1000
M. Thevis et al.
the present study, synthesized reference material and com-
mercially obtained substances of steroid-17-O-glucuronides
and androstanediol-3- and 17-O-glucuronides (Fig. 1) were
characterized by NMR and GC/MS after derivatization to
per-TMS derivatives and to the corresponding methyl ester
trimethylsilyl ethers. Common and individual fragmenta-
tion patterns of the steroidal and glycosidic moiety were
obtained and the generation of fragment ions is proposed
based on selected reaction monitoring and different deu-
terium labelling experiments.
the aqueous layer was adjusted to 2.5 by addition of 3 M HCl
and extracted twice with 20 ml of ethyl acetate. The com-
bined organic layers were evaporated to dryness yielding
the pure 3-O- or 17-O-glucuronides in amounts of 71–79%
of the theory. In the case of the reduction of 1˛-methyl-5˛-
androstane-17ˇ-ol-3-one-17-O-glucuronide, the 3-keto group
yielded the 3˛- and 3ˇ-configurations.
Preparation of 17-O-glucuronides of androstane-3˛,
17ˇ-diol and d5-androstane-3˛,17ˇ-diol
Protection of the 3-hydroxy group. A 150 mg amount of andros-
terone or d5-androsterone, respectively, was dissolved in
25 ml of a mixture of acetic anhydride, acetonitrile and
pyridine (10 : 10 : 5, v/v/v) and heated in a water-bath at
EXPERIMENTAL
Chemicals and steroids
°
60 C while stirring. After 5 h, complete acetylation of the
N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was
purchased from Chem. Fabrik Karl Bucher (Waldstetten,
Germany) and distilled before use, ammonium iodide
(purum, p.a.), silver carbonate (purum, p.a.) and toluene
(puriss., absolute over molecular sieve) from Fluka (Buchs,
Switzerland), sodium hydroxide (p.a.), potassium car-
bonate (p.a.), glacial acetic acid (p.a.) and iodomethane
(for synthesis) from Merck (Darmstadt Germany) and
ethanethiol (97%) from Aldrich (Deisendorf, Germany) and
methyl 1-bromo-1-desoxy-2,3,4-triacetylglucopyranuronate
was synthesized by the procedure of Bollenback et al.7
The reference substances testosterone glucuronide and
the unconjugated steroids of androsterone, testosterone,
epitestosterone, metenolone, mesterolone, nortestosterone
and methyltestosterone were purchased from Sigma (St.
Louis, MO, USA). The metabolite of nortestosterone,
19-norandrosterone, and the deuterated counterparts of
nortestosterone, testosterone, 19-norandrosterone and an-
drosterone were synthesized in our laboratory.8,9
hydroxy group at C-3 was achieved, and 20 ml of doubly
distilled water were added. The pH was adjusted to 7 by
carefully adding solid sodium carbonate and the solution
was extracted twice with 40 ml of tert-butyl methyl ether.
The organic layers were combined, evaporated to dryness
°
at 50 C under reduced pressure and the residue was stored
in a desiccator over phosphorus pentoxide and potassium
hydroxide under reduced pressure for 10 h.
Reduction of the 17-keto group. According to Rao et al.,10 the
reduction of the 17-keto group was performed by solving the
crude product in 50 ml of a mixture of ethanol and doubly
disstilled water (80 : 20, v/v) and the addition of a 1.5 molar
excess of sodium borohydride, obtaining the 17ˇ-OH group.
Conjugation reaction. The glucuronidation and subsequent
hydrolysis of the protecting groups of the glycosidic moiety
were performed by means of the modified Koenigs–Knorr
reaction11–14 as described earlier.6
Synthesis of steroid glucuronide conjugates
The monohydroxylated steroids testosterone, d3-testoster-
one, nortestosterone, d3-nortestosterone, mesterolone,
metenolone and epitestosterone were conjugated to steroid
glucuronides according to the method described earlier.6
The selectively conjugated bis-hydroxylated steroids were
prepared as described below.
Hydrolysis of the protecting group at C-3. The obtained
3-acetylated glucuronide conjugate was dissolved in 10 ml
of methanol, 1 ml of 2 M aqueous NaOH was added and the
°
solution was stirred for 3 h in a water-bath at 60 C, yielding
complete hydrolysis of the acetyl group at C-3. After cooling
to ambient temperature, the solution was evaporated to
dryness and the residue was dissolved in 20 ml of doubly
distilled water and extracted with tert-butyl methyl ether and
ethyl acetate as described above. The main product obtained
was the 17-O-glucuronide of androstane-3˛,17ˇ-diol or d5-
androstane-3˛,17ˇ-diol, respectively.
Preparation of 3-O-glucuronides of androstane-3˛,
17ˇ-diol, 19-norandrostane-3˛,17ˇ-diol, deuterated
analogues and 17-O-glucuronide of 1˛-methyl-5˛-
androstane-3˛/ˇ,17ˇ-diol
Amounts of 20–100 mg of androsterone glucuronide, 19-
norandrosterone glucuronide or their deuterated counter-
parts, respectively, were dissolved in 20 ml of a mixture of
ethanol and doubly distilled water (80 : 20, v/v). While stir-
ring, a 1.5 molar excess of sodium borohydride was added
and the solution was stirred at ambient temperature for 1 h.
Yields
The crude substances were finally purified by HPLC
fractionation with overall yields of 22.4 and 18.3% of theory,
respectively.
Nuclear magnetic resonance (NMR) analysis
°
The reaction mixture was evaporated to dryness at 50 C
NMR analyses were performed on a Bruker DPX 300
or Bruker DRX 500 instrument. Amounts of 5 mg
of the glucuronides of testosterone, d3-testosterone,
epitestosterone, nortestosterone, d3-nortestosterone, methyl-
testosterone, metenolone, mesterolone, androstane-3˛,17ˇ-
diol, d5-androstane-3˛,17ˇ-diol, 19-norandrostane-3˛,17ˇ-
diol and d4-19-norandrostane-3˛,17ˇ-diol were dissolved in
under reduced pressure by means of a rotary evaporator and
the residue was dissolved in 20 ml of doubly distilled water.
The pH was adjusted to 10–11 (if necessary) by addition
of 1 M aqueous NaOH and the solution was extracted twice
with 20 ml of tert butyl methyl ether to remove unconjugated
steroids possibly generated by hydrolysis. Then, the pH of
Copyright 2001 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2001; 36: 998–1012
Thevis, Mario
