Characterization of desoxymethyltestosterone main urinary metabolite produced from cultures of human fresh hepatocytes

Article abstract of DOI:10.1016/j.steroids.2012.02.008

Desoxymethyltestosterone (DMT; 17β-hydroxy-17α-methyl-5α- androst-2-ene) is a designer steroid present in hormonal supplements distributed illegally as such or in combination with other steroids, for self-administration. It figures on the list of substances prohibited in sports and its detection in athlete's urine samples is based upon the presence of the parent compound or the main urinary metabolite, which has not been characterized yet. Following its isolation from cultures of human fresh hepatocytes and S9 fractions of liver homogenates, we were able to identify this metabolite as being 17α-methyl-2β,3α,17β-trihydroxy-5α-androstane. Other minor metabolites were also characterized. The production, isolation, NMR, mass spectral analyses and chemical synthesis are presented.

Full text of DOI:10.1016/j.steroids.2012.02.008

Steroids 77 (2012) 635–643
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Characterization of desoxymethyltestosterone main urinary metabolite
produced from cultures of human fresh hepatocytes
Julie Gauthier a, Donald Poirier b, Christiane Ayotte a,
⇑
a INRS – Institut Armand-Frappier, Laval, Canada
b CHUQ-CHUL Research Center and Université Laval, Québec, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Desoxymethyltestosterone (DMT; 17b-hydroxy-17a-methyl-5a-androst-2-ene) is a designer steroid
Received 24 November 2011
Received in revised form 7 February 2012
Accepted 8 February 2012
present in hormonal supplements distributed illegally as such or in combination with other steroids,
for self-administration. It figures on the list of substances prohibited in sports and its detection in ath-
lete’s urine samples is based upon the presence of the parent compound or the main urinary metabolite,
which has not been characterized yet. Following its isolation from cultures of human fresh hepatocytes
Available online 17 February 2012
and S9 fractions of liver homogenates, we were able to identify this metabolite as being 17
2b,3 ,17b-trihydroxy-5 -androstane. Other minor metabolites were also characterized. The production,
isolation, NMR, mass spectral analyses and chemical synthesis are presented.
Ó 2012 Elsevier Inc. All rights reserved.
a-methyl-
Keywords:
a
a
Desoxymethyltestosterone
Metabolites
Synthesis
Hepatocytes
GC–MS
1. Introduction
yldrostanolone (2a,17a-dimethyl-17b-hydroxy-5a-androstan-3-
one), two other designer steroids [1]. In 2007, Diel et al. reported
powerful anabolic properties with potential risk of toxic side effects
to be expected from the administration of products containing this
steroid [7].
Desoxymethyltestosterone (DMT, 1), a mixture composed prin-
cipally but not uniquely of 17b-hydroxy-17
2-ene and to a lesser extent of the 3-ene isomer, was identified in
a bottle bearing the inscription ‘‘New Stuff ’’, seized at the Cana-
a-methyl-5a-androst-
D
Desoxymethyltestosterone figures on the list of prohibited sub-
stances published by the World Anti-Doping Agency [8]. Its
detection in athlete’s samples depends upon the identification of
characteristic, long-lasting metabolites. To that end, DMT must first
be synthesized or purified from the low quality preparations
available, which combined to the absence of clinical data limits
the feasibility of the administration to volunteers. The formation
of metabolites from DMT was therefore only partially studied.
Sekera et al. reported in the baboon the excretion of a dihydroxyl-
ated metabolite formed from incubations of pooled human liver
microsomes [9], while Rodchenkov et al. described extensive
metabolism from an administration to a human volunteer, leading
to the formation of several mono- and dihydroxylated urinary
metabolites; none were identified [10].
dian customs in 2003 [1,2]. This clandestine product was since then
introduced in so-called designer hormonal supplements as such
(Ergomax LMG, PheraPlex) or in combination with other molecules
(Halodrol). Even if the distribution of products containing DMT is
illegal in many countries such as Canada and USA, it has not fully
stopped. Not only are these products prepared and commercialized
without the required quality controls, but also they have not gone
through any clinical trials; effects and health problems are discov-
ered a posteriori [3–6]. In earlier work, we have studied the hor-
monal properties of DMT using mammalian-based reporter gene
bioassays. More specifically, the androgenic activity was deter-
mined using CAMA-1 breast cancer cells transiently transfected
with a reporter plasmid, which comprises the androgen-responsive
mouse mammary tumor virus promoter in front of the luciferase
gene (MMTV-luc). DMT was found in these assays to be androgenic,
although less potent than tetrahydrogestrinone (13-ethyl-17-hy-
Different hepatic models were used to mimic phases I and II
metabolism of similar molecules [11–14]. The production of metab-
olites by fresh human hepatocytes was evaluated for androstenedi-
one (androst-4-ene-3,17-dione), norandrostenedione (estr-4-ene-3,
droxy-18,19-dinor-17a-pregna-4,9,11-trien-3-one) and 17a-meth-
17-dione), gestrinone (13-ethyl-17-hydroxy-18,19-dinor-17a-pre-
gna-4,9,11-trien-20-yn-3-one) and tetrahydrogestrinone [15–17],
correlating to a certain degree with the results of excretion studies.
Microsomal and S9 fractions of human liver homogenates were uti-
lized for the formation of phase I and II metabolites of 17-alkylated
⇑
Corresponding author. Address: INRS – Institut Armand-Frappier, 531, boul. des
Prairies Laval, Québec, Canada H7V 1B7. Tel.: +1 (450) 686 5442; fax: +1 (450) 686
5614.
E-mail address: christiane.ayotte@iaf.inrs.ca (C. Ayotte).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2012.02.008

636
J. Gauthier et al. / Steroids 77 (2012) 635–643
steroids such as methyltestosterone (17b-hydroxy-17
androst-4-en-3-one) and methandienone (17b-hydroxy-17
methyl-androsta-1,4-dien-3-one) [12]. We also reported the char-
acterization of the metabolites of drostanolone (17b-hydroxy-2
methyl-5 -androstan-3-one) and its 17-methylated analog isolated
a
-methyl-
2.2. Reverse phase HPLC
a
-
The steroids extracted from the incubation media were purified
by reverse phase HPLC on a HP1100 chromatograph equipped with
a photodiode array detector, a quaternary pump and with a Zorbax
a
-
a
from cryopreserved human hepatocytes [18]. However in our
hands, desoxymethyltestosterone (DMT) remained almost intact
following incubations with cryopreserved hepatocytes only traces
of metabolites being formed, which precluded any further investi-
gations at the time.
In this paper, we wish to report the characterization of the main
urinary metabolite of DMT reported previously, produced in cul-
tures of human fresh hepatocytes and isolated from liver S9
fractions.
SB-C18 column (250 mm  9.4 mm  5
lm) (Agilent Technologies,
New Castle, USA). Fractions were collected during the run made
with the following mobile phase: acetonitrile:water (60%) for the
first 12 min to 95% (rate: 1 min) held for 5 min. The metabolite
of interest was recovered in the fraction collected between 8.0
and 8.4 min.
2.3. GC–MS
Analyzes in the full scan mode (50–750 amu) with EI ionization
at 70 eV were performed on an Agilent 6890 gas chromatograph
coupled to a 5973 mass selective detector equipped with an
5973 auto-sampler with instrument control and data analysis by
the Enhance ChemStation software. The chromatograph was
2. Experimental
2.1. Chemicals and reagents
equipped with
a
DB-5MS column (SGE) (30 m  0.22 mm
id  0.25 m film thickness) (Agilent, New Castle, USA) and the
l
Distilled water was purified on a Milli-Q Gradient system from
Millipore (Fisher Scientific, Whithy, Canada). The following reagents
were purchased from Sigma–Aldrich (Oakville, Canada): tryptan
blue, trimethylsilyliodide (TMIS), tetrahydrofuran (THF), N-
analyzes were carried out with the following temperature program
of the oven: 100 °C (0.5 min) to 240 °C at 20 °C/min to 278 °C at
6 °C/min and 320 °C (10 °C/min, held 4 min) with He as the carrier
gas (BOC, Montréal, Canada). The temperature of the injector and
the transfer line was, respectively 270 and 310 °C.
methyl-N-(trimethylsilyl)-trifluoracetamide
grade
purissimo
(MSTFA, Fluka), b-glucuronidase from Escherichia coli, Percoll solu-
tion, sodium chloride (NaCl), pyridine, trimethylsilylchloride
(TMCS), triethylamine, NAPD, glucose-6-phosphate, glucose-6-
phosphate dehydrogenase, dimethylsulfoxide, chromium (VI)
oxide, 2-propanol, dioxane, deuterated chloroform, deuterated
methanol, lithium bromide, lithium carbonate, lithium chloride,
hydrazine monohydrate, 2-propanol, sodium borohydrate, Pd/C
10%, methyl magnesium bromide, pyridium tribromide, zinc dust,
N-bromosuccinimide, perchloric acid and chromium trioxide. Etha-
nethiol was obtained from Fisher Scientific (Whithy, Canada) and
acetonitrile, diethyl ether, methanol, hexane, chlorhydric acid,
sulfuric acid and dichloromethane from Caledon (Georgetown,
Canada), all the others from EMD (Gibbstown, USA). Deuterared
N,O-bis(trimethyl-d9-silyl)acetamide and chlorotrimethyl-d9-si-
lane were from C/D/N Isotopes (Pointe-Claire, Canada).
2.4. NMR
1H NMR spectra were recorded at 400 MHz on a Brucker AVANCE
400 spectrometer (Billerica, MA, USA). The chemical shifts (d) are
expressed in ppm and referenced to chloroform (7.26 ppm).
2.5. Incubations with fresh human hepatocytes
According to the protocol suggested by the supplier [20,21], the
cells were first washed on Percoll gradient following which they
were suspended in 2.5 mL of Krebs–Henseleit buffer (KHB). The
vial contained at least 5 Â 106 cells and their viability was assessed
with tryptan blue. As described previously [18], DMT (1) was dis-
solved in ethanol (0.01 M); further dilution in KHB to a concentra-
Sodium phosphate monobasic, sodium bicarbonate grade ACS
and sodium carbonate were obtained from VWR (Batavia, USA).
The Krebs–Henseleit buffer, the HT medium, pooled human male
liver S9 fractions and fresh hepatocytes were from Celsis In Vitro
Technologies (Baltimore, USA). The following products were ob-
tained from MAT (Québec, Canada): hydrogen peroxide 33%, so-
dium hydroxide, celite, K2CO3, Na2SO4 and NH4Cl.
Anhydrous reactions were performed in oven-dried glassware
under positive argon pressure using commercially available anhy-
drous solvents (Gibbstown, USA), except THF distilled from so-
dium/benzophenone ketyl under argon. Chemical reagents for
synthesis were purchased from Aldrich Chemical Co. (Milwaukee,
USA). Flash chromatography was performed on Silicycle 60 230–
400-mesh silica gel (Québec, Canada).
tion of 100 lM was freshly made just prior to the experiment. Two
fifty microliters of the cell suspension and the DMT solution were
sampled in 24-well plates and the incubation was carried out at
37 °C for 4 h under an atmosphere of 95% O2 to CO2. The enzymatic
reactions were stopped by the addition of 500 lL of acetonitrile,
the content was transferred to Eppendorf tubes which were vor-
texed and centrifuged at 14,000 rpm for 10 min. The metabolites
were purified as described previously by solid phase extraction
on Sep Pak C18 cartridges (Waters, Montréal, Canada), and selective
hydrolysis of the glucuroconjugates and sulfates was carried out by
enzymatic hydrolysis with b-glucuronidase from E. coli and chem-
ical solvolysis, respectively. The steroids were converted to TMS-
ether, TMS-enol derivatives and the mixture was analyzed as such
by GC/MS [22].
The synthesis of DMT was described previously [1]. 17b-Acetyl-
oxy-5
a
-androst-2-en-4-one (4) and 4n,17b-diacetyloxy-5
a
-and-
2.6. Incubations with S9 human liver fractions
rost-2-ene (6) were a kind gift from Pr. John F. Templeton,
Professor Emeritus, Faculty of Pharmacy, University of Manitoba,
The incubations were performed according to the recommenda-
tions of the supplier [23]. The frozen vials containing the S9 frac-
tions (30 mg per vial) were placed in a water bath at 37 °C; when
17b-hydroxy-17
was synthesized earlier by Pr. Donald Poirier [19]. 3b-Hydroxy-
-androstan-17-one (8) was purchased from Aldrich, androst-4-
ene-3,17-dione (17) from Steraloids. Two capsules of ‘‘Halodrol’’
and one aliquot of ‘‘New Stuff ’’ were provided by the Laboratory
a
-methyl-2,3
a-spirooxirane-5
a-androstane
(2)
5a
thawed, 550
50 mM were added to test tubes cooled in an ice water bath, fol-
lowed by the addition of 55 L of a solution of DMT in ethanol at
lL of this suspension and 3.52 mL of Tris buffer at
D
l
and Scientific Services Directorate of the Canada Border Services
Agency.
5 mM. In parallel, 170 mg of NADP (2.3 mmol) and 600 U of
glucose-6-phosphate dehydrogenase (6 U/mL) were added to

J. Gauthier et al. / Steroids 77 (2012) 635–643
637
100 mL of an aqueous solution of NaHCO3 at 2% (W:V). Both prep-
arations were placed in a water bath at 37 °C for 5 min following
which 1.375 mL of the second solution were added to the first
one and incubations were carried out at 37 °C during 1 h. After
the addition of 5 mL of acetonitrile to each tube, the solvent was
transferred and evaporated to dryness. After the extraction of the
metabolite as described previously (solid phase and liquid–liquid
3.64 (dd, 1H, CH-17), 6.00 (dd, J1 = 2.92 Hz, J2 = 10 Hz, 1H, CH-3),
6.8 (m, 1H, CH-2).
2.8.3. Synthesis of 4n,17b-dihydroxy-5
a
-androst-2-ene (7)
-androst-2-ene (6)
To solution of 4n,17b-diacetyloxy-5
a
a
(0.0147 g, 0.039 mmol) in methanol (1 mL) was added K2CO3
(0.1 g, 0.72 mmol) and the reaction was stirred overnight at room
temperature. The reaction mixture was then poured in water and
extracted three times with ethyl acetate. The organic phase was
washed with brine and treated as described previously. The
resulting product was purified by flash chromatography using
ethyl acetate:hexane (20:80 V:V) as eluent to give 4n,17b-dihy-
extraction), the residue was dissolved in 30 lL of methanol and
45 lL of acetonitrile and injected on the HPLC. Fractions containing
DMT metabolites were pooled and analyzed by 1H NMR and GC/MS
as their TMS-ether or TMS-enol, TMS-ether derivatives.
2.7. Reference human urine samples
droxy-5a
-androst-2-ene (7) (4 mg, 39%). 1H NMR (CDCl3): 0.75
(s, 3H, CH3-18), 0.97 (s, 3H, CH3-19), 3.63 (dd, CH-17), 3.97 (d,
A urine sample obtained following the ingestion of DMT was
kindly provided by the Anti-doping Centre in Moscow: one capsule
of ‘‘PheraPlex’’ from Anabolic Xtreme containing 10 mg of DMT was
administered to a healthy male volunteer and all specimens pro-
vided in the following 24 h were pooled. The second reference was
an athlete’s sample collected during an international event and
received for testing that was found to contain the main urinary
metabolite of desoxymethyltestosterone, along with those of
17-methyldrostanolone and dehydrochlormethyltestosterone; the
athlete admitted to the use of ‘‘Halodrol’’ which is listed as contain-
ing DMT. The main urinary metabolites detected in these specimens
were those researched in the cultures of hepatocytes and liver S9
fractions.
J = 3.92 Hz, 1H, CH-4), 5.82, (m, 2H, CH-2 and CH-3).
2.8.4. Synthesis of 1
(14) and 1b,17b-dihydroxy-17
2.8.4.1. 3b,17b-Dihydroxy-17 -methyl-5
to the method described by Ferraboschi et al. [24], to a solution of
3b-hydroxy-5 -androstan-17-one (8) (3.0153 g, 10.3 mmol) in
a
,17b-dihydroxy-17
-methyl-5
-androstane (9). According
a
-methyl-5
a-androst-2-ene
a
a
-androst-2-ene (16)
a
a
a
anhydrous toluene (84 mL) was added a solution of CH3MgBr in
toluene:THF (75:25 V:V) (1.4 M, 117.6 mmol) at room temperature
under argon. The reaction was stirred for 4 h at reflux and left over-
night at room temperature. An aqueous solution of ammonium
chloride (2.6 M) was added dropwise at 0 °C to the reaction mix-
ture; the aqueous phase was extracted eight times with chloro-
form. The organic phase was washed with water and treated as
described previously. The resulting crude product was used for
the next step without any further purification (2.31 g, 73%). 1H
NMR (CDCl3): 0.82 (s, 3H, CH3-18), 0.84 (s, 3H, CH3-19), 1.21 (s,
3H, CH3-20), 3.59 (m, 1H, CH-3).
2.8. Synthesis of proposed DMT metabolites
A structure was proposed for the most intense DMT metabolite
(2-en isomer) purified by HPLC on the basis of the 1H NMR and
mass spectra. The synthesis of 17a-methyl-2b,3a,17b-trihydroxy-
5
a
-androstane (3) and that of other minor metabolites (hydroxyl
2.8.4.2. 17b-Hydroxy-17
ing to the method described by Ferraboschi et al. [24], to a solution
of 3b,17b-dihydroxy-17 -methyl-5 -androstane (9) (2.2917 g,
a-methyl-5a-androstan-3-one (10). Accord-
or ketone in C-1 or C-4) was carried out as described in the follow-
ing sections.
a
a
9.84 mmol) in acetone (60 mL) was added dropwise Jones reagent
(5.46 mL, 14.75 mmol) at room temperature under argon, until the
orange color persisted. The reaction was stirred at room tempera-
ture for 30 min and 2-propanol was added dropwise until the green
color persisted. The reaction mixturewas filteredunder vacuumon a
celite pad and the solvent was evaporated under vacuum. The result-
ing crude product was used as such (2.1 g, 92%). 1H NMR (CDCl3):
0.88 (s, 3H, CH3-18), 1.03 (s, 3H, CH3-19), 1.21 (s, 3H, CH3-20).
2.8.1. Synthesis of 17
(3)
a
-methyl-2b,3
a
,17b-trihydroxy-5
a-androstane
A solution of 17b-hydroxy-17
a
-methyl-2,3 -spirooxirane-5a-
a
androstane (2) (0.0125 g, 0.041 mmol) in THF (0.77 mL) was trea-
ted with 0.5 M H2SO4 (0.51 mL, 0.1027 mmol) and stirred at room
temperature for 4 h. After neutralization with a saturated NaHCO3
solution, the reaction mixture was extracted three times with ethyl
acetate. The organic layer was dried over MgSO4 and treated as de-
scribed previously. The resulting product was purified by flash
chromatography using ethyl acetate:hexane (70:30 V:V) as eluent
2.8.4.3.
(11). According to the method described by Ferraboschi et al. [24],
to solution of 17b-hydroxy-17 -methyl-5 -androstan-3-one
2a-Bromo-17b-hydroxy-17a-methyl-5a-androstan-3-one
to give 17
a
-methyl-2b,3
a,17b-trihydroxy-5
a-androstane (3)
a
a
a
(0.0099 g, 75%). 1H NMR (CDCl3): 0.85 (s, 3H, CH3-18), 1.01 (s,
3H, CH3-19), 1.21 (s, 3H, CH3-20), 3.87 (q, 1H, CH-3), 3.91 (q, 1H,
CH-2). 13C NMR (CDCl3): 14.03 (C-18), 14.58 (C-19), 20.48, 23.19,
25.81 (C-20), 28.09, 29.69, 39.9, 35.77 (C-8), 38.96, 39.02 (C-5),
40.56 (C-1), 50.63 (C-9), 55.14 (C-14), 70.56 (C-3), 71.77 (C-2).
(10) (2.09 g, 6.89 mmol) in anhydrous ethanol (41.8 mL) and anhy-
drous methylene chloride (20.9 mL) was added pyridium tribro-
mide (2.2 g, 6.9 mmol) at 40 °C under argon. The reaction was
stirred at 40 °C for 2 h and pyridium tribromide was added
(0.94 g, 2.9 mmol) to complete the reaction, which was stirred for
another hour at 40 °C. The reaction mixture was poured in an aque-
ous solution of NaHCO3 (87 g/L) and extracted three times with
dichloromethane. The organic phase was washed with brine and
treated as described previously. The resulting product was purified
by flash chromatography using toluene:hexane (70:30 V:V) as elu-
2.8.2. Synthesis of 17b-hydroxy-5
a-androst-2-ene-4-one (5)
To solution of 17b-acetyloxy-5
a
a-androst-2-en-4-one (4)
(0.15 g, 0.45 mmol) in methanol (10 mL) was added K2CO3 (1 g,
7.24 mmol) and the reaction was stirred overnight at room tem-
perature. The reaction mixture was poured in water and extracted
three times with ethyl acetate. The organic phase was washed with
brine, dried over Na2SO4, filtered and evaporated under vacuum.
The resulting product was purified by flash chromatography on sil-
ica gel, using the following combination of ethyl acetate:hexane:
200 mL 30:70 V:V, 200 mL 40:60 V:V and 100 mL 50:50 V:V) as
ent to give 2
one (11) (1.31 g, 50%). 1H NMR (CDCl3): 0.87 (s, 3H, CH3-18), 1.11
(s, 3H, CH3-19), 1.21 (s, 3H, CH3-20), 1.81 (m, 1H, CH-1 ), 2.41 (m,
a-bromo-17b-hydroxy-17a-methyl-5a-androstan-3-
a
2H, CH2-4), 2.64 (dd, J1 = 6.34 Hz, J2 = 12.87 Hz, 1H, CH-1b), 4.45
(dd, J1 = 6.31 Hz, J2 = 13.34 Hz, 1H, CH-2).
eluent to give 17b-hydroxy-5
a
-androst-2-en-4-one (5) (92 mg,
2.8.4.4. 17b-Hydroxy-17
ding to the method described by Ferraboschi et al. [24], to a
a-methyl-5a-androst-1-en-3-one (12). Accor-
71%). 1H NMR (CDCl3): 0.74 (s, 3H, CH3-18), 0.86 (s, 3H, CH3-19),

638
J. Gauthier et al. / Steroids 77 (2012) 635–643
solution of 2
a
-bromo-17b-hydroxy-17
a
-methyl-5
a
-androstan-3-
borohydride (0.0186 g, 0.479 mmol) at room temperature under ar-
gon followed by stirring for 1 h. The reaction mixture was poured
in water and extracted three times with ethyl acetate. The organic
phase was washed with brine, and treated as described previously.
The resulting crude product was purified by flash chromatography
using ethyl acetate:hexane (30:70 V:V) as eluent to give a mixture
one (11) (1.38 g, 3.61 mmol) in anhydrous DMF (27.4 mL) were
added lithium bromide (2.06 g, 23.7 mmol) and lithium carbonate
(1.79 g, 26.03 mmol) at room temperature under argon. The reac-
tion was stirred at reflux during 1 h, and then the mixture was
poured in water and extracted five times with diethyl ether. The
organic phase was washed with water and brine, and treated as de-
scribed previously. The resulting product was purified by flash
chromatography using ethyl acetate:hexane (30:70 V:V) as eluent
of the two isomers of 1,17b-hydroxy-17
ene (14 and 16). 1H NMR (CDCl3): 0.74 (1
, s, 3H, CH3-19), 0.81 (1b,
s, 3H, CH3-19), 0.86 (s, 3H, CH3-18), 1.21 (s, 3H, CH3-20), 3.71 (1
a-methyl-5a-androst-2-
a
a,
to give 17b-hydroxy-17
a
-methyl-5
a
-androst-1-en-3-one (12),
d, J1 = 4.24 Hz, 1H, CH-1), 3.96 (m, 1H, CH-1), 5.39 (d, J1 = 9.23 Hz,
1H, CH), 6.84 (m, 2H, CH-2 and CH-3).
(0.5274 g, 48%), which was utilized as such. 1H NMR (CDCl3):
0.87 (s, 3H, CH3-18), 1.01 (s, 3H, CH3-19), 1.20 (s, 3H, CH3-20),
2.20 (dd, J1 = 4.04 Hz, J2 = 17.68 Hz, 1H, CH-4), 2.35 (dd,
J1 = 14.16 Hz, J2 = 17.66 Hz, 1H, CH-4), 5.83 (d, J1 = 10.18 Hz, 1H,
CH-2), 7.13 (d, J1 = 10.21 Hz, 1H, CH-1).
2.8.5. Synthesis of 4n,17b-dihydroxy-17
(23)
a-methyl-5a-androst-2-ene
2.8.5.1. Androst-3-en-17-one (18). As adapted from Tavares da Silva
et al. [27], to a solution of androst-4-ene-3,17-dione (17) (5.005 g,
17.48 mmol) in acetic acid (300 mL) was added zinc dust (30 g,
461 mmol) at reflux under argon over a period of 10 min, followed
by refluxing with stirring for 1.5 h. The suspension was filtered, the
residue was washed with acetic acid and the filtrate was evapo-
rated to dryness. The residue was dissolved in water and extracted
three times with diethyl ether. The organic phase was washed
three times with a solution of NaHCO3, three times with water,
2.8.4.5. 17b-Hydroxy-17
one (13). As described by Counsell et al. [25], to a solution of 17b-
hydroxy-17 -methyl-5 -androst-1-en-3-one (12) (0.5074 g,
a-methyl-1a,2a-spirooxirane-5a-androst-3-
a
a
1.68 mmol) in anhydrous methanol (8.5 mL) were added simulta-
neously hydrogen peroxide 30% (0.547 mL, 5.3 mmol) and sodium
peroxide (10% in MeOH) (0.142 mL, 0.51 mmol) at 0 °C under ar-
gon. The reaction was stirred at 0 °C under argon during 2 h and
at room temperature for 3 h; it was then poured in water and ex-
tracted three times with ethyl acetate. The organic phase was
washed with brine and treated as described previously. The
resulting product was purified by flash chromatography using
ethyl acetate:hexane (30:70 V:V) as eluent to give 17b-hydroxy-
and treated as described previously to give a mixture of the 5
a
and 5b-olefin; the former (18) (2.3 g, 49%) was precipitated after
standing in heptane at À15 °C for 48 h. 1H NMR (CDCl3): 0.80
(s, 3H, CH3-18), 0.88 (s, 3H, CH3-19), 5.29 (ddd, J1 = 1.89 Hz,
J2 = 9.86 Hz, 1H, CH-4), 5.57 (ddd, J1 = 3.25 Hz, J2 = 6.55 Hz,
J3 = 9.89 Hz, 1H, CH-3).
17a-methyl-1a,2a-spirooxirane-5a-androst-3-one (13) (0.4868 g,
91%). 1H NMR (CDCl3): 0.88 (s, 3H, CH3-18), 0.91 (s, 3H, CH3-19),
1.24 (s, 3H, CH3-20), 2.26 (dd, J1 = 13.81 Hz, J2 = 18.61 Hz, 1H, CH-
4), 3.24 (d, J1 = 4.15 Hz, 2H, CH-1), 3.53 (d, J1 = 4.14 Hz, 1H, CH-2).
2.8.5.2. 17b-Hydroxy-17a-methyl-5a-androstan-3-ene (19). As ada-
pted from Klimstra [28], to a solution of androst-3-en-17-one
(18) (2.2785 g, 8.38 mmol) in anhydrous toluene (30.71 mL) was
added dropwise a solution of CH3MgBr in diethyl ether (3 M,
30.71 mL, 92.13 mmol) at room temperature under argon. The
mixture was left stirring at room temperature for 16 h and refluxed
for 4 h. A saturated solution of NH4Cl in water (37.2 g in 100 mL)
was added dropwise at 0 °C, the aqueous phase was extracted eight
times with chloroform. The organic phase was washed with brine
and treated as described previously. The product obtained was
used without any further purification (2.28 g, 89%). 1H NMR
(CDCl3): 0.79 (s, 3H, CH3-18), 0.87 (s, 3H, CH3-19), 1.22 (s, 3H,
CH3-20), 5.28 (ddd, J1 = 1.9 Hz, J2 = 7.9 Hz, J3 = 11.8 Hz, 1H, CH-4),
5.56 (ddd, J1 = 3.2 Hz, J2 = 6.6 Hz, J3 = 9.8 Hz, 1H, CH-3).
2.8.4.6.
adapted from the method described by Klimstra [26], a solution of
17b-hydroxy-17 -methyl-1 ,2 -spirooxirane-5 -androst-3-one (13)
1a,17b-Dihydroxy-17a-methyl-5a-androst-2-ene (14). As
a
a
a
a
(0.2983 g, 0.938 mmol) in hydrazine monohydrate (4.71 mL,
91.2 mmol) was stirred at room temperature for 30 min, at 90 °C for
15 min and at 100 °C for 10 min. The reaction mixture was then poured
in water and extracted three times with ethyl acetate. The organic phase
was washed with brine and treated as described previously. The result-
ing product was purified by flash chromatography using ethyl ace-
tate:hexane (40:60 V:V) as eluent to give 1
a,17b-dihydroxy-17a-
methyl-5
a
-androst-2-ene (14) (0.1512 g, 53%). 1H NMR (CDCl3): 0.73
(s, 3H, CH3-19), 0.86 (s, 3H, CH3-18), 1.21 (s, 3H, CH3-20), 3.71 (d,
J1 = 4.36 Hz, 1H, CH-1), 5.82 (m, 2H, CH-2 and CH-3).
2.8.5.3.
3
a-Bromo-4n,17b-dihydroxy-17
a-methyl-5a-androstane
(20). To a solution of 17b-hydroxy-17a-methyl-5a-androstan-3-
2.8.4.7. 17b-Hydroxy-17
a
-methyl-5
a
-androst-2-en-1-one (15). To a
ene (19) (0.9086 g, 2.89 mmol) in water and THF (20% V:V, 4 mL)
was added at room temperature N-bromosuccinimide freshly
recrystallized from water (0.5906 g, 3.32 mmol), followed by stir-
ring overnight. The reaction mixture was poured in water and ex-
tracted three times with ethyl acetate. The organic phase was
washed with brine and treated as described previously. The
resulting product was purified by flash chromatography using
solution of 1 ,17b-dihydroxy-17
a
a
-methyl-5 -androst-2-ene (14)
a
(0.092 g, 0.302 mmol) in acetone (3 mL) was added dropwise Jones
reagent (0.23 mL, 0.58 mmol) at room temperature under argon
until an orange color persisted. The reaction was stirred for
30 min at room temperature and 2-propanol was added dropwise
until a green color persisted. The reaction mixture was filtrated un-
der vacuum on a celite pad and the solvent was evaporated under
vacuum. The resulting crude product was purified by flash chroma-
tography using ethyl acetate:hexane (40:60 V:V) as eluent to give
ethyl acetate:hexane (10:90 V:V) as eluent to give 3
a
-bromo-
4n,17b-dihydroxy-17a-methyl-5a-androstane (20) (0.2251 g,
19%). 1H NMR (CDCl3): 0.85 (s, 3H, CH3-18), 1.04 (s, 3H, CH3-19),
1.21 (s, 3H, CH3-20), 2.36 (m, 1H, CH-5), 3.87 (td, 1H, CH-3), 4.36
(dd, 1H, CH-4).
17b-hydroxy-17a-methyl-5a-androst-2-en-1-one (15) (0.0713 g,
78%). 1H NMR (CDCl3): 0.88 (s, 3H, CH3-18), 1.08 (s, 3H, CH3-19),
1.23 (s, 3H, CH3-20), 5.80 (d, J1 = 9.60 Hz, 1H, CH-2), 6.65 (d,
J1 = 9.65 Hz, 1H, CH-3).
2.8.5.4.
(21). As adapted from Klimstra [28], to a solution of 3
4n,17b-dihydroxy-17 -methyl-5 -androstane (20) (0.1842 g,
3a-Bromo-17b-hydroxy-17a-methyl-5a-androstan-4-one
a-bromo-
2.8.4.8.
1b,17b-dihydroxy-17
of 17b-hydroxy-17
1
a
,17b-Dihydroxy-17
a
a
-methyl-5
-methyl-5 -androst-2-ene (16). To a solution
-androst-2-en-1-one (15) (0.0495 g,
a
-androst-2-ene (14) and
a
a
a
0.447 mmol) in acetic acid (14 mL) was added dropwise a solution
of chromium trioxide (0.1174 g, 1.17 mmol) in acetic acid (2.5 mL)
at 0 °C, followed by stirring overnight at room temperature. The
a-methyl-5a
0.1639 mmol) in anhydrous methanol (5 mL) was added sodium

J. Gauthier et al. / Steroids 77 (2012) 635–643
639
reaction mixture was poured in aqueous NaHCO3 (87 g/L) and ex-
(metabolite of 17
methyl-5 -androst-1-ene (metabolite of delta-1-methyltestoster-
one), androst-4-ene-3,17-dione as well as isomeric 3-hydroxy-5-
androst-4-ene-17-one. Traces of 1 ,17b-dihydroxy-17 -methyl-
-androst-2-ene (14) and 17b-hydroxy-17 -methyl-5 -androst-
a-methyltestosterone), 3a,17b-dihydroxy-17a-
tracted three times with ethyl acetate. The organic phase was washed
with brine and treated as described previously. The resulting product
was purified by flash chromatography using ethyl acetate:hexane
a
a
a
(30:70 V:V) as eluent to give 3
a
-bromo-17b-hydroxy-17
a-methyl-
5a
a
a
5a
-androstan-4-one (21) (0.1055 g, 58%). 1H NMR (CDCl3): 0.75 (s,
2-en-4-one (22), synthesized within the scope of this project were
3H, CH3-18), 0.85 (s, 3H, CH3-19), 1.22 (s, 3H, CH3-20), 3.09 (dd,
also found in the mixture.
J1 = 3.1 Hz, J2 = 12.2 Hz, 1H, CH-5), 4.3 (dd, 1H, CH-3).
‘‘Halodrol’’ from Gaspari Nutrition, is another supplement prod-
uct claimed to ‘‘induce maximal muscle hypertrophy’’ labeled as
containing ‘‘Polydehydrogenated, polyhydroxylated halomethetio-
allocholane’’ but known to contain steroids such as DMT. A capsule
of Halodrol was crushed and dissolved in methanol. The solvent was
evaporated, the residue dissolved in KOH and extracted with hex-
ane. After evaporation, the content was analyzed as described pre-
viously by GC/MS. At least 12 steroids were detected of which
nine appeared to be derived from dehydrochlormethyltestosterone
2.8.5.5. 17b-Hydroxy-17
adapted from Klimstra [28], to a solution of 3
xy-17 -methyl-5 -androstan-4-one (21) (0.0927 g, 0.226 mmol)
a
-methyl-5a-androstan-2-en-4-one (22). As
a-bromo-17b-hydro-
a
a
in anhydrous DMF (6 mL) were added lithium chloride (0.0482 g,
1.14 mmol) and lithium carbonate (0.0324 g, 0.47 mmol) at room
temperature under argon, followed by stirring at reflux for 3 h.
The reaction mixture was poured in water and extracted five times
with ethyl ether. The organic phase was washed with water and
brine and treated as described previously. The resulting product
was purified by flash chromatography using ethyl acetate:hexane
(DHCMT, 17b-hydroxy-17a-methyl-4-chloroandrosta-1,4-diene-3-
one), itself present in the mixture. The mass spectra of these chlori-
nated compounds show molecular ions increased by 2, 4 and 6 amu,
hence consistent with the result of a non selective hydrogenation of
(20:80 V:V) as eluent to give 17b-hydroxy-17a-methyl-5a-and-
rost-2-en-4-one (22) (0.0424 g, 62%). 1H NMR (CDCl3): 0.86 (s,
3H, CH3-18), 0.88 (s, 3H, CH3-19), 1.22 (s, 3H, CH3-20), 5.99 (dd,
J1 = 2.8 Hz, J2 = 10.0 Hz, 1H, CH-3), 6.79 (m, 1H, CH-2).
DHCMT. The two isomers of DMT and 17
were also major in that product.
a-methyldrostanolone
3.2. Comparison of metabolites found in incubations and reference
human urine samples
2.8.5.6. 4n,17b-Dihydroxy-17
a
-methyl-5a-androstan-2-ene (23). To
a
solution of 17b-hydroxy-17
a
-methyl-5a
-androst-2-en-4-one
Under the conditions employed, four metabolites mostly in the
free form were formed from the incubation with fresh human
hepatocytes in low but sufficient amount to permit the acquisition
of their mass spectrum as their pertrimethylsilylated derivatives.
Metabolite M1 (molecular ion at m/z 448) appeared to be hydrox-
ylated in C-16 showing the characteristic ions at m/z 231 and 218
from D-ring fragmentation [29]. This metabolite expected from 17-
alkylated steroids was barely detectable in the reference urines
and therefore, whether it belonged to the 2-en or 3-en isomer
was not determined.
The mass spectrum of the TMS-derivative of the second metab-
olite (M2) suggested the presence of a keto group in the A-ring
with an intact D-ring (m/z 179, 193, 195, 143), while closely eluting
M3 could be the corresponding hydroxylated metabolite (molecu-
lar ions at m/z 446 and 448 respectively). The chemical synthesis of
the isomeric compounds with the keto or hydroxyl group in C-1
and C-4 was undertaken for the main isomer of DMT with the dou-
ble bond in C-2. The corresponding products from the non methyl-
(22) (0.0267 g, 0.088 mmol) in anhydrous methanol (2.5 mL) was
added sodium borohydride (0.018 g, 0.48 mmol) at room tempera-
ture under argon, followed by stirring for 3 h. The reaction mixture
was poured in water and extracted three times with ethyl acetate.
The organic phase was washed with brine and treated as described
previously. The resulting product was purified by flash chromatog-
raphy using ethyl acetate:hexane (25:75 V:V) as eluent to give a
mixture of the two isomers in C-4 of 4n,17b-dihydroxy-17
methyl-5 -androstan-2-ene (23) (0.0219 g, 81%). Both were iso-
lated by semi-preparative reverse phase HPLC: the residue was dis-
solved in acetonitrile and water (50% V:V, 50 L) and injected. The
elution was carried out with varying proportions of acetonitrile
and water each containing formic acid (0.1% V:V) starting at 41%
(1 mL/min) for 3 min, to 42.3% in 0.1 min (0.5 mL/min), raised to
43.2% in 8.9 min and finally to 80% in 0.1 min, held for 0.4 min.
One isomer was isolated with sufficient purity in the fraction elut-
ing between 9.5 and 10.3 min; the configuration of the hydroxyl
group in C-4 was not determined. 1H NMR (CDCl3): 0.86 (s, 3H,
CH3-18), 0.97 (s, 3H, CH3-19), 1.21 (s, 3H, CH3-20), 3.97 (m, 1H,
CH-4), 5.82 (m, 2H, CH-2 and CH-3).
a-
a
l
ated analog, 17b-hydroxy-5
dihydroxy-5 -androst-2-ene (7) were also prepared from their
acetates as described earlier, to compare their mass spectra.
17b-Hydroxy-17 -methyl-5 -androst-2-en-1-one (15) and
1,17b-dihydroxy-17 -methyl-5 -androst-2-ene (14 and 16) were
obtained as described above and summarized in Fig. 1 from 3b-hy-
droxy-5 -androstan-17-one (8).
a-androst-2-en-4-one (5) and 4n,17b-
a
a
a
3. Results
a
a
3.1. Content of the DMT (‘‘New Stuff
of ‘‘Halodrol’’
D
’’) preparation and of a capsule
a
The methyl group in C-17 was added by a Grignard reaction,
which was followed by the oxidation of the hydroxyl group in C-
3 and the addition of a bromine atom in C-2 to give (11). Following
elimination in the presence of lithium bromide and carbonate, the
1a,2a-spirooxirane (13) was formed by Weitz–Scheffer reaction,
which begins with the addition of the peroxide ion on the double
The oily preparation labeled ‘‘New Stuff
D’’ was diluted and
analyzed by GC/MS as such and as its trimethylsilyl derivatives.
The most abundant products were characterized as being the 2-
and 3-en isomers of DMT (approximately 3:1 mixture) [1]. The lack
of sufficient purification was attested by the presence of other ste-
bond in C-1. This addition occurring preferentially on the less hin-
roids. Of these, the probable chemical precursor utilized 5
a-and-
dered side of the molecule, the 1
a
,2a-spirooxirane was obtained.
rost-2-en-17-one and the 3-en isomer (signal 20% in height of
the most intense peak) and 17b-epimers of DMT that could have
resulted from a poorly controlled methylation. From the complex
mixture of hydroxyl and ketonic compounds present were identi-
The latter, by a Wharton reaction, produced the C-1
a
-hydroxyle
(14), with the retained configuration. This alcohol was oxidized
to the conjugated ketone (15), in turn reduced with sodium
borohydride to afford a mixture of both isomers of the C-1-hydrox-
ylated metabolite (14, 16). The mass spectra of the TMS-derivatives
fied inter alii 17
-androst-1-en-3-one also known as delta-1-methyltestosterone
and its 17-epimer, 3 ,17b-dihydroxy-17 -methyl-5 -androstane
a-methyltestosterone, 17b-hydroxy-17a-methyl-
5a
of 17b-hydroxy-17
a
-methyl-5 -androst-2-en-1-one (15) and
a
a
a
a
1a,17b-dihydroxy-17
a-methyl-5a-androst-2-ene (14) (proposed

640
J. Gauthier et al. / Steroids 77 (2012) 635–643
O
OH
OH
CH3MgBr toluene
Jones Reagent
1) 80oC 4h
2) rt 16h
73%
rt, 30 min
92%
O
HO
HO
10
9
8
Py H Br3 EtOH, CH2Cl2
40oC, 2h
50%
OH
OH
OH
H2O2 30%
MeOH
LiBr Li2CO3
DMF
O
Br
Rt
2h 0oC
3h rt
Reflux, 1h
91%
O
O
O
12
13
11
N2H4
1)30 min, rt
2)15 min, 90oC
3)15 min, 90oC
53%
OH
OH
OH
OH
O
OH
NaBH4
MeOH
Jones Reagent
rt
rt
30 min
1 h
78%
14
15
-androst-2-ene (14) and 1b,17b-dihydroxy-17
14 and 16
-methyl-5a-androst-2-ene (16).
Fig. 1. Synthesis of 1
a,17b-dihydroxy-17a-methyl-5
a
a
structure) are shown in Fig. 2 along with the proposed fragmenta-
tion pathways leading to the most intense ions. These two com-
pounds do not appear to be direct metabolites of DMT however
the reference urine samples. A minor isomer with the same molec-
ular ion is also present in both. The mass spectrum of its pertrim-
ethylsilyl derivative is not that informative apart from indicating
the presence three hydroxyl groups, with a weak molecular ion
at m/z 538 and ions at m/z 448, 358 and 269, while the base peak
at m/z 143 excludes hydroxylation sites on the D-ring. Scaling up
with repeated incubations of human liver S9 fractions permitted
the NMR analysis required for establishing the configuration of
the hydroxyl groups prior to its synthesis.
the 1
‘‘New Stuff
17b-Hydroxy-17
4n,17b-dihydroxy-17
a
,17b-diol (14) was identified in the crude oily preparation
D
’’.
a
-methyl-5
a
-androstan-2-en-4-one (22) and
a-methyl-5a-androstan-2-ene (23) were ob-
tained from androst-4-ene-3,17-dione (17) as described earlier
and summarized in Fig. 3.
The methylation of (18) was followed by the formation of bro-
mohydrine the alkene in C-3 which resulted in the formation of
several by-products, the desired compound being obtained in
low yield. Oxidation followed by elimination produced the conju-
gated ketone (22) which was finally reduced with sodium borohy-
dride in methanol to afford several products including fully
reduced alcohols; the diol (23) was purified from this mixture by
semi-preparative reverse phase HPLC. The stereochemistry of the
hydroxyl group in C-4 was not determined. The 1H NMR spectrum
obtained showed a signal at 3.97 ppm for the proton attached to
the carbon bearing the hydroxyl group in C-4, while the two vinylic
protons in C-2 and C-3 were found at 5.82 ppm. The mass spectra
of the TMS-derivatives of the two ketones (5) and (22) are similar,
exhibiting intense ions corresponding to the loss of a methyl group
and the fragmentation of the B-ring (ions at m/z 195, 194, 193 and
179) (Fig. 4). The mass spectrum of the TMS-derivative of the diol
(23), also shown, is characterized by the presence of a weak molec-
ular ion at m/z 448 and the fragment ions at m/z 433 (–CH3), 358,
343, 268, 253 (sequential losses of HOTMS and CH3), with the base
peak at m/z 143 (D-ring). These two compounds correspond to
metabolites M2 and M3, but the ketone (22) was also identified
in the crude preparation of DMT.
The presence of a signal at 3.9 ppm (2H) suggested a 2b,3a-
dihydroxy steroid, based upon this chemical shift [30–32]. This
hypothesis is also supported by the absence of an ion at m/z 191
and the relatively weak ion at m/z 147 in the mass spectrum of
M4 expected to be formed with vicinal diols in the cis configura-
tion (TMSO-CH = OTMS+).
17a-Methyl-2b,3a,17b-trihydroxy-5a-androstane (3) was ob-
tained from the opening in acidic conditions of the spirooxirane
(2); the retention time and mass spectrum of its TMS-derivative
matched the metabolite present in the reference urines and the
incubation mixtures with fresh hepatocytes and S9 fractions as
shown in Fig. 5.
The NMR, mass spectra and retention times were identical to
metabolite M4, which confirms its structure. The HSQC NMR spec-
trum showed two signals at 71.77 and 70.56 ppm for the two car-
bon atoms C-2 and C-3, while their two protons formed
quadruplets with respective chemical shifts of 3.87 (CH-3) and
3.91 (CH-2) in the NOESY NMR spectrum. These two protons in
C-2 and C-3 being in an equatorial orientation, their coupling with
their respective vicinal protons in C-1 and C-4 was shown to be of
equal intensity, which would not have been the case, had it been in
the axial configuration. Accordingly, in the 1H NMR spectrum, the
two quadruplets having a coupling constant inferior to 4 Hz sug-
gests two equatorial protons in C-2 and C-3 [33].
The major metabolite M4 isolated from the incubations with
fresh hepatocytes and S9 fractions was also the principal one in

J. Gauthier et al. / Steroids 77 (2012) 635–643
641
Fig. 2. Mass spectra and proposed fragmentation pathways of the TMS-derivatives of synthesized 17b-hydroxy-17
a
-methyl-5
a-androst-2-en-1-one (15) (upper panel) and
1a
,17b-dihydroxy-17a-methyl-5
a-androst-2-ene (14, proposed structure) (lower panel).
O
O
OH
CH3MgBr
Toluene
Zn dust
acetic acid
Reflux
1h30
48%
rt, 16h
89%
O
17
18
21
19
20% H2O/THF
NBS
19%
OH
OH
OH
LiCl
Li2CO3
DMF
rt, 16h
CrO3
AcOH
rt, 16h
58%
3h
reflux
62%
Br
20
OH
Br
22
O
O
NaBH4
MeOH
rt
81%
OH
3h
23
OH
Fig. 3. Synthesis of 4n,17b-dihydroxy-17a-methyl-5a-androstan-2-ene (23).
employed for its synthesis and the stereoselectivity and purifica-
tion of the 17-methylation reaction [1,9]. Therefore, locating and
identifying the specific metabolites in the urine matrix itself com-
plex, was rightly described as difficult [10]. Incubations with fresh
hepatocytes and liver S9 fractions were most helpful in that re-
gards, having been done with DMT of known purity from synthesis
[1].
4. Discussion
The ‘‘hormonal’’ products containing DMT are in fact, mixtures
of several steroids, some of closely related structure of same
molecular weight. The 2-en isomer of DMT is predominant but
always accompanied by the 3-en isomer in varying proportions
(5–8:1), which reflects the composition of the precursor steroid

642
J. Gauthier et al. / Steroids 77 (2012) 635–643
Fig. 4. Mass spectra of the TMS-derivatives of synthesized 17b-hydroxy-17
a-methyl-5a-androstan-2-en-4-one (22) (upper panel), 17b-hydroxy-5a-androst-2-en-4-one (5)
(middle panel) and 4n,17b-dihydroxy-17 -methyl-5 -androstan-2-ene (23) (lower panel).
a
a
In humans, according to Rodchenkov et al. the urinary metab-
olites of DMT appeared to be mostly mono-hydroxylated with the
double bond intact or fully reduced or dihydroxylated [10]. In
fact, our results show that from the several compounds with mass
spectra showing the expected molecular ions at m/z 450 and 448
(TMS derivatives), many were either metabolites of other steroids
or already present in the starting material. Having compared the
reference urine samples with incubation mixtures and considered
the steroids present in the starting material analyzed, we con-
cluded that the main glucuroconjugated metabolite attributed
However, although the designer steroid itself could have been
invisible to anti-doping controls, some of the other steroids present
in the product such as methyltestosterone and DHCMT were most
certainly picked up by the highly sensitive contemporary assays
(LC or GC–MSn).
Acknowledgments
This project has been carried out in part with the support of the
World Anti-Doping Agency and of NSERC (Grant to Julie Gauthier).
We are grateful to Pr. John Templeton for the gift of precious refer-
ence steroids, to Cathy Copeland, Paul Loo and Philippe St-Amour
of the Laboratory and Scientific Services Directorate of the Canada
Border Services Agency for their contribution in uncovering DMT
and providing preparations and to Celsis In Vitro Technologies
for the gift of fresh human hepatocytes. The assistance of Philippe
Räss and Patrick Allard for scaling up and purifying DMT main
strictly to DMT major 2-en isomer, was 17
a-methyl-2b,3a,17b-
trihydroxy-5 -androstane (3). The structure of the other isomer
a
eluting earlier in the GC/MS chromatogram has not been charac-
terized but is postulated to be the 3,4-diol arising from 3-en DMT.
Another metabolite was exclusively sulfoconjugated and its char-
acterization will be reported elsewhere.
The characterization of the main urinary glucuroconjugated
metabolite of DMT will enable its detection in athlete’s samples.

J. Gauthier et al. / Steroids 77 (2012) 635–643
643
143
8000000
7000000
6000000
5000000
4000000
3000000
2000000
1000000
73
269
523
105
100
448
215
343
169
394
194
200
242
250
303
300
420
400
51
50
365
350
475
0
150
450
500
m/z-->
Fig. 5. Mass spectrum of the TMS-derivative of 17a-methyl-2b,3a,17b-trihydroxy-5a-androstane (3).
[16] Levesque JF, Gaudreault M, Houle R, Chauret N. Evaluation of human
hepatocyte incubation as new tool for metabolism study of
metabolite, of René Maltais and Marie-Claude Trottier with the
chemical syntheses and NMR analyses is gratefully acknowledged.
a
androstenedione and norandrostenedione in a doping control perspective. J
Chromatogr B 2002;780(1):145–53.
[17] Levesque JF, Templeton E, Trimble L, Berthelette C, Chauret N. Discovery,
biosynthesis, and structure elucidation of metabolites of a doping agent and a
direct analogue, tetrahydrogestrinone and gestrinone, using human
hepatocytes. Anal Chem 2005;77(10):3164–72.
[18] Gauthier J, Goudreault D, Poirier D, Ayotte C. Identification of drostanolone and
17-methyldrostanolone metabolites produced by cryopreserved human
hepatocytes. Steroids 2009;74(3):306–14.
[19] Poirier D, Maltais R, Roy J. 2-(N-substituted piperazinyl) steroid derivatives,
WO 2010/060215 A1; 2010.
[20] Celsis IVT. Fresh Hepatocyte Suspension. 2007. Available from: <http://www.
celsis.com/media/pdf/filelib/ps-fresh-human-animal-hepatocyte-suspensions.
pdf>.
References
[1] Ayotte C, Goudreault D, Cyr D, Gauthier J, Ayotte P, Larochelle C, Poirier D.
Characterisation of chemical and pharmacological properties of new steroids
related to doping of athletes. In: Manfred Donike Workshop, 24th Cologne
Workshop on Dope Analysis. Cologne; 2006.
[2] WADA. WADA announces discovery of new designer steroid; 2005. Available
from: <http://www.wada-ama.org/en/newsarticle.ch2?articleID=3115089>.
[3] HealthCanada, Health Canada advises consumers not to use unauthorized
products containing anabolic steroids. Health Canada: Ottawa; 2006.
[4] Hartgens F, Kuipers H. Effects of androgenic–anabolic steroids in athletes.
Sports Med 2004;34(8):513–54.
[5] Bahrke MS, Yesalis CE. Abuse of anabolic androgenic steroids and related
substances in sport and exercise. Curr Opin Pharmacol 2004;4(6):614–20.
[6] Kutscher EC, Lund BC, Perry PJ. Anabolic steroids: a review for the clinician.
Sports Med 2002;32(5):285–96.
[7] Diel P, Friedel A, Geyer H, Kamber M, Laudenbach-Leschowsky U, Schanzer W,
Thevis M, Vollmer G, Zierau O. Characterisation of the pharmacological profile
of desoxymethyltestosterone (Madol), a steroid misused for doping. Toxicol
Lett 2007;169(1):64–71.
[8] WADA. 2006. Available from: <http://www.wada-ama.org/rtecontent/
document/Explanatory_Note_2006.pdf>.
[9] Sekera MH, Ahrens BD, Chang YC, Starcevic B, Georgakopoulos C, Catlin DH.
Another designer steroid: discovery, synthesis, and detection of ‘madol’ in
urine. Rapid Commun Mass Spectrom 2005;19(6):781–4.
[10] Rodchenkov G, Sobolevsky T, Sizoi V. New Designer Anabolic Steroids from
Internet. In: Manfred Donike Workshop, 24th Cologne Workshop on Dope
Analysis. Cologne; 2006.
[11] Kuuranne T, Kurkela M, Thevis M, Schanzer W, Finel M, Kostiainen R.
Glucuronidation of anabolic androgenic steroids by recombinant human
UDP-glucuronosyltransferases. Drug Metab Dispos 2003;31(9):1117–24.
[12] Kuuranne T, Leinonen A, Thevis M, Schanzer W, Pystynen KH, Kostiainen R.
Metabolism of ‘‘new’’ anabolic steroids: development of in vitro methodology
in metabolite production and analytical techniques. In: Manfred Donike
Workshop 24th Cologne Workshop on Dope Analysis. Cologne; 2006.
[13] Hintikka L, Kuuranne T, Aitio O, Thevis M, Schanzer W, Kostiainen R. Enzyme-
assisted synthesis and structure characterization of glucuronide conjugates of
eleven anabolic steroid metabolites. Steroids 2008;73(3):257–65.
[14] Rendic S, Nolteernsting E, Schanzer W. Metabolism of anabolic steroids by
recombinant human cytochrome P450 enzymes. Gas chromatographic–mass
[21] Celsis-In Vitro, T. Products > Cryopreserved Hepatocytes. Available from:
<http://www.invitrotech.com/cryo_hepatocytes.cfm>.
[22] Ayotte C, Goudreault D, Charlebois A. Testing for natural and synthetic
anabolic agents in human urine. J Chromatogr B 1996;687(1):3–25.
[23] Celsis IVT. Human and Animal Liver S9; 2007. Available from: <http://
www.celsis.com/media/pdf/filelib/ps-human-animal-s9.pdf>.
[24] Ferraboschi P, Colombo D, Prestileo P. A convenient synthesis of oxandrolone
through a regioselective Candida antarctica lipase-catalyzed transformation.
Tetrahedron: Asymmetry 2003;14(18):2781–5.
[25] Counsell RE, Klimstra PD. Anabolic agents: derivatives of 2-halo 5alpha-
androst-1-ene. J Med Pharm Chem 1962;91:477–83.
[26] Klimstra PD, Optionally 17-alkylated-3alpha-methyl- and derivatives thereof,
USP Office, editor. USA; 1968.
[27] Tavares da Silva EJ, Roleira FMF, Sa e Melo ML, Campos Neves AS, Paixao JA, de
Almeida MJ, Silva MR, Andrade LCR. X-ray and deuterium labeling studies on
the abnormal ring cleavages of a 5[beta]-epoxide precursor of formestane.
Steroids 2002;67(3–4):311–9.
[28] Klimstra PD, Optionnally 17-alkylated 17-oxygenated 5alpha-2-en-4-ones and
intermediates thereto, USP Office, editor. USA; 1965.
[29] Masse R, Bi HG, Ayotte C, Dugal R. Studies on anabolic-steroids. 2. Gas-
chromatographic–mass-spectrometric characterization of oxandrolone
urinary metabolites in man. Biomed Environ Mass 1989;18(6):429–38.
[30] Cruz Silva MM, Riva S, Sá e Melo ML. Regioselective enzymatic acylation of
vicinal diols of steroids. Tetrahedron 2005;61(12):3065–73.
[31] Horvath A, Skoda-Foldes R, Maho S, Berente Z, Kollar L. Facile ring opening of
2,3-epoxy-steroids with aromatic amines in ionic liquids. Steroids
2006;71(8):706–11.
[32] Santos GA, Murray GA, Pujol CA, Damonte EB, Maier MS. Synthesis and
antiviral activity of sulfated and acetylated derivatives of 2beta,3alpha-
dihydroxy-5alpha-cholestane. Steroids 2003;68(2):125–32.
[33] Karplus M. Vicinal proton coupling in nuclear magnetic resonance. J Am Chem
Soc 1963;85(18):2870–1.
spectrometric
1999;735(1):73–83.
determination
of
metabolites.
J
Chromatogr
B
[15] Levesque JF, Gaudreault M, Aubin Y, Chauret N. Discovery, biosynthesis, and
structure elucidation of new metabolites of norandrostenedione using in vitro
systems. Steroids 2005;70(4):305–17.