New insights into themetabolism ofmethyltestosterone and metandienone: Detection of novel a-ring reducedmetabolites

Article abstract of DOI:10.3390/molecules26051354

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl- 17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone (“oral turinabol”), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, 3α,5β-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for antidoping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

Full text of DOI:10.3390/molecules26051354

molecules
Article
New Insights into the Metabolism of Methyltestosterone and
Metandienone: Detection of Novel A-Ring Reduced Metabolites
1
1
1
1
2
2
Steffen Loke , Lingyu Liu
, Maxi Wenzel , Heike Scheffler , Michele Iannone , Xavier de la Torre
,
Nils Schlörer 3 , Francesco Botrè , Annekathrin Martina Keiler
Maria Kristina Parr *
2,4
5,6
, Matthias Bureik 7 and
1
,
1
Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry, Freie Universität Berlin,
Königin-Luise-Straße 2+4, 14195 Berlin, Germany; s.loke@fu-berlin.de (S.L.); lingyu.liu@fu-berlin.de (L.L.);
maxi.wenzel@fu-berlin.de (M.W.); heike.scheffler@fu-berlin.de (H.S.)
Laboratorio Antidoping FMSI, Largo Giulio Onesti 1, 00197 Rome, Italy; micheleiannone14@gmail.com (M.I.);
Xavier.delatorre@fmsi.it (X.d.l.T.); francesco.botre@unil.ch (F.B.)
Institute for Organic Chemistry, Universität zu Köln, Grenstraße 4, 50939 Cologne, Germany;
nils.schloerer@uni-koeln.de
REDs–Research and Expertise in Antidoping Sciences, ISSUL–Institute del Sciences du Sport de l’Université
de Lausanne, 1015 Lausanne, Switzerland
Institute of Doping Analysis & Sports Biochemistry Dresden, Dresdner Str. 12, 01731 Kreischa, Germany;
a.keiler@idas-kreischa.de
2
3
4
5
6
7
Environmental Monitoring & Endocrinology, Faculty of Biology, Technische Universität Dresden,
Zellescher Weg 20b, 01217 Dresden, Germany
School of Pharmaceutical Science and Technology, Tianjin University, 92 Weijin Road, Nankai District,
Tianjin 300072, China; matthias@tju.edu.cn
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Loke, S.; Liu, L.; Wenzel,
M.; Scheffler, H.; Iannone, M.; de la
Torre, X.; Schlörer, N.; Botrè, F.; Keiler,
A.M.; Bureik, M.; et al. New Insights
into the Metabolism of
*
Correspondence: maria.parr@fu-berlin.de; Tel.: +49-30-838-57686
Abstract: Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a
17 -methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis.
Following the previously reported detection of long-term metabolites with a 17 -hydroxymethyl-
-methyl-18-nor-5 -androst-13-en-3 -ol structure in the chlorinated metandienone analog de-
hydrochloromethyltestosterone (“oral turinabol”), in this study we investigated the formation of
similar metabolites of metandienone and 17 -methyltestosterone with a rearranged D-ring and a
fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference com-
pounds, two diastereomeric substances, viz. 17 -hydroxymethyl-17 -methyl-18-nor-5 -androst-
3-en-3 -ol and its 5 -analog, were identified following an administration of methyltestosterone.
In post-administration urines of metandienone, only the 5 -metabolite was detected. Additionally,
,5 -tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides
the classical metabolites included in the screening procedures. Besides their applicability for anti-
doping analysis, the results provide new insights into the metabolism of 17 -methyl steroids with
α
Methyltestosterone and
ξ
Metandienone: Detection of Novel
A-Ring Reduced Metabolites.
1
7
ξ
ξ
ξ
Molecules 2021, 26, 1354. https://
doi.org/10.3390/molecules26051354
α
α
β
β
Academic Editors: Christoph Müller
and Franz Bracher
1
α
α
β
3
α β
Received: 17 February 2021
Accepted: 27 February 2021
Published: 3 March 2021
α
respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations
to the D-ring.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: 17α-methyl steroids; long-term metabolites; gas chromatography-mass spectrometry;
1
7-hydroxymethyl-17-methyl-18-nor; D-ring alteration; doping control; metabolism
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
1. Introduction
Metandienone (17
available in supplement S1) and methyltestosterone (17
-one, MT, 18) are anabolic-androgenic steroids. They were introduced to the market in
960 (MD) [ ] and 1939 (MT) [ ] as orally active anabolic androgenic steroids. Although
β
-hydroxy-17
α
-methyl-androsta-1,4-dien-3-one, MD, 12; list of steroids
β
-hydroxy-17 -methylandrost-4-en-
α
3
1
1
2
there is no approved drug available anymore, they are still widely marketed and misused
as performance-enhancing drugs in sports, even though they are prohibited in and outside
4
.0/).
Molecules 2021, 26, 1354. https://doi.org/10.3390/molecules26051354
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1354
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of competition by the World Anti-Doping Agency [3]. Many so-called “adverse analytical
findings” (AAFs) in doping control have been reported, and their numbers in the last 17 years
are displayed in Figure 1 Over the last few years, metandienone and methyltestosterone
represent 10% and 1% of all AAFs in the class of anabolic agents. In 2018, only five other
substances out of all prohibited compound classes were identified more frequently than
metandienone (n = 131), with clenbuterol giving the highest number (n = 320).
Figure 1. Adverse analytical findings of methyltestosterone and metandienone between 2003 and
2
018, according to [4].
Due to the 17
α
-methyl group, the steroids become orally active, because it prevents
-hydroxy group sterically,
the first-pass metabolism by hindering the oxidation of the 17
β
while the introduction of a double bond in position 1 was intended to avoid aromatization
and reduced the activity of A-ring-reducing enzymes [5–7].
Many metabolites related to the intake of metandienone are reported in the literature,
and a number of these have been known for decades. These are generated by both phase
I and phase II drug metabolizing enzymes. Phase I reactions include the introduction of
a double bond at position 6, the reduction of double bonds in the A-ring, the reduction
of the 3-oxo group, hydroxylations in positions 6, 11, 12, 16, or 18, epimerization in
position 17, and rearrangement of the D-ring [
both glucuronidation and sulfonation have been reported [16
investigations on long-term metabolites of MD (12) identified further metabolites with 17
hydroxymethyl-17 -methyl-13-ene structure [18 21]. The known metabolites are shown
8–
15]. With respect to phase II reactions,
,17]. In recent years, new
β-
α
–
in Figure 2. Anti-doping laboratories mostly target the parent compound (12), 6-OH-
metandienone (13), epi-metandienone (14), epi-metendiol (15), nor-epi-metendiol (16), and
20βOH-nor-metandienone (17) [18,22,23].
The intake of methyltestosterone leads to several metabolites, which derive from
hydroxylations in positions 2, 4, 6, 11, or 20, reduction of the 4,5-double bond, reduction of
the 3-oxo group, oxidation yielding a 1,2- or a 6,7-double bond, epimerization in position
1
7, and rearrangement of the D-ring [24
to both glucuronides and sulfates [26
shown in Figure 3. Laboratories mainly screen for the parent compound itself (18) and two
reduced derivatives (3 -THMT, 19; 3 -THMT, 20). The metabolites of both substances
–
28]. Subsequent phase II reactions are also leading
,
29]. The structures of metabolites of MT (18) are
α
5
α
α5β
are frequently monitored by gas chromatography-mass spectrometry after hydrolysis of
the glycosidic bond of glucuronides as aglycons [22,23].
For other steroids with a similar structure, such as dehydrochloromethyltestosterone,
there is a metabolite described with a fully reduced A-ring and a rearranged D-ring [30],
which was synthesized in 2018 [31,32]. This metabolite led to an extended detection time

Molecules 2021, 26, 1354
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of the intake for this substance and thereby increased the number of adverse analytical
findings.
As the chemical structures of metandienone (12) and methyltestosterone (18) are
similar to dehydrochloromethyltestosterone, it is conceivable that intake of these substances
results in metabolites with a related structure. The discovery of such new metabolites
may help in extending the time of detection after the intake of metandienone (12) or
methyltestosterone (18), which would be a considerable contribution to the fight against
doping, as cheating may be traced back over a longer period. Additionally, such findings
may help to further elucidate the metabolism of synthetic steroids and therefore improve
the understanding of human biotransformation.
Figure 2. Chemical structures of phase I metabolites of metandienone reported in the literature.

Molecules 2021, 26, 1354
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Figure 3. Chemical structures of phase I metabolites of methyltestosterone reported in the literature.
2
2
2
. Results
.1. Synthesis and Characterization of Reference Steroids
.1.1. 17-Hydroxymethyl-17-methyl-18-nor-13-enes
Different diastereomeric 17α-hydroxymethyl-17β-methyl-18-nor-5ξ-androst-13-en-3ξ-
ols were synthesized using 3-hydroxyandostan-17-ones as starting material by modifying
the D-ring. The method was adapted from Kratena et al. [33] but started with regu-
larly C13β-CH configured androstanes in contrast to the ent-configurated (C13α-CH3)
3
androstanes used by Kratena et al. As the first step of synthesis, attachment of an addi-
tional carbon-atom at C17 was achieved using Nysted reagent. The epoxidation of the
newly introduced 17(20) double bond and subsequent acid catalyzed ring-opening was
accompanied by the stereoselective Wagner–Meerwein rearrangement, resulting in 17
hydroxymethyl-17 -methyl-18-nor-5 -androst-13-en-3 -ols as the major product, while
the 17 -hydroxymethyl-17 -methyl analogs were obtained as minor side products. The
reaction scheme is displayed in Figure 4. The preceding synthesis of etiocholanolone (
α-
β
ξ
ξ
β
α
5)
is described in the supplementary material (S2). The other educt androsterone (5a) was
obtained from commercial sources.

Molecules 2021, 26, 1354
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Figure 4. Reaction scheme for 17α-hydroxymethyl-17β-methyl-18-nor-5ξ-androst-13-en-3ξ-ol steroids.
As is common in diastereomers, all yielded very similar mass spectra. As an exam-
ple, the spectrum of 17 -hydroxymethyl-17 -methyl-18-nor-5 -androst-13-en-3 -ol ( ) is
α
β
β
α
8
displayed in Figure 5. Using electron ionization at low energy (15 eV, low energy electron
ionization, LEI) the molecular ion, which was literally invisible at regular ionization energy
(viz. 70 eV), was detected at the accurate mass m/z 448.3162. The retention time of the
bis-trimethylsilyl (TMS) derivatives of the diastereomers are given in Table 1. Further struc-
ture confirmation was achieved by 1D and 2D-NMR analysis. Assignments are provided
in Table 2.
Figure 5. Mass spectrum (GC-EI-QTOF-MS, 70 eV) of 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (8),
bis- trimethylsilyl) (TMS (x-axis: m/z; y-axis: relative abundance).
•
+
Table 1. Retention times (GC-QQQ-MS), molecular ions (M ) in low electron ionization (LEI,
+
•
0 eV), and mass difference to exact mass (m/zther 448.3187, C H O Si ) of diasteromeric 17α-
26 48 2 2
2
hydroxymethyl-17β-methyl-18-nor-5ξ-androst-13-en-3ξ-ols as per-TMS derivatives.
Stereochemical
Assignment
Molecular Ion
(LEI)
No.
8
RT [min]
∆m/z [ppm]
3α, 5β, 17α-CH OH
9.80
10.13
448.3162
448.3164
−5.6
−5.1
2
8
a
3α, 5α, 17α-CH OH
2

Molecules 2021, 26, 1354
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Table 2. 1H and 13C NMR spectral data of 17
-ol ( ) and 17 -hydroxymethyl-17 -methyl-18-nor-5
signals indicated as singlet (s), doublet (d).
α
-hydroxymethyl-17
β
-methyl-18-nor-5
β
-androst-13-en-
3α
8
α
β
α
-androst-13-en-3α-ol (8a). Multiplicity of
1
7α-hydroxymethyl-17β-methyl-18-
nor-5β-androst-13-en-3α-ol
8)
17α-hydroxymethyl-17β-methyl-18-
nor-5α-androst-13-en-3α-ol
(8a)
(
δ
δ
δ
δ
H
C
H
C
α: 1.92
β: 1.06
α: 1.35
β: 1.58
1
35.18
31.93
α: 1.37
β: 1.72
α: 1.66
β: 1.75
2
3
4
5
6
30.64
71.75
36.59
41.75
27.63
28.93
66.43
35.70
39.03
28.86
β: 3.66
β: 4.08
α: 1.75
β: 1.56
α: 1.41
β: 1.53
β: 1.47
α: 1.60
α: 1.35
β: 1.93
α: 1.25
β: 1.30
α: 1.72
β: 1.21
α: 1.04
β: 1.95
7
26.08
31.47
8
9
37.41
38.44
34.67
β: 2.14
α: 1.67
-
36.97
52.03
36.13
β: 2.10
α: 1.01
-
1
0
α: 1.79
β: 1.14
α: 1.90
β: 1.16
1
1
1
22.43
22.65
22.14
22.59
α: 1.83
β: 2.02
α: 1.80
β: 2.01
2
1
1
3
4
135.94
141.76
-
-
135.85
141.81
-
-
α: 2.32
β: 2.12
α: 2.33
β: 2.11
1
1
5
6
30.60
34.20
30.61
34.13
α: 1.58
β: 1.97
α: 1.97
β: 1.58
1
1
7
9
51.66
22.93
21.72
-
51.54
10.61
21.75
-
0.93 (s)
1.00 (s)
0.78 (s)
0.99 (s)
2
0βCH3
3
.34 (d)
3.31 (d)
3.42 (d)
2
0α
CH OH
68.97
68.99
2
3.44 (d)
2
.1.2. 17β-Methyl-5β-androstane-3α,17α-diol (11)
Additionally, the diastereomeric 17 -methyl-5
thesized using epi-methyltestosterone (17
educt. After reduction of the 4,5-double bond and the 3-oxo group, the four fully reduced
products (11 11a 11b 11c) were obtained as shown in Figure 6. In parallel, reduction of
the 3-oxo group in epi-mestanolone (17 -hydroxy-17 -methyl-5 -androstan-3-one, 10a)
β
ξ
-androstane-3
ξ
,17
α
-diols were syn-
α
-hydroxy-17β-methyl-androst-4-en-3-one, 9) as
,
,
,
α
β
α
yielded the two products 11a and 11c. Assignment of the stereochemistry was based on
the known stereoselectivity of the reductions, the comparison of the two reactions, and
the elution order of the bis-TMS derivatives in GC-MS [24,34]. As a major product 3α,5β-
epi-tetrahydromethyltestosterone (11) was obtained (Figure 7). The mass spectrum of its
bis-TMS derivative is displayed in Figure 8. In LEI the molecular ion was detected at m/z

Molecules 2021, 26, 1354
7 of 18
+
•
(exact mass
4
50.3352 (accurate mass), confirming the elemental composition C H O Si
26
50
2
2
m/z 450.3344, difference ∆m/z = 1.78 ppm).
Figure 6. Synthesis route for 17β-methyl-5β-androstane-3α,17α-diol (11).
Figure 7. Mass spectrum (GC-EI-QTOF-MS, 70 eV) of 17
y-axis: relative abundance).
β-methyl-5β-androstane-3α,17α-diol (11), bis TMS (x-axis: m/z;
2.2. Post-Administration Urines
Urine samples from the administration trials were analyzed with a GC-QTOF-MS and
GC-QQQ-MS after per-TMS derivatization.
The common metabolites of MT (18) and MD (12) were detected by comparison of re-
tention time and quantifier and qualifier transitions, as reported in Table 3. Corresponding
chromatograms are available as supplemental material (S3).
Monitoring of the ion transitions m/z 345.3
159.0, selected for the 17 -hydroxymethyl-17
isomers, resulted in the detection of two signals at RT
→
255.0, m/z 345.3
-methyl-18-nor-5
= 9.56 min and RTmetabolite B
→
173.0, and m/z 345.3
→
ξ
ξ
ξ
-androst-13-en-3 -ol
ξ
metabolite A
=
(
=
9.80 min in a case of metandienone (12). In the post-administration (p.a.) samples of MT
18), three signals were detected—one in addition to the two mentioned above (RTmetabolite A
9.56 min, RTmetabolite B = 9.80 min, and RTmetabolite C = 10.13 min). The comparison with
the synthesized reference compounds assigned the metabolites common for MD (12) and
MT (18) to 17 -hydroxymethyl-17 -methyl-18-nor-5 -androst-13-en-3 -ol ( ) and 3α,5β-
α
β
β
α
8
epi-tetrahydromethyltestosterone (11). The additional metabolite in MT administration
was assigned to 17α-hydroxymethyl-17β-methyl-18-nor-5α-androst-13-en-3α-ol (8a).
The 3α,5β-epi-tetrahydromethyltestosterone was identified as the first peak in positive
urine samples of metandienone and methyltestosterone at 9.56 min (Figure 8).
Another substance with a slightly different structure as compound 11, namely 3α,5α-
epi-tetrahydromethyltestosterone (11a), has almost the same retention time as compound 8.
•
+
However,
8
does not show the transition m/z 450
→
345 because of its structure (M as

Molecules 2021, 26, 1354
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TMS-derivative: m/z 448). As this transition is present in the urine sample, the 3
α5α-epi-
tetrahydromethyltestosterone (11a) can be excluded as the metabolite at 9.80 min (Figure 9).
Another closely eluting metandienone metabolite, 17β-methyl-5β-androst-1-ene-3α,17α-
diol (15, RT15 = 9.87 min, M+ = 448), was mainly separated and identified by the selective
•
ion transitions given in Table 3.
Table 3. Retention times (GC-QQQ-MS) and ion transitions of currently targeted metabolites in
anti-doping analysis.
Ion Transitions (m/z) &
Collision Energies
Compound (Parent Compound)
RT [min]
3
3
3
58.0 → 301.0 (10 eV)
58.0 → 169.0 (30 eV)
58.0 → 196.0 (10 eV)
1
7β-methyl-5β-androst-1-ene-3α,17α-diol
9
.87
358.0 → 194.0 (10 eV)
(
15)
2
2
2
16.0 → 159.0 (5 eV)
68.0 → 211.0 (10 eV)
16.0 → 187.0 (5 eV)
5
5
17.5 → 229.0 (5 eV)
17.5 → 297.0 (5 eV)
17.5 → 205.0 (30 eV)
6
β,17β-dihydroxy-17α-methyl-androsta-1,4-
dien-3-one
16.19
13.77
5
5
(
13)
17.5 → 429.4 (5 eV)
4
4
3
44.4 → 206.0 (10 eV)
44.4 → 191.0 (30 eV)
39.0 → 270.0 (20 eV)
1
7α-hydroxy-17β-methyl-androsta-1,4-dien-
-one
14)
3
(
4
44.4 → 283.0 (30 eV)
2
53.0 → 185.0 (20 eV)
1
7,17-dimethyl-18-nor-5β-androsta-1,13-
dien-3α-ol
253.0 → 197.0 (20 eV)
253.0 → 105.0 (30 eV)
216.0 → 131.0 (20 eV)
6.19
(
16)
2
16.0 → 145.0 (20 eV)
2
36.0 → 133.0 (5 eV)
39.0 → 193.0 (20 eV)
42.4 → 243.0 (15 eV)
42.4 → 133.0 (15 eV)
39.0 → 133.0 (20 eV)
3
4
4
3
1
7β-hydroxymethyl-17α-methyl-18-nor-
androsta-1,4,13-trien-3-one
13.84
(
17)
3
39.0 → 243.0 (20 eV)
2
28.0 → 174.0 (5 eV)
70.0 → 157.0 (30 eV)
70.0 → 171.0 (30 eV)
2
2
1
7α-methyl-5β-androstane-3α,17β-diol (20)
7α-methyl-5α-androstane-3α,17β-diol (19)
13.36
13.22
2
70.0 → 199.0 (30 eV)
3
3
18.0 → 199.0 (10 eV)
18.0 → 187.0 (10 eV)
1
318.0 → 182.0 (10 eV)
4
50.4 → 365.0 (10 eV)
4
50.4 → 261.0 (10 eV)

Molecules 2021, 26, 1354
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Figure 8. GC-QQQ-MS chromatograms (MRM, m/z 345 → 173).

Molecules 2021, 26, 1354
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Figure 9. Chromatograms (MRM, m/z 450
methyltestosterone; red: synthesized substances.
→
345); black: positive urine sample after intake of
Two more diastereomers with 17
α
-methyl-17β-hydroxy configurations (17α-methyl-
5
α
-androstane-3 ,17 -diol (19) and 17
α
β
α
-methyl-5
β
-androstane-3 ,17 -diol (20)) were
α
β
commercially available and used for retention time comparison and urinary metabolite
identification.
3. Discussion
3.1. Chemical Syntheses and Characterization of Reference Material
The described syntheses starting from etiocholanolone (
5
) or androsterone (5a) led
-hydroxy-5 -; 8a: 3-hydroxy-
-methyl-18-nor-13-ene). As ex-
pected from the reactions, stereochemistry at C3 and C5 was retained unchanged. Due
to the commonly known remaining stereochemistry of the 13 -methyl group during the
Wagner–Meerwein rearrangement, the 17 -hydroxymethyl-17 -methyl products were
to androstane derivatives with a fully reduced A-ring (
8: 3
α
β
5
α
-) and a rearranged D-ring (17 -hydroxymethyl-17β
α
β
α
β
the major products as expected. In GC-EI-MS, using common ionization energy of 70 eV,
literally no molecular ions were obtained, due to extensive fragmentation. As the domi-
+
nant fragment, [M-CH -OTMS] (accurate mass m/z 345.2607, exact mass m/z 345.2608,
2
∆
m/z = −0.29 ppm) was found. The loss of 103 Da is considered characteristic for the
TMS derivatized 17 -hydroxymethyl-17 -methyl-18-nor-13-ene steroids [18 35]. The base
peak with an accurate mass m/z 255.2107 (exact mass m/z 255.2107, m/z = 0.00 ppm)
α
β
,
∆
corresponds to an additional loss of TMSOH. This transition was selected as target in the
+
GC-QQQ-MS method. As qualifiers the transitions to m/z 159 (C H , accurate mass
12
15
+
m/z 159.1168, exact mass m/z 159.1168, ∆m/z = 0.00 ppm) and m/z 173 (C H , accurate
13 17
mass m/z 173.1324, exact mass m/z 173.1325, ∆m/z = −0.56 ppm) are monitored.
NMR data confirmed the structure assignments. In 17α-hydroxymethyl-17β-methyl-
1
8-nor-5
β
-androst-13-en-3
α
-ol (
8
) stereochemistry at C5 was assigned by the downfield
> 22 ppm are known to be character-
shifted C19 (
δ
C19
= 22.93 ppm) signal. C19 shifts
δ
C19
istic for 5β-androstanes [36]. Configuration at C3 was deduced from the multiplicity of
H3 ( = 3.66 ppm, dddd, J = 11/11/5/5 Hz). The diaxial coupling with H-4ax and H-2ax
δ
H3
substantiated the axial orientation of H3(
The NMR data for the residues attached to C17 (
β
), thus confirming 3
α
-hydroxy configuration.
= 1.00 ppm
δ
= 21.72 ppm,
δ
H20-CH3
C20-CH3
and
δ
= 68.97 ppm,
δ
= 3.34 ppm and 3.44 ppm) together with NOESY
C20-CH2OH
H20-CH2OH
experiments confirmed the 17α-hydroxymethyl-17β-methyl assignment.
In case of 17 -hydroxymethyl-17 -methyl-18-nor-5 -androst-13-en-3
chemistry at C5 was assigned by the upfield shifted C19 ( = 10.61 ppm) signal. C19
α
β
α
α-ol (8a), stereo-
δ
C19
shifts
δ
C19
< 17 ppm are known to be characteristic in 5
α
-androstanes [36]. Configuration at
C3 was deduced from the multiplicity of H3 (
δ
H3
= 4.08 ppm, dddd, J = 3/3/3/3 Hz) repre-
senting coupling constants of H-3eq with H-2eq, H-2ax, H-4eq and H-4ax. This substantiated
the orientation of H3 , thus confirming 3 -hydroxy configuration. Further confirmation
β
α

Molecules 2021, 26, 1354
11 of 18
was achieved by selective NOE experiments (irradiation of H19,
δ
= 0.78 ppm). The
H19
NMR data for the residues attached to C17 (
δ
= 21.7 ppm,
δ
= 0.97 ppm
C20-CH3
H20-CH3
and
δ
= 21.7 ppm,
δ
= 0.97 ppm) together with NOESY experiments
C20-CH2OH
H20-CH2OH
confirmed the 17α-hydroxymethyl-17β-methyl assignment.
In comparison to the administered drug, the product of the last synthesis (17β-methyl-
5
β
-androstane-3
methyltestosterone (17
using hydrogen gas and palladium on charcoal as catalyst leads to the 5
with huge excess [37]. The subsequent reduction of the 3-oxo group of 5
methyltestosterone (10) with sodium borohydride mainly results in the 3
-OH:3 -OH according to Schänzer et al. [24]; 11). Stereochemistry at C17 is retained
during these reactions and thus assigned to 17 -hydroxy-17 -methyl. Due to the collision
energy of 70 eV, nearly no molecular ion is found, whereas prominent fragments occur. The
two dominant fragments of the above mentioned 17 -hydroxymethyl-17 -methyl steroids,
α
,17
α
-diol, 11) has a different stereochemistry at C17. Starting from epi-
-hydroxy-17 -methylandrost-4-en-3-one, ), the first reduction
-product (10
-dihydro-epi-
-isomer (88:12,
α
β
9
β
)
β
α
3
α
β
α
β
α
β
m/z 345 and m/z 255, are present in the spectrum of epi-tetrahydromethyltestosterone as
+
well. The signal at m/z 345 is caused by [M-CH -HOTMS] (accurate mass m/z 345.2619,
3
exact mass m/z 345.2608,
∆
m/z = 3.19 ppm), that at m/z 255 by another loss of TMSOH
m/z = 3.13 ppm), and the base
(accurate mass m/z 255.2115, exact mass m/z 255.2107,
∆
peak at m/z 143 by a characteristic D-ring fragment of 17-methyl steroids (accurate mass
m/z 143.0892, exact mass m/z 143.0887, ∆m/z = 3.49 ppm).
3.2. Urinary Metabolites
As is common in several doping control laboratories, glucuronidated metabolites
are enzymatically cleaved and determined as their aglycons together with their analogs
that are excreted as unconjugated compounds. Due to the low abundance of some of the
target analytes, GC-QQQ-MS in MRM mode is considered as a better-suited technique
for metabolite detection after optimization of the ion transitions. As described in the
literature [
20, MT M1) following the administration of both steroids, MD (12) and MT (18), in all
samples. Its 3 ,5 -analog (19, MT M2) was detected following the administration of MT
18), while in MD (12) p.a. samples, only very minor corresponding signals were detectable
8,24,38], GC-QQQ-MS analysis detected 17α-methyl-5β-androstane-3α,17β-diol
(
α
α
(
in the 48 h urine and remained unconfirmed due to the low signal-to noise ratio of the
qualifier transitions. According to earlier studies, these two metabolites are considered as
longest detectable by GC-QQQ-MS after MT (18) administration in GC-MS [38].
Exclusively after MD (12) administration, the parent compound (12), epimetendiol (15
M1: EMD), 6-hydroxymetandienone (13, M2: 6OH-MD), epimetandienone (14, M3: EpiMD),
normetendiol (17,17-dimethyl-18-nor-5β-androsta-1,13-dien-3α-ol, 16, M4: NorEMD), and
,
the long-term metabolite 17β-hydroxymethyl-17α-methyl-18-nor-androsta-1,4,13-trien-3-
one (17, M6: 20βOH-NorMD) were detected, which is in agreement with earlier find-
ings [8,12,13,18].
In addition to the commonly monitored metabolites, the two synthesized 17-hydroxy
methyl-17-methyl steroids were found in p.a. urines of methyltestosterone (17 -hydroxy
methyl-17 -methyl-18-nor-5 -androst-13-en-3 -ol, 8a, 17 -hydroxymethyl-17 -methyl-
8-nor-5β-androst-13-en-3α-ol, 8) by GC-MS comparison.
Aberrantly, only 17 -hydroxymethyl-17 -methyl-18-nor-5
was confirmed in the p.a. urines of metandienone (12). The stereochemistry at C17 is
the opposite of the currently monitored long-term metabolite of MD and also to 17
hydroxymethyl-17 -methyl-18-nor-androsta-4,13-dien-3-one, which was detected earlier
after administration of MT [38]. They are also different from the majority of metabolites of
α
β
α
α
α
β
1
α
β
β-androst-13-en-3α-ol (8)
β-
α
analogous 17-methyl steroids [30
methyl metabolites of metandienone, methyl-1-testosterone (17
androst-1-en-3-one) and oxandrolone [13 41], as well as the recently identified 4-chloro-17
hydroxymethyl-17 -methyl-18-nor-androsta-4,13-dien-3
,
31
,
38
–
42]. Only less abundant 17
α
-hydroxymethyl-17
β
α
α
-
-
-
β-hydroxy-17
α
-methyl-5
,
β
β-ol (named “M4” by Sobolevsky

Molecules 2021, 26, 1354
12 of 18
in 2012) as metabolite of 4-chlorometandienone (dehydrochloromethyltestosterone, active
component in Oral Turinabol) [33,43] have a similar stereochemistry at C17.
Interestingly, the structure of the long-term metabolite of 4-chlorometandienone with
modified D-ring structure and a fully reduced A-ring (Sobolevsky’s “M3”) was assigned
to 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androst-13-en-3α-ol by Forsdahl
et al. [31]. The metabolites proposed for MT and MD as described above show an inverse
stereochemistry at the D-ring in comparison to these assignments.
Additionally, the product of the last synthesis (17
) has an inverse D-ring at C17 in comparison to the parent compounds and the fully
A-ring reduced metabolites, 17 -methyl-5 -androstane-3 ,17 -diol (20, MT M1) and 17
methyl-5 -androstane-3 ,17 -diol (19, MT M2). The latter are formed through reduction
β-methyl-5β-androstane-3α,17α-diol,
11
α
β
α
β
α-
α
α
β
of the 1,2- and 4,5-double bond and the 3-oxo group. The 17-epimer was found in the
urines after the intake of both mentioned anabolic-androgenic steroids. In the case of
MT administration, the metabolite 11 was also described earlier, but found with shorter
detection times than the 17
was also found earlier, but with a problem in separation of the four diastereomers [15].
The epimerization of position 17 is a common reaction of 17 -methyl steroids and
was first described in 1971 [44]. In humans, it is generated through sulfonation of the
tertiary 17-hydroxy group and its subsequent hydrolysis [ ]. Besides 17-epimerization,
α-methyl analogs 19 and 20 [38]. After the intake of MD, this
α
9
the sulfate may also undergo an elimination of sulfuric acid and concomitant Wagner–
Meerwein rearrangement, leading to 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one.
This may undergo A-ring reduction, leading to the metandienone metabolite normetendiol
(
17,17-dimethyl-18-nor-5β-androsta-1,13-dien-3α-ol, 16, M4: NorEMD) [11].
Generation of 17 -hydroxymethyl-17 -methyl-18-norandrosta-1,4,13-trien-3-one is gen-
α
β
erated from the intermediate 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one by CYP3A4
catalyzed hydroxylation [20], while CYP21A1-catalyzed hydroxylation leads to the formation
of a 17β-hydroxymethyl-17α-methyl analog [20].
The stereoselectivity of the A-ring reduction is dependent on the parent compound.
For metandienone, there is only very limited generation of metabolites with a 5
This is is likely due to the 1,2-double bond, which inhibits the activity of 5 -reductase [45].
In contrast, methyltestosterone is metabolized to 5 - and 5 -isomers. This substantiates
α-structure.
α
α
β
our hypothesis of metabolite generation due to the A-ring structure with a double bond
in position 4 and its already saturated positions 1 and 2 in methyltestosterone, while
MD (12) has an unsaturated A-ring (i.e., 3-oxo-1,4-diene). Thus, it is reasonable that
the 17
α
-hydroxymethyl-17
β
-methyl-18-nor-5
α
-androst-13-en-3
α
-ol-derivative (8a) is only
-analog ( ) is observed
detectable in p.a. samples of methyltestosterone (18), while the 5
β
8
after MT or MD administration. This supports our concept of the order of reductions: if
the 1,2-double bond was reduced before the 4,5-double bond, there would have also been
5α-metabolites in p.a. urines of metandienone [8].
Thus, the order of the following two reductions of metandienone (1,2-double bond,
-oxo group) is not yet confirmed, but it seems to be more likely that the formation of the
-hydroxy group takes place before the hydrogenation of the 1,2-double bond, because
3
3
there are known metabolites of metandienone with a 3-hydroxy-1-ene structure but not
with a 3-oxo group in a fully reduced A-ring. Both potential ways represent the last step
of the proposed formation of the metabolites
8 and 11. They are displayed in Figure 10.
The other reactions of the metabolism of both investigated compounds are displayed in
Figure 11.

Molecules 2021, 26, 1354
13 of 18
Figure 10. Potential ways of A-ring reduction.
OH
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
SO3-
O
OH
5
OH
OH
OH
H
H
H
H
ß-reductase
AKR1C2/4
???
H
H
H
H
H
H
H
H
O
O
HO
HO
H
H
H
OH
OH
OH
OH
H
H
H
H
CYP3A4
5ß-reductase
AKR1C2/4
???
H
H
H
H
O
O
HO
HO
H
H
H
5a-reductase
OH
OH
H
H
AKR1C2/4
H
H
O
HO
H
H
Figure 11. Proposed metabolism of methyltestosterone (black, 18) and metandienone (red, 12) to the found metabolites
17α
-hydroxymethyl-17
β-methyl-18-nor-5
β-androst-13-en-3
α-ol (8), 17α-hydroxymethyl-17β-methyl-18-nor-5α-androst-
1
3-en-3 -ol (8a), and 17
α
β
-methyl-5 -androstane-3
β
α
,17 -diol (11) except last step of metandienone. The question marks
α
represent reactions whose enzymes have not been elucidated yet.
Based on preliminary data, the mentioned substances are detected for at least 48 h after
the intake of parent compounds. Excretion studies with a higher number of volunteers and
prolonged sample collection will be performed in the near future to evaluate the detection
windows of the new metabolites.
In addition to that, a potential next step will be the investigation of the substrate
specificity of 5α-reductase towards 1,2-ene steroids by means of molecular modelling to
elucidate structural requirements for generation of 5α-metabolites of androgenic steroids.
The detection and structure identification of the above-mentioned substances in the
urine samples help to gain further insights into human metabolism of metandienone and
1
7α
-methyltestosterone. Due to the similarity of other anabolic androgenic steroids to the
investigated compounds, it is probable that other metabolites with related structures may

Molecules 2021, 26, 1354
14 of 18
be found in further 17
α
-methyl steroids. Finally, the results may support the fight against
doping by introducing new analytes for screening in anti-doping analysis.
4
4
4
. Materials and Methods
.1. Instrumentation
.1.1. GC-MS/MS
The gas chromatographic-tandem mass spectrometric analysis was performed on
an Agilent 7890A gas chromatographic system coupled to an Agilent 7000 GC/MS triple
quadrupole mass spectrometer (Agilent Technologies, Milano, Italy). The following condi-
tions for the analysis of the intermediates and products were applied: Agilent HP1 column
◦
(
+
17 m, 0.20 mm, 0.11
µ
m), carrier gas: helium, oven program: 188 C, hold for 2.5 min,
◦
◦
◦
◦
◦
◦
3 C/min to 211 C, hold for 2.0 min, +10 C/min to 238 C, +40 C to 320 C, hold for
◦
3
(
.2 min, injection volume: 2
EI): 70 eV, transitions: m/z 345
Prior to injection, samples were treated with 50
µ
L, split: 20:1, injection temperature: 280 C, electron ionization
→
255 (5 eV), m/z 345
L of trimethyliodosilane (TMIS) reagent
N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/ethanethiol/ammonium iodide,
→
173 (20 eV), m/z 345
→
159 (10 eV).
µ
(
1
◦
000:6:4, v:v:w) at 75 C for 20 min before analysis to generate the per-TMS derivatives.
4.1.2. GC-QTOF-MS
High resolution accurate mass analyses were performed on an Agilent GC-QToF
890B/7250 (Agilent Technologies, Milano, Italy), equipped with an Agilent HP1 column
7
(
17 m, 0.20 mm; 0.11
µm) with helium as carrier gas. Injection was performed in split mode
◦
◦
with a 1:10 ratio at 280 C. The oven program had the following heating rates: 188 C
hold for 2.5 min, 3 C/min to 211 C and hold for 2 min, 10 C/min to 238 C, 40 C/min
to 320 C, and hold for 3.2 min. The coupled QToF was operated in full scan with an
◦
◦
◦
◦
◦
◦
ionization energy of 70 eV. Aberrantly, in LEI an ionization energy of 15 eV was applied.
Ions were detected from m/z 50 to 750.
4.1.3. HPLC Purification
The purification of the synthesized reference steroids was performed by semi-preparative
HPLC using an Agilent 1260 Infinity Quaternary HPLC system coupled to an Agilent Infinity
260 diode array detector (Agilent Technologies GmbH, Waldbronn, Germany). Chromato-
graphic separation was achieved on a Hypersil ODS C18 column (pore size: 120 Å, 250 mm
length, 10 mm ID, 5 m particle size, Thermo Scientific, Schwerte, Germany). Isocratic
1
µ
elution was accomplished at a flow rate of 3 mL/min using acetonitrile:water (7:3, v:v) as the
mobile phase. The UV signal was monitored at 194 nm.
4
.1.4. Nuclear Magnetic Resonance
1
The nuclear magnetic resonance (NMR) analyses were performed at 500 MHz ( H
13
NMR) and 125 MHz ( C NMR) at 296 K on a Bruker (Rheinstetten, Germany) Avance
III instrument equipped with a nitrogen-cooled 5 mm inverse TCI cryoprobe with ac-
tively shielded z-gradient coil. Chemical shifts are reported in
δ values (ppm) relative to
tetramethylsilane. Solutions of about 5 mg of each compound in deuterated dimethylsul-
1
13
foxide (d -DMSO) were used for conducting H; H,H COSY; C; edited HSQC; HMBC,
6
selective NOE and NOESY experiments. Two-dimensional experiments were recorded in
non-uniform sampling (NUS) mode.
4.2. Chemicals and Reagents
Androst-4-ene-3,17-dione (
1
) was purchased from TCI (Tokyo, Japan), androsterone
-methyltestosterone
18), palladium on charcoal, nysted reagent, K-Selectride and meta-chloroperoxybenzoic
-Methyltestosterone (epi-MT, ) was ob-
tained from Santa Cruz Biotechnology (Heidelberg, Germany). 17 -methyl-5 -androstane-
,17 -diol (20) and 17 -methyl-5 -androstane-3 ,17 -diol (19) were purchased from the
(
(
5a), and TiCl from Acros Organics (Fair Lawn, New Jersey, USA), 17α
4
acid from Aldrich (Steinheim, Germany). 17
β
9
α
β
3
α
β
α
α
α
β

Molecules 2021, 26, 1354
15 of 18
National Measurement Institute (North Ryde, Australia). Benzene was delivered from
Thermo Fisher (Karlsruhe, Germany), hexane, ethyl acetate, methanol, dichloromethane,
diethyl ether, hydrochloric acid, sodium bicarbonate, and potassium carbonate from Fisher
Scientific (Loughborough, United Kingdom). MSTFA was obtained from from Chemis-
che Fabrik Karl Bucher GmbH (Waldstetten, Germany). THF, monosodium phosphate,
sodium borohydride, and p-toluenesulfonic acid were purchased from Merck (Darmstadt,
Germany), while TBME, potassium hydroxide, and sodium carbonate were bought from
Carl Roth (Darmstadt, Germany). Hydrogen gas was provided by Air Liquide (Düsseldorf,
Germany) and
β-glucuronidase from Roche Diagnostics (Mannheim, Germany). All other
chemicals were purchased from VWR (Darmstadt, Germany).
4.3. Synthesis of Reference Steroids
4.3.1. Diastereomeric 17-hydroxymethyl-17-methyl-18-nor-5-androst-13-en-3-ols
17-Methylene-5ξ-androstan-3ξ-ol (6, 6a)
A flask was flushed with argon gas and held under an argon atmosphere. After
◦
cooling to 0 C, Nysted reagent (20%) was diluted with absolute tetrahydrofurane (THF
abs.), and titanium tetrachloride was added dropwise. After 15 min of stirring, the mixture
was brought to room temperature. The precursor steroid (5, or 5a) was dissolved in 10 mL
THF (abs.) and was added dropwise to the mixture. The reaction was held under these
◦
conditions overnight. After cooling down the mixture to 0 C, aqueous hydrochloric acid
(2 M) and ice-cold water were added, and it was extracted four times with diethyl ether.
The organic phases were combined, washed with sodium hydrogen carbonate and brine,
dried over sodium sulfate, and evaporated to dryness. Detailed amounts of reactants and
solvents are available in the supplements (S4).
0
Spiro[5 ξ-androstan-17,2 -oxirane]-3ξ-ol (7, 7a)
The crude substance was dissolved in dichloromethane, and potassium hydrogen
carbonate and meta-chloroperoxybenzoic acid were added. The solution was stirred for
3
h at ambient temperature. Afterwards, the mixture was poured into water and extracted
three times with dichloromethane. The organic phases were washed with brine and then
dried over sodium sulfate. Further details are disclosed as supplemental information.
17α-Hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (8)
The intermediate product was dissolved in 5 mL methanol plus 5 mL aqueous
7
hydrochloric acid (1 M). The solution was stirred overnight. Then, 10 mL water were
added, and the mixture was extracted three times with 20 mL t-butyl methyl ether. The
organic phases were combined and dried over sodium sulfate. The product was purified
by column chromatography (silica gel 60, 300 mm
using hexane/ethyl acetate (3:2, v:v) followed by HPLC fractionation. The finally purified
product (8) was obtained in a total amount of 16 mg (yield: 1.51%, purity >98%).
×
30 mm, particle size 40–63 µm),
17α-Hydroxymethyl-17β-methyl-18-nor-5α-androst-13-en-3α-ol (8a)
The intermediate product 7a was dissolved in 20 mL of methanol and 20 mL of
aqueous hydrochloric acid (1 M). The solution was stirred overnight. Then, 30 mL of water
was added, and the mixture was extracted three times with 50 mL t-butyl methyl ether. The
organic phases were combined and dried over sodium sulfate. The product was purified
by column chromatography (silica gel 60, 300 mm
hexane/ethyl acetate (3:2, v:v) followed by HPLC fractionation. A total amount of 263 mg
yield: 57.1%, purity >98%) of the final product (8a) was obtained.
×
30 mm, particle size 40–63 µm), using
(
4
.3.2. Epi-Tetrahydromethyltestosterones
7β-Methyl-5β-androstane-3α,17α-diol (11)
A mixture of 450 L methanol and 50 L potassium hydroxide solution (5 M) was
prepared, and 100 g epi-methyltestosterone ( ) was dissolved. A spatula tip of palladium
1
µ
µ
µ
9
on charcoal was added, and hydrogen gas flushed through the solution for 5 min. After
adding 2 mL of water, the mixture was extracted three times with 3 mL of hexane and
evaporated to give the product 10. The residue was dissolved in methanol/water (9:1, v:v)

Molecules 2021, 26, 1354
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and a spatula tip of sodium borohydride was added. The solution was stirred for one hour
at room temperature. After adding ammonium chloride to stop the reaction, potassium
hydroxide solution (1 M) was added to yield alkaline solution. Then, the solution was
extracted three times with dichloromethane and evaporated to give the product 11.
1
7β-Methyl-5α-androstane-3α,17α-diol (11a)
A spatula tip of epi-mestanolon (10a) was dissolved in 2 mL of absolute THF, 80
of K-Selectride was added and the mixture was stirred for 1 h at ambient temperature.
Afterwards, 100 L of aqueous hydrochloric acid (1 M) was added until there was no
formation of bubbles anymore. Then, 150 L of potassium hydroxide solution (1 M) was
µL
µ
µ
added and the mixture was extracted three times with 5 mL of hexane. The hexane-phase
was evaporated to give the product 11a.
4.4. Human Administration Trial
Urine samples out of the stock of the anti-doping laboratory in Rome were available
for analysis. Samples collected before and after an oral administration of either MD or MT
were used for evaluation of the excretion of the hypothized metabolites. The excretion
study with MT was carried out by a healthy male volunteer (Caucasian, 50 years old,
®
8
0 kg and normal body mass index). A single oral dose of 10 mg of MT (Metadren ,
Novartis, Basel, Switzerland) was administered. For investigation of MD metabolism, a
®
single oral dose of 5 mg MD (Dianabol , Ciba-Geigy, Basel, Switzerland) was administered
to a healthy male volunteer (Caucasian, 45 years old, 82 kg and normal body mass index).
4.5. Urine Sample Preparation
An aliquot of 6 mL urine was used for the following analysis. As internal standard
methyltestosterone (50
50 L of phosphate buffer (0.8 M) and 50
at 55 C for 60 min. Afterwards, 500
µ
L of a solution of 100
µ
g/mL) was added. After the addition of
-glucuronidase, the mixture was incubated
L of carbonate buffer (20%) was added and the
7
µ
µL β
◦
µ
mixture was extracted with 10 mL of TBME. After evaporation, 50
added to the sample and the mixture was treated at 75 C for 20 min before analysis to
generate the per-TMS derivatives.
µ
L of TMIS reagent was
◦
Supplementary Materials: The following are available online: Supplement S1: Table of steroids, Sup-
plement S2: Preceding synthesis of etiocholanolone, Supplement S3: Chromatograms of urine
samples. Supplement S4: Detailed amounts of reactants and solvents in the synthesis of 17-
hydroxymethyl-17-methyl-18-nor-5-androst-13-en-3-ols.
Author Contributions: Conceptualization, M.K.P., S.L., A.M.K. and X.d.l.T.; methodology, M.K.P.,
S.L. and X.d.l.T.; investigation, S.L., L.L., H.S., M.W., M.I., X.d.l.T. and N.S.; resources, M.K.P., N.S.,
A.M.K., M.B. and F.B.; data curation, M.K.P., S.L., M.I., X.d.l.T. and N.S.; writing—original draft
preparation, S.L.; writing—review and editing, M.K.P.; visualization, M.K.P., S.L., M.I., X.d.l.T. and
N.S.; supervision, M.K.P., M.B. and F.B.; project administration, M.K.P.; funding acquisition, M.K.P.,
A.M.K., M.B. and F.B. All authors have read and agreed to the published version of the manuscript.
Funding: The publication of this article was funded by Freie Universität Berlin.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Ethics Committee of the School of Pharmaceutical
Science and Technology, Tianjin University (date of approval 9 December 2020).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Raw data are stored at the authors.
Acknowledgments: We acknowledge support by the Open Access Publication Fund of the Freie
Universität Berlin.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are kept amongst the authors.

Molecules 2021, 26, 1354
17 of 18
References
1
.
Whitelaw, M.J.; Foster, T.N.; Graham, W.H. Methandrostenolone (Dianabol): A controlled study of its anabolic and androgenic
effect in children. J. Pediatrics 1966, 68, 294–295. [CrossRef]
2
3
.
.
Foss, G.L.; Simpson, S.L. Oral methyltestosterone and jaundice. Br. Med. J. 1959, 1, 259–263. [CrossRef] [PubMed]
World Anti-Doping Agency. The 2021 Prohibited List. Available online: https://www.wada-ama.org/sites/default/files/
resources/files/2021list_en.pdf (accessed on 16 February 2021).
4
.
World Anti-Doping Agency. Anti-Doping Testing Figures—Laboratory Report; World Anti-Doping Agency: Montreal, QC, Canada,
2
019.
5
6
.
.
Kicman, A.T. Pharmacology of anabolic steroids. Br. J. Pharmacol. 2008, 154, 502–521. [CrossRef] [PubMed]
Kicman, A.T.; Gower, D.B. Anabolic steroids in sport: Biochemical, clinical and analytical perspectives. Ann. Clin. Biochem. 2003
,
4
0, 321–356. [CrossRef]
7
8
.
.
Joseph, J.F.; Parr, M.K. Synthetic androgens as designer supplements. Curr. Neuropharmacol. 2015, 13, 89–100. [CrossRef] [PubMed]
Schanzer, W.; Geyer, H.; Donike, M. Metabolism of metandienone in man: Identification and synthesis of conjugated excreted uri-
nary metabolites, determination of excretion rates and gas chromatographic-mass spectrometric identification of bis-hydroxylated
metabolites. J. Steroid. Biochem. Mol. Biol. 1991, 38, 441–464. [CrossRef]
9
1
.
Schanzer, W. Metabolism of anabolic androgenic steroids. Clin. Chem. 1996, 42, 1001–1020. [CrossRef] [PubMed]
0. Schanzer, W.; Horning, S.; Donike, M. Metabolism of anabolic steroids in humans: Synthesis of 6 beta-hydroxy metabolites of
-chloro-1,2-dehydro-17 alpha-methyltestosterone, fluoxymesterone, and metandienone. Steroids 1995, 60, 353–366. [CrossRef]
4
1
1
1
1
1. Schanzer, W.; Delahaut, P.; Geyer, H.; Machnik, M.; Horning, S. Long-term detection and identification of metandienone and
stanozolol abuse in athletes by gas chromatography-high-resolution mass spectrometry. J. Chromatogr. B Biomed. Appl. 1996, 687,
9
3–108. [CrossRef]
2. Schänzer, W.; Geyer, H.; Horning, S. 17,17-Dimethyl-18-nor-5a-androst-1,13-dien-3a-ol (18-normetenol) in longterm detection and
confirmation of positive metandienone cases. In Recent Advances in Doping Analysis (6); Schänzer, W., Geyer, H., Gotzmann, A.,
Mareck-Engelke, U., Eds.; Sport und Buch Strauß: Cologne, Germany, 1999; pp. 37–52.
3. Parr, M.K.; Zollner, A.; Fussholler, G.; Opfermann, G.; Schlorer, N.; Zorio, M.; Bureik, M.; Schanzer, W. Unexpected contribution
of cytochrome P450 enzymes CYP11B2 and CYP21, as well as CYP3A4 in xenobiotic androgen elimination—Insights from
metandienone metabolism. Toxicol. Lett. 2012, 213, 381–391. [CrossRef] [PubMed]
4. Pozo, O.J.; Lootens, L.; Van Eenoo, P.; Deventer, K.; Meuleman, P.; Leroux-Roels, G.; Parr, M.K.; Schanzer, W.; Delbeke, F.T.
Combination of liquid-chromatography tandem mass spectrometry in different scan modes with human and chimeric mouse
urine for the study of steroid metabolism. Drug Test Anal. 2009, 1, 554–567. [CrossRef] [PubMed]
1
1
1
1
5. Massé, R.; Bi, H.; Ayotte, C.; Du, P.; Gélinas, H.; Dugal, R. Studies on anabolic steroids. J. Chromatogr. B Biomed. Appl. 1991, 562,
3
23–340. [CrossRef]
6. Esquivel, A.; Pozo, O.J.; Garrostas, L.; Balcells, G.; Gomez, C.; Kotronoulas, A.; Joglar, J.; Ventura, R. LC-MS/MS detection of
unaltered glucuronoconjugated metabolites of metandienone. Drug Test Anal. 2017, 9, 534–544. [CrossRef] [PubMed]
7. Gomez, C.; Pozo, O.J.; Garrostas, L.; Segura, J.; Ventura, R. A new sulphate metabolite as a long-term marker of metandienone
misuse. Steroids 2013, 78, 1245–1253. [CrossRef] [PubMed]
8. Schanzer, W.; Geyer, H.; Fussholler, G.; Halatcheva, N.; Kohler, M.; Parr, M.K.; Guddat, S.; Thomas, A.; Thevis, M. Mass
spectrometric identification and characterization of a new long-term metabolite of metandienone in human urine. Rapid Commun.
Mass. Spectrom. 2006, 20, 2252–2258. [CrossRef]
1
2
9. Parr, M.K.; Zoellner, A.; Bureik, M.; Dragan, C.A.; Schlörer, N.; Peters, F.; Maurer, H.; Schänzer, W. Production of metandienone
longterm-metabolite, 17β-hydroxymethyl-17α-methyl-18-nor-androsta-1,4,13-trien-3-one, using S. pombe based biotransforma-
tion assay. In Recent Advances in Doping Analysis (17); Schänzer, W., Geyer, H., Gotzmann, A., Mareck, U., Eds.; Sport und Buch
Strauß: Cologne, Germany, 2009; pp. 95–96.
0. Zollner, A.; Parr, M.K.; Dragan, C.A.; Dras, S.; Schlorer, N.; Peters, F.T.; Maurer, H.H.; Schanzer, W.; Bureik, M. CYP21-catalyzed
production of the long-term urinary metandienone metabolite 17beta-hydroxymethyl-17 alpha-methyl-18-norandrosta-1,4,13-
trien-3-one: A contribution to the fight against doping. Biol. Chem. 2010, 391, 119–127. [CrossRef] [PubMed]
1. Kratena, N.; Enev, V.S.; Gmeiner, G.; Gartner, P. Synthesis of 17beta-hydroxymethyl-17alpha-methyl-18-norandrosta-1,4,13-trien-
2
2
2
3
-one: A long-term metandienone metabolite. Steroids 2016, 115, 75–79. [CrossRef]
2. Sobolevsky, T.; Krotov, G.; Dikunets, M.; Nikitina, M.; Mochalova, E.; Rodchenkov, G. Anti-doping analyses at the Sochi Olympic
and Paralympic Games 2014. Drug Test Anal. 2014, 6, 1087–1101. [CrossRef] [PubMed]
3. Mazzarino, M.; Orengia, M.; Botre, F. Application of fast gas chromatography/mass spectrometry for the rapid screening of
synthetic anabolic steroids and other drugs in anti-doping analysis. Rapid. Commun. Mass. Spectrom. 2007, 21, 4117–4124.
[CrossRef]
2
2
2
4. Schänzer, W.; Donike, M. Metabolism of anabolic steroids in man: Synthesis and use of reference substances for identification of
anabolic steroid metabolites. Anal. Chim. Acta 1993, 275, 23–48. [CrossRef]
5. Schänzer, W.; Opfermann, G.; Donike, M. 17-Epimerization of 17α-methyl anabolic steroids in humans: Metabolism and synthesis
of 17α-hydroxy-17β-methyl steroids. Steroids 1992, 57, 537–550. [CrossRef]
6. Gomez, C.; Pozo, O.J.; Marcos, J.; Segura, J.; Ventura, R. Alternative long-term markers for the detection of methyltestosterone
misuse. Steroids 2013, 78, 44–52. [CrossRef] [PubMed]

Molecules 2021, 26, 1354
18 of 18
2
2
2
7. Martinez-Brito, D.; Iannone, M.; Tatangelo, M.A.; Molaioni, F.; de la Torre, X.; Botre, F. A further insight into methyltestosterone
metabolism: New evidences from in vitro and in vivo experiments. Rapid. Commun. Mass. Spectrom. 2020, 34, e8870. [CrossRef]
PubMed]
8. Pozo, O.J.; Van Eenoo, P.; Deventer, K.; Lootens, L.; Van Thuyne, W.; Parr, M.K.; Schanzer, W.; Sancho, J.V.; Hernandez, F.;
Meuleman, P.; et al. Detection and characterization of a new metabolite of 17alpha-methyltestosterone. Drug Metab. Dispos. 2009
7, 2153–2162. [CrossRef] [PubMed]
9. Balcells, G.; Pozo, O.J.; Esquivel, A.; Kotronoulas, A.; Joglar, J.; Segura, J.; Ventura, R. Screening for anabolic steroids in sports:
Analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem
mass spectrometry. J. Chromatogr. A 2015, 1389, 65–75. [CrossRef]
[
,
3
3
0. Sobolevsky, T.; Rodchenkov, G. Detection and mass spectrometric characterization of novel long-term dehydrochloromethyl-
testosterone metabolites in human urine. J. Steroid. Biochem. Mol. Biol. 2012, 128, 121–127. [CrossRef]
3
1. Forsdahl, G.; Geisendorfer, T.; Goschl, L.; Pfeffer, S.; Gartner, P.; Thevis, M.; Gmeiner, G. Unambiguous identification and
characterization of a long-term human metabolite of dehydrochloromethyltestosterone. Drug Test Anal. 2018. [CrossRef]
[PubMed]
3
3
3
2. Kratena, N.; Pilz, S.M.; Weil, M.; Gmeiner, G.; Enev, V.S.; Gartner, P. Synthesis and structural elucidation of a dehydrochloromethyl-
testosterone metabolite. Org. Biomol. Chem. 2018, 16, 2508–2521. [CrossRef]
3. Kratena, N.; Pfeffer, S.; Enev, V.S.; Gmeiner, G.; Gartner, P. Synthesis of human long-term metabolites of dehydrochloromethyl-
testosterone and oxymesterone. Steroids 2020, 164, 108716. [CrossRef]
4. Parr, M.K.; Zapp, J.; Becker, M.; Opfermann, G.; Bartz, U.; Schanzer, W. Steroidal isomers with uniform mass spectra of their
per-TMS derivatives: Synthesis of 17-hydroxyandrostan-3-ones, androst-1-, and -4-ene-3,17-diols. Steroids 2007, 72, 545–551.
[CrossRef]
3
5. Stoll, A.; Loke, S.; Joseph, J.F.; Machalz, D.; de la Torre, X.; Botre, F.; Wolber, G.; Bureik, M.; Parr, M.K. Fine-mapping of the
substrate specificity of human steroid 21-hydroxylase (CYP21A2). J. Steroid Biochem. Mol. Biol. 2019, 194, 105446. [CrossRef]
[PubMed]
3
3
6. Blunt, J.W.; Stothers, J.B. 13C NMR spectra of steroids—A survey and commentary. Org. Magn. Reson. 1977, 9, 439–464. [CrossRef]
7. Nishimura, S.; Shimahara, M.; Shiota, M. Stereochemistry of the Palladium-Catalyzed Hydrogenation of 3-Oxo-4-ene Steroids1. J.
Org. Chem. 1966, 31, 2394–2395. [CrossRef]
3
3
4
4
4
4
4
4
8. Parr, M.K.; Fußhöller, G.; Gütschow, M.; Hess, C.; Schänzer, W. GC-MS(/MS) investigations on long-term metabolites of 17-methyl
steroids. In Recent Advances in Doping Analysis (18); Schänzer, W., Geyer, H., Gotzmann, A., Mareck, U., Eds.; Sport und Buch
Strauß: Cologne, Germany, 2010; pp. 64–73.
9. Liu, J.; Chen, L.; Joseph, J.F.; Nass, A.; Stoll, A.; de la Torre, X.; Botre, F.; Wolber, G.; Parr, M.K.; Bureik, M. Combined chemical and
biotechnological production of 20betaOH-NorDHCMT, a long-term metabolite of Oral-Turinabol (DHCMT). J. Inorg. Biochem.
2
018, 183, 165–171. [CrossRef] [PubMed]
0. Sobolevsky, T.; Rodchenkov, G. Mass spectral characterization of novel dehydrochloromethyltestosterone and oxandrolone
metabolites after HPLC clean-up. In Recent Advances in Doping Analysis (19); Schänzer, W., Geyer, H., Gotzmann, A., Mareck, U.,
Eds.; Sportverlag Strauß: Cologne, Germany, 2011; p. 49.
1. Guddat, S.; Fussholler, G.; Beuck, S.; Thomas, A.; Geyer, H.; Rydevik, A.; Bondesson, U.; Hedeland, M.; Lagojda, A.; Schanzer, W.;
et al. Synthesis, characterization, and detection of new oxandrolone metabolites as long-term markers in sports drug testing.
Anal. Bioanal. Chem. 2013, 405, 8285–8294. [CrossRef] [PubMed]
2. Fernández-Álvarez, M.; Jianghai, L.; Cuervo, D.; Youxuan, X.; Muñoz-Guerra, J.A.; Aguilera, R. Detection of new Oral-Turinabol
metabolites by LC-QToF. In Recent Advances in Doping Analysis (22); Schänzer, W., Geyer, H., Gotzmann, A., Mareck, U., Eds.;
Sportverlag Strauß: Cologne, Germany, 2014; pp. 182–187.
3. Parr, M.K.; Piana, G.L.; Stoll, A.; Joseph, J.F.; Loke, S.; Schlörer, N.; Torre, X.d.l.; Botrè, F. Tracing Back Drug Misuse—Proper
Metabolite Identification Requires Synthesis. In Proceedings of the TIAFT 2019: The 57th Annual Meeting of the International
Association of Forensic Toxicologists, Birmingham, UK, 6 September 2019.
4. Macdonald, B.S.; Sykes, P.J.; Adhikary, P.M.; Harkness, R.A. The identification of 17
as a metabolite of the anabolic steroid drug 17 -hydroxy-17-methyl-1,4-androstadien-3-one in man. Steroids 1971, 18, 753–766.
CrossRef]
5. Galletti, F.; Gardi, R. Metabolism of 1-dehydroand rostanes in man. Steroids 1971, 18, 39–50. [CrossRef]
α-hydroxy-17-methyl-1,4-androstadien-3-one
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