Seized designer supplement named "1-Androsterone": Identification as 3β-hydroxy-5α-androst-1-en-17-one and its urinary elimination

Article abstract of DOI:10.1016/j.steroids.2011.02.001

New analogues of androgens that had never been available as approved drugs are marketed as "dietary supplement" recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids. In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product "1-Androsterone" of the brand name "Advanced Muscle Science" was labeled to contain 100 mg of "1-Androstene-3b-ol,17-one" per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3β-hydroxy-5α-androst-1-en-17- one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3β-hydroxy-5α- androst-1-en-17-one in the capsules as labeled. Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17β-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17- one, 5α-androst-1-ene-3α,17β-diol, and 5α-androst-1-ene- 3β,17β-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of "1-Androsterone". Especially the ratios of androsterone/ etiocholanolone and 5α-/5β-androstane-3α,17β-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule.

Full text of DOI:10.1016/j.steroids.2011.02.001

Steroids 76 (2011) 540–547
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Seized designer supplement named “1-Androsterone”: Identification as
3␤-hydroxy-5␣-androst-1-en-17-one and its urinary elimination
Maria K. Parra,∗, Georg Opfermanna, Hans Geyera, Folker Westphalb, Frank D. Sönnichsenc,
Josef Zappd, Dorota Kwiatkowskae, Wilhelm Schänzera
a Center for Preventive Doping Research, Institute of Biochemistry, German Sport University Cologne, Am Sportpark Muengersdorf 6, 50933 Cologne, Germany
b State Bureau of Criminal Investigation Schleswig-Holstein, Section Narcotics and Toxicology, 24116 Kiel, Germany
c Otto-Diels-Institute of Organic Chemistry, Christian-Albrechts-University, 24098 Kiel, Germany
d Institute of Pharmaceutical Biology, Saarland University, 66041 Saarbruecken, Germany
e Department of Anti-Doping Research, Institute of Sport, 01-982 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
New analogues of androgens that had never been available as approved drugs are marketed as “dietary
supplement” recently. They are mainly advertised to promote muscle mass and are considered by the
governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as
being pharmacologically and/or chemically related to anabolic steroids.
In the present study, we report the detection of a steroid in a product seized by the State Bureau of
Criminal Investigation Schleswig-Holstein, Germany. The product “1-Androsterone” of the brand name
“Advanced Muscle Science” was labeled to contain 100 mg of “1-Androstene-3b-ol,17-one” per capsule.
The product was analyzed underivatized and as bis-TMS derivative by GC–MS. The steroid was iden-
tified by comparison with chemically synthesized 3␤-hydroxy-5␣-androst-1-en-17-one, prepared by
reduction of 5␣-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by
nuclear magnetic resonance. Semi-quantitation revealed an amount of 3␤-hydroxy-5␣-androst-1-en-
17-one in the capsules as labeled.
Following oral administration to a male volunteer, the main urinary metabolites were monitored.
1-Testosterone (17␤-hydroxy-5␣-androst-1-en-3-one), 1-androstenedione (5␣-androst-1-ene-3,17-
dione), 3␣-hydroxy-5␣-androst-1-en-17-one, 5␣-androst-1-ene-3␣,17␤-diol, and 5␣-androst-1-ene-
3␤,17␤-diol were detected besides the parent compound and two more metabolites (up to now not finally
identified but most likely C-18 and C-19 hydroxylated 5␣-androst-1-ene-3,17-diones). Additionally, com-
mon steroids of the urinary steroid profile were altered after the administration of “1-Androsterone”.
Especially the ratios of androsterone/etiocholanolone and 5␣-/5␤-androstane-3␣,17␤-diol and the con-
centration of 5␣-dihydrotestosterone were influenced. 3␣-Hydroxy-5␣-androst-1-en-17-one appears
to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was
detectable in screening up to nine days after a single administration of one capsule.
Received 14 September 2010
Received in revised form
23 December 2010
Accepted 2 February 2011
Available online 16 February 2011
Keywords:
1-Androsterone
Metabolism
Steroid profile
Doping control
GC–MS
NMR
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
introduced on the dietary supplement market as prohormone
of 1-testosterone (17␤-hydroxy-5␣-androst-1-en-3-one), quickly
For a few years, several steroids have been available on the
Internet as sport supplements, often sold by bodybuilding sites.
Following the classification of the so-called prohormones of testos-
terone and nandrolone as schedule III controlled substances by
the anabolic steroids control act [1] in the USA, more and more
products appeared on the market, which contain steroids that
have never been marketed as approved drugs [2–6]. Already
in 2001, “1-Androstenediol” (5␣-androst-1-ene-3␰,17␤-diol) was
followed by 1-androstenedione (5␣-androst-1-ene-3,17-dione).
Only very limited information is available on the metabolism
of these steroids. Information on the in vitro metabolism of
5␣-androst-1-ene-3,17-dione and 5␣-androst-1-ene-3␤,17␤-diol
is available, albeit with the limited focus on the determina-
tion of the 17␤-hydroxy and the 3␤,17␤-dihydroxy analogues,
testosterone, dihydrotestosterone and androst-4-ene-3,17-dione
[7]. An excretion study with 1-testosterone (1) reported the
identification of 17␤-hydroxy-5␣-androst-1-en-3-one (1) and 5␣-
androst-1-ene-3,17-dione (2) as metabolites [8]. The structure
of the main metabolite in all volunteers remained unconfirmed
up to now. Further, while considerations on the influences of
∗
Corresponding author. Tel.: +49 221 4982 4960; fax: +49 221 497 32 36.
E-mail address: m.parr@biochem.dshs-koeln.de (M.K. Parr).
0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.02.001

M.K. Parr et al. / Steroids 76 (2011) 540–547
541
the steroid administration on the levels on some endogenous
2.4. Instrumentation
steroids were made in this investigation, they focused only on
the testosterone/epitestosterone (T/EpiT) ratio. As the product “1-
Androsterone” appeared to contain an active content such as these
three above mentioned 1-ene steroids that are covered by the cur-
rent drug regulations in several countries, e.g. the US and Germany,
the product was confiscated. Herein we report the identification of
its active content. For elucidation of the urinary elimination post-
administration urines are analyzed and the elimination profiles of
several metabolites as well as the alterations in the urinary steroid
profile are monitored.
2.4.1. NMR spectroscopy
The NMR data were obtained using a Bruker DRX 500 instru-
ment, equipped with a 5 mm inverse probehead with actively
shielded z-gradient coil. Chemical shifts were given in ı values
(ppm) relative to tetramethylsilane. The spectra were recorded at
500 MHz (1H) and 125 MHz (13C) at 298 K using solutions of about
5 mg of each compound in deuterated chloroform. For confirmation
of the assumed structures 1H, 13C, and DEPT spectra were measured
together with 2D HH-gCOSY, HSQC and HMBC experiments.
2.4.2. GC–MS analyses
2. Experimental
The GC–MS analyses of the TMS derivatives were performed
on an Agilent 6890N gas chromatograph coupled to an Agilent
5973 inert mass selective detector (MSD) GC–MS system applying
the following parameters: column: Agilent Ultra-1 (polysiloxane,
17 m; 0.20 mm i.d.; 0.11 ␮m film thickness), carrier gas: helium,
head pressure: 1 bar, oven temperature program: 0 min 183 ◦C,
+3 ◦C/min, 0 min 232 ◦C, +40 ◦C/min, 2 min 310 ◦C, injection vol-
ume: 3 ␮L, split 1:16, injection temperature: 300 ◦C, ionization:
70 eV, EI, full scan mode, 40–800 Da or in case of the urine
extract analysis selected ion monitoring. Additionally, the under-
ivatized compounds were analyzed on a GC Agilent 6890 coupled
to a MSD Agilent 5973, injection volume: 2 ␮L, splitless, injec-
tion temperature: 300 ◦C, column: Macherey-Nagel Optima-P-XLB
column (diphenyl dimethyl silarylene, 30 m, 0.25 mm i.d., 0.5 ␮m
film thickness), carrier gas: helium, 3 mL/min, oven temperature
program: 3 min 60 ◦C, +40 ◦C/min, 0 min 260 ◦C, +5 ◦C/min, 0 min
330 ◦C, ionization: 70 eV, EI, data acquisition: full scan mode,
40–400 Da.
2.1. Supplement
The product “1-Androsterone” of the brand name “Advanced
Muscle Science” was seized and provided by the State Bureau
of Criminal Investigation Schleswig-Holstein (Kiel, Germany). It
was labeled to be a dietary supplement containing 100 mg of “1-
Androstene-3b-ol,17-one” per capsule.
2.2. Chemicals and reagents
Testosterone
androstane-3␣,17␤-diol
(Bdiol), and dehydroepiandrosterone (3␤-hydroxyandrost-
5-en-17-one) were obtained from NMI (Sydney, Australia).
Epitestosterone
etiocholanolone
androsterone
(17␤-hydroxyandrost-4-en-3-one),
5␣-
(Adiol),
5␤-androstane-3␣,17␤-diol
(EpiT,
(ETIO,
(AND,
17␣-hydroxyandrost-4-en-3-one),
3␣-hydroxy-5␤-androstan-17-one),
3␣-hydroxy-5␣-androstan-17-one),
(17␤-hydroxy-17␣-methylandrost-4-en-
methyltestosterone
2.5. Preparation for GC–MS measurement
3-one), LS-selectride (lithium tris-isoamylborohydride, 1 M in
tetrahydrofuran), was purchased from Sigma–Aldrich GmbH
(Steinheim, Germany), dihydrotestosterone (DHT, 17␤-hydroxy-
5␣-androstan-3-one) from Serva (Heidelberg, Germany),
1-testosterone (1, 17␤-hydroxy-5␣-androst-1-en-3-one) from
Thinker Chemicals (Hanghzou, China) and 1-androstenedione
(2, 5␣-androst-1-ene-3,17-dione) from Steraloids (Wilton,
USA). d3-Testosterone, d3-epitestosterone, d4-etiocholanolone,
d5-androsterone-glucuronide, and 3␣-hydroxy-5␤-androst-1-
en-17-one were synthesized in our laboratory. ␤-Glucuronidase
from Escherichia coli (E. coli, >140 U/mL) was obtained from Roche
Diagnostics (Mannheim, Germany), N-methyl-N-trimethylsilyl-
trifluoroacetamide (MSTFA) from Chem. Fabrik Karl Bucher
(Waldstetten, Germany).
The final residues of sample preparation were either derivatized
with TMIS reagent (MSTFA/NH4I/ethanethiol, 1000:2:3, v:w:v) by
heating for 20 min at 60 ◦C as introduced by Donike [10] and Geyer
et al. [11] or reconstituted in acetone and injected into the GC–MS.
As reference, solutions of the respective steroids in methanol were
evaporated to dryness and processed in the same manner.
Relative retention times (RRT) were calculated using testos-
terone as reference.
2.6. Supplement analysis
For identification of the steroid ingredient 1.0 g of the capsule
content was extracted with n−hexane in a Soxhlet apparatus. After
crystallization from n−hexane the steroid was characterized by
GC–MS and NMR.
All other reagents and solvents were of analytical grade and
obtained from Merck (Darmstadt, Germany).
2.3. Synthesis of reference material
2.7. Administration study
Several isomers of 5␰-androst-1-ene-3␰,17␰-diol, including 5␣-
androst-1-ene-3␣,17␤- (5) and 3␤,17␤-diol (6), were synthesized
by reduction from the respective oxo-analogues as described else-
where [9].
An administration study using one capsule of the above-
mentioned product was performed in one healthy male volunteer
(68 years, 85 kg, p.o.) and urine samples were collected for two
weeks. It was approved by Research Ethical Committee at the Sports
Institute, Warsaw. The samples were prepared according to the
routine steroid screening procedure used in our laboratory [11]. In
brief, after addition of 40 ␮l of the internal standard solution con-
taining methyltestosterone, d3-testosterone, d3-epitestosterone,
d4-etiocholanolone and d5-androsterone-glucuronide 2 mL of
urine were incubated at pH 7 with ␤-glucuronidase from E. coli
at 50 ◦C for 1 h. The steroids were extracted with 5 mL of TBME
at pH 9.6, the organic layer was evaporated to dryness and ana-
lyzed by GC–MS. For quality control blank samples are analyzed in
2.3.1. 3␰-Hydroxy-5˛-androst-1-en-17-one (3 and 4)
To a stirred solution of 1 mg of 5␣-androst-1-ene-3,17-dione
(2) in 1 mL of absolute diethyl ether 10 ␮L of LS-selectride solution
were added. After 1 h at ambient temperature 1 mL of an aqueous
ammonium chloride solution (10% in H2O) was added, pH∼10 was
adjusted by adding solid sodium hydrogencarbonate/potassium
carbonate (2:1, w:w) and the mixture was extracted with 5 mL of
t-butyl methyl ether (TBME).

542
Abund.
M.K. Parr et al. / Steroids 76 (2011) 540–547
Abund.
OTMS
194
1000
800
600
400
200
0
73
1000
800
600
400
200
0
OTMS
417
432
73
TMSO
432
275
290
H
TMSO
H
206
179
417
403
129
169
91
80
327
320
403
400
195
200
342
143
[m/z]
80
120 160 200 240 280 320 360 400
OH
[m/z]
288
40
120
160
240
280
360
Abund.
1000
O
Abund.
1000
246
218
122
288
800
600
400
200
0
800
600
400
200
0
O
161
H
79
HO
91
H
190
204
41
122
120
55
149
173
259
40
80
120
160
200
240
[m/z]
[m/z]
40
80
160
200
240
Fig. 1. Mass spectra of 17␤-hydroxy-5␣-androst-1-en-3-one (1-testosterone, (1)),
upper bis-TMS, M+ = 432, lower underivatized, M+ = 288.
Fig. 3. Mass spectra of 3␤-hydroxy-5␣-androst-1-en-17-one (3), upper bis-TMS,
M+ = 432, lower underivatized, M+ = 288.
the same manner with every batch to check for the absence of the
analytes.
The stereochemistry at C-3 and C-17 of 5␣-androst-1-ene-
3␣,17␤-diol (5) and -3␤,17␤-diol (6) could be deduced from their
NMR data: The 1H and 13C NMR shifts of their ring C and D protons
and carbons were similar, indicating the same orientation of the 17-
hydroxy group. Comparison of this data with those of 17-hydroxy
epimeric sterols from the literature [12] clearly identified both
compounds as 17␤-hydroxy derivatives. The 1H NMR of (6) showed
for H-3 the same diaxial coupling with H-4ax as found for (3). There-
fore, (6) is 5␣-androst-1-ene-3␤,17␤-diol. This implicated (5) to
be its 3␣-hydroxy epimer, which was furthermore proven by the
coupling constants of H-3eq with H-4eq and H-4ax, both 4.5 Hz.
The mass spectra of the TMS derivatives of the androstanes
with 3-hydroxy-1-ene structure (Figs. 3–5) show fragments with
m/z 142 and 143 (corresponding to C1–C4 of the A-ring) and
M+-142 (corresponding to a neutral loss of C1–C4) and M+-15-
3. Results and discussion
3.1. Synthesis and characterization of reference material
Some of the androst-1-enes, either commercially available or
synthesized by reduction from the respective oxo-analogues were
analyzed by GC-EI-MS to identify the compounds detected (mass
spectra in Figs. 1–5). The generation of the 5␰-androst-1-ene-
3␰,17␰-diols was already reported previously. All these epimers
were found to reveal very similar mass spectra [3,9]. The retention
times of the steroids (as per-TMS derivatives and as underivatized
compounds) are listed in Table 1 and the NMR data of selected
compounds are shown in Table 2.
Abund.
Abund.
OTMS
73
1000
800
600
400
200
0
415
1000
800
600
400
200
0
OTMS
417
432
73
TMSO
143
H
275
290
TMSO
H
430
169
327
194
91
195
403
400
147
169
223
45
40
325
[m/z]
80
120
160
200
O
240
280
320
360
[m/z]
286
80
120 160 200 240 280 320 360 400
O
Abund.
1000
Abund.
1000
288
122
244
218
800
600
400
200
0
800
600
400
200
0
HO
O
91
190
H
H
161
79
67
259
122
120
202
41
40
41
40
147
80
160
200
240
280 [m/z]
[m/z]
280
80
120
160
200
240
Fig. 2. Mass spectra of 5␣-androst-1-ene-3,17-dione (2), upper bis-TMS, M+ = 430,
Fig. 4. Mass spectra of main metabolite, identified as 3␣-hydroxy-5␣-androst-1-
en-17-one (4), upper bis-TMS, M+ = 432, lower underivatized, M+ = 288.
lower underivatized, M+ = 286.

M.K. Parr et al. / Steroids 76 (2011) 540–547
543
Table 1
Molecular ions (M+) and relative retention times (RRT) of steroids used as reference (calculated using testosterone, RT(bis-TMS) = 13.01 min, RT(underiv.) = 17.76 min).
Analyte
Bis-TMS
RRT
Underivatized
RRT
M+
M+
17␤-Hydroxy-5␣-androst-1-en-3-one (1)
5␣-Androst-1-ene-3,17-dione (2)
3␤-Hydroxy-5␣-androst-1-en-17-one (3)
3␣-Hydroxy-5␣-androst-1-en-17-one (4)
5␣-Androst-1-ene-3␣,17␤-diol (5)
5␣-Androst-1-ene-3␤,17␤-diol (6)
0.91
0.88
0.90
0.89
0.91
0.92
432
430
432
432
434
434
0.95
0.95
0.88
0.87
0.92
0.93
288
286
288
288
290
290
Table 2
1H and 13C NMR spectral data of 3␤-hydroxy-5␣-androst-1-en-17-one (3), 5␣-androst-1-ene-3␣,17␤-diol (5) and 5␣-androst-1-ene-3␤,17␤-diol (6).
3␤-Hydroxy-5␣-androst-1-en-17-one (3)
5␣-Androst-1-ene-3␣,17␤-diol (5)
5␣-Androst-1-ene-3␤,17␤-diol (6)
ıC
ıH
ıC
ıH
ıC
ıH
1
2
3
4
137.62
129.19
68.89
5.906 dd (10/2)
5.510 ddd (10/2/2)
4.312 dddd (9.5/7/2/2)
1.78 dddd (12/7/1/1)
1.52 m
1.43 m
1.81 m
1.02 m
140.39
126.11
64.81
6.078 d (10)
5.652 ddd (10/4.5/1.5)
4.097 dd (4.5/4.5)
1.79 m
1.51 m
1.56 m
137.94
128.74
68.85
5.917 dd (10/2)
5.492 ddd (10/2/2)
4.303 dddd (9.5/7/2/2)
1.76 m
1.50 m
1.45 m
1.69 m
0.90m
35.91
34.84
35.88
5
6
43.64
30.86
39.04
31.49
43.56
31.43
1.70 m
0.94 m
7
27.95
1.41 m
1.41 m
1.60 ddd (11/11/4)
0.89 m
–
1.81 m
1.40 m
1.83 m
1.26 m
27.86
1.37 m
1.33 m
1.44 m
0.87 m
27.98
1.35 m
1.35 m
1.47 m
0.84 m
–
1.74 m
1.38 m
1.82 m
1.07 m
–
0.99 m
1.59 m
1.26 m
2.06 m
1.43 m
3.634 dd (8.5/8.5)
0.747 s
0.920 s
8
9
10
11
35.32
51.59
38.34
20.68
35.83
51.13
38.13
20.72
35.66
51.59
38.17
20.86
–
1.75 m
1.37 m
1.83 m
1.07 m
12
31.66
36.71
36.68
13
14
15
48.04
51.72
21.87
–
43.11
51.13
23.41
–
43.13
50.08
23.34
1.30 ddd (13/11/6)
1.93 ddd (12/9/6)
1.50 m
2.44 dd (19/9)
2.07 ddd (19/19/9)
–
0.99 m
1.59 m
1.26 m
2.16 m
1.47 m
3.646 dd (8.5/8.5)
0.753 s
16
35.93
30.54
30.53
17
18
19
221.18
14.07
15.80
81.87
11.28
13.87
81.89
11.27
15.70
0.876 s
0.938 s
0.813 s
Abund.
1000
142. In case of 17-hydroxy structures (Figs. 1 and 5), the A-ring
fragments are quite abundant, while the B/C/D-ring fragments
are abundant in the 17-oxo steroids (Figs. 2–4). Additionally, the
loss of 15 Da is very dominant in the 17-oxo androstanes. The
spectra of the TMS-derivatized 3-oxo-1-enes show characteristic
fragments at m/z 179, 194 and 206 corresponding to the A/B-ring
(Figs. 1 and 2).
The mass spectra of the underivatized steroids show a loss of
42 Da for the 3-oxo-1-enes (Figs. 1 and 2), 59 Da for the 1-ene-3,17-
diols (Fig. 5) and 70 Da in case of 3␤-hydroxy-5␣-androst-1-en-17-
one (Fig. 3) generating the base peak in the spectra. Additionally,
all underivatized 1-enes with an oxo group either at C-3 or C-17
show m/z 122 in their spectra.
OTMS
434
800
600
400
200
0
73
143
TMSO
H
405
419
202
200
377
343
292
280
[m/z]
80
120
160
240
320
360 400
Abund.
1000
OH
231
290
800
600
400
200
0
3.1.1. 3␰-Hydroxy-5˛-androst-1-en-17-one (3 and 4)
HO
The reduction of 5␣-androst-1-ene-3,17-dione (2) with LS-
selectride yielded the two isomeric 3␤- (3) and 3␣-hydroxy (4)
analogues in a ratio of 85:15. In accordance to the literature [13,14]
the reduction of 3-oxo-5␣-androst-1-enes with complex borohy-
drides mainly resulted in 3␤-hydroxy isomers which show longer
retention times than the 3␣-hydroxy isomers. The GC–MS reten-
tion times and spectra of the 3␤-isomer (3) in the mixture and
the steroid extracted from the supplement matched and thus con-
firmed the assignment. Because of very low amounts prepared,
separation of the isomers was only performed by GC–MS. Thus, 3␣-
H
79
55
123
120
163
199
200
257
[m/z]
40
80
160
240
Fig. 5. Mass spectra of metabolite, identified as 5␣-androst-1-ene-3␣,17␤-diol (5),
upper bis-TMS, M+ = 434, lower underivatized, M+ = 290.

544
M.K. Parr et al. / Steroids 76 (2011) 540–547
Abund.
73
1000
800
600
400
200
0
415
179
222
103
100
518
[m/z]
147
310
300
200
400
500
Abund.
1000
503
73
800
600
400
200
0
Fig. 6. 1H NMR spectrum (500 MHz, CDCl3, 298 K) of 3␤-hydroxy-5␣-androst-1-en-
17-one (3), extracted from the product “1-Androsterone”.
518
147
hydroxy-5␣-androst-1-en-17-one (4) was not amenable to NMR
analysis.
193
200
415
400
295
300
100
500 [m/z]
Fig. 7. Mass spectra of hydroxylated metabolites as tris-TMS derivatives upper:
3.2. Supplement analysis
HO-Dione1 (M+ = 518, RRT = 1.14), lower HO-Dione2 (M+ = 518, RRT = 1.26).
The total capsule content was determined with 555 mg of pow-
der per capsule. The depletive extraction of 1 g resulted in 205.9 mg
of steroid, corresponding to 115 mg/capsule. It was identified as
3␤-hydroxy-5␣-androst-1-en-17-one (3) by NMR and GC–EI–MS
(NMR spectrum and data in Fig. 6 and Table 2, GC retention times in
Table 1 and mass spectra in Fig. 3). All data of the extracted material
complied to that of the synthesized reference (3).
1000
800
600
400
200
0
10000
8000
6000
4000
2000
0
Its 1H and 13C NMR data (Table 2) revealed the structure of
a hydroxy androstenone. The position of the functional groups
could be deduced from the 2D NMR HHCOSY, HSQC and HMBC
spectra and led to a 3-hydroxy-androst-1-en-17-one. The H-3
coupling pattern exhibits a pseudo diaxial coupling with H-4ax
,
JH-3ax/H-4ax = 9.5 Hz, indicating a pseudo equatorial position for the
hydroxyl group, which therefore is 3␤-orientated. The stereochem-
istry at C-5 followed directly from the comparison of the NMR
data with those of 3␤-hydroxy-5␰-androst-1-enes that were syn-
thesized some years ago in our lab [14]. The values for C-3/H-3
and C-5/H-5 matched perfectly with the 5␣ series. Therefore, the
structure of the steroid was identified as 3␤-hydroxy-5␣-androst-
1-en-17-one (3).
0
48
96
144
Mean time of collection [h]
Fig. 8. Elimination kinetics of 3␤-hydroxy-5␣-androst-1-en-17-one (3, ꢀ, left y-
axis), 17␤-hydroxy-5␣-androst-1-en-3-one (1, ꢀ, right y-axis), and 5␣-androst-1-
ene-3,17-dione (2, ꢁ, left y-axis), after one capsule of “1-Androsterone”, data shown
using midpoints of collection periods and concentrations in 1000-fold peak area of
analyte divided by area of internal standard.
3.3. Administration study
respectively. The 5␣-androst-1-ene-3␰,17␤-diols (5) and (6) were
unambiguously identified for about one (3␤-) and three days (3␣-)
due to their elution in a region of relatively high background in the
chromatograms. An optimized sample pretreatment may eliminate
this in case of confirmation of a positive screening result. Posi-
tive screening results were obtained for about six (1), eight (2),
four (5), and three days (6). Additionally, two more metabolites
were detected in the early post-administration urines (HO-Dione1
and HO-Dione2). Based on their mass spectra (Fig. 7), structures of
androst-1-ene-3,17-diones hydroxylated most likely at position C-
18 (OH-Dion1) and C-19 (OH-Dion2) are proposed due to the loss
of 103 u (TMS-O-CH2•) from the molecule resulting in m/z 415.
The elimination kinetics of the above mentioned androst-1-enes
are displayed in Figs. 8–11.
Following the oral administration of one capsule of “1-
Androsterone”, corresponding to 115 mg of 3␤-hydroxy-5␣-
androst-1-en-17-one (3), to one healthy male volunteer the
combined unconjugated and glucuronidated metabolite fractions
were analyzed as per-TMS derivatives as used in routine sports
doping control. The parent compound (3) was detectable in the
urines for about two days with the highest concentration detected
in the 0–3 h urine. Its 3␣-isomer (4) was identified by com-
parison with the unpurified reference obtained by reduction of
1-androstenedione with LS-Selectride. It was found to be the
metabolite detectable for the longest time. The highest concen-
tration was found in the 3–7.5 h urine, while its presence could
still be confirmed up to seven days (ions used: m/z 432, 275,
and 169) according to the regulations applicable in human sports
doping control [15]. Suspicious results in screening analysis were
possible for two more days. Applying these criteria, 5␣-androst-
1-ene-3,17-dione (m/z 415, 430, and 194) and 1-testosterone (m/z
194, 432, and 417) were detectable for six and almost four days,
In addition, the influence of the administration on the
endogenous urinary steroids was monitored. The steroid
profile ratios of the corresponding 5␣/5␤-steroids, namely
androsterone/etiocholanolone
(AND/ETIO)
and
5␣-/5␤-
androstane-3␣,17␤-diol (Adiol/Bdiol), were significantly increased
(Fig. 12). Concomitantly increased concentrations of epiandros-

M.K. Parr et al. / Steroids 76 (2011) 540–547
545
Table 3
Selected steroid profile data of the post-administration urines and criteria indicative for DHT administration in males.
Collection period [h]
c (DHT) [ng/mL]
>21a
Adiol/Bdiol
>1.5
AND/ETIO
>2.9
DHT/ETIO
>8.2
DHT/EpiT
Decision limit
>0.73
Sample 01
Sample 02
Sample 03
Sample 04
Sample 05
Sample 06
Sample 07
Sample 08
Sample 09
Sample 10
Sample 11
Sample 12
Sample 13
Sample 14
Sample 15
Sample 16
Sample 17
Sample 18
Sample 19
Sample 20
Sample 21
0:00–2:50
2:50–7:30
20.7
142.6
152.5
101.8
143.2
94.4
96.1
57.7
18.2
66.2
98.9
63.1
27.1
28.4
10.7
22.2
14.8
11.8
13.6
26.0
24.4
0.4
1.1
3.3
2.9
2.9
2.5
2.0
1.6
1.5
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.4
0.4
0.4
0.4
4.6
18.7
6.7
5.1
4.8
4.6
4.8
4.5
4.3
2.6
2.4
2.3
2.2
2.0
1.7
1.4
1.2
1.2
1.1
1.0
1.2
18.2
279.5
116.1
98.1
92.7
89.4
102.8
90.4
88.9
60.0
51.5
46.0
36.5
29.9
27.2
29.4
30.4
36.0
30.9
24.2
19.8
0.3
0.9
1.8
2.0
2.6
2.3
2.4
2.1
1.4
1.5
1.7
1.5
1.0
0.8
0.6
0.7
0.8
0.8
0.7
0.8
0.6
7:30–11:00
11:00–18:00
18:00–20:30
20:30–24:40
24:40–28:10
28:10–30:10
30:10–31:20
31:20–42:30
42:30–44:40
44:40–46:20
46:20–48:20
48:20–52:30
52:30–54:30
54:30–58:10
58:10–60:20
60:20–62:10
62:10–67:00
67:00–69:40
69:40–74:30
a
Adjusted for specific gravity of 1.020 g/mL.
20000
750
500
250
15000
10000
5000
0
0
0
0
72
144
216
24
48
72
96
Mean time of collection [h]
Mean time of collection [h]
Fig. 9. Elimination kinetics of 3␣-hydroxy-5␣-androst-1-en-17-one (4, ᭹, main
metabolite) after one capsule of “1-Androsterone”, data shown using midpoints of
collection periods and concentrations in 1000-fold peak area of analyte divided by
area of internal standard.
Fig. 10. Elimination kinetics of 5␣-androst-1-ene-3␣,17␤-diol (5, ꢂ) and 5␣-
androst-1-ene-3␤,17␤-diol (6, ꢁ) after one capsule of “1-Androsterone”, data shown
using midpoints of collection periods and concentrations in 1000-fold peak area of
analyte divided by area of internal standard.
O
OH
OH
O
(3)
(EpiA)
HO
HO
HO
HO
HO
(6)
(3β-Adiol)
H
H
H
H
O
O
O
O
O
O
H
(HO-Dione1/2)
O
HO
HO
HO
HO
(AND)
(4)
(5)
(2)
H
H
H
H
HO
OH
OH
OH
OH
O
H
O
O
(Adiol)
(1)
H
H
H
(DHT)
Scheme 1. Proposed metabolism of 3␤-hydroxy-5␣-androst-1-en-17-one (3).

546
M.K. Parr et al. / Steroids 76 (2011) 540–547
4000
3000
2000
1000
800
tracing back the administered substance to these steroids. The
proposed metabolism is illustrated in Scheme 1. As these data
are based on only one administration study future research is
needed to reinforce these findings. Additionally the analysis of
the sulfoconjugated metabolites has to be addressed to future
research.
600
400
200
0
4. Conclusions
The present results demonstrate that another “prohormone”
with 1-ene structure is now available on the market. It is advertised
to be “pro anabolic” and displays “even improved properties com-
pared to the old 1-Andro or 1-Test products”. While 1-testosterone,
5␣-androst-1-ene-3,17-dione, and 5␣-androst-1-ene-3␰,17␤-diol
are covered by general legislation in several countries, the clas-
sification of 3␤-hydroxy-5␣-androst-1-en-17-one is not explicitly
specified. In sports it is covered by the WADA list of prohibited sub-
stances [18] by the wording “and other substances with a similar
chemical structure or similar biological effect(s)”, and therefore, its
use by athletes is prohibited.
0
0
24
48
Mean time of collection [h]
Fig. 11. Elimination kinetics of HO-Dione1 (*, left y-axis) and HO-Dione2 (×, right
y-axis) after one capsule of “1-Androsterone”, data shown using midpoints of col-
lection periods and concentrations in 1000-fold peak area of analyte divided by area
of internal standard.
4
3
2
1
0
20,0
15,0
10,0
5,0
Acknowledgements
The Manfred Donike Institut für Doping Analytik e.V., Cologne,
and the Federal Ministry for the Interior, Berlin, are acknowledged
for financial support of the study.
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