5
46
steroids 7 2 ( 2 0 0 7 ) 545–551
[
2–10]. In recent investigations different isomers of 5-
2.2.
Syntheses of reference substances
androst-1-ene-3,17-diol, androst-4-ene-3,17-diol, and 17-
hydroxy-5-androstan-3-one were detected [10–12]. Besides
that, 5␣-androst-1-ene-3,17-diol occurs naturally in the fat
of pigs [13]. Its 3␣ isomer is a metabolite of 5␣-androst-
2.2.1. Catalytic hydrogenation
The reduction of C–C double bonds in the 3-oxo steroids was
performed with hydrogen (or deuterium) using methanol as
solvent and palladium on charcoal (Pd/C, 10%) as catalyst.
In case of the hydrogenation of androsta-1,4-diene-3,17-
dione methanol/6 M sodium hydroxide (20:1, v/v) was used
as solvent. After filtration from the catalyst the mixture was
evaporated and the residue was extracted with t-butyl methyl
ether (TBME). The products were separated using column
chromatography on silica gel 60 (Merck, 35–70 mesh ASTH,
bed 30 mm × 400 mm). n-Hexane/ethyl acetate was used as
mobile phase applying a gradient starting with 80% n-hexane.
After 1.5 L the ratio was changed to 70% n-hexane and after
further 1.5 L 60% n-hexane were used. The crude product
resulting from the hydrogenation of androsta-1,4-diene-3,17-
dione was further purified using HPLC (Hewlett Packard HP
1090 with UV detection at 200 nm, Macherey-Nagel VP 250/10
1
-ene-3,17-dione and 17-hydroxy-5␣-androst-1-ene-3-one
(
also named 1-testosterone) [14], which have also been mar-
keted as dietary supplements. After ingestion of the anabolic
steroid boldenone (17-hydroxyandrosta-1,4-dien-3-one), 5-
androst-1-ene-3␣,17-diol is excreted together with other
metabolites [15,16]. 17-Hydroxy-5␣-androstan-3-one, better
known as 5␣-dihydrotestosterone, is a biologically active
metabolite of testosterone.
In sports anabolic androgenic steroids are prohibited
as doping substances according to the regulations of the
World Anti-Doping Agency (WADA) [17]. According to their
regulation the identification should be based on reten-
tion time and on mass spectrometric characteristics [18].
The detection of the misuse of anabolic steroids is usu-
ally performed after enzymatic cleavage of the glucuronides
with -glucuronidase from Escherichia coli. The aglycons
are then detected by means of gas chromatography–mass
spectrometry (GC–MS) as per-trimethylsilyl (TMS) derivatives
NUCLEOSIL 100-7 C18, 3 mL/min, A: H O, B: acetonitrile, gra-
dient: 0–16 min 50% B to 90% B, 16–17 min 90% B to 50% B,
17–22.5 min 50% B).
2
[
19].
Those per-TMS derivatives of the title compounds were
2.3.
Reduction of 3- and/or 17-oxo groups
found to have very similar mass spectra and are therefore
hard to discriminate. Thus, the availability of reference com-
pounds for the correct identification of suspicious peaks is
essential.
The reduction of the oxo groups was performed with K-
Selectride (∼10-fold surplus) in absolute TBME. After 1 h at
ambient temperature the reduction was stopped by addi-
tion of H O and the mixture was extracted three times with
2
During the present study, the stereoisomers of 5-androst-
TBME. After evaporation of the solvent the products were
1
-ene-3,17-diol (8 isomers), androst-4-ene-3,17-diol (4
separated using HPLC (Hewlett Packard HP 1090 with UV detec-
tion at 200 nm, Macherey-Nagel VP 250/10 NUCLEOSIL 100-7
isomers), and 17-hydroxy-5-androstan-3-one (4 isomers)
were synthesized by reduction of the corresponding ␣,-
unsaturated oxo steroids and Kovac indices and mass
spectrometric data are provided.
C18, 3 mL/min, A: H O, B: acetonitrile, gradient: 0–16 min 50%
2
B to 90% B, 16–17 min 90% B to 50% B, 17–22.5 min 50% B,
in case of the 5␣-androst-1-ene-3,17-diols separation was
achieved on a Thermo 250/10 ODS HYPERSIL column, gradi-
ent: 0–25 min 50% B to 100% B, 25–25.5 min 100% B to 50%
B, 25.5–32 min 50% B). For the separation of the 5-androst-
2
.
Experimental
1-ene-3,17-diols the substances were pre-separated using
column chromatography (Silica gel 60, Merck, 35–70 mesh
ASTH, bed 30 mm × 400 mm). As mobile phase n-hexane/ethyl
acetate was used applying a gradient starting with 80% n-
hexane. After 2 L the ratio was changed to 70% n-hexane.
2
.1.
Chemicals
1
7-Hydroxyandrost-4-en-3-one (testosterone), 17␣-hydrox-
yandrost-4-en-3-one (epitestosterone), 17-hydroxy-5␣-and-
rostan-3-one (5␣-dihydrotestosterone), 17-hydroxy-5-and-
rostan-3-one (5-dihydrotestosterone), 5␣-androstane-3␣,
2.4.
Identification of the products by reduction to the
corresponding 5ꢀ-androstane-3ꢀ,17ꢀ-diols
1
7-diol, 5␣-androstane-3,17-diol, 5-androstane-3␣,17-
diol, and K-Selectride (potassium tri-2-butylborohydride,
M in THF) were purchased from Sigma–Aldrich (Steinheim,
Germany). 5␣-Androstane-3␣,17␣-diol, 5␣-androstane-3,17␣-
diol, 5-androstane-3␣,17␣-diol, 5-androstane-3,17-diol,
All synthesized isomers were identified by further reduction
as described above yielding the corresponding 5-androstane-
3,17-diols. Comparing the GC–MS characteristics (mass
spectra and retention times) with commercially available
reference compounds as per-TMS derivatives identified the
resulting products.
1
5␣-androst-1-ene-3,17-dione
(1-androstenedione),
17-
hydroxy-5␣-androst-1-en-3-one (1-testosterone), androst-
4
3
-ene-3␣,17␣-diol, androst-4-ene-3,17␣-diol, androst-4-ene-
␣,17-diol, and androst-4-ene-3,17-diol were obtained
2.5.
NMR analyses
from Steraloids (Wilton, USA). N-Methyl-N-trimethylsilyl-
trifluoroacetamide (MSTFA) was purchased from Chem.
Fabrik Karl Bucher (Waldstetten, Germany). Other reagents
and solvents were obtained from Merck (Darmstadt,
Germany).
The nuclear magnetic resonance (NMR) spectra of the iso-
mers (<5 mM solute) were recorded in CDCl3 at 500 MHz ( H
NMR) and 125 MHz (13C NMR) at 298 K on a Bruker Avance 500
1
spectrometer. Chemical shifts were given in ı values (ppm)