Mass spectrometric identification and characterization of urinary metabolites of isopropylnorsynephrine for doping control purposes

Abstract

Isopropylnorsynephrine (isopropyloctopamine, deterenol, 4‐(1‐hydroxy‐2‐(isopropylamino)ethyl)phenol), a beta‐selective and direct‐acting adrenergic agonist, has been reported in the past as declared as well as non‐declared ingredient of dietary supplements. The proven biological activity and the structural similarity of isopropylnorsynephrine to substances classified as prohibited compounds according to the World Anti‐Doping Agency's (WADA's) regulations could necessitate the inclusion of this sympathomimetic amine into routine doping control analytical assays. Therefore, information on urinary metabolites is desirable in order to allow for an efficient implementation of target compounds into existing multi‐analyte testing procedures, enabling the unequivocal identification of the administration of isopropylnorsynephrine by an athlete. In a pilot study setting, urine samples were collected prior to and after the oral application of ca. 8.7 mg of isopropylnorsynephrine, which were subjected to liquid chromatography‐high resolution/high accuracy (tandem) mass spectrometry. The intact drug, hydroxylated and/or glucurono‐ or sulfo‐conjugated isopropylnorsynephrine were detected up to 48 h post‐administration, with isopropylnorsynephrine sulfate representing the most abundant urinary target analyte. No relevant amounts of the dealkylation product (octopamine) were observed, indicating that merely moderate adaptations of existing test methods (or data evaluation strategies) are required to include isporpoylnorsynephrine in antidoping analytics, if required.

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334 Analytical Science Advances
Full Article
doi.org/10.1002/ansa.202100004
Received: 14 January 2021
Revised: 18 January 2021
Accepted: 18 January 2021
Mass spectrometric identification and characterization of
urinary metabolites of isopropylnorsynephrine for doping
control purposes
Oliver Krug1,2Andreas Thomas1Mario Thevis1,2
1Center for Preventive Doping Research –
Institute of Biochemistry, German Sport
University Cologne, Cologne, Germany
2European Monitoring Center for Emerging
Doping Agents (EuMoCEDA), Cologne/Bonn,
Germany
Correspondence
Mario Thevis, PhD, Center for Preventive
Doping Research – Institute of Biochemistry,
German Sport University Cologne, Am Sport-
park Müngersdorf 6, 50933 Cologne, Germany.
Email: thevis@dshs-koeln.de
Abstract
Isopropylnorsynephrine (isopropyloctopamine, deterenol, 4-(1-hydroxy-2-
(isopropylamino)ethyl)phenol), a beta-selective and direct-acting adrenergic agonist,
has been reported in the past as declared as well as non-declared ingredient of
dietary supplements. The proven biological activity and the structural similarity of
isopropylnorsynephrine to substances classified as prohibited compounds accord-
ing to the World Anti-Doping Agency’s (WADA’s) regulations could necessitate the
inclusion of this sympathomimetic amine into routine doping control analytical assays.
Therefore, information on urinary metabolites is desirable in order to allow for an
efficient implementation of target compounds into existing multi-analyte testing
procedures, enabling the unequivocal identification of the administration of isopropyl-
norsynephrine by an athlete. In a pilot study setting, urine samples were collected prior
to and after the oral application of ca. 8.7 mg of isopropylnorsynephrine, which were
subjected to liquid chromatography-high resolution/high accuracy (tandem) mass
spectrometry. The intact drug, hydroxylated and/or glucurono- or sulfo-conjugated
isopropylnorsynephrine were detected up to 48 h post-administration, with isopropy-
lnorsynephrine sulfate representing the most abundant urinary target analyte. No
relevant amounts of the dealkylation product (octopamine) were observed, indicat-
ing that merely moderate adaptations of existing test methods (or data evaluation
strategies) are required to include isporpoylnorsynephrine in antidoping analytics, if
required.
KEYWORDS
doping, mass spectrometry, sport, stimulants
1INTRODUCTION
The selective beta-adrenergic activity of isopropylnorsynephrine
(IPNS, isopropyloctopamine, deterenol, 4-(1-hydroxy-2-
(isopropylamino)ethyl)phenol, WIN 833, Figure 1) was identified in the
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late 1940s,1and specifically the mechanism of its in vitro-observed
lipolytic effect on adipose tissue was continuously investigated
since,2,3 attributed to a substantial β3-adrenoceptor agonism of the
substance.4While in vitro and animal study data (eg, on its toxicity5)
exist, clinical data are scarce, and yet IPNS was reported as ingredient
334 wileyonlinelibrary.com/journal/ansa Anal Sci Adv. 2021;2:334–341.

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FIGURE 1 Structures of isopropylnorsynephrine (IPNS, center) and tentatively identified metabolites
of dietary supplements on several occasions,6–10 indicating its facile
availability and, thus, potential presence also in doping control sam-
ples. IPNS is currently not explicitly listed on the World Anti-Doping
Agency’s (WADA’s) Prohibited List11; however, octopamine is classi-
fied as stimulant and, consequently,information on urinary metabolites
of IPNS are desirable to enable the implementation of the substance
into anti-doping testing programs as well as to probe for the poten-
tial biotransformation of IPNS into octopamine and/or octopamine
sulfate. Here, a reporting limit of 1000 ng/mL applies for the sum of
urinary octopamine and its phase-II sulfate metabolite,12 above which
octopamine concentrations constitute an adverse analytical finding.
Hence, pilot studies were conducted with a commercially available
dietary supplement containing IPNS. Following a single oral dose of
50% of the recommended amount of the supplement, the formation
of urinary metabolites of IPNS and their elimination profiles were
investigated by chromatographic – mass spectrometric approaches,
and structural information on selected major metabolites derived from
high resolution/accurate mass measurements as well as derivatization
experiments was used to suggest tentatively assigned structures to the
observed metabolic products.
2MATERIALS AND METHODS
2.1 Chemicals and reagents
Iodomethane (purum), potassium carbonate (p.a.), IPNS (Deterenol
acetate), 3-chloroperbenzoic acid (<77%), acetone, lithium aluminium
hydride (powder, 95%), potassium carbonate (>99%), sulfur trioxide
pyridine complex (97%), and octopamine hydrochloride (analytical
standard) were obtained from Sigma-Aldrich (Deisendorf, Germany),
acetonitrile, formic acid, and ammonium acetate (all analytical grade)

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were purchased from Merck (Darmstadt, Germany), and solid-phase
extraction cartridges (OASIS HLB, 3 mL, 60 mg sorbent) were obtained
from Waters (Eschborn, Germany). The internal standard IPNS-
d7(deterenol-d7) was provided by LGC Standards GmbH (Wesel,
Germany).
2.2 Liquid chromatography-mass spectrometry
All analyses were conducted using a Thermo Fisher Scientific (Dreieich,
Germany) Vanquish LC system interfaced via electrospray ionization
(ESI) to an Exploris 480 quadrupole/orbitrap mass spectrometer. The
LC was equipped with a Thermo Accucore C-8 (100 ×2.1 mm, 2.7 µm
particle size) analytical column. The LC method employed 5 mM aque-
ous ammonium acetate (containing 0.1% acetic acid, solvent A) and
acetonitrile (solvent B) and gradient elution starting with 99.5% A for
0.5 min, decreasing to 98% A in 4.5 min, decreasing further to 92% A
in 3 min and then 50% A in 2 min, before flushing the column at 0% A
for 1.5 min and subsequent re-equilibration at starting conditions for
3.5 min. The overall run time was 15 min. The ESI source was operated
in positive mode using a spray voltage of 3.0 kV, and full scan (m/z 100-
800) as well as product ion scan experiments were conducted at a res-
olution of 30.000 (full width at half maximum at m/z 200). Here, the iso-
lation window of the quadrupole for the product ion experiments was
set to 1.3 Da. The collision gas was nitrogen (provided by a CMC nitro-
gen generator, CMC Eschborn, Germany), and the collision energy was
set to 25 eV. The system was calibrated using the manufacturer’s cali-
bration procedure ensuring mass accuracies better 2 ppm.
2.3 Dietary supplement testing
A dietary supplement labeled to contain 20 mg of IPNS per serving
(11 g) was obtained via Internet order. The material consisted of water-
soluble powder and, after homogenization, 55 mg were dissolved in
50 mL of deionized water/acetonitrile (9:1, v/v). Eight aliquots of 10 µL
each were prepared, seven of which were spiked with either 10, 25, 50,
75, 100, 150, or 200 ng of IPNS reference substance (10, 25, 50, 75,
100, 150, or 200 µL of an aqueous solution containing 1 µg/mL), and
all eight samples were finally topped-up to 1 mL with deionized water.
The content of IPNS was calculated from the resulting standard addi-
tion curve. Analogously, the supplement was tested for the presence of
octopamine.
2.4 Elimination study
Following informed written consent, two healthy male volunteers (46
and 47 years) ingested 5.5 g of the dietary supplement (i.e. 50% of the
recommended dose and corresponding to ca. 8.7 mg of IPNS as deter-
mined by standard addition analysis), dissolved in 150 mL of tab water.
Blank urine samples were collected immediately before the supple-
ment consumption, and post-administration specimens were sampled
up to 48 h. The study was conducted with approval of the local ethics
committee of the German Sport University Cologne (#167/2020).
2.5 Sample preparation
Urine samples (0.5 mL) were aliquoted into 2 mL Eppendorf tubes and
prepared for analysis by the addition of 100 ng of the internal stan-
dard IPNS-d7, followed by dilution of the sample with deionized water
(1:1, v/v), thorough vortexing for 10 s, and subsequent centrifugation
at 3300 x g. A volume of 200 µL of the sample was finally transferred to
a glass vial for LC-MS(/MS) analysis.
In order to allow for methylating phenolic hydroxyl functions, car-
boxyl and amino residues for structure elucidation,13,14 urine (1 mL)
was solid-phase extracted on a HLB cartridge after conditioning of the
adsorber resin with 2 mL of methanol and 2 mL of water. After wash-
ing with another volume of 2 mL of water, the retained analytes were
eluted with 2 mL of methanol, which was evaporated to dryness under
reduced pressure at 40◦C in a centrifuge. The dry residue was reconsti-
tuted in 200 µL of a mixture containing 185 µL of acetonitrile and 15 µL
of iodomethane, and 200 mg of potassium carbonate was added prior
to heating the mixture to 50◦C for 60 min. After cooling, the super-
natant was transferred to a new test tube, evaporated to dryness under
reduced pressure at 40◦C in a centrifuge, and reconstituted in 200 µL
of acetonitrile/water (1:1, v/v) for LC-MS(/MS) analysis.
2.6 Estimation of urinary IPNS concentrations
Urinary IPNS concentrations observed in post-administration samples
were estimated by means of a calibration curve prepared by spiking
a blank urine sample with IPNS reference material at 0.01, 0.02, 0.1,
0.5, 5, and 1 µg/mL. The samples were prepared and analyzed in accor-
dance to the above-mentioned ‘dilute-and-inject’ protocol, and peak
area ratios of IPNS and the internal standard were utilized.
3RESULTS AND DISCUSSION
3.1 Mass spectrometric characterization of major
urinary metabolites
The obtained dietary supplement was labeled to contain 20 mg of IPNS
per serving (11 g), and standard-addition analyses confirmed an IPNS
concentration of ca. 1.6 mg/g. The resulting dosage per recommended
serving matches roughly earlier reports concerning IPNS-containing
tablets from 2014,6where ca. 20–40 mg per serving (tablet) was
found in the context of follow-up investigations into serious adverse
events. Urine samples collected after the ingestion of ca. 8.7 mg of
IPNS yielded signals attributed to intact and unmodified IPNS, IPNS
sulfate, hydroxylated IPNS, and two species of hydroxylated and
glucurono-conjugated IPNS (Figure 1) as supported by product ion
mass spectra illustrated in Figure 2. The protonated molecule of IPNS

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(A) (B)
(C)
(E) (F)
(D)
FIGURE 2 ESI product ion mass spectra of (A) isopropylnorsynephrine (IPNS) ([M+H]+=m/z 196.13); (B) hydroxylated
isopropylnorsynephrine (M1) ([M+H]+=m/z 212.13); (C) hydroxylated and glucurono-conjugated isopropylnorsynephrine (M2a) ([M+H]+=m/z
388.16, RT =1.9 min); (D) hydroxylated and glucurono-conjugated isopropylnorsynephrine (M2b) ([M+H]+=m/z 388.16, RT =8.1 min); (E)
sulfo-conjugated isopropylnorsynephrine (M3) ([M+H]+=m/z 276.09), and (F) sulfo-conjugated isopropylnorsynephrine N-oxide (M4)
([M+H]+=m/z 290.07)
at m/z 196 was suggested to eliminate water (18 u) and propene (42
u) to form the product ions at m/z 178 and 136, respectively, which
was proposed to further dissociate to m/z 119 and 91 by consecutive
losses of ammonia (17 u) and carbon monoxide (28 u) (Figure 2A,
Scheme 1) as supported by the corresponding accurate masses of
the observed ions as well as by pseudoMS3experiments using m/z
178, 136, or 119 as in-source CID-generated precursor ions (data
not shown). The ion at m/z 58 was assigned to propan-2-imimium.
Hydroxylated IPNS (M1) gave rise to a protonated molecule at m/z
212, and its dissociation pathway suggested the location of the newly
introduced hydroxyl group at the phenylethanolamine residue by
mass shifts of 16 u observed with all product ions proposed to include
the norsynephrine structure (Figure 2B). The presence of a catechol
moiety was excluded based on pseudoMS3experiments with nore-
pinephrine, where [M+H]+-18atm/z 152 was used as precursor,
which yielded a product ion mass spectrum deviating from that of m/z
152 obtained from M1 under pseudoMS3conditions (Figure 3). While
the hydroxylated IPNS (M1) eliminated a hydroxyl radical from m/z 152
to form m/z 135, norepinephrine exclusively eliminated ammonia to
yield m/z 135 (Figure 3). Therefore, in case of M1, the formation of m/z
152 as an N-hydroxylated species must be taken into consideration,
either ab initio (cf. methamphetamine15–18) or by rearrangement of

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SCHEME 1 Proposed dissociation pathway of protonated isopropylnorsynephrine (IPNS)
a metabolically introduced phenolic hydroxyl group. The existence
of a N-hydroxylated species is further supported by the presence of
m/z 100 (elemental composition: C5H10NO) in the product ion mass
spectrum of M1 (Figure 2B) as well as the appearance of m/z 58 (ele-
mental composition: C2H4NO) in both the MS/MS and the pseudoMS3
mass spectrum of m/z 152 of M1, attributable to 4H-1,2-oxazet-2-ium,
and the formation of the product ion at m/z 119 (C8H7O, Figure 3),
the generation of which necessitates the elimination of hydroxy-
lamine (NH2OH, 33 u) as observed in earlier studies investigating
metabolites of N-hydroxylated secondary amine structure.19 Further
corroborating evidence for the metabolic formation of N-hydroxyl iso-
propylnorsynephrine was obtained by chemical microsynthesis of the
putative metabolite. Following established synthetic routes,20 IPNS
was first converted into its N-oxide by means of 3-chloroperbenzoic
acid, and subsequently reduced by lithium aluminium hydride to
yield the corresponding N-hydroxide. The product ion mass spec-
trum of the obtained product is depicted in Figure 3C,which
plausibly matches the product ion mass spectrum of M1 shown in
Figure 2B.
Two species of glucuronic acid conjugates of M1 were detected
with protonated molecules at m/z 388, separated chromatographically
by 6.2 min (Figure 2C: M2a;Figure2D: M2b, typical chromatogram
depicted in Figure 5A). The earlier eluting metabolite M2a was of sub-
stantially lower abundance compared to the later eluting M2b, and fea-
tured [M+H]+-18 at m/z 370 in contrast to M2b, which (in analogy
to IPNS) suggested the site of glucuronidation at a phenolic hydroxyl
group. Conversely, the glucuronidation of M1 at the alcoholic hydroxyl
group is proposed to result in M2b that, upon protonation and col-
lisional activation, eliminates immediately glucuronic acid (194 u) to
form the product ion at m/z 194.
A signal attributed to the sulfo-conjugate of IPNS (M3)was
detected, representing the most abundant metabolite of the sympath-
omimetic amine. The product ion mass spectrum derived from [M+H]+
at m/z 276 is illustrated in Figure 2E, exhibitinga product ion at m/z 258,
which indicates a (predominant) conjugation at the phenolic hydroxyl
group. By means of a microsynthesis of sulfo-conjugated IPNS in accor-
dance to established protocols,21 a reference spectrum was obtained
as illustrated in Figure 4A, which plausibly matches the spectrum gen-
erated from M3 (Figure 2E). Further investigations into the conjuga-
tion site were conducted by selective methylation of the metabolic
products isolated from elimination study urine samples, affecting the
secondary amine and phenolic but not alcoholic hydroxyl groups.
The presumed sulfo-conjugate of IPNS (M3) produced a dimethylated
derivative with both newly introduced methyl groups located at the
nitrogen atom, forming an aminium residue (and thus a M+at m/z 304)
as evidenced by the presence of the product ion at m/z 88, attributed to
N,N-dimethylpropan-2-aminium (Figure 4B). A free phenolic hydroxyl
group would be methylated under the chosen conditions, yielding a M+
precursor ion at m/z 318 and, consequently, an asulfate product ion at
m/z 238 rather than the ion at m/z 224 seen in Figure 4B.Thecom-
bination of a precursor ion at m/z 318 and a product ion at m/z 238
was found but only at a relative abundance of ca. 2% in comparison to
the precursor/product ion pair of m/z 304/224, indicating that a minor
share of sulfo-conjugation at the alcoholic hydroxyl group might also
exist (data not shown).
Also, a signal attributable to the sulfo-conjugate of IPNS N-oxide
(M4) was observed with a product ion mass spectrum shown in Fig-
ure 2F, supported by the characteristic product ions observed at m/z
248 (- propene), m/z 210 (-SO3), and m/z 203 (assigned to sulfo-
conjugated hydroxyl(4-hydroxyphenyl)methylium).

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(A)
(B)
(C)
FIGURE 3 pseudoMS3mass spectra of (A) norepinephrine
([M+H]+=m/z 170.13 →152.07), (B) hydroxylated
isopropylnorsynephrine (M1) ([M+H]+=m/z 212.13 →152.07), both
recorded at collision energies of 30 eV, and product ion mass spectrum
of (C) synthesized N-hydroxylated IPNS ([M+H]+=m/z 212.12)
3.2 Urinary excretion profile
The extracted ion chromatograms of major target analytes (ISTD, IPNS,
M2 and M3), measured in product ion scan mode and presented by
means of accurate masses of product ions ±0.02 m/z, of a urine sample
collected 5.5 h post-administration are illustrated in Figure 5A.Below
the top pane, which depicts the signal of the internal standard, the peak
of IPNS is shown at 2.39 min. The signal at 2.01 min in the same ion
trace is attributed to the partial in-source dissociation of IPNS sulfate
(M3), which is corroborated by the intact M3 presented at identical
retention time in the third pane of Figure 5. The bottom pane exhibits
(A)
(B)
FIGURE 4 Product ion mass spectra of (A) synthesized
sulfo-conjugated IPNS (M3)with[M+H]+=m/z 276.09; and b)
dimethylated sulfo-conjugated IPNS (M3)with[M+H]+=m/z 304.12,
presenting a product ion at m/z 224.16 that matches the asulfate of
M3 bearing two methyl groups at the nitrogen atom
three signals, all tentatively assigned to hydroxylated and glucurono-
conjugated IPNS with M2a at 1.87 min and M2b at 8.10 min. The lat-
ter features a minor additional signal at 7.96 min with identical product
ion mass spectrum (data not shown), which is suggested to represent a
stereoisomer of M2b.
By means of an external calibration curve and internal standard, the
urinary concentration of IPNS was estimated in post-administration
samples as depicted in Figure 5B. Peak concentrations (specific gravity-
adjusted) of ca. 2.3 µg/mL were observed 1 h after oral administration
of ca. 8.7 mg of IPNS contained in the dietary supplement, declining
below 50 ng/mL within approximately 12 h. In the absence of refer-
ence material for the tentatively identified metabolites M2-M3,peak
area ratios of the analytes/internal standard were employed to plot
elimination profiles (Figure 5C), outlining the comparably high abun-
dance of IPNS sulfate (M3)andOH-IPNS(M2b), which contributes to
qualifying as target analytes in routine doping controls. Of note, only
negligible (if any) amounts of octopamine and octopamine sulfate were
detected. Hence, is appears unlikely that the use of IPNS will result in
an adverse analytical finding for octopamine in doping control samples;
yet, probing for the presence of diagnostic metabolites of IPNS such as
e.g. the aforementioned M2b and M3 complements the analytical data
and supports subsequent result managing processes.

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(A)
(B)
(C)
FIGURE 5 (A) extracted ion chromatograms obtained from a post-administration urine sample collected 5.5 h after ingestion of ca. 8.7 mg of
IPNS; (B) estimated urinary concentrations of IPNS in post-administration samples collected over a period of 48 h; (C) elimination profiles of IPNS
main metabolites, presented as peak area ratios of the target analytes/internal standard
4CONCLUSION
Isopropylnorsynephrine has overtly been advertised as active ingre-
dient in commercially available dietary supplements, and adverse
health events were associated with the use of products labeled to
contain, amongst other pharmacologically active components, IPNS.
Albeit chemically closely related to octopamine, the presented data
do not suggest a substantial conversion of IPNS into octopamine in
both individuals tested within this study; instead, IPNS itself plus diag-
nostic phase-II metabolites are eliminated into urine that enable the

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unequivocal detection of an IPNS administration at dosages commonly
suggested by supplement manufacturers.
ACKNOWLEDGMENTS
The authors thank the Manfred-Donike-Institute for Doping Analysis
(Cologne, Germany) and the Federal Ministry of the Interior, Commu-
nity and Building of the Federal Republic of Germany (Berlin, Germany)
for support.
DATA SHARING
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
ORCID
Mario Thevis https://orcid.org/0000-0002-1535-6451
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How to cite this article: Krug O, Thomas A, Thevis M. Mass
spectrometric identification and characterization of urinary
metabolites of isopropylnorsynephrine for doping control
purposes. Anal Sci Adv. 2021;2:334–341.
https://doi.org/10.1002/ansa.202100004