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Please cite this article in press as: M. Thevis, W. Schänzer, Analytical approaches for the detection of emerging therapeutics and non-

approved drugs in human doping controls, J. Pharm. Biomed. Anal. (2014), http://dx.doi.org/10.1016/j.jpba.2014.05.020

ARTICLE IN PRESSG Model

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Journal of Pharmaceutical and Biomedical Analysis xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journa l homepage: www.elsevier .com/ locate / jpba

Review

Analytical approaches for the detection of emerging therapeutics andnon-approved drugs in human doping controls

Mario Thevis a,∗, Wilhelm Schänzerb

a Center for Preventive DopingResearch – Institute of Biochemistry, GermanSport University Cologne, Am SportparkMüngersdorf 6,

50933 Cologne, Germanyb EuropeanMonitoringCenter 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 test-

ing, 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 summa-

rizes literature published over the past 5 years on new drug entities, discontinued therapeutics, and

‘tailored’ compounds classified 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.modified 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, S-

107and ARM036/aladorian,etc.) as well as inorganic (cobalt) nature are considered and discussed in terms

of specific 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 biochemi-

cal mechanisms at cellular and molecular levels, more and more

optionsfor pharmacologicalinterventions havebeen identified that

suggest newpaths to desired therapiespotentiallyallowing curefor

severe if not fatal diseases. The flipside of such research is the mis-

use potentialoffered by a subsetof newdrug candidates, especially

those that promote muscle growth, stimulate erythrocyte produc-

tion, or enhance physical stamina and athletic performance via

other routes [1]. Such drug candidates have been offered and soldvia Internet-based providers for years, despite the lack of clinical

approval and, in some cases, discontinuation of their development

dueto severeside effects. Thetargeted ‘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 Müngersdorf 6,

50933 Cologne, Germany. Tel.: +49 221 4982 7070; fax: +49 221 4982 7071.

E-mail address: [email protected] (M. Thevis).

World Anti-Doping Agency (WADA) [2] These regulations as pre-

sented in WADA’s 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 labo-

ratories update, expand, and improve their portfolio of analytical

assays, mostof which relyon chromatographic-mass spectrometric

approaches[3,4]; however,the implementation of newcompounds

into sports drug testing protocols requires a substantial amount

of information including therapeutic dosage, pharmacokinetics,

metabolism, and elimination. Moreover, specific physicochemi-

cal properties might necessitate dedicated sample collection and

transport conditions, sample preparation or analytical procedures

to ensure the required sensitivity and specificity to detect the tar-

get 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 fitness-for-purpose

as appropriate initial testing procedure has to be demonstrated.

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2 M. Thevis, W. Schänzer / Journal of Pharmaceutical andBiomedical Analysis xxx(2014) xxx–xxx

While formerly drug classes dictated the composition of analyti-

cal assays, nowadaysthe available analytical equipmentcommonly

governs the employed test strategies [3]. To date, routine doping

control matrices are urine, serum and blood, occasionally comple-

mented by alternative matrices such as hair potentially providing

supporting evidence. The collection protocols follow stringent reg-

ulationsand require trained doping control officers/phlebotomists;

transport times and conditions have to be controlled and docu-

mentedespeciallyincaseofbloodsamplesfortheAthleteBiological

Passport (ABP), where also time limits for transport and analy-

sis 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 identification 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 perfor-

mance, the substances considered in the followingare divided into

the categories of low and high molecular mass products.

2.1. Lowmolecular mass substances

2.1.1. Ryanodine receptor-calstabin-complex stabilizers (Rycals)

Studies on cardiac arrhythmia as well as sarcopenia (as defined

as the age-related loss of muscle mass, force, and exercise capac-

ity) 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 func-tion. Substantial research on mechanisms of post-translational

modifications 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

[6–8]. A potential therapy is based on benzothiazepine-derived

drug candidates such as the first- and second-generation thera-

peutics 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. Conse-

quently, 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 wasstudied in extensousing electrospray ionization (ESI) and collision-

induced dissociation (CID) [10] as well as electron ionization (EI)

[11] employing high resolution/high accuracy mass spectrome-

try, stable isotope labeling and, in case of ESI-CID, H/D-exchange

experiments. By means of the obtained information, test meth-

ods for urine [10,11] and plasma [12] were developed enabling

the detection of the intact molecules at LODs of 0.1–6ng/ml. In

case of blood plasma, peak concentrations of the drug candidates

after therapeutic dosing were expected at approximately 40 ng/ml,

whichwas well withinthe detection windowof the developed test

method. In the absence of data on the metabolism and(renal)elim-

ination of the benzothiazepines, urine samples were subjected to

enzymatic hydrolysis followed by liquid–liquid extraction (LLE) of

the target analytes and subsequent detection by means of liquid

chromatography–(tandem) mass spectrometry (LC–MS/MS) or gas

chromatography–mass spectrometry (GC–MS). In order to further

complement the analytical approach with putative metabolites,

phase-I and phase-II metabolic reactions were simulated for S-

107 in vitro, yielding predominantly N- and S-oxygenated species

as well as N- and O-demethylated metabolites. Moreover, glu-

curonic acid conjugates of the intact drug and its O-demethylated

phase-I metabolic product were identified representing viable tar-

gets for future doping controls [13]. Moreover, the development of

next-generation benzothiazepine-derived compounds needs fur-

ther consideration, e.g . with regardto thephase-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 first-in-class compounds were identified in 1998, predom-

inantly 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 substan-

tial interest in new drug entities with SARM-like properties is

still on the incline according to recent reviews [16,18] and pub-lications on advances in SARM-related research [19–21]. With

the increasing amount of possible non-steroidal and steroidal

SARM drug candidates, examples of which are illustrated in Fig. 2

(4–13), 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 appro-

priate 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 demon-

strated with the first adverse analytical findings (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 identified and

characterized in in vitro [28] and in vivo studies [29–31]. Despite

modest structural similarities between some SARMs comprising

e.g. a 4-substituted aniline moiety, a substantial heterogeneity of

pharmacophores is present in currentlyinvestigated SARMsinclud-

ing (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 identified and character-

ized target compounds.

All SARMs recently studied in a doping control context demon-

strated good or excellent ionization properties using electrospray,

thus supporting the sensitive detection of these compounds andrelated metabolic products in sports drug test samples employ-

ing LC–MS/MS-based strategies [3,32]. Arylpropionamide-derived

SARMs were among the first 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 arylpropionamide-

derived SARMs S-4 and S-22 (Fig. 2, 4 and 5, respectively)

predominantly yielded the glucuronic acid conjugates of the intact

drugs andcorrespondingmono-hydroxylated metabolites as viable

analytesfor routine doping controls[29,30] withLODs forthe intact

drug candidates found below 1 ng/ml. Complementary, LODs of

0.05–20ng/ml [27,33] and 10ng/ml [26] were determined in DBS

and plasma, respectively, for the intact therapeutics. Substance

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M. Thevis, W. Schänzer / Journal of Pharmaceutical andBiomedical Analysis xxx(2014) xxx–xxx 3

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 collisionenergy of 25eV.

characterization by mass spectrometric techniques, particularly

LC–MS/MS employing high resolution/high accuracy mass spec-

trometry, 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. Simi-

larly, 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) SARM Pharmacophore Elemental composition Molecular mass (Da) Refs.

S-1 Arylpropionamide C17 H14F4N2O5 402.0839 [165]

4 S-4 (Andarine) Arylpropionamide C19 H18F3N3O6 441.1148 [18]

S-9 Arylpropionamide C17 H14ClF3N2 O5 418.0543 [165]

5 S-22 (Ostarine) Arylpropionamide C19 H14F3N3O3 389.0987 [18]

S-23 Arylpropionamide C18 H13ClF4N2 O3 416.0551 [165]

S-24 Arylpropionamide C18 H14F4N2O3 382.0941 [165]

6 LGD-2226 Quinolinone C14 H9F9N2O 392.0571 [18]LGD-2941 Quinolinone C17 H16F6N2O2 394.1116 [18]

LGD-3303 Quinolinone C16 H14ClF3N2 O 342.0747 [18]

7 LG-121071 Quinolinone C15 H15F3N2O 296.1136 [166]

8 S-40503 Tetrahydroquinoline C15 H23N3 O3 293.1739 [18]

S-49288 Tetrahydroquinoline C25 H26N4 O 398.2107 [20]

9 S-101479 Tetrahydroquinoline C26 H24F2N4O3 478.1816 [16]

JNJ-28330835 Phenyl-pyrazol-carboxyamide C14 H10F6N4O 364.0759 [18]

10 BMS-564929 Hydantoin C14 H12ClN3O3 305.0567 [18]

JNJ-37654032 Benzoimidazole C11 H7Cl2 F3N2O 309.9888 [18]

11 RAD140 Phenyl-oxadiazole C20H16ClN5O2 393.0993 [18]

12 ACP-105 Tropanol C16 H19ClN2O 290.1186 [18]

AC-262536 Tropanol C18 H18N2 O 278.1419 [18]

13 LGD-4033 Pyrrolidinyl-benzonitrilea C14 H12F6N2O 338.0854

RAD35010 Indole C13 H11ClF3NO 289.0481 [18]

Ly2452473 Indole C23 H23N3 O2 373.1790 [16]

FTBU-1 Benzoimidazole C19 H16FN5OS 381.1060 [18]

2-FPA Pyridinylmethanamide C17 H19FN2O 286.1481 [18]

GLPG0634 Diarylimidazolidinedione C19 H14F3N3O3 389.0987 [16]

MK-3984 Phenylmethanamide C17 H12F7NO2 395.0756 [16]

NEP28 Thiophene C10H10BrF3N2S 325.9700 [21]

MK-0773 Steroidal C27 H34FN5O2 479.2697 [18]

Cl-4-AS-1 Steroidal C26 H33ClN2O2 440.2231 [18]

TFM-4AS-1 Steroidal C27 H33F3N2O2 474.2494 [18]

YK11 Steroidal C25 H34O6 430.2355 [18]

S-42 Steroidal C21 H28O 296.2140 [18]

a Unconfirmed.

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 uri-

nary target compound for doping control purposes as only modest

metabolic reactions to a monohydroxylated analog were observedand RAD140 was detected in post-administration study urine sam-

ples 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-trifluoro-1-hydroxyethyl)pyrrolidin-

1-yl)-2-(trifluoromethyl)-benzonitrile (Fig. 2, 13) to it. Despite the

absence of confirmation, the drug entity is part of LIGAND PHAR-

MACEUTICAL’s patents on compounds with SARM-like properties

[38] and thus also a candidate for doping controls. Hence, detec-tion 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. Production mass spectrum of thedeprotonated molecule [M−H]−

at m/z 337 of LGD-4033, recorded at a collision energy of 15eV.


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2.1.3. Sirtuin-1 activators

Sirtuins (1–7) 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

suchas forkhead box proteins (FOXO) and peroxisomeproliferator-

activated receptor coactivator 1 (PGC-1) [39]. Since thediscovery of resveratrol’s SIRT1-activating effect with significant

impact on skeletal muscle mitochondrial content, extended life

span and anti-aging as well as antidiabetogenic properties in ani-

mal 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) respon-

sible for the observed beneficial effects [42–46], 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 first-

generation STACs and additional model compounds in blood and

urine. Moreover, invitroand invivobiotransformation studies were

conducted to provide information on viable targets for extended

detection windows in routine sports drug testing.Commercially available and synthesized STAC reference sub-

stances 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 100l of specimenwas 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 LC–ESI-MS/MS in multiple reaction monitoring (MRM)

mode, allowing for detection limits between 0.1 and 1ng/ml.

Considering the reported plasma concentrations of STACsin clinical

trials reaching up to 390ng/ml, the accomplished LODs demon-

strate the fitness-for-purpose of the presented approach. In order

to complement the approach with analyses conducted from urine,

target analytes were generated using in vitro biotransformationsystems [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 lim-

its for the target analytes in urinary matrix of 0.5 ng/ml. It remains

to be clarified 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 factorof mitochondrial biogenesis andfunc-tion [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 mitochon-

drial 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 scle-

rosis, 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 cell’s‘powerhouse’, whichis affected by stressors such as caloric restric-

tion and exercise [52]. The activation of PGC-1 can be triggeredthroughgene expressionas wellas post-translationalmodifications

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 SIRT1via deacetylation and

capable of inducinggene expressionrequiredfor mitochondriogen-

esis. This interconnection, the relevance of AMPK andits activation

through potent agonists suchas 5-amino-4-imidazolecarboxamide

ribonucleoside (AICAR, Fig. 4, 16) [53] have been the rationale to

include this compound and related substances to WADA’s Pro-

hibited List in 2009, first 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 500mg/kg/day was shown to effectively activate the

AMPK signaling pathway resulting in an improved endurance of

untrained mice by 23–44% [54]. This supportedthe necessityof the

compound’s 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 hassupposedly found its way

into the world of sport meanwhile [55,56].

Due to the natural occurrence of AICAR in human urine, pro-

viding 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/isotope-

dilution tandem mass spectrometry (LC-IDMS/MS), naturally

occurring urinary concentrations of AICAR werequantified in a pop-

ulation of 499 athletes, demonstrating a significant 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 sam-

ples collected fromrecreationalathletes,a tentative thresholdlevel

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, par-

ticularly due to substantial intra- and inter-individual variations;

such information will nevertheless enable and trigger further anal-

yses supporting or disproving a possible AICAR administration and

eventually revealing the source of the compound (endogenous or

exogenous).

An alternative matrix presumably conserving administeredAICAR 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

efflux 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 (10–500ng/ml) of the analyte [58].

By means of in vitro incubation reactions, the administration of

pharmacological doses of AICAR were simulated, demonstratingan increase of intra-erythrocytic AICAR-ribotide concentrations of

1–10g/ml providing proof-of-concept for this approach. Moredata for reference ranges and authentic administration study

samples will be required to assess the significance of these num-

bers; however, the intra-individual profile of AICAR concentrations

might be a sensible contribution to the Athlete Biological Passport

(ABP) to allow flagging an unusual blood parameter finding.

Conclusive test results concerning the endogenous or exoge-

nousorigin of a naturallyoccurring substance in an athlete’sdoping

control sample is commonly obtained by isotope-ratio mass spec-

trometry (IRMS) [59]. Thistechnical approachhas beensuccessfully

employed in steroid analyses for over a decade in doping con-

trols, 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), Roflumilast (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 introduc-

ing the substance into the gas chromatography-combustion-IRMS

(GC/C/IRMS) system, andthe isolationof 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 3ml of urine and currently

urine samples with AICAR levels higher than 1500ng/ml are rec-

ommended 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 sig-

nature; 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 revis-

ited. Phosphodiesterases comprise a family of 11 currently known

members with specific characteristics and stimulating or inhibi-

ting agents. PDE4, a major target in chronic obstructive pulmonary

disease (COPD) treatment [61–63], catalyzes the conversion of


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3-5-cyclic adenosine monophosphate (cAMP) to 5-AMP. Its inhi-

bition was shown to result in a cascade of consequences allowing

to explain the beneficial effects of the unspecific PDE inhibitor

resveratrol in animal test models [50,64] including, amongst oth-

ers, increased mitochondrial biogenesis and function associated

with improved fatutilizationand,last butnot least,enhanced exer-

cise 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 identified. 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 therapeu-

tic agents with PDE4-inhibiting properties [65]; however, to date

only one (roflumilast, 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, roflumilast, and cilomilast was noticed and first analyt-ical 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 roflumilast and

cilomilast eachyieldedmainlyone reasonably abundant metabolite

invitro (roflumilast-N -oxide andhydroxylatedcilomilast), rolipram

was converted into six intense metabolic products resulting from

hydroxylation and dealkylation reactions. Employing an estab-

lished sample preparation protocol for urine specimens consisting

of enzymatichydrolysisfollowed by LLEand subsequentLC–MS/MS

analysis, LODs of 1–5 ng/ml for the compounds of interest were

accomplished, and proof-of-concept data were achieved with a

patient’s urine sample collected after therapeutic use of roflumi-

last. Due to the substantial increase in performance as observedwith rodents on a treadmill test (time-to-exhaustion) and the evi-

dently elevated number of mitochondria in skeletal muscle of the

test animals resulting from PDE4-inhibitor administrations, mis-

use 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 arguablyperformance-enhancing)effects

of human growth hormone (hGH), primarily mediated via insulin-

like 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 activegrowth hormone secretagogues (GHS) based on various different

pharmacophores including e.g. benzolactame, 4-spiropiperidine,

capromorelin, and oxindole derivatives such as L-739943, MK-

0677, CP-424391, andSM-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 hor-

mone (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 dis-

orders and age-related decline. Sustainedincreases in plasma IGF-1

were reported in GHSadministrationstudies; however, none of the

leaddrug candidates hasyet receivedclinical approval,possiblydue

to limited benefits 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 methodolo-

gies such as mass spectrometry (Fig. 5a). In addition to these orally

available GHS, a considerable variety of growth hormone releas-

ing 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 [73–75]. One representa-

tive (Pralmorelin, GHRP-2) received clinical approval 2004in Japan

as a diagnostic tool for GH deficiency in adults [76]. Whilst the

majority of GHRPs, a selection of which is summarized in Table 2,

does currentlynot holdapproval by anyhealth authority for human

therapeutic use, numerous black-market sources have been iden-

tified 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 (125I-ghrelin), which is displaced by (urinary)GHS in a dose-dependentmanneras demonstratedwith 7 synthetic

GHS in a proof-of-concept study. The responses or minimal positive

concentrations for GHS in urine varied from 1.5E−10M to1.0E−06

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 4.5h for this particular drug. Confirma-

tion of the presence of a prohibited substance belongingto the class

of GHS was recommended and conducted viadedicated LC–MS/MS

assays.

Such a mass spectrometry-based methodology focusing par-

ticularly on the detection of GHRP-2 and its main metabolite

(d-Ala-d(-naphthyl)-Ala-Ala-OH) in human urine was published

in2010 byOkanoet al. [81]. Here, an isotope-labeledGHRP-2 inter-nal standard was used to ensure appropriate sample preparation

andanalysisconditions before the specimenwas subjectedto solid-

phase extraction (SPE) and subsequent LC–MS/MS analysis. The

assay enabled LODs of 20–50 pg/ml and was applied to an excre-

tion study with ten volunteers who received an intravenous bolus

of 100g of GHRP-2dihydrochloride. While theintact GHRP-2wasfoundup to13 h,the aforementionedmetabolitewas detectedup to

24h 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

LC–MS 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 microborereversed-phase LC separation and full scan high resolution/high

accuracymass spectrometry,which enabled LODs of 0.2–0.5ng/ml.

The applicability of the developed method to authentic urine sam-

ples was tested with an elimination study with 10mg 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-upstudy, the

instrumental setupwas modified 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

detectionof 2–10pg/ml of eachsubstance.Since DMPKdata of most

GHRPs are not available but of great importance in terms of doping

controls, animal invivo studies and invitrosimulations 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 528of MK-0677, recorded at a collision energy of 25eV;(b) product ion mass spectrum of

the protonated molecule [M+H]+ at m/z 655 of GHRP-3, recorded at a collision energy of20 eV; (c) full scanMS 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 com-

pounds 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 confirmed by human in vitro simula-

tions andwillextend the initial testing options forGHRPs 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 arepresented in 3-letter code, larger peptides in 1-letter code.

Compound Amino acid sequence Molecular mass

monoisotopic

[Da]

GHRP-1 Ala-His-d-Nal-Ala-Trp-d-Phe-Lys-NH2

954.5

GHRP-2 d-Ala-d-Nal-Ala-Trp-d-Phe-

Lys-NH2

817.4

GHRP-3 Aib-d-Trp-d-Pro-d-Ile-Arg-

NH2

654.4

GHRP-4 d-Trp-Ala-Trp-d-Phe-NH2 607.3

GHRP-5 Tyr-d-Trp-Ala-Trp-d-Phe-NH2 770.4

GHRP-6 His-d-Trp-Ala-Trp-d-Phe-Lys-

NH2

872.4

Alexamorelin Ala-His-d-Mrp-Ala-Trp-d-Phe-

Lys-NH2

957.5

Hexarelin His-d-Mrp-Ala-Trp-d-Phe-Lys-

NH2

886.5

Ipamorelin Aib-His-d-2-Nal-d-Phe-Lys-

NH2

711.4

GHRH YADAIFTNSY RKVLGQLSAR

KLLQDIMSRQ QGESNQERGA

RARL

5037.6

Sermorelin YADAIFTNSY RKVLGQLSAR

KLLQDIMSR-NH2

3355.8

CJC-1288 YADAIFTNSY RKVLGQLSAR

KLLQDIMSR K*-NH2

3634.9

CJC-1293 Y dA DAIFTNSY RKVLGQLSAR

KLLQDIMSR K*-NH2

3634.9

CJC-1295 Y dA DAIFTQ SY RKV A GQLSAR

KLLQDIL SR K*-NH2

3437.9

IGF-1 GPETLCGAEL VDALQFVCGD

RGFYFNKPTG YGSSSRRAPQ

TGIVDECCFR SCDLRRLEMY

CAPLKPAKSA

7643.6

des(1-3)-IGF-1 TLCGAELVDA L QFVCGDRGF

YFNKPTGYGS SSRRAPQTGI

VDECCFRSCD LRRLEMYCAP

LKPAKSA

7360.5

R 3-IGF-1 GPR TLCGAEL VDALQFVCGD



CAPLKPAKSA

7670.6

long-R 3-IGF-1 MFPAMPLSSL FVNGPR TLCG

AELVDALQFV CGDRGFYFNK

PTGYGSSSRR APQTGIVDEC

CFRSCDLRRL EMYCAPLKPAKSA

9105.4

MGF YQPPSTNKNT KSQRRKGSTF

EERK-NH2

2865.5

‘full length’ MGF GPETLCGAEL VDALQFVCGD



CAPLKPAKSA RSVRAQRHTD

MPKTQKYQPP STNKNTKSQR

RKGSTFEEH

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 phar-

macokinetic data for GHRP-6 were presented by Cabrales et al. in

2012/2013 [85,86]. Employing an isotope-dilution mass spectro-

metric approach, GHRP-6 was determined from plasma with an

LOQ of 5 ng/ml. Samples were enriched with 13 C-labeled GHRP-6,

plasma proteinswere depleted by acetone-facilitated precipitation,

and the target analyte was quantifiedfrom the concentratedsuper-

natant by nanoLC-Q/TOF MS using a monolithic nano LC column.

Following an i.v. bolus dose of 100, 200, or 400g/kg of body-weight, plasma samples were collected up to 72h, and GHRP-6

concentrations were found to fall below the LOQ (5ng/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 modified synthetic analogs received an enormous stimulus

for illicit and mostly Internet-based sales [77,87,88]. In contrast to

GHS, GHRHs actvia receptorsat the anteriorpituitary, andclinically

approved representatives of GHRHs are tesamorelin and sermore-

lin.In addition, research concerning newpotent analogs of GHRHas

therapeutic agents has beencontinued 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 modifications at 5 positions

as well as a 3-maleimido-propionic acid for in vivo bioconjuga-

tion to albumin [90], its efficacy in rats with extended plasma

half-life was reported. Hence, complementary to GHS test meth-

ods, 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 immunoaffinity purification followed by

nanoLC–MS/MS analysis was presented in 2012 [91]. By means of

preconcentration of 5 ml of urine using SPEand subsequent extrac-

tion of the target analytes GHRH (1–29, sermorelin) and CJC-1295(withoutthe maleimidopropionic acidmoiety), the highly sensitive

and specific analysis of the compounds was accomplished allow-

ing for detection limits of 5 and 1 pg/ml, respectively. It remains to

be clarified, however, whether the intact peptides or correspond-

ing metabolites will be most efficient as target analytes in routine

doping controlswhen usingurineas primarydopingcontrolmatrix.

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. referringto IGF-1Eb (orEcin humans) mRNA, pro-IGF-1Eb, as well as the synthetic MGF pep-

tide, 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 prolif-

eration 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 regeneration-

promoting effects of MGF and a dedicated MGF receptor has yet to

be identified to support and corroborate the commonly acceptedMGF hypothesis [94].

To date, no dedicated detection assay for doping control pur-

poses has been reported but findings 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 men-

tioned questionable efficacy. 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

beenpostulated forthe potentially naturallyoccurringhumanMGF.

2.2.3. Anti-myostatin antibody MYO-029

Myostatin, also known as growth and differentiation factor-

8 (GFD-8), is a highly conserved member of the transforming

growth factor beta (TGF-ˇ) superfamily which acts as a negative


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regulator of skeletal muscle mass [96,97]. In myostatin knock-

out mice (Mstn−/−), a substantial increase in muscle mass was

observed resulting from both an elevated volume (hypertrophy)

and number (hyperplasia) of skeletal muscle fibers [97–99]. Hence,

selective blocking of the myostatin signaling pathway has been

considered as a therapeuticmeans to counteract or cure severedis-

eases such as muscular dystrophies but also other arenas such as

improving livestock production were mentioned as desirable goals

[98,100–102]. Approaches specifically targeting myostatin include

injectable myostatin-binding proteins such as the GDF-8 pro-

peptide [103,104] as well as recombinant antibodies [104–106].

In a mouse model of muscular dystrophy, the inhibition of endoge-

nous myostatin by specific antibodies considerably improved the

dystrophic phenotype of the animals as their muscle mass, mus-

cle size, muscle strength and body weight were significantly

increased [105]. Consequently, antibody-based drug candidates

for humans were developed with MYO-029, also referred to as

stamulumab, being first in class in 2005 [107–109]. Despite dis-

continuation from clinical development, a considerable misuse

potential is eminent and test methods on immunological or mass

spectrometric platforms are desirable. With the improving capa-

bility of high resolution/high accuracy mass spectrometry also the

direct analysis of the (purified) 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 recog-

nized in numerous fields of medicinal treatment, particularly

where the temporary knock-down of negative regulators is desir-

able. Here, a frequently referenced example is the inhibition of

myostatin, the prominent negative regulator of myogenesis, the

elimination of which has resulted in significant increases of mus-

cle mass and strength in animal models and, thus, suggesting

a viable means to cure myopathies such as Duchenne muscu-lar dystrophy (DMD) [111–113]. 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, modified ribonucleic acids were introduced such as

2-fluoronucleotides, 2-O-methylated nucleotides, phosphorothi-

oate nucleotides, or locked nucleic acids, effectively protecting the

siRNAfrom hydrolysis and earlyeliminationfrom 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 thera-

peutic approach bearing the risk abuse in sport have been initiated

[115,116].

A major step toward demonstrating the fitness-for-purpose of developed anti-doping methods was provided in 2013, when ani-

mal 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 specific 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 prod-

ucts), or chemical hydrolysis and LC-HRMS(/MS) determination

of synthetically mono- and oligonucleotides. Further to these, a

combination of gel electrophoresis followed by mass spectromet-

ric analysis in a bottom-up approach was demonstrated to provide

the desired information whether chemically modified RNA was

present in a urine sampleor not. Overall,screening forsiRNA by gel

electrophoresis followed by confirmatory measurements employ-

ing LC-HRMS was found to be suitable for doping control purposes

offering detection limits of 25pmol/ml of urine and detection win-

dowsof 24 h whensingle 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 anti-

doping authorities have been urged to expand their analytical

scope beyond approved erythropoiesis-stimulating agents (ESAs)

such as erythropoietin (EPO). These have been successfully cov-

ered in the past by continuously improved and updated isoelectric

focusing (IEF) and sodium dodecylsulfate polyacrylamide gel elec-

trophoresis (SDS-PAGE) methods [118–121]; 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 ery-

thropoiesis 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 hydrox-

ylation 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 advan-

tage 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 substancesis 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 com-

pounds such as FG-4592 (Fig. 7a), in vitrometabolism elucidations,

and the development of comprehensive analytical approaches

[127–131]. Since most of the potential HIF stabilizers comprise

structures that suggest physico-chemical properties suitable for

chromatographic and mass spectrometric analyses, LC–MS/MS has

been the method of choice in most of the published analyti-

cal approaches. By means of targeted MRM-based measurements,detection limits for FG-2216 and related (model) compounds

between 1 and 10ng/ml were accomplished [130], which is suffi-

cientlysensitive consideringthe expectedtherapeuticamountsand

resulting urinary concentrations of the drugs’ metabolites. More-

over, in order to account for the yet largely unknown structures of

drug candidates and respective metabolic products, a mass spec-

trometric peculiarity of isoquinoline-derived HIF stabilizers was

exploited to allow for a specific initial testing approach. In sev-

eral studies, the combined elimination of methylenamine (29Da)

or carbon monoxide(28 Da)accompaniedby theaddition ofa water

molecule (18 Da) was observed, resulting in a nominal loss of 11 or

10Da, respectively. Hence, a neutral loss scan for this rather spe-

cific mass difference can support and broaden the screening for

current and future drug candidates with similar structural features


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Table 3

Structure characteristics of selected HIF stabilizers.

No. (Fig. 6) HIF stabilizer/company Pharmacophore Elemental composition Molecular mass (Da)

24 FG-2216 Isoquinoline-glycineamide C12 H9ClN2O4 280.0251

25 FG-4592 Isoquinoline-glycineamide C19 H16 N2O5 352.1059

26 Fibrogen Thienopyridine-glycineamide C10 H7ClN2O4S 285.9815

Fibrogen Thiazolopyridine-glycineamide C9H6ClN3O4 S 286.9738

Fibrogen isothiazolopyridine-glycineamide C24 H18 N4O4S 458.1049

27 GSK360A Quinoline-glycineamide C17 H17 FN2O5 348.1121

28 GSK Tetrahydropyrimidine-glycineamide C19 H27 N3O6 393.190029 GSK Dihydropyrimidine-glycineamide C21 H19 N3O5 393.1325

GSK Dihydropyrazolopyrimidine-glycineamide C16 H14 N4O5 342.0964

GSK Pyridopyrimidine-glycineamide C11 H8BrN3O5 340.9647

30 Amgen Naphthyridine-glycineamide C12 H11 N3O5 277.0699

31 Amgen Dihydropyridopyrimidine-glycineamide C17 H13 FN4O5 372.0870

32 Bayer Pyrazol-picolinonitrile C15 H11 N5O 277.0964

33 Bayer Pyrazol-nicotinic acid C14 H9BrN4O3 359.9858

34 Janssen Benzoimidazol C13 H11 ClN4O2S 322.0291

35 Merck Naphthyridine-glycineamide C18 H13 F3N4 O5 422.0838

Merck Tetrahydropyrrolo-glycineamide C22 H17 F3N4 O6S 522.0821

although this mass spectrometric scan mode is not particularly

sensitive.

3.2. Cobaltous chloride

An arguablyoutdated andold-fashionedoptionto stimulateery-

thropoiesis is the administration of cobaltous (II) chloride [132].

Having been used since the early 1950s, it later served as refer-

ence to assess the activity of one international unit of EPO [133].

Technically,cobaltous chloridecan be categorized among the group

of HIF stabilizers as its erythropoetic effect is, at least partially,

attributed to an HIF stabilizationviadisplacementof iron inthe cat-

alytically 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 abusein sport for the very same reasons cannot be excluded [134]. In the

contrary, particularly in animal sport, the abuse of cobaltous chlo-

ride has been raised very recently [135] and analytical approaches

such as LC–MS/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 ele-

ment in doping control specimens [137–139]. 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 1400g/day havebeen reported to be acceptably safe for humans [140–142].

3.3. Erythropoietin fusion protein EPO-Fc

Despite the fact that EPO has been clinically approved, exten-

sively used in therapeutic settings, and further developed for 25

years, alternatives with either improved pharmacokinetics, pro-

files of undesirable effects, and/or easier routes of administration

have been explored in the past [123,143–146]. Among these, the

fusion protein composed of EPO and the fragment crystallizable

(Fc) part of immunoglobulin G (IgG), commonly referred to as EPO-

Fc,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 wasenabled as supportedby thepresence of airway-

epithelial 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 first and compre-

hensive study concerning the traceability of EPO-Fc in human

serum was done in 2012 [148]. Both screening and confirmatoryapproaches were suggested allowing the detection of EPO-Fc at

concentrations as low as 5pg/ml of serum employing ELISA and

gel electrophoretic/immunological approaches. The ELISA-based

methodology exploited the fact that the Fc-part of the drug can-

didate 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 confirmatory analyses if a measurable signal indi-

cating the presence of EPO is obtained [148]. The confirmation

of an EPO-Fc finding is ideally included in routine doping control

protocols such as IEF or SDS-PAGE, both followed by Western blot-

ting. The IEF properties of EPO-Fc under routine doping control

conditions were found to be unfavorable; however, the apparent

molecular mass of 60kDa of EPO-Fc as determined by SDS-PAGEyielded 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 first

medicinal reports were published in 1996 on the capability of

EMPs to bind to and stimulate the EPO receptor [149]. A first-in-

class approved EMP-based drug referred to as peginesatide was

launched in 2012, comprising a homodimeric EMP structure linked

t o a 2×20 kDa polyethylene glycol (PEG) support (Fig.8). However,

in early 2013, the manufacturer voluntarily recalled the therapeu-

ticagent as safetyendpoint data of cardiovascularevents anddeathwere worse for peginesatide than for the comparator EPO prod-

uct, and until now it has not been re-introduced to the market.

Nevertheless, the availability of this ESA and, thus, its abuse can-

not be excluded requiring appropriate test methods as established

between 2011 and 2012 [150–153].

In order to allow for comprehensive doping controls, viable

matrices including serum/plasma, urine, and dried blood spots

(DBS)were evaluated, demonstratingthat the drug can be detected

if therapeutic amounts have been administered. When using MS-

basedmethodologies, precipitationof highabundant proteins from

serum (or plasma) was followed by enzymatic hydrolysis of the

peptidic moiety using subtilizin, yielding a diagnostic pentapep-

tide [GPIT(1-nal)] for LC–MS/MS analysis [151]. The assay allowed

for detection limits of 1n g/ml, which was found appropriate


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Fig. 6. Structures of selectedHIF stabilizers including FG-2216 (24),FG-4592 (25), and GSK360A (27). Further details are given in Table 3.

consideringtherapeuticplasmaconcentrations of up to 1000ng/ml.

Due to the considerably high blood concentrations, DBS analysis

was pursued offering a faster and cheaper sample collection strat-

egy [152]. Here, essentially the same sample preparation strategy

was applied including extraction of the DBS, subtilizin digestion,

and subsequent LC–MS/MS measurement. However, as a result of

the limited sample volume (approximately 20l of DBS), detec-tion limits were determined at 10ng/ml, which nevertheless will

serve the purpose of sports drug testing. As urine is the most fre-

quently collected doping control specimen, animal in vivo study

urine samples were used toprobefor 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 optimizedfor 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 0.5ng/ml [153]. The ELISA consisted

of a sandwich-like test method employing an immobilized mono-

clonal anti-PEG antibody and a monoclonal biotinylated antibody

directed against the peptidic moiety of peginesatide. Using serum

samples from an administration study with 50g/kg bodyweight,the drug was detectable up to 10 days post-administration using

the ELISA-based initial test method. Confirmatory analyses without

mass spectrometry were then suggested by means of gel elec-

trophoretic determination of immunoprecipitated peginesatide.

Here, a different monoclonal antibody targeting a different epitope


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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 30eV; (b) product ion mass spectrum of

M+ at m/z 355 of thecobalt-diethyldithiocarbamate (DDC) complex as analyzed with ESI-MS/MSat a collision energy of 40eV.

of thepeptide residue wasused to capture and extract theESA from

500l of serum. Subsequently, conventional SDS-PAGE with dou-ble Western blotting was conducted, enabling the visualization of

0.5ng/ml of the target analyte.

4. Other compounds

As the questfor faster recovery,better athleticperformance,and

optimized bodycomposition continues, new substances advertised

with such properties have frequently been observed with Internet-

based suppliers as well as in products confiscated at customs.

4.1. TB-500

An arguablynaturaland chemical-freesubstance for horse treat-

ment 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 confiscated

material was characterized by mass spectrometry [154], chemi-

cal synthesis and NMR [155]. Advertised as a synthetic analog to

thymosin beta 4 (T4), it was identified as an N -terminally acety-lated heptapeptide, representing the amino acid sequence 17–23

(the major actin-binding site) of T4. This sequence was shown topossess (corneal) tissue-repair supportive and angiogenic proper-

ties in animal models [156,157], hence a potentialbenefit to human

and/or animal athletes was attributed. Due to the non-natural com-

position of the product, its traceability in doping control sampleswas demonstrated for plasma and urine specimens [155]. Samples

were subjected to SPE (in case of plasma, high abundant proteins

were first precipitated), and extracts were analyzed by LC–MS/MS,

allowing for detection limits of 500pg/ml. Even though no human

clinical study data have been published for TB-500, extrapolation

Fig. 8. Structure cartoon of peginesatide.


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Fig. 9. (a)Product ionmass spectrum of theprotonated molecule [M+H]+ at m/z 889 of TB-500, recorded at a collisionenergy of 45eV; (b)product ionmass spectrum of the

doubly charged precursor ion [M+2H]2+ at m/z 908 of AOD-9604, measured at a collisionenergy of 30eV.

fromrecommended dosing (10mg) 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 1815D a.

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]. Since2013, AOD-9604 has been considered as prohibited according

to WADA’s anti-doping regulations [162]; however, similarly to

other non-approved substances, its metabolism has not been fully

investigated or publicized. A first 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 iso-

form test is specific and not affected by AOD-9604 [163]. Due to

the peptidic nature of AOD-9604, its implementation into rou-

tine doping controls has been accomplished according analytical

strategies employed e.g . for GHRPs (vide supra); it remains how-

ever to be shown whether urine, serum, and/or DBS are the matrix

of choice for this substance, if the intact drug or metabolic prod-

ucts are viable target analytes, and if the presumably limited

performance-affecting properties of AOD-9604 will be of suffi-

cient temptation to athletes to conduct anti-doping rule violations

[164]. Clearly, trafficking of AOD-9604 via illicit and black-market

providershas been monitoredin the past [154], requiring attention

of doping and customs controls.

5. Conclusion

Preventive and proactive anti-doping work, particularly con-

cerning analytical strategies, is of paramount importance consider-

ingthe enormous breadth of newemergingas 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 specific

analytical approaches, the development of which requires state-of-

the-art instrumentation as well as insights into pharmacology and

metabolicpathways of these compounds, which spanfrom low(est)

molecular mass analytes such as cobalt to intact antibodies consti-

tuting organic molecules of more than 150kDa. 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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