Studies on the collision-induced dissociation of adipoR agonists after electrospray ionization and their implementation in sports drug testing

Article abstract of DOI:10.1002/jms.3545

AdipoR agonists are small, orally active molecules capable of mimicking the protein adiponectin, which represents an adipokine with antidiabetic and antiatherogenic effects. Two adiponectin receptors were reported in the literature referred to as adipoR1 and adipoR2. Activation of these receptors stimulates mitochondrial biogenesis and results in an improved oxidative metabolism (via adipoR1) and increased insulin sensitivity (via adipoR2). Hence, adipoR agonists are potentially performance enhancing substances and targets of proactive and preventive anti-doping measures. In this study, two adipoR agonists termed AdipoRon and 112254 as well as two isotopically labeled internal standards (ISTDs) were synthesized in three-step reactions. The products were fully characterized by nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS) and density functional theory (DFT) computation. Collision-induced dissociation pathways following electrospray ionization were suggested based on the determined elemental compositions of product ions, comparison to product ions derived from labeled analogs (ISTDs), H/D-exchange experiments and the results of DFT calculations. The most abundant product ions were found at m/z 174, tentatively assigned to protonated 1-benzyl-1,2,3,4-tetrahydropyridine for AdipoRon, and m/z 207, suggested as protonated 1-(4-methoxybenzyl)piperazine, for 112254. Notably, the loss of the heterocyclic ring (i.e. piperazine and piperidine, respectively) in a supposedly intramolecular elimination reaction was observed in both cases. A qualitative determination of both AdipoR agonists in human plasma was established and fully validated for doping control purposes. Validation items such as recovery (86-89%), specificity, linearity, lower limit of detection (1ng/ml), intraday (3-18%) and interday (5-16%) precision as well as ion suppression or enhancement were determined. Based on these findings adipoR agonists can be implemented in sports drug testing procedures.

Full text of DOI:10.1002/jms.3545

Research article
Received: 2 September 2014
Revised: 15 October 2014
Accepted: 12 November 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3545
Studies on the collision-induced dissociation of
adipoR agonists after electrospray ionization
and their implementation in sports drug testing
Josef Dib,a Nils Schlörer,b Wilhelm Schänzera and Mario Thevisa,c*
AdipoR agonists are small, orally active molecules capable of mimicking the protein adiponectin, which represents an adipokine
with antidiabetic and antiatherogenic effects. Two adiponectin receptors were reported in the literature referred to as adipoR1
and adipoR2. Activation of these receptors stimulates mitochondrial biogenesis and results in an improved oxidative metabolism
(via adipoR1) and increased insulin sensitivity (via adipoR2). Hence, adipoR agonists are potentially performance enhancing sub-
stances and targets of proactive and preventive anti-doping measures. In this study, two adipoR agonists termed AdipoRon and
112254 as well as two isotopically labeled internal standards (ISTDs) were synthesized in three-step reactions. The products were
fully characterized by nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS) and density functional theory
(DFT) computation. Collision-induced dissociation pathways following electrospray ionization were suggested based on the deter-
mined elemental compositions of product ions, comparison to product ions derived from labeled analogs (ISTDs), H/D-exchange
experiments and the results of DFT calculations. The most abundant product ions were found at m/z 174, tentatively assigned to
protonated 1-benzyl-1,2,3,4-tetrahydropyridine for AdipoRon, and m/z 207, suggested as protonated 1-(4-methoxybenzyl)piper-
azine, for 112254. Notably, the loss of the heterocyclic ring (i.e. piperazine and piperidine, respectively) in a supposedly intramo-
lecular elimination reaction was observed in both cases. A qualitative determination of both AdipoR agonists in human plasma
was established and fully validated for doping control purposes. Validation items such as recovery (86–89%), specificity, linearity,
lower limit of detection (1 ng/ml), intraday (3–18%) and interday (5–16%) precision as well as ion suppression or enhancement
were determined. Based on these findings adipoR agonists can be implemented in sports drug testing procedures. Copyright ©
2015 John Wiley & Sons, Ltd.
Keywords: doping; sport; mass spectrometry; HRMS; mitochondrial biosynthesis
exercise-mimetic for muscle cells,[13] indicating adiponectin’s po-
tential as a doping agent. This conclusion is supported by two pat-
Introduction
Adiponectin is a protein secreted exclusively in adipocytes. The
structure, containing a collagen-like domain and a globular domain,
is similar to the complement factor C1q.[1–4] Plasma concentrations
of monomeric adiponectin were reported to range from 1.9 to
17 μg/ml.[5] YAMAUCHI et al. demonstrated binding affinities of
adiponectin to two receptors referred to as adipoR1 and adipoR2.
AdipoR2 is abundantly expressed in liver cells. AdipoR1, however,
while ubiquitously expressed, is found mainly in skeletal muscle
cells.[6]
Adiponectin has antidiabetic and antiatherogenic effects. In
mice, the plasma level of adiponectin is decreased in obesity lead-
ing to insulin resistance and type 2 diabetes,[7–9] which is amelio-
rated by the replenishment of adiponectin. The substrate acts via
two major pathways. One relies on the stimulation of the peroxi-
some proliferator-activated receptor alpha (PPARα) in liver and skel-
etal muscle (through adipoR2) which leads to an increased insulin
sensitivity.[8] The second pathway operates via adipoR1 and the ac-
tivation of 5′ adenosine monophosphate-activated protein kinase
(AMPK).[10–12] Adiponectin increases the concentration of AMP in
the muscle, which leads to the increased activity of AMPK.[10] An-
other effect of adiponectin binding to adipoR1 is the influx of extra-
cellular Ca2+ into the cell. These effects lead to an increased
mitochondrial biogenesis and improved oxidative metabolism in
skeletal muscle. In sum, adipoR1 activated by adiponectin is an
ents published in 2013, which aim at finding adipoR-agonists for
adipoR1 and the pseudo-exercise effect these agonists might
have.[14,15] The screening studies made by this group were focused
on the ability of a molecule to activate AMPK. The most effective hit
in this study is named AdipoRon (1). A second, less active hit was
termed 112254 (2). AdipoRon (1) and 112254 (2) are small, orally
active molecules, which act as adiponectin mimetics. Due to its
substantially higher activity, most subsequent studies were re-
stricted to AdipoRon (1). Treatment of C2C12 myotubes with con-
centrations of 5–50μM increases the phosphorylation of AMPK,
the expression of PGC-1α and the mitochondrial DNA content in a
dose dependent manner. In mice, oral administrated AdipoRon
*
Correspondence to: Mario Thevis, Institute of Biochemistry—Center for
Preventive Doping Research, German Sport University Cologne, Am Sportpark
Müngersdorf 6, 50933 Cologne, Germany. E-mail: thevis@dshs-koeln.de
a Center for Preventive Doping Research—Institute of Biochemistry, German Sport
University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany
b Department of Chemistry, Institute of Organic Chemistry, University of Cologne,
Greinstr. 4, 50939, Cologne, Germany
c
European Monitoring Center for Emerging Doping Agents, Cologne/Bonn,
Germany
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J. Dib et al.
(50 mg/kgÀ1 body weight) leads to a maximal plasma concentra-
tion of 11.8 μM (5.1 mg/mlÀ1). It activates both the adipoR1-
AMPK-PGC-1α- and adipoR2-PPAR-α-pathway,[16,17] similar to other
drugs and drug candidates banned in sports by the Prohibited List
of the World Anti-Doping Agency (WADA) as for instance AICAR.[18]
Hence, AdipoRon (1), 112254 (2) and two isotopically labeled an-
alogs (3 and 4, respectively, Fig. 1) used as internal standards (ISTD)
were synthesized and characterized by nuclear magnetic reso-
nance spectroscopy (NMR) and high-resolution/high-accuracy tan-
dem mass spectrometry (MS/MS). Furthermore, a fully validated
test method, including specificity, lower limit of detection (LLOD),
identification capability, robustness, carryover, matrix interferences,
recovery, interday and intraday precision and linearity was
established for human plasma employing liquid chromatography-
tandem mass spectrometry (LC/MS/MS).
Deionized water used for synthesis and sample preparation was
of MilliQ quality. For validation (except specificity), commercially
available plasma obtained from Octapharma GmbH (Langenfeld,
Germany) was used. For specificity, plasma samples were obtained
from healthy volunteers without known medications.
Synthesis and characterization
Synthesis of AdipoRon and 13C2-labeled AdipoRon
AdipoRon was synthesized in a three-step reaction (Scheme 1). First a
nucleophilic substitution of 4-hydroxybenzophenone (a) and
methyl bromoacetate (b) was conducted to yield methyl 2-(4-
benzoylphenoxy)-acetate (c). Compound c was then saponified to
obtain 2-(4-benzoylphenoxy)-acetic acid (d). Finally, d and 1-
benzylpiperidin-4-amine (e) were converted to AdipoRon (1) by am-
ide formation with 1-hydroxybenzotriazole hydrate (HOBt) and N-(3-
dimethylaminopropyl)-N′-ethylcarbo-diimide hydrochloride (EDCl) as
coupling reagents and triethylamine (TEA) as base. The synthesis of
the ISTD 3 was performed analogously, but 13C2-labeled ethyl
bromoacetate was used instead of methyl bromoacetate (b).
Experimental
Chemicals, reagents and plasma samples
All chemicals were commercially available and were used without
further purification. 1-Hydroxybenzotriazole hydrate (99%), sodium
methanolate (0.5 N in methanol), N-(3-dimethylaminopropyl)-N′-
ethylcarbo-diimide hydrochloride (99%), 4-amino-1-benzylpi-
peridine (98%), β-alanine methyl ester hydrochloride (98%),
4-hydroxybenzophenone (98%), 1-(4-methoxybenzyl)piperazine
(97%), piperazine-2,2,3,3,5,5,6,6-d8 dihydrochloride (98 atom% D,
98%), 1-(chloromethyl)-4-methoxybenzene (98%) and ethyl
bromoacetate-2-13C (99 atom% 13C, 98%), methyl bromoacetate
(97%) and triethylamine (99%) were purchased from Sigma-Aldrich
(Taufkirchen, Germany). Sodium hydroxide (99%) and 4-tert-
butylbenzoic acid (99%) were obtained from Merck (Darmstadt,
Germany). All solvents used (methanol, dimethylacetamide, N,N-
dimethylformamide, dichloromethane and tetrahydrofurane) were
of analytical grade and were used without further purification.
Synthesis of 112254 and D8-labeled 112254
The synthesis of 112254 was also a three-stage synthesis (Scheme 1).
At first, an amide formation between 4-tert-butylbenzoic acid (f) and
β-alanine methyl ester hydrochloride (g) with HOBt and EDCl as
coupling reagents and TEA as a base gave methyl 3-(4-(tert-butyl)-
benzamido)-propanoate (h). This was saponified to obtain 3-(4-(tert-
butyl)-benzamido)-propanoic acid (i) followed by the formation of
an amide between iand the piperazine-derivate j yielding the desired
product 112254 (2). The synthesis of ISTD 4 was conducted
accordingly but using a labeled piperazine-derivative prepared via
nucleophilic substitution reaction from piperazine-2,2,3,3,5,5,6,6-d8
dihydrochloride and 1-(chloromethyl)-4-methoxybenzene.
Characterization
The structure of compound 1 and 2 was confirmed by NMR 1H-, H,H-
COSY-, H,C-HMQC- and 13C-ATP experiments. Spectra were
recorded employing a Bruker Avance III 500 instrument (Bruker,
Karlsruhe, Germany) equipped with a 5-mm inverse probe head
(z-gradient coil). The spectra were recorded at room temperature
from solutions at concentration levels of approximately 10 mg/ml.
The elemental composition and dissociation patterns of the sub-
stances weredetermined usinghigh-resolution/high-accuracy mass
spectrometry using a LTQ Orbitrap (Thermo, Bremen, Germany). De-
tailed information and further parameters are described below.
Stock and working solutions
All stock solutions were prepared with a concentration of 1 mg/ml
in acetonitrile and stored at À8 °C. Over a period of four weeks no
degradation was observed. Working solutions for validation pur-
poses were prepared freshly at the required concentrations by dilu-
tion of stock solutions.
Density functional theory calculations
DFT calculations were carried out to determine the most probable
position of protonation of compounds 1 and 2. Preoptimization
was conducted for all compounds by MM2 force field cal-
culations.[19] Then, all computations were performed with ORCA
freeware (version 2.9.1)[20] at the PBE[21]/def2-TVZP[22] level of the-
ory. All structures were fully optimized, and proton affinities were
Figure 1. Chemical structures of AdipoRon (1, M = 428 g/molÀ1), 112254 (2,
M = 437 g/molÀ1),13C2-labeled AdipoRon (3, M = 430 g/molÀ1) and D8-labeled
112254 (4, M = 446 g/molÀ1). Numbers in brackets indicate deuterated
AdipoRon (5, M = 429 g/molÀ1
) ) as
and 112254 (6, M = 438 g molÀ1
obtained by H/D-exchange experiments.
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Analysis of adipoR agonists in doping controls
Scheme 1. Route of synthesis of AdipoRon (1) and 112254 (2).
calculated as the difference between the total energy of the proton-
ated species and the neutral compound. Thermal and vibrational
contributions were not considered during computation as they
were expected to play a minor role in calculating relative rather
than absolute proton affinities.
Liquid chromatography-tandem mass spectrometry
All analyses were performed using an Agilent 1290 Infinity liquid
chromatograph linked to an Agilent 6550 iFunnel QTOF LC/MS with
electrospray ionization (Waldbronn, Germany). The column used
was a Thermo Scientific C8 Hypersil Gold (100 × 2.1 mm, 3 μm parti-
cle size). The eluents were water (mobile phase A) and acetonitrile
(mobile phase B), both containing 0.1% formic acid. Gradient elu-
tion was employed and started at 2% B increasing to 100% B within
6 min, maintained for 1 min and followed by re-equilibration at 2%
B for 3 min. The flow rate was set to 250μl/min during chromatog-
raphy and 400μl/min during re-equilibration. The ion source was
operated in positive mode with a temperature of 290 °C, and the
spray voltage was 3500 V. The FWHM was 18 000 (at m/z 400). Both
compounds and the ISTD (1 to 4) were detected by means of char-
acteristic product ions formed from protonated molecules by
collision-induced dissociation (CID). Nitrogen was used as sheath
and collision gas delivered from CMC nitrogen generator (CMC In-
struments, Eschborn, Germany).
Electrospray ionization-tandem mass spectrometry and MS3-
experiments
ESI-MS(/MS) and MS3-experiments were performed on a LTQ
Orbitrap mass spectrometer. The instrument, using a HESI-I (heated
electrospray ionization-I) source, was operated in positive ionization
mode. For calibration, the manufacturer’s calibration mixture,
consisting of caffeine, the tetrapeptide MRFA and ultramark (mix-
ture of fluorinated phosphazenes), was used. Compounds were in-
troduced into the mass spectrometer via syringe pump at a flow
rate of 15 μl/min. They were dissolved in acetonitrile/water (1:1,
v/v) and at concentrations of 1 μg/ml. Ionization voltage was
5000 V, and the capillary temperature was set to 275 °C. Protonated
precursors were isolated using a width of 1.0Da in the MSn experi-
ments, and the protonated species were dissociated at normalized
collision energies between 20 and 25 (arbitraty units, Xcalibur soft-
ware version 2.0, Bremen, Germany). For high-resolution mass spec-
trometry (HRMS), a full width at half maximum (FWHM) of 30 000 (at
m/z 400) was used, and mass accuracies <5 ppm were accom-
plished for the period of analysis. Damping gas in the linear ion trap
was helium (purity grade 5.0), and gas supplied to the curved linear
ion trap (CLT) was nitrogen, obtained from a CMC nitrogen genera-
tor (CMC Instruments, Eschborn, Germany)
Sample preparation
Ten microliters of a 0.1 μg/mlÀ1 solution containing both ISTDs was
placed in a 1.5-ml polypropylene tube, and the solvent was re-
moved under reduced pressure. Then, 100 μl of plasma was added,
followed by the addition of 100 μl of water and 400μl of acetoni-
trile. The sample was gently mixed. Precipitated proteins were re-
moved by centrifugation for 5 min at 17 000 g. The supernatant
was transferred and evaporated under reduced pressure. The
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J. Dib et al.
residue was dissolved in 100-μl acetonitrile/water (2:8 v:v). The solu-
tion was transferred to a HPLC vial, and an aliquot of 10 μl was
injected into the LC/MS-system.
calculated by mean peak area ratios of plasma samples and sam-
ples with solvent only.
Recovery
The loss of target compounds during sample preparation was de-
termined by calculating the recovery. Therefore, six samples were
fortified with the target analytes at 50ng/ml before, and another
set of samples after preparation of the specimens. To both sample
batches, the ISTDs 3 and 4 were spiked at the end of the precipita-
tion protocol. Recoveries were calculated by the mean peak area ra-
tios of the compound and the corresponding ISTD.
H/D-exchange experiments
In order to provide more information for the product ion character-
ization and dissociation pathways via mass spectrometry,
hydrogen/deuterium-exchange experiments were performed for
AdipoRon (1) and 112254 (2). Ten microliters of the stock solution
was dissolved in 1 ml of D2O. After incubation at RT for 3 h, a solu-
tion of the deuterated compounds (Fig. 1, 5, 6) with the concentra-
tion of 1 ppm in acetonitrile/D2O (1:1 v:v) was prepared and
introduced into the LTQ Orbitrap mass spectrometer via the syringe
pump. The measurements were done under identical conditions as
applied to the compounds 1 and 2 without H/D-exchange.
Intraday and interday precision
Within one day (intraday) as well as on three consecutive days
(interday), six plasma samples of compound 1 and 2 of low (1ng/
ml), medium (50 ng/ml) and high (200 ng/ml) concentration
(n = 54) were prepared and analyzed. Both intraday and interday
precision were calculated for each concentration level.
Assay validation
The qualitative determination of the two AdipoR agonists 1 and 2
was validated regarding specificity, LLOD, identification capability,
robustness, carryover and matrix interferences according to the
International Standard for Laboratories (ISL) of the WADA for non-
threshold substances[23] and the WADA technical document with
identification criteria for qualitative assays.[24] Additionally, recov-
ery, interday and intraday precision and linearity were determined.
Linearity
Linearity was tested with six fortified plasma samples at the concen-
tration levels 1, 10, 50, 100, 200 and 300 ng/ml.
Results and discussion
Specificity
Characterization of adipoR agonists
Ten plasma samples (five male und five female donors) were pre-
pared as described above at 50 ng/ml to probe for interfering peaks
in the ion chromatograms for analyte 1 and 2.
The overall yields for the synthesis of compounds 1 to 4 range
from 24% to 56% (Scheme 1). Only AdipoRon (1) and 112254
(2) were characterized by NMR spectroscopy, since the labeled
compounds 3 and 4 were prepared analogously. The experimen-
tal data found for AdipoRon (1) and 112254 (2) are consistent with
the expected chemical shifts and coupling constants and confirm
the structures:
Lower limit of detection
The LLOD was defined as the ‘lowest content that can be measured
with a responsible statistical certainty’[25] at a signal-to-noise ratio
≥3. Six plasma samples were fortified with 1 ng/ml of analyte 1
and 2 providing the data to determine the LLOD.
AdipoRon (1)
1H-NMR (500 MHz, CDCl3) δ = 7.97 (s, 1H, H17), 7.81 (d, J = 8.7Hz, 2H,
H9, H13), 7.71 (d, J = 7.1Hz, 2H, H1, H3), 7.54 (t, J = 7.4Hz, 1H, H2),
7.44 (t, J = 7.6Hz, 2H, H4, H6), 7.33–7.26 (m, 2H, H27, H29), 7.24–
7.13 (m, 2H, H26, H30), 6.95 (d, J = 8.8Hz, 2H, H10, H12), 6.34 (d,
J = 8.0Hz, 1H, H28), 4.50 (s, 2H, H15), 3.93–3.84 (m, 1H, H18), 3.46
(s, 2H, H24), 2.77 (d, J = 10.8 Hz, 2H, H22), 2.11 (t, J = 11.0 Hz, 2H,
H20), 1.89 (d, J = 10.6 Hz, 2H, H23), 1.57–1.43 (m, 2H, H19) ppm;
13C-NMR (126 MHz, CDCl3) δ = 195.71 (C7), 167.00 (C16), 160.80
(C11), 138.27 (C5, C8), 133.06 (C9, C13), 132.58 (C6, C4), 132.00
(C25), 130.16 (C1, C3), 129.46 (C2, C28), 128.68 (C30), 128.65 (C26),
127.50 (C29), 127.46 (C27), 114.66 (C12, C10), 67.70 (C15), 63.36
(C24), 52.48(C20, C22), 46.68 (C18), 32.48 (C19, C23) ppm.
Identification capability
Following the WADA technical document with identification criteria
for qualitative assays, the retention time of the analyte shall not
differ more than 0.1 min from that of the same substance.[24] To
determine this, six plasma samples fortified with both analytes
(50 ng/ml) were analyzed.
Robustness
In order to determine the robustness of the established methodol-
ogy, six plasma samples fortified with all analytes at 50 ng/ml were
prepared as described. The subsequent analyses were conducted
using a Thermo Scientific C8 Accucore X2 (100 ×3 mm, 4 μm parti-
cle size).
112254 (2)
1H NMR (500 MHz, CDCl3) δ = 7.71 (d, J = 8.4 Hz, 2H, H3, H5), 7.42 (d,
J = 8.4Hz, 2H, H2, H6), 7.20 (d, J = 8.5Hz, 2H, H24, H28), 6.85 (d,
J = 8.6Hz, 2H, H25, H27), 3.79 (s, 3H, H30), 3.76–3.70 (m, 2H, H20),
3.63–3.61 (br, 2H, H16), 3.44 (br, 2H, H13), 3.43–3.40 (m, 2H, H22),
2.60 (t, J = 5.5 Hz, 2H, H14), 2.42–2.36 (m, 4H, H17, H19), 1.32 (s,
9H, H8–10) ppm; 13C NMR (126 MHz, CDCl3) δ = 170.15 (C16),
167.14 (C11), 158.90 (C4), 154.83 (C1, C23), 131.61 (C26), 130.32
(C24, C28), 126.83 (C3, C5), 125.40 (C2, C6), 113.70 (C25, C27),
62.19 (C14), 55.26 (C30), 52.79 (C19), 52.50 (C17), 45.28 (C22),
41.56 (C16), 35.46 (C20), 34.89 (C7), 32.79 (C17), 31.18 (C8–10) ppm.
Carryover
The carryover was determined by analyzing six blank samples
between samples fortified with 1, 50 and 200 ng/ml.
Matrix interference
To estimate matrix effects causing ion suppression or enhance-
ment, four plasma samples and four samples with solvent (acetoni-
trile:water, 8:2, v/v), fortified with both compounds 1 and 2, were
prepared (50ng/ml) and analyzed. The ion suppression was
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Analysis of adipoR agonists in doping controls
Scheme 2. Proposed dissociation pathway of AdipoRon (1).
Electrospray ionization-tandem mass spectrometry
Dissociation of the protonated molecule of 1 under given CID
conditions yields the most abundant product ion at m/z 174
(Fig. 2a), which is suggested to represent the protonated species
of 1-benzyl-1,2,3,4-tetrahydropyridine (Scheme 2e). Both m/z 174
and m/z 172, tentatively assigned to protonated 1-benzyl-1,2-
dihydropyridine, further dissociate to the ion at m/z 91, which
is proposed to consist of a cycloheptatrienyl cation, generated
by the elimination of the respective partially hydrated pyridine
systems (À83 u and À81u) as corroborated by MS3 experiments
(Scheme 2e).
The product ion at m/z 307 nominally results from the loss of
benzoic acid (122 u) from the protonated molecule of 1. Its genera-
tion remains unclear; however, 13C2-labeling (compound 3, Fig. 2c)
and H/D-exchange experiments (compound 5, Fig. 2e) indicate the
presence of both 13C-labels (m/z 309) and one deuterium in the
product ion. Hence, a complex intramolecular rearrangement is re-
quired that arguably involves the benzoyl moiety (carbons 1–7) and
the amide oxygen at C-16, suggesting a product ion structure as
proposed in Scheme 2c.
The CID behavior of compounds 1 to 6 was studied using high-res-
olution/high accuracy (tandem) mass spectrometry using a hybrid
linear ion trap-orbitrap system. Product ion mass spectra of synthe-
sized compounds are illustrated in Fig. 2, and accurate masses and
calculated elemental compositions of precursor and product ions
are listed in Table 1. Proposed dissociation pathways for the proton-
ated molecules of AdipoRon (1) and 112254 (2) are depicted in
Schemes 2 and 3, substantiated by stable isotope labeling (com-
pound 3 and 4) and H/D-exchange experiments (compound 5
and 6).
AdipoRon (1)
According to DFT calculations, the highest proton affinity of
AdipoRon (1) is given at position 21 (Fig. 1), namely the nitrogen
of the piperidine-system, which is favored by 76 kJ/molÀ1 over
the oxygen at position 16. The resulting equilibrium structure is
shown in Fig. 3a.
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J. Dib et al.
Figure 2. Electrospray ionization product ion mass spectra acquired in the linear ion trap (low resolution) of adipoR agonists 1 to 4 and corresponding
spectra following H/D-exchange (5 and 6) measured on an LTQ Orbitrap (arbitrary units = a. u., collision energy = CE): a) AdipoRon (1), CE = 23 a. u.; b)
13C2-AdipoRon (3), CE = 23 a. u.; c) 112254 (2), CE = 20 a. u.; d) D8-112254 (4), CE = 20 a. u.; e) compound 5, CE = 23 a. u.; f) compound 6, CE = 23 arbitrary
units. Spectra of MS3 experiments are shown as inserts in MS/MS product ion spectra.
The loss of ammonia (17 u) from the protonated molecule of 1
leads to m/z 412 and, subsequently, the elimination of two ethine
units (in sum 54 u) yields the product ion at m/z 358 (Scheme 2a).
The proposed route is supported by analyses of ISTD 3 and 1 fol-
lowing H/D-exchange (Fig. 2c/e), where both 13C-labels were
retained, and both introduced deuterium atoms were removed
from the product ion during formation.
The second most abundant product ion (m/z 322) represents the
result of the elimination of benzylamine (Scheme 2d). Also here,
supporting evidence is provided by the labeled ISTD (3, Fig. 2c)
and compound 5 (Fig. 2e), demonstrating the presence of two
13C- and one deuterium label.
intramolecularly eliminated and the benzyl residue rearranged to
connect to the amide nitrogen (Scheme 2c). Several examples for
intramolecular elimination reactions under CID conditions are given
in the literature such as, e.g. the elimination of SO2 in thiazide-
based diuretics.[26] The difference, however, is that such elimination
reactions were observed to occur when (intermediately formed) cy-
clic systems were available. In the present case, equilibrium struc-
ture determined by DTF calculation (Fig. 3a) does not suggest any
cyclic structure; nevertheless, the release of the tetrahydropyridine
residue is undisputed and corroborated by the product ion at m/z
348 of both the labeled AdipoRon 3 (Fig. 2c) and compound 5 as
a result of H/D exchange (Fig. 2e). In order to elucidate the underly-
ing mechanism, further studies are required.
An unusual ion was formed from the protonated molecule of 1
with the product observed at m/z 346. With the elemental compo-
sition of C22H20NO3 it is suggested that the entire piperidine ring is
The product ion at m/z 105, assigned to positively charged
benzaldehyde, was shown to be generated from both m/z 346
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Analysis of adipoR agonists in doping controls
Table 1. Elemental composition of protonated molecules of 1 to 6 and product ions (>5% of relative abundance) using high-resolution/high mass ac-
curacy MSn experiments acquired on an LTQ Orbitrap
Compound
Precursor
Elemental
Error
Collision energy Product ion
Elemental
composition (exp.)
Error
[ppm]
Cleaved
species
ion (m/z) composition (exp.) [ppm]
[arb. units]
(m/z) (exp)
AdipoRon (1)
429.2164
C
27H29N2O3
À1.99
35
35
35
35
35
35
35
35
35
20
20
20
20
20
20
35
35
35
35
35
35
35
35
35
20
20
20
20
20
20
20
35
35
35
35
35
35
35
35
35
35
20
20
20
20
20
20
20
412.1895
358.1426
346.1431
322.1433
307.1797
174.1275
172.1119
105.0377
91.0544
C27H26NO3
C23H20NO3
C22H20NO3
À3.00
À3.22
À2.04
À1.52
À2.58
À1.46
À1.18
2.04
NH3
C4H6
C5H9N
C20H20NO
C7H9N
C20H23N2O
C7H6O2
C15H13NO3
C15H15NO3
C13H15NO2
C5H9N
C12H16
C12H14
C7H5O
C7H7
N
N
2.13
112254 (2)
438.2748
431.2235
C26H36N3O3
À0.75
À1.03
352.1905
316.2016
232.1329
207.1490
161.0959
121.0648
414.1969
360.1502
348.1501
324.1501
309.1861
174.1276
172.1119
105.0337
91.0546
C22H26NO3
C18H26N3O2
À0.64
À1.09
À1.32
À1.04
À0.89
À0.18
À1.18
À0.83
À1.04
À1.30
À1.27
À0.98
À0.80
2.42
C4H10N2
C8H10
O
C
14H18NO2
12H19N2O
C12H18N2O
C14H17NO2
C7H13N3O
C4H10N2
NH3
C
C11H13O
C8H9O
13C2-AdipoRon (3)
C
C2H29N2O3
C
C
C
C2H26NO3
C2H20NO3
C2H20NO3
13
25
13
25
13
21
13
20
C4H6
C5H9N
C1138C2H20NO3
C1138C2H23N2O
C7H9N
C7H6O2
13
C12H16
C12H14
C7H5O
C7H7
N
C
C
C
C2H13NO3
C2H15NO3
C2H15NO2
13
13
13
13
11
N
3.89
C133C2H9N
C4H2D8N2
C8H9DO
D8-112254 (4)
446.3247
431.2288
C26H28D8N3O3
À1.53
À2.29
352.1904
323.2455
232.1330
233.1395
215.1993
161.0960
121.0648
413.1960
358.1427
359.1484
348.1554
323.1493
308.1861
174.1270
172.1119
105.0337
91.0545
C22H26NO3
À0.82
À1.11
À0.95
À0.04
À0.67
À0.62
0.15
C18H19D7N3O2
C
C
C
14H18NO2
C12H10D8N2O
C12H11D7N2O
C14H17NO2
C7H6D7N3O
C4H2D8N2
NH2D
14H17DNO2
12H11D8N2O
C11H13O
C8H9O
D2-AdipoRon (5)
C27H27D2N2O3
C27H25DNO3
À2.34
À2.99
À4.61
À0.88
À2.20
À0.61
À1.47
À1.19
1.62
C23H20NO3
C4H5D
C23H19DNO3
C4H8N
C22H18D2NO3
C5H9N
C
20H19DNO
20H22DN2O
C7H8DN
C
C7H5DO2
C15H11D2NO3
C15H13D2NO3
C13H14DNO2
C5H9N
C12H16
C12H14
C7H5O
C7H7
N
N
3.14
D2-112254 (6)
440.2867
C26H34D2N3O3
À2.14
352.1900
318.2016
232.1329
233.1392
209.1615
161.0958
121.0648
C22H26NO3
À1.93
À1.95
À1.47
À1.28
À1.25
À1.55
À0.23
C4H8D2N2
C18H24D2N3O2
C8H10O
C14H18NO2
C12H16D2N2O
C12H17D1N2O
C14H17NO2
C7H11N3O
C14H17DNO2
C12H17D2N2O
C11H13O
C8H9O
C4H8D2N2
(À241 u) and m/z 322 (À217 u, Scheme 2c/d, Table 1) as identified
well as the carbonyl-oxygen at position 15. The equilibrium struc-
tures are shown in Figs. 3b and 3c, respectively, and despite sub-
stantially different three-dimensional features, the protonation at
position 18 (nitrogen) is only favored by 3 kJ/molÀ1. Hence, both lo-
cations are considered as similarly preferred as initial ionization
sites. Under CID conditions, the predominant product ion of the
protonated molecule of 2 is m/z 207, suggested to represent the
MS3 experiments.
112254 (2)
DFT computation of 112254 yielded highest proton affinities of the
molecule at the nitrogen of the piperazine moiety (position 18) as
J. Mass Spectrom. 2015, 50, 407–417
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J. Dib et al.
Scheme 3. Proposed dissociation pathway of 112254 (2).
protonated 1-(4-methoxybenzyl)piperazine as presumably gener-
ated by cleavage of the bond between position 15 and 21 (231 u,
Scheme 3b). This ion is confirmed both by the protonated labeled
ISTD 4 (Fig. 2d), where the eight deuterium atoms are retained,
and by the H/D exchange experiment (6, Fig. 2f), where the two
hydrogen atoms attached to the piperazine ring are substituted
by deuterium.
By analogy with AdipoRon (1), the protonated molecule of 2
forms the product ion at m/z 352, suggesting that the piperazine
ring is expelled per intramolecular elimination (À86 u, Scheme 3a).
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J. Mass Spectrom. 2015, 50, 407–417

Analysis of adipoR agonists in doping controls
and resulting structures (Fig. 3b/c) equally, which is reflected by the
identical relative abundances of the two product ions as shown in
Fig. 2d.
The product ion at m/z 316 as formed from the protonated
molecule of 2 is proposed to be generated by the elimination of
1-methoxy-4-methylbenzene (Scheme 3d). This structure is corrob-
orated by both the protonated labeled ISTD 4 (Fig. 2d), where seven
deuteriums are left at m/z 323 and by the H/D exchange experi-
ment (6, Fig. 2f), where the hydrogen attached at the piperazine
ring and the hydrogen of the amide bond are substituted by
deuterium.
Both m/z 232 (À71 u) and m/z 316 (À155 u) yield a positively
charged 4-(tert-butyl)benzaldehyde with m/z 161 in MS3 experi-
ments (Scheme 3c/d).
Assay validation
An assay for the qualitative determination of compounds 1 and 2
was established and validated for doping control purposes. Results
are summarized in Table 2, and typical extracted ion chromato-
grams and corresponding mass spectra are illustrated in Fig. 4. In
the absence of authentic administration study samples, only spiked
specimens were used to provide proof-of-concept data, and the
applicability of the developed methodology was demonstrated
by common validation and method characterization parameters
as outlined below.
Specificity
At expected retention times, no interfering signals were observed
for both target analytes (1, 2, Fig. 4a)
Figure 3. Equilibrium structures after DFT calculation (grey: carbon, white:
hydrogen, red: oxygen, blue: nitrogen, dashed line: hydrogen bond): a)
AdipoRon (1) protonated at position 21; b) 112254 (2) protonated at
position 18; c) 112254 (2) protonated at position 15.
Lower limit of detection
Both AdipoRon (1) and 112254 (2) were identified at an estimated
LLOD of 1 ng/ml (Table 2). As mentioned before, the maximal
plasma concentration of AdipoRon (1) in mice found after oral ad-
In case of 112254 (2), some details are of considerable interest and
result from the fact that all protonated molecules of 2, the labeled
ISTD 4, and compound 6 (H/D experiment) yield a product ion at
m/z 352 (Figs. 2b/d/f). It appears plausible that the entire piperazine
moiety is eliminated since no deuterium atom of compound 4 re-
mains in the charged species at m/z 352. Also, the deuterium at
the nitrogen in position 12 of 6 is replaced by hydrogen during
the dissociation process. Since this hydrogen must have its origin
in the molecule itself, the elimination route is likely to necessitate
a more complex mechanism than depicted in Scheme 3a, and a
cyclic transition state is probable, especially as the protonated mol-
ecule of 2 can be ‘folded’ due to the formation of a hydrogen bond
as illustrated in Fig. 3b. In MS3 experiments, both m/z 352 (À231 u)
and m/z 207 (À86 u) yield the methoxycycloheptatrienyl cation m/z
121 (Fig. 2b, Scheme 3a/b).
Another interesting product ion is m/z 232. It is suggested that, if
the protonation occurs at the oxygen in position 15 (Fig. 3c), a neutral
loss of the 1-(4-methoxybenzyl)piperazine accompanied by a hydro-
gen shift from position 12, 13 or 14 towards the leaving group fol-
lows. Alternatively, if the initial protonation is at position 18
(Fig. 3b), a McLafferty-like rearrangement can contribute to a charge
transfer from the piperazine residue to the carbonyl moiety, followed
by the aforementioned elimination of 1-(4-methoxybenzyl)pipera-
zine to form the ion at m/z 232 (Scheme 3c). These postulated path-
ways are supported by product ions at m/z 232 and m/z 233
obtained from the protonated molecule of the isotopically labeled
compound 4 (Fig. 2d) bearing 8 deuterium atoms in the piperazine
residue. Moreover, DFT calculations favored both protonation sites
ministration (50 mg/kgÀ1 body weight) was 11.8μM (5.1 mg/mlÀ1
)
after 2 h.[16] This is considerably more than the found LLOD. To
the best of our knowledge, no other studies have been published
about pharmacokinetic or pharmacodynamic data yet but substan-
tially lower plasma values are expected in humans. Hence, best pos-
sible sensitivity is desirable for sports drug testing purposes.
Table 2. Summary of assay validation results
Compound
LLOD
[ng/ml]
Recovery [%] at
50 ng/ml
Ion suppression
[%]
1
2
1
1
89
86
26
<10
Precision:
Intraday precision
(n = 18)
Interday precision
(n = 54)
Compound Concentration
[ng/ml]
CV [%]
CV [%]
1
1
50
14–18
3–6
16
7
200
1
6–7
6
2
16–17
4–7
12
5
50
200
4–8
6
J. Mass Spectrom. 2015, 50, 407–417
Copyright © 2015 John Wiley & Sons, Ltd.
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J. Dib et al.
Figure 4. Extracted ion chromatograms of a) blank plasma containing both ISTDs (3, 4) only; b) AdipoRon (1), precursor: [M + H] + = 429.2173, targeted product
ion: m/z 91.0542; 13C2-AdipoRon (2), precursor: [M + H] + = 431.2240, targeted product ion: m/z 91.0542; 112254 (3), precursor: [M + H] + = 438.2751, targeted
product ion: m/z 121.0648; D8-112254 (3), precursor: [M + H] + = 446.3253, targeted product ion: m/z 121.0648; all at 1 ng/ml; c) product ion mass spectra
(HRMS2) of all measured analytes.
Identification capability
Conclusion
The requirement for the identification capability, the retention time
Preventive doping research is a crucial method to incorporate new,
of the analyte shall not differ more than 0.1min, is fulfilled for both
emerging drugs into sports drug testing as soonas a potential abuse
analyte 1 and 2.
of new therapeutics can be anticipated. An impediment is the avail-
ability of the new drugs. Reference material is crucial to establish re-
liable and sensitive detection assays. Since novel substances,
Robustness
especially in early stages of development, are not readily available,
chemical synthesis and full characterization are necessary.
The method’s robustness was demonstrated by employing a differ-
ent HPLC column and packing material with slightly different di-
mensions, which did not negatively influence peak resolution or
peak form.
In the present study, adipoR agonists belonging to a new sub-
stance class based on the adipokine adiponectin were prepared
and implemented in doping control procedures as these therapeu-
tics might possess potential for misuse due to their capability to in-
crease the mitochondrial content in muscle cells. Two compounds
and two corresponding labeled analogs of this substance class were
synthesized, fully characterized by NMR, DFT computationand mass
spectrometry, and a test method was validated for human plasma.
The data obtained will be available for future in vitro/in vivo studies,
the identification of metabolites and the development of an analyt-
ical assay to detect these compounds in urine.
Carryover
At 200 ng/ml, a marginal carryover of 0.6% for AdipoRon (1) and
0.1% for 112254 (2) were determined. For lower concentrations,
no carryover was observed.
Matrix interference
Four plasma samples were prepared and analyzed as described. For
AdipoRon (1) an ion suppression of 26% was determined. 112254
(2) has shown an ion suppression of less than 10% at expected
retention time (Table 2).
Acknowledgements
The project was supported by Anti-Doping Switzerland (Berne,
Switzerland), the German Federal Ministry of the Interior and the
Manfred-Donike-Institute for Doping Analysis (Cologne, Germany).
Recovery
The recovery was determined to be 89% for AdipoRon (1) and 86%
for 112253 (3) as shown in Table 2.
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