Identification of steroid isoxazole isomers marketed as designer supplement

Article abstract of DOI:10.1016/j.steroids.2008.11.006

The product Orastan-A from Gaspari Nutrition was analyzed for its steroid content. According to the labeling, it is supposed to contain "5a-Androstano[2,3-c]furazan-17b-tetrahydropyranol ether", also called furazadrol-THP ether. The GC-MS analyses of the

Full text of DOI:10.1016/j.steroids.2008.11.006

Steroids 74 (2009) 322–328
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Identification of steroid isoxazole isomers marketed as designer supplement
Maria K. Parra,∗, Michael Gütschowb, Jörg Danielsc, Georg Opfermanna,
Mario Thevisa, Wilhelm Schänzera
a Center for Preventive Doping Research, Institute of Biochemistry, German Sport University Cologne, Carl-Diem-Weg 6, 50933 Cologne, Germany
b Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Germany
c Institute for Inorganic Chemistry, University of Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 17 November 2008
The product Orastan-A from Gaspari Nutrition was analyzed for its steroid content. According to the label-
ing, it is supposed to contain “5a-Androstano[2,3-c]furazan-17b-tetrahydropyranol ether”, also called
furazadrol-THP ether. The GC–MS analyses of the liberated steroids (after extraction from the capsule
matrix and cleavage of the THP ether, TMS-derivative and underivatized) revealed mass spectra of two
components, both inconsistent with the labeling. Thus, the steroids were characterized by different ana-
lytical techniques such as mass spectrometry, nuclear magnetic resonance spectroscopy and X-ray crystal
structure analysis. They were identified as 17␤-hydroxyandrostano[3,2-c]isoxazole and -[2,3-d]isoxazole.
© 2008 Elsevier Inc. All rights reserved.
Keywords:
17␤-Hydroxyandrostanoisoxazoles
Designer supplement
GC–MS
NMR
X-ray
1. Introduction
the other androstanoisoxazoles are covered by the wording “and
other substances with a similar chemical structure or similar bio-
During the last few years more and more products appeared
on the market containing steroids that were never approved as
therapeutic drugs mostly without proper labeling of the contents
[1–7]. Recently, preparations containing stanozolol analogues, e.g.
Prostanozol (17␤-hydroxy-5␣-androstano[3,2-c]pyrazole), were
also advertised on the Internet market for sport supplements [5,8].
In this study, we report the detection of another unap-
logical effect(s)”.
2. Experimental
2.1. Chemicals and reagents
Androisoxazol was obtained from Prof. Manfred Donike
(Cologne, Germany), Danazol from Thinker Chemical (Hangzou,
China). 5␣-Dihydrotestosterone (17␤-hydroxy-5␣-androstan-3-
one) and hydroxylamine hydrochloride were purchased from
Sigma–Aldrich GmbH (Steinheim, Germany) and N-methyl-N-
trimethylsilyl-trifluoroacetamide (MSTFA) from Chem. Fabrik Karl
Bucher (Waldstetten, Germany). All other reagents and solvents
were of analytical grade and obtained from VWR (Darmstadt, Ger-
many).
proved steroid in
a “dietary supplement”. Two isomers of
17␤-hydroxyandrostanoisoxazole (structure formula in Fig. 1) were
identified by different analytical techniques. The synthesis of
these steroids was already described in the 1960s [9]. The [2,3-
d]-isomer was reported to show anabolic properties after s.c.
application while the [3,2-c]-isomer has lower activity [9,10].
Following oral administration, both isomers were found to be inac-
tive [10]. The 17-methylated Androisoxazol ([3,2-c]isoxazole) is
listed under the therapeutic category “Anabolic” by the Merck
Index and the only approved androstanoisoxazole, Danazol (17␣-
ethinyl-17␤-hydroxyandrost-4-eno[2,3-d]isoxazole), is classified
as antigonadotropin [11]. Danazol (e.g. Danatrol®, Cyclomen®,
Danol®) is available in several countries, especially for the treat-
ment of endometriosis [12]. According to the doping regulations
of the World Anti-Doping Agency (WADA), anabolic androgenic
steroids are prohibited substances for use in sports [13]. While
Danazol is explicitly mentioned in the list of prohibited substances,
2.2. Instrumentation
2.2.1. GC–MS analyses
For GC–MS analyses the residues were either dissolved in cyclo-
hexane or derivatized with trimethyliodosilane (TMIS) reagent
(MSTFA/NH4I/ethanethiol, 1000:2:3, v:w:v) by heating for 20 min
at 60 ◦C and injected into the GC–MS.
The analyses of the underivatized compounds were carried out
on a gas chromatograph (GC) Agilent 6890 coupled to a mass selec-
tive detector (MSD) Agilent 5973, injection volume: 2 ␮L, splitless,
injection temperature: 220 ◦C, column: Macherey-Nagel Optima ı3
(20 m, 0.25 mm i.d., 0.25 ␮m film thickness), carrier gas: helium,
∗
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 © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.11.006

M.K. Parr et al. / Steroids 74 (2009) 322–328
323
Fig. 1. Structures of the isolated steroids characterized as 17␤-hydroxyandrostano[3,2-c]isoxazole (1) and -[2,3-d]isoxazole (2), and the steroid Furazadrol-THP ether labeled
on the Orastan-A preparation but proven to be absent.
1.2 mL/min, oven temperature program: 1.5 min 60 ◦C, +40 ◦C/min,
0 min 240 ◦C, +2 ◦C/min, 0 min 260 ◦C, +40 ◦C/min, 1.5 min 300 ◦C,
ionization: 70 eV, EI, data acquisition: full scan mode (m/z 40–800),
sampling rate: 22.
retention indices the GC oven was programmed as follows: 0 min
160 ◦C, +5 ◦C/min, 4 min 320 ◦C.
2.2.2. HPLC fractionation
The TMS-derivatives were analyzed on an Agilent 6890 GC cou-
pled to an Agilent 5973 MSD using the following parameters:
injection volume: 2 ␮L, split 1:10, injection temperature: 300 ◦C,
column: Agilent HP5MS (16.5 m, 0.25 mm inner diameter (i.d.),
0.25 ␮m film thickness), carrier gas: helium, 0.8 mL/min, oven
temperature program: 0 min 140 ◦C, +20 ◦C/min, 2 min 320 ◦C, ion-
ization: 70 eV, electron ionization (EI), data acquisition: full scan
mode (m/z 40–800), sampling rate: 22. For determination of the
The HPLC fractionation was performed on an Agilent 1090
equipped with an ODS Hypersil column (Thermo Electron,
10 mm × 250 mm, particle size 5 ␮m) using the following parame-
ters: injection volume: 50 ␮L, mobile phase A: H2O, B: acetonitrile,
gradient: 0–25 min 50% B to 100% B, 15–32 min 100% B, 2 min re-
equilibration, flow: 3 mL/min, detector wavelength: 228 nm.
The fractions were collected from 12.9 to 13.8 min (isomer 1)
and 14.1 to 15.0 min (isomer 2). After evaporation of the solvent
Fig. 2. Mass spectra (EI) of isomer 1 (upper left: underivatized, M+ = 315, lower left: TMS-derivative, M+ = 387) and isomer 2 (upper right: underivatized, M+ = 315, lower right:
TMS-derivative, M+ = 459).

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M.K. Parr et al. / Steroids 74 (2009) 322–328
Table 1
Retention indices (methylene units) of androstanoisoxazoles (underivatized and as per-TMS-derivatives).
Analyte
Methylene units (Optima ␦3) underivatized
Methylene units (HP5MS) after silylation
17␤-Hydroxyandrostano[3,2-c]isoxazole (1)
17␤-Hydroxyandrostano[2,3-d]isoxazole (2)
17␤-Hydroxyandrostano[3,2-c]isoxazole (1)-acetate
17␤-Hydroxyandrostano[2,3-d]isoxazole (2)-acetate
17␤-Hydroxyandrostano[3,2-c]isoxazole (1)-THP
17␤-Hydroxyandrostano[2,3-d]isoxazole (2)-THP
Androisoxazol ([3,2-c]isoxazole)
3041
3071
3380
3410
3571
3579
3077
3367
2888
3040
n.d.
n.d.
n.d.
n.d.
3070
3137
Danazol ([2,3-d]isoxazole)
n.d. value not determined.
under reduced pressure, the residues were crystallized from ethyl
acetate (m.p. isomer 1: 161–162 ◦C, Lit [3,2-c]: 156–158 ◦C, isomer
2: 184–187 ◦C, Lit [2,3-d]: 184–186 ◦C [14]).
2.2.3. NMR spectroscopy
The NMR data were obtained using a Bruker DRX 500 instru-
ment, equipped with a 5 mm inverse probehead with 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 DMSO. For confirmation of the assumed
structures 1H, H,H COSY, DEPT, APT, H,C HMQC, H,C HMBC and H,H
NOESY spectra were recorded.
2.2.4. X-ray crystal structure analysis
Suitable single crystals of isomer
2
(17␤-hydroxy-
androstano[2,3-d]isoxazole, 2) were obtained from ethyl acetate
by evaporation. X-ray data were collected with a Nonius KappaCCD
diffractometer [15,16] equipped with a low temperature device
at 100 K by using graphite-monochromated Mo K␣ radiation
(ꢀ = 0.71073 Å). The structure was solved by direct methods using
SHELXS-97 [17] and the non-hydrogen atoms were refined by
full-matrix least-squares methods using SHELXL-97 [18]. The
positional parameters of the H atoms were obtained geometrically,
with C–H distances fixed and refined as riding on their respective
C atoms with Uiso H = 1.2 Ueq C.
Crystal data: C20H29NO2, M = 315.46, colourless, crystal dimen-
sions 0.46 mm × 0.31 mm × 0.11 mm, monoclinic, space group P21,
Z = 4, a = 9.7180(7) Å, b = 7.5511(5) Å, c = 23.227(2) Å, ˇ = 98.295(4)◦,
V = 1686.6(2) Å3, DX = 1.242 g cm−3
, ꢁ ,
(Mo K␣) = 0.079 mm−1
T = 100(2) K, transmission factors (min/max) 0.9646/0.9914, ana-
lytical absorption correction based on the indexing of the crystal
faces [19], 2ꢂ(max) = 51◦, number of unique data 4790, Rint 0.0558, R1
[I > 2ꢂ(I)] = 0.0712, w(R2) all data 0.1669, final R = 0.0924, goodness
of fit 1.062, ꢃꢄ (max/min) = 0.261/−0.363 e Å−3
.
2.2.5. High-resolution high-accuracy mass spectrometry
For the verification of the elemental composition high-
resolution high-accuracy mass spectrometry was performed on a
Thermo LTQ Orbitrap mass spectrometer (Bremen, Germany). The
instrument was operated in positive ionization mode and calibrated
using the manufacturer’s calibration mixture (containing caffeine,
MRFA, and ultramark that yield a total of 7 reference masses).
Mass accuracies < 2 ppm (calculated from 30 averaged spectra) at a
resolution of 30,000 (full width half maximum, FWHM) were deter-
mined before and after the period of analysis of target compounds.
Working solutions were introduced into the mass spectrometer
using a syringe pump at a flow rate of 5 ␮L/min. The ionization
voltage was 3.0 kV, and the capillary temperature was set to 275 ◦C.
Damping gas in the linear ion trap was helium (purity grade 5.0),
and gas supplied to the curved linear ion trap (CLT) was nitro-
gen obtained from a CMC nitrogen generator (CMC Instruments,
Eschborn, Germany).
Fig. 3. Mass spectra (EI) of steroid compounds of Orastan-A, underivatized, (A)
acetate of isomer 1, (B) acetate of isomer 2, (C) THP ether of isomer 1, (D) THP
ether of isomer 2.

M.K. Parr et al. / Steroids 74 (2009) 322–328
325
Fig. 5. Mass spectra (EI) of Danazol (upper: underivatized, M+ = 337, lower: TMS-
Fig. 4. Mass spectra (EI) of Androisoxazol (upper: underivatized, M+ = 329, lower:
derivative, M+ = 481).
TMS-derivative, M+ = 401).
of pyridine as solvent. After refluxing for 3 h the mixture was
evaporated to dryness, re-dissolved in water and extracted with
t-butyl methyl ether. Evaporation of the solvent resulted in 17␤-
hydroxyandrostano[3,2-c]isoxazole (1).
2.3. Supplement analysis
The product Orastan-A from the company Gaspari Nutri-
tion was obtained by Internet order from the sport supplement
market, classified as “dietary supplement” according to its
labeling. It was labeled to contain “5a-Androstano[2,3-c]furazan-
17b-tetrahydropyranol ether”, also called furazadrol-THP ether
(structure formula in Fig. 1).
The homogenized content of one capsule was suspended in 5 mL
of methanol. After shaking for 5 min and centrifugation for 5 min
at 800 × g, the methanolic layer was separated. Aliquots were ana-
lyzed by GC–MS.
For characterization of the steroid moiety the content of 30
capsules was extracted with n-hexane in a Soxhlet apparatus.
After evaporation of the solvent, the residue was hydrolyzed for
1 h in refluxing aqueous acetic acid (30%). Aliquots were ana-
lyzed by GC–MS (underivatized and as TMS-derivatives). The
co-crystallizing (from ethyl acetate) steroids were separated by
semi-preparative HPLC. After crystallization from ethyl acetate,
the steroids were characterized by GC–MS, NMR, X-ray and high-
resolution/high-accuracy mass spectrometry for the identification
of the molecular structure.
2.4. Synthesis of reference material
2.4.1. 17ˇ-Hydroxyandrostano[3,2-c]isoxazole (1)
For the synthesis of 17␤-hydroxyandrostano[3,2-c]isoxazole (1),
2-hydroxymethylene-5␣-dihydrotestosterone (4) was prepared by
reaction of 5␣-dihydrotestosterone (3) with ethyl formate and
sodium methylate in absolute pyridine as described by Schänzer
[20]. The isoxazole ring was formed by condensation of 33 mg
of 4 with 27 mg of hydroxylamine hydrochloride using 3.5 mL
Fig. 6. UV spectra (HPLC-DAD) of isomer 1 (upper) and isomer 2 (lower).

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M.K. Parr et al. / Steroids 74 (2009) 322–328
Table 2
1H and 13 C NMR spectral data (d6-DMSO) of 17␤-hydroxyandrostano[3,2-c]isoxazole (1) and 17␤-hydroxyandrostano[2,3-d]isoxazole (2).
17␤-hydroxyandrostano[3,2-c]isoxazole (1)
17␤-hydroxyandrostano[2,3-d]isoxazole (2)
ıC
ıH
ıC
ıH
1
2
30.951
2.011/2.652
–
33.567
2.034/2.419
–
114.136
158.672
24.581
41.333
28.738
32.088
35.456
53.159
35.672
20.477
36.678
42.508
50.585
23.249
29.998
80.175
11.324
11.382
154.812
–
111.176
165.617
26.069
41.860
28.360
30.931
35.378
53.174
36.120
20.584
36.665
42.533
50.525
23.226
29.972
80.151
11.320
11.463
150.315
–
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
–
–
2.222/2.674
1.504
1.319/1.535
0.852/1.646
1.316
0.816
–
1.337/1.554
0.984/1.758
–
2.230/2.651
1.564
1.328/1.515
0.854/1.642
1.312
0.822
–
1.355/1.534
0.986/1.757
–
0.885
0.887
1.160/1.500
1.333/1.826
3.436
0.639
0.661
1.161/1.496
1.334/1.823
3.432
0.641
0.669
19
isoxazole CH
OH
8.511
4.397
8.298
4.399
2.4.2. 17ˇ-Hydroxyandrostano[2,3-d]isoxazole (2)
steroids (underivatized and as TMS-derivatives) are shown in Fig. 2.
In disagreement with the product label the mass spectra of neither
compound identified matched that of furazadrol. However, isomer
1 showed analogous mass spectra compared with Androisoxazol
(17␣-methyl-17␤-hydroxy-5␣-androstano[3,2-c]isoxazole, Fig. 4),
lacking the 17-methyl group. High-resolution high-accuracy mass
spectrometry ([M + H]+exp. = 316.2273, [M + H]+theor. = 316.2271,
error 0.56 ppm) deduced the elemental composition C20H29NO2
for both isomers. While isomer 1 yielded a mono-TMS-derivative,
a bis-TMS-derivative was obtained from isomer 2 following deriva-
tization with TMIS reagent. Analogous findings were obtained for
Androisoxazol ([3,2-c]-) and Danazol ([2,3-d]-isoxazole) deriva-
tized with TMIS reagent (mass spectra in Figs. 4 and 5). From these
findings structures of 17␤-hydroxyandrostano[3,2-c]isoxazole and
-[2,3-d]isoxazole were assumed.
Unfortunately, both steroids co-crystallized and had to be
separated by semi-preparative HPLC. The obtained UV spectra
(HPLC-DAD, Fig. 6) showed maxima at 222 nm (isomer 1) and
228 nm (isomer 2). Comparing the melting points and UV maxima
of the two isomers with data from the literature [9,14,21] supported
the assignment of the [3,2-c]isoxazole for isomer 1 and of the [2,3-
d]isoxazole for isomer 2.
For the synthesis of 17␤-hydroxyandrostano[2,3-d]isoxazole
(2) the isoxazole ring was formed by condensation of 3.3 mg
of 2-hydroxymethylene-5␣-dihydrotestosterone (4) with 30 ␮L of
an aqueous solution of hydroxylamine hydrochloride (90 mg/mL)
using 1 mL of ethanol as solvent. After refluxing for 3 h the mix-
ture was evaporated to dryness, redissolved in water and extracted
with t-butyl methyl ether. 17␤-Hydroxyandrostano[2,3-d]isoxazole
(2) was obtained by evaporation of the solvent [14].
3. Results and discussion
3.1. Supplement analysis
The GC–MS analyses of the underivatized methanolic extract of
Orastan-A revealed three pairs of steroidal compounds which were
identified as two isomeric steroid alcohols, their corresponding
acetates and tetrahydropyranyl (THP) ethers (mass spectra in
Figs. 2 and 3, retention indices in Table 1). As the THP ethers
represented the main ingredients, the steroids were released by
acidic hydrolysis prior to further characterization, yielding both
steroids in a ratio of about 1:2. The mass spectra of the liberated
Fig. 7. Representation of the structure of 17␤-hydroxyandrostano[2,3-d]isoxazole (isomer 2) as determined by X-ray crystallography.

M.K. Parr et al. / Steroids 74 (2009) 322–328
327
Scheme 1. Synthesis of 17␤-hydroxyandrostano[3,2-c]isoxazole (1) and -[2,3-d]isoxazole (2).
The 1H NMR spectra of the purified steroids (detailed data in
Table 2) show a 1H singlet at 8.51 ppm (isomer 1) and 8.30 ppm
(isomer 2), representing the isoxazole CH. As described by Fahren-
holtz et al. [22], these signals for [3,2-c]isoxazoles are shifted
downfield compared to [2,3-d]isoxazoles. The corresponding 13C
chemical shift was determined at 154.8 and 150.3 ppm. Further
significant differences occurred for C-2 (114.1 ppm vs. 111.1 ppm)
and C-3 (158.7 ppm vs. 165.6 ppm). X-ray crystal structure anal-
ysis of isomer 2 (Fig. 7) finally confirmed the structures of the
assignment 17␤-hydroxyandrostano[3,2-c]isoxazole for isomer 1
and 17␤-hydroxyandrostano[2,3-d]isoxazole for isomer 2.
Acknowledgements
The German Federal Ministry of the Interior and the Manfred
Donike Institute for Doping Analyses e.V., Cologne, are acknowl-
edged for their financial support.
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In the product “Orastan-A” the two isomeric steroids, 17␤-
hydroxyandrostano[3,2-c]isoxazole (1) and -[2,3-d]isoxazole (2),
were identified. They were mainly present as THP-ether. Following
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hydrolysis by gastric acid [23].
The present study again shows that products, containing unap-
proved steroids that have already been synthesized in the 1960s,
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as “dietary supplement”, even though the products have to be clas-
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aware of the doping risks connected with the use of such products.
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studies on the urinary metabolism are required in the future. Pre-
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