers as well as in products conscated at customs.
4.1. TB-500
An arguably natural and chemical-free substance for horse treatment is TB-500 (Fig. 9a). Following anecdotal evidence of its abuse
in human sport, the substance was in fact seized from possessions

of a professional athletes team entourage [56], and conscated
material was characterized by mass spectrometry [154], chemical synthesis and NMR [155]. Advertised as a synthetic analog to
thymosin beta 4 (T4), it was identied as an N-terminally acetylated heptapeptide, representing the amino acid sequence 1723
(the major actin-binding site) of T4. This sequence was shown to
possess (corneal) tissue-repair supportive and angiogenic properties in animal models [156,157], hence a potential benet to human
and/or animal athletes was attributed. Due to the non-natural composition of the product, its traceability in doping control samples
was demonstrated for plasma and urine specimens [155]. Samples
were subjected to SPE (in case of plasma, high abundant proteins
were rst precipitated), and extracts were analyzed by LCMS/MS,
allowing for detection limits of 500 pg/ml. Even though no human
clinical study data have been published for TB-500, extrapolation
Fig. 8. Structure cartoon of peginesatide.
G Model
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ARTICLE IN PRESS
Fig. 9. (a) Product ion mass spectrum of the protonated molecule [M+H]+ at m/z 889 of TB-500, recorded at a collision energy of 45 eV; (b) product ion mass spectrum of the
doubly charged precursor ion [M+2H]2+ at m/z 908 of AOD-9604, measured at a collision energy of 30 eV.
from recommended dosing (10 mg) and horse administration study

results [158] suggest that such LODs are adequate also for human
doping controls.
4.2. AOD-9604
The peptidic compound referred to as AOD-9604 consists
of 16 amino acids (Fig. 9b), largely identical to the primary
structure found within the C-terminal region of human growth
hormone (hGH), with an average molecular mass of 1815 Da.
Due to reported lipolytic and anti-lipogenic properties [159], it
has been under development for assisting anti-obesity treatments
but has not reached full clinical approval yet [160,161]. Since
2013, AOD-9604 has been considered as prohibited according
to WADAs anti-doping regulations [162]; however, similarly to
other non-approved substances, its metabolism has not been fully
investigated or publicized. A rst concern as to doping control
analyses was whether this substance could have any impact on
the antibody-based detection assay for hGH, and studies were
conducted demonstrating that the so-called growth hormone isoform test is specic and not affected by AOD-9604 [163]. Due to
the peptidic nature of AOD-9604, its implementation into routine doping controls has been accomplished according analytical
strategies employed e.g. for GHRPs (vide supra); it remains however to be shown whether urine, serum, and/or DBS are the matrix
of choice for this substance, if the intact drug or metabolic products are viable target analytes, and if the presumably limited
performance-affecting properties of AOD-9604 will be of sufcient temptation to athletes to conduct anti-doping rule violations
[164]. Clearly, trafcking of AOD-9604 via illicit and black-market
providers has been monitored in the past [154], requiring attention
of doping and customs controls.
5. Conclusion
Preventive and proactive anti-doping work, particularly concerning analytical strategies, is of paramount importance considering the enormous breadth of new emerging as well as discontinued
drugs and drug candidates. A constantly increasing plethora of
substances with abuse potential has been recognized over the
past years, necessitating comprehensive, sensitive, and specic
analytical approaches, the development of which requires state-ofthe-art instrumentation as well as insights into pharmacology and
metabolic pathways of these compounds, which span from low(est)
molecular mass analytes such as cobalt to intact antibodies constituting organic molecules of more than 150 kDa. Hence, continuous
research and improvements in sports drug testing are indicated to
ensure an adequate support of the clean athlete.
Acknowledgments
The authors thank the Federal Ministry of the Interior of the
Federal Republic of Germany and the Manfred-Donike Institute for
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ARTICLE IN PRESS
Doping Analysis for supporting the presented work. In addition,

the authors wish to thank Dr. Cyril Y. Bowers for invaluable help in
extending doping control test options for GHRPs.
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PBA-9583; No. of Pages 18

Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxxxxx

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

Analytical approaches for the detection of emerging therapeutics and

Corresponding author at: Institute of Biochemistry Center for Preventive

PBA-9583; No. of Pages 18

chromatography(tandem) mass spectrometry (LCMS/MS) or gas

characterization by mass spectrometric techniques, particularly

PBA-9583; No. of Pages 18

Molecular mass (Da)

analytes as the administered compound was detected intact only

have assigned 4-(2-((S)-2,2,2-triuoro-1-hydroxyethyl)pyrrolidin1-yl)-2-(triuoromethyl)-benzonitrile (Fig. 2, 13) to it. Despite the

2.1.3. Sirtuin-1 activators

believed to be subsequently primed by SIRT1 via deacetylation and

successful IRMS analysis of AICAR, namely the low volatility of

reported weight gain due to appetite-stimulating effects of GHS.

compounds for sports drug testing approaches [84]. Focusing on

and ipamorelin) yielded at least 3 urinary metabolites each after

PBA-9583; No. of Pages 18

Amino acid sequence

2.2. High(er) molecular mass substances

present in a urine sample or not. Overall, screening for siRNA by gel

PBA-9583; No. of Pages 18

Molecular mass (Da)

although this mass spectrometric scan mode is not particularly

Despite missing clinical approval, doping control test methods

considering therapeutic plasma concentrations of up to 1000 ng/ml.

the methodology as adapted and optimized for urine was validated,

in human sport, the substance was in fact seized from possessions

Fig. 8. Structure cartoon of peginesatide.

from recommended dosing (10 mg) and horse administration study

Doping Analysis for supporting the presented work. In addition,

[26] A. Thomas, S. Guddat, M. Kohler, O. Krug, W. Schnzer, M. Petrou, M.

[80] A. Pinyot, Z. Nikolovski, J. Bosch, G. Such-Sanmartin, S. Kageyama, J. Segura, R.

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