Mass spectrometry of steroid glucuronide conjugates. I. Electron impact fragmentation of 5α-/5β-androstan-3α-ol-17-one glucuronides, 5α-estran-3α-ol-17-one glucuronide and deuterium-labelled analogues

Article abstract of DOI:10.1002/jms.117

Owing to the developments of analytical instruments and interfaces (e.g. coupling high-performance liquid chromatography to mass spectrometry), there has been increased interest in new reference materials, for example in doping analysis with steroid glucuronide conjugates. The synthesized reference material has to pass several characterization steps including the use of gas chromatography/mass spectrometry (GC/MS) for its structure confirmation. In the present study, the fragmentation and mass spectrometric behaviour of several steroid glucuronide conjugates of endogenous and anabolic steroids after derivatization to pertrimethylsilylated products and to methyl ester pertrimethylsilylated products were investigated using GC/MS ion trap and GC/MS quadrupole instruments. The mass spectra of the derivatives of androsterone glucuronide, d₅-androsterone glucuronide, epiandrosterone glucuronide, etiocholanolone glucuronide, 11β-hydroxy etiocholanolone glucuronide, 19-norandrosterone glucuronide, d₄-19-norandrosterone glucuronide and 1α-methyl-5α-androstan-3α-ol-17-one glucuronide are presented and the origin of typical fragment ions of the glycosidic and steroidal moieties is proposed, based on different derivatization techniques including derivatization with d₁₈-bistrimethylsilylacetamide, methyl ester and trimethylsilyl ester derivatization and selected reaction monitoring. Typical fragmentation patterns which are related to the steroid structure are discussed. Copyright

Full text of DOI:10.1002/jms.117

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2001; 36: 159–168
Mass spectrometry of steroid glucuronide conjugates. I.
Electron impact fragmentation of 5a-/5b-androstan-
3a-ol-17-one glucuronides, 5a-estran-3a-ol-17-one
glucuronide and deuterium-labelled analogues
1
2
1
Mario Thevis,1∗ Georg Opfermann, Hans Schmickler and Wilhelm Schanzer
¨
1
Institute of Biochemistry, German Sports University, Cologne, Germany
Institute of Organic Chemistry, University of Cologne, Germany
2
Received 2 November 2000; Accepted 4 December 2000
Owing to the developments of analytical instruments and interfaces (e.g. coupling high-performance
liquid chromatography to mass spectrometry), there has been increased interest in new reference
materials, for example in doping analysis with steroid glucuronide conjugates. The synthesized reference
material has to pass several characterization steps including the use of gas chromatography/mass
spectrometry (GC/MS) for its structure confirmation. In the present study, the fragmentation and mass
spectrometric behaviour of several steroid glucuronide conjugates of endogenous and anabolic steroids
after derivatization to pertrimethylsilylated products and to methyl ester pertrimethylsilylated products
were investigated using GC/MS ion trap and GC/MS quadrupole instruments. The mass spectra of
the derivatives of androsterone glucuronide, d5-androsterone glucuronide, epiandrosterone glucuronide,
etiocholanolone glucuronide, 11b-hydroxy etiocholanolone glucuronide, 19-norandrosterone glucuronide,
d4-19-norandrosterone glucuronide and 1a-methyl-5a-androstan-3a-ol-17-one glucuronide are presented
and the origin of typical fragment ions of the glycosidic and steroidal moieties is proposed, based on
different derivatization techniques including derivatization with d18-bistrimethylsilylacetamide, methyl
ester and trimethylsilyl ester derivatization and selected reaction monitoring. Typical fragmentation
patterns which are related to the steroid structure are discussed. Copyright  2001 John Wiley & Sons, Ltd.
KEYWORDS: anabolic steroids; Koenigs–Knorr synthesis; gas chromatography; mass spectrometry; structure confirmation;
mass spectra interpretation; trimethylsilyl derivatives
steroids in doping analysis but the confirmation of results
obtained by measurements with these techniques requires
reference materials which are only seldom commercially
available.
The synthesized reference substances have to pass
different characterization steps (e.g. NMR and MS), which
can partly be performed by GC/MS. The interpretation of
the mass spectra of unconjugated and underivatised steroids
was described in detail by Budzikiewicz et al.5 Several reports
dealing with unconjugated steroids derivatized for GC/MS
analysis6–10 and the characterization of methyl and acetyl
derivatives of arylglucuronic acid conjugates have been
published.11
In this study, the mass spectrometric behaviour of com-
mercially available steroid glucuronides and synthesized
reference material (Fig. 1) as pertrimethylsilyl (per-TMS) and
methyl ester per-TMS derivatives is presented and propos-
als for the origin of characteristic fragment ions generated
by the glycosidic or steroidal moieties are described. These
data contain different information to those obtained by e.g.
LC/MS analyses, and complete identification and structural
INTRODUCTION
The detection of endogenous and anabolic steroid metabo-
lites in human urine is one of the main tasks in doping
analysis. These compounds can be excreted as phase I (not
conjugated) and/or phase II (conjugated) metabolites, of
which the latter can be sulphates or glucuronides.1 Usually
hydrolysis of these conjugates is performed, yielding the free
steroids which are derivatized and then identified by means
of gas chromatography/mass spectrometry (GC/MS).2 The
improvements in interfacing high-performance liquid chro-
matography (HPLC) with mass spectrometry (e.g. electro-
spray ionization, atmospheric pressure chemical ionization)
allow the sensitive detection and quantification of non-
hydrolysed and underivatized metabolites3 4 by avoiding
several sample preparation steps. In the future, LC/MS and
LC/MS/MS analyses of conjugates may be an alternative for
the qualitative and quantitative determination of anabolic
ŁCorrespondence to: M. Thevis, Deutsche Sporthochschule Ko¨ln,
Institut fu¨r Biochemie, Carl-Diem-Weg 6, 50933 Cologne, Germany.
E-mail: mario@biochem.dshs-koeln.de
Contract/grant sponsor: Bundesinstitut fu¨r Sportwissenschaft.
Copyright  2001 John Wiley & Sons, Ltd.

160
M. Thevis et al.
O
O
O
D
D
COOH
COOH
COOH
O
O
O
O
O
O
D
H
H
H
D
D
OH
OH
OH
HO
HO
HO
II
III
I
OH
OH
OH
O
O
O
HO
COOH
COOH
COOH
O
O
O
O
O
O
H
H
H
OH
OH
OH
HO
HO
HO
IV
V
VI
O
O
OH
OH
OH
D
D
COOH
COOH
O
O
O
O
H
H
D
D
OH
OH
HO
HO
VII
VIII
OH
OH
Figure 1. Structural formulae of (I) androsterone glucuronide, (II) d5-androsterone glucuronide, (III) epiandrosterone glucuronide, (IV)
etiocholanolone glucuronide, (V) 11 -hydroxy etiocholanolone glucuronide, (VI) 19-norandrosterone glucuronide, (VII) d4-19-noran-
drosterone glucuronide and (VIII) 1 -methyl-5 -androstan-3 -ol-17-oneglucuronide.
characterization of synthesized steroid glucuronide conju-
gates by the generation of abundant and typical ions based
on the presence of the glucuronic acid moiety in addition to
those originating from the steroid.
sieve) with 500 mg (1.26 mmol) of freshly prepared methyl-1-
bromo-1-deoxy-2,3,4-triacetylglucopyranuronate in the pres-
ence of 280 mg (1 mmol) of silver carbonate as catalyst.18
After consumption of the steroid, depending on the position
of glucuronidation and steric circumstances, the catalyst is
removed by filtration through a medium-porosity frit and the
solution is evaporated to dryness by means of a rotary evap-
orator under reduced pressure. The residue is resolved in
10 ml of methanol, 1 ml of 1 M aqueous NaOH is added and,
by frequent control of the pH, the hydrolysis of the methyl
and acetyl groups at the glycosidic moiety is achieved. The
solution is evaporated to dryness, a volume of 20 ml of dou-
bly distilled water is added and the solution is extracted
twice with 40 ml of tert-butyl methyl ether to remove the
remainder of the unconjugated steroid. The aqueous layer is
then adjusted to pH 2.5 with 6 M aqueous HCl and extracted
three times with 50 ml of ethyl acetate. The combined organic
phases are evaporated to dryness, the residue is dissolved
in 2 ml of glacial acetic acid and the steroid glucuronide is
purified by semipreparative HPLC fractionation.
EXPERIMENTAL
Chemicals and steroids
N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was
purchased from Chemische Fabrik Karl Bucher (Waldstet-
ten, 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 carbon-
ate (p.a.), glacial acetic acid (p.a.) and iodomethane (for
synthesis) from Merck (Darmstadt, Germany), ethanethiol
(97%) from Aldrich (Deisendorf, Germany), acetonitrile (p.a.)
from Roth (Karlsruhe, Germany) and tert-butyl methyl
ether and ethyl acetate (distilled before use) from Krae-
mer & Martin (St. Augustin, Germany). Methyl-1-bromo-
1-deoxy-2,3,4-triacetylglucopyranuronate was synthesized
by the procedure of Bollenback et al.12 The reference sub-
stances epiandrosterone glucuronide, etiocholanolone glu-
curonide and 11 -hydroxyetiocholanolone glucuronide were
purchased from Sigma (St. Louis, MO, USA) and the
steroid metabolites norandrosterone, d4-norandrosterone,
1 -methyl-5 -androstan-3 -ol-17-one and d5-androsterone
were synthesized in our laboratory.13 14
Purification of synthesized steroid glucuronide
conjugates
HPLC fractionation
A glacial acetic acid solution of the steroid glucuronides
is injected in portions of 150 µl into an HP 1090 liquid
chromatograph system (Hewlett-Packard) equipped with
a 250 µl syringe and a VP 250/10 Nucleosil 100-7 C18
preparative column (Macherey–Nagel, Du¨ren, Germany).
The mobile phase is a mixture of (A) phosphoric acid in
water (pH 2.5) and (B) acetonitrile starting with 30% solvent
B increasing to 100% in 10 min and the column is washed
for another 5 min with acetonitrile. Three wavelengths were
Synthesis of steroid glucuronide conjugates
The synthesis of steroid glucuronides is performed by means
of a modification of the Koenigs–Knorr reaction,15–17 treating
75 mg of a steroid in absolutely dry toluene (over molecular
Copyright  2001 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2001; 36: 159–168

Mass spectrometry of steroid glucuronide conjugates
161
°
detected (198, 208 and 228 nm) and, because of the unknown
elution time of the desired products, the elutates of all
detected signals of the first injection (50 µl) are collected
and tested by LC/MS or GC/MS for the glucuronide
conjugate. All subsequent injections are fractionated only
in the expected time range.
v/v/v/w) and the sample is heated for 20 min at 60 C.
With this procedure no enolization of the 17-keto group
is observed but all hydroxy groups are derivatized to d9-
trimethylsilyl ethers, resulting in increments of fragments
by steps of nine. The additional enolization is performed
by evaporation of the d18-BSA solution and derivatization
as described in step B. Here, a partial exchange of a d9-TMS
goup by an unlabelled TMS group was observed with the
TMS ester derivatives; the methyl ester derivatives were
stable under the conditions of step B.
Solid-phase extraction
The combined fractions are diluted with a 10-fold amount
of doubly distilled water and the steroid glucuronides are
adsorbed on Amberlite XAD2 –polystyrene resin. The XAD2
columns (pipettes of 0.8 cm i.d. closed with a glass pearl,
bed height 5 cm) are finally washed with 5 ml of doubly
distilled water and then eluted twice with 5 ml of methanol.
The methanolic eluate is evaporated to dryness, yielding
the pure steroid glucuronide conjugate with amounts of
22.0–40.1 mg (17.6–31.8% of theory).
GC/MS parameters
All the spectra presented were generated with a Finni-
gan GCQ ion trap (for molecular masses 800) and with
a Hewlett-Packard HP 6890/5973A GC/MSD system with
°
the following parameters: injector, ATAS Optic 2, 300 C
(ion trap only); column, HP Ultra1 (Hewlett-Packard), film
thickness 0.11 µm, i.d. 0.2 mm, length 14 m; carrier gas,
helium at 1.5 ml minꢀ1; injection volume; 2 µl (100 ng of
analyte); split ratio, 1:10; oven temperature, increased from
The structures of most glucuronides were additionally
confirmed by two-dimensional 1H and 13C NMR spec-
troscopy.
ꢀ1
°
°
°
180 C at 20 C min to 320 C, maintained for 5 min at
°
°
320 C; interface temperature, 320 C; ion source tempera-
Derivatization for GC/MS analysis
°
ture, 225 C; mass range (full scan), 70–920 (ion trap) and
70–800 (5973A MSD); ionization, electron impact ionization
(EI) (70 eV).
The steroid glucuronides are derivatized to per-TMS-
products (including a TMS-ester group at the glycosidic
moiety) by step B (see below), and to methyl ester per-TMS-
products by steps A and B, allowing a comparison of the
mass spectra and providing information about fragment
ion generation. Further, perdeuterotrimethylsilylation is
performed without enolization of the 17-keto group by means
of d18-bistrimethylsilylacetamide (d18-BSA, step C). The
enolization of the 17-keto group was additionally achieved
by evaporation of the d18-BSA solution and derivatisation
with the same procedure described with step B.
High-resolution mass spectrometry parameters
The exact masses of fragment ions were determined with
a Hewlett-Packard HP 5890 gas chromatograph coupled to
a Finnigan MAT 95 instrument with the following param-
eters: column, HP Ultra1 (Hewlett-Packard), film thickness:
0.11 µm, i.d. 0.2 mm, length 17 m; carrier gas, helium at
1.5 ml minꢀ1; injection volume, 2 µl (100 ng of analyte);
°
split ratio, 1:10; oven temperature, increased from 185 C
ꢀ1
ꢀ1
°
°
°
°
at 5 C min to 260 C then at 25 C min to 310 C; interface
Step A. Methylation of the carboxylic group
°
°
temperature, 300 C; ion source temperature, 240 C; ioniza-
tion, EI (65 eV); scan mode, Escan; scan range, 285–325;
resolution, 5000; scan rate, 0.3 s per scan; calibration gas,
perfluorophenanthrene; calibration masses, 292.9824 and
316.9824.
5 µg of the steroid glucuronide are solved in a test tube in
200 µl of acetonitrile, 20 µl of iodomethane and 0.5 mg of
potassium carbonate are added and the sample is carefully
°
closed and heated for 60 minutes at 60 C. After cooling
to ambient temperature the organic layer is transferred to
a fresh tube, evaporated to dryness by means of a rotary
evaporator and the residue is trimethylsilylated as described
below.
Nuclear magnetic resonance (NMR) analysis
NMR analyses were performed on Bruker DPX 300
and Bruker DRX 500 instruments. Amounts of 5 mg of
the glucuronides of androsterone, d5-androsterone, 19-
norandrosterone and d4-19-norandrosterone were dissolved
in CD3OD and 1H, 13C (DEPT, distortionless enhancement by
polarization transfer), H,H-COSY (homonuclear correlated
spectroscopy), HQMC (heteronuclear multiple quantum
correlation) and NOESY (nuclear overhauser effect spec-
troscopy) were performed to confirm the structures of the
synthesized substances.
Step B. Trimethylsilylation
A 5 µg amount of the steroid glucuronide is dissolved
in a test-tube in 100 µl of a mixture of N-methyl-N-
trimethylsilyltrifluoroacetamide, ammonium iodide and
ethanethiol (100 : 0.2 : 0.6, v/w/v) and heated for 20 min
°
at 60 C. The presence of NH4I in the reaction mixture is used
for the in situ preparation of trimethyliodosilane, a highly
sensitive and unstable compound owing to its tremendous
reactivity, which enables a rapid per-trimethylsilylation with
enolization of keto groups.
RESULTS AND DISCUSSION
Step C. Perdeuterotrimethylsilylation
With the derivatization of steroid glucuronide conjugates to
methyl ester-TMS and per-TMS products, stable compounds
are obtained suitable for GC/MS analysis which are not
thermally degradated during the measurements. The EI
A 5 µg amount of the steroid glucuronide is dissolved in
100 µl of a mixture of d18-bistrimethylsilylacetamide, acetoni-
trile, ethanethiol and ammonium iodide (1000:4000:20:0.1,
Copyright  2001 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2001; 36: 159–168

162
M. Thevis et al.
A)
OTMS
Abundance
504
m/z 345
900
800
700
600
500
400
345
217
COOTMS
O
TMSO
TMSO
O
H
TMSO
375
305
255
329
73
489
292
300
204
149
233
200
100
105
449
391
577
631
0
m/z-->
100 150 200 250 300 350 400 450 500 550 600 650 700 750
B)
OTMS
Abundance
217
900
800
700
600
500
400
300
200
100
345
COOCH3
O
204
TMSO
TMSO
O
H
TMSO
73
m/z 345
234
255
329
317
147
169
129
375
390
100 150 200 250 300 350 400 450 500 550 600 650 700 750
504
275
768
489
753
0
m/z-->
Figure 2. EI mass spectra of (A) androsterone glucuronide
per-TMS (Mr D 826) and (B) androsterone glucuronide methyl
ester per-TMS (Mr D 768).
Figure 4. EI mass spectra of (A) 19-norandrosterone
glucuronide per-TMS (Mr D 812) and (B) 19-norandrosterone
glucuronide methyl ester per-TMS (Mr D 754).
A)
A)
OTMS
OTMS
Abundance
Abundance
509
494
D
D
217
D
D
900
800
700
600
500
400
300
200
100
900
800
700
600
500
400
300
200
100
COOTMS
217
O
COOTMS
O
TMSO
TMSO
O
TMSO
TMSO
D
H
O
D
D
TMSO
H
305
TMSO
D
D
73
204
m/z 350
305
350
m/z 335
335
245
204
73
292
260
233
292
479
449
143
333
494
319
147
169
375
380
365
449
91
582
831
654
816
423
639
568
726
0
0
m/z-->
m/z-->
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
B)
B)
OTMS
Abundance
350
OTMS
900
800
700
600
500
400
300
200
100
D
217
D
Abundance
D
COOCH3
O
217
COOCH3
D
TMSO
TMSO
O
O
900
800
700
600
TMSO
TMSO
D
204
H
O
D
D
TMSO
380
H
TMSO
D
D
m/z 350
73
335
333
305
494
m/z 335
509
494
234
260
234
500 73
147169
773
758
204
400
300
200
100
0
245
452
407
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
0
m/z-->
479
149
758
743
273
Figure 3. EI mass spectra of (A) d5-androsterone glucuronide
per-TMS (Mr D 831) and (B) d5-androsterone glucuronide
methyl ester per-TMS (Mr D 773).
365
94
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
563
423
650
m/z-->
Figure 5. EI mass spectra of (A) d4-19-norandrosterone
glucuronide per-TMS (Mr D 816) and (B) d4-19-norand-
rosterone glucuronide methyl ester per-TMS (Mr D 758).
mass spectra of the prepared and commercially obtained
analytes show common and very specific fragmentations,
as shown in the Figs 2–7. The basic fragmentation pattern
is presented with androsterone glucuronide (Fig. 2) and is
applied to the spectra of the other substances. All glucuronide
conjugates were analysed with both instruments (ion trap
Copyright  2001 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2001; 36: 159–168

Mass spectrometry of steroid glucuronide conjugates
163
A)
General fragment ions of the glucuronic acid
moiety
OTMS
TMSO
The ions of m/z 375, 305, 292, 217, 204 and 169 are found
in all spectra of per-TMS derivatives of the investigated
steroid glucuronides and additionally m/z 465 and 449 are
observed in many cases the origin of which is proposed to be
the glycosidic moiety. A spectrum of pertrimethylsilylated
glucuronic acid is presented in Fig. 8 showing fragment
ions comparable to those obtained from the glucuronide
conjugates.
Abundance
217
343
COOTMS
O
900
800
700
600
500
400
300
200
100
0
TMSO
TMSO
O
H
149
169
TMSO
433
253
592
73
m/z 433
305
204
292
233
375
449
463
914
899
824
809
752
719
502
630
m/z-->
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900
Abundance
217
B)
900
800
700
600
500
400
300
200
100
OTMS
TMSO
TMSO
O
OTMS
COOTMS
OTMS
Abundance
217
TMSO
305
292
73
900
800
700
600
500
400
300
200
100
343
73
204
COOCH3
O
169
204
449
450
147
169
150
359
TMSO
TMSO
O
191
234
331
H
421
149
TMSO
433
233
250
375
253
285
300
m/z 433
539
449
0
m/z-->
100
200
350
400
500
317
592
856
841
283
Figure 8. EI mass spectrum of glucuronic acid per-TMS
(Mr D 554).
359
766
740
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900
676
549
0
m/z-->
Figure 6. EI mass spectra of (A) 11 -hydroxy etiocholanolone
glucuronide per-TMS (Mr D 914) and (B) 11 -hydroxy etio-
cholanolone glucuronide methyl ester per-TMS (Mr D 856).
A)
OTMS
6COO-R
O
5
A)
4
1
OTMS
2
3
OTMS
COO-R
TMSO
Abundance
359
O
O
H
OTMS
R = TMS
900
−90
OTMS
518
217
800
6COO-R
TMSO
m/z 465
m/z 407
COOTMS
R = CH3
700
OTMS
O
O
5
TMSO
TMSO
O
600
500
400
300
200
100
H
4
1
OTMS
2
269
TMSO
305
343
3
m/z 359
73
292
OTMS
m/z 375
204
169
233
503
R = TMS
R = CH3
131
389
m/z 317
449
591
645
0
m/z-->
100 150 200 250 300 350 400 450 500 550 600 650 700 750
B)
C)
H
TMSO
OTMS
B)
4
H
C
OTMS
2
C
OTMS
TMSO
C
3
m/z 305
m/z 217
TMSO
H
Abundance
359
900
800
700
600
500
400
300
200
100
217
COOCH3
O
TMSOOC
TMS
H
OTMS
H
TMSO
O
TMSO
OTMS
C
C
H
OTMS
TMSO
m/z 359
343
269
m/z 204
m/z 292
234
518
169
73
Figure 9. (A) Proposed generation of m/z 465 of per-TMS
glucuronide conjugates by fission of the O-glycosidic bond.
Consecutive loss of TMS-OH (ꢀ90) leads to m/z 375. The
substitution of the TMS ester by a methyl ester produces
m/z 407 and 317, respectively. (B) Proposed composition and
structure of the fragment ions of m/z 292 and 305. Masses
calculated (mcal) and found (mexp): m D 292 135,
317
782
767
105
503
447
100 150 200 250 300 350 400 450 500 550 600 650 700 750
0
m/z-->
Figure 7. EI mass spectra of (A) 1 -methyl-5 -androstan-3 -
ol-17-one glucuronide per-TMS (Mr D 840) and (B) 1 -methyl-
5 -androstan-3 -ol-17-one glucuronide methyl ester per-TMS
(Mr D 782).
mexp D 292 136, m D 305 142, mexcpalD 305 144.
cal
and quadrupole) but no significant differences due to the
analysis techniques were observed in the spectra.
(C) Postulated structure of the fragment ions of m/z 217 and
204.
Copyright  2001 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2001; 36: 159–168

164
M. Thevis et al.
of the methyl ester derivatives of steroid glucuronides
show a shift of 58 u from m/z 292 to 234, owing to an
exchange of a TMS group by a methyl group; (b) the
fragment is incremented by 27 u owing to three TMS groups
in deuterium-labelling experiments (Table 1); (c) m/z 305
does not include the ester group and is also detected in
methyl ester derivatives with decreased intensity; (d) the
ion shifts to m/z 332 due to three deuterium-labelled TMS
groups (Table 1); and (e) high-resolution mass spectrometry
confirmed the postulated composition of C11H28Si3O3 and
C12H29Si3O3 for m/z 292 and 305, respectively. Their
proposed structures are presented in Fig. 9B. The fragments
of m/z 217 and 204 are known from other sugar derivatives19
and their postulated structures are shown in Fig. 9(C). These
are consistent with the labelling experiments of this study
and the fragment increment with respect to the introduced
deuteria (Table 1). The ion of m/z 169 is also generated by
the glycosidic moiety as shown in the spectrum of glucuronic
acid per-TMS but can also originate from some steroid
moieties from the D-ring bearing an O-TMS group after
enolization of a keto group (Fig. 1).
m z 465 ! 375, m z 407 ! 317, m/z 449
A fission of the O-glycosidic bond between the steroid oxygen
and the glucuronic acid moiety [Fig. 9(A)] results in a leaving
group consisting of a steroid radical and in a fragment of
m/z 465, which generates m/z 375 by a neutral loss of TMS-
OH (ꢀ90). Their counterparts in spectra of the methyl ester
derivatives are detected with m/z 407 (465–58) and m/z 317
(375–58). Evidence for the presence of three and two TMS
groups, respectively, in these fragments is obtained with the
spectra of TMS-deuterium-labelled glucuronide conjugates,
where m/z 375 shifts to m/z 402 (C27) and m/z 317 to m/z
335 (C18). The abundance of the secondary ions (m/z 375,
317) varies with the general steroid structure.
The loss of the whole steroid from the molecular ion with
a hydrogen abstraction produces m/z 464, which loses CH3
ž
a radical (ꢀ15) forming the fragment of m/z 449. The latter
is also present in the spectrum of glucuronic acid per-TMS
where the same ion can be generated by the loss of TMS-OH
ž
from C-1 and a subsequent loss of CH3 .
m/z 305, 292, 217, 204, 169
The fragment ions of m/z 305 and 292 were not detected
in any product ion spectrum taken from any ion having an
abundance higher than 10% of the base peak. Proposals for
their generation and structure are based on the following
data: (a) m/z 292 comprises the ester group because spectra
Proposed fragmentation pattern of androsterone
glucuronide per-TMS (Mr = 826)
The spectrum of androsterone glucuronide-per-TMS (Fig. 2)
contains the typical ions originating from the glucuronic acid
Table 1. Selected fragment ions of androsterone glucuronide (AG) derivatives and their
counterparts in deuterium labelling experiments
Fragment ions
of AG-per-TMS
Fragment ions of AG-methyl
ester-tetrakis-TMS
(m/z)
Aa
Ba
(m/z)
Ca
Da
b
—
—
—
—
—
768
753
—
504
489
—
375d
345
329
317
—
773
758
—
509
494
—
380
350
333/334
317
—
795
780
—
513
498
—
375
345
329
335
—
—
577
504
489
375c
375d
345
329
—
305
292
255
—
582
509
494
375
380
350
333/334
—
305
292
260
—
595
513
498
402
375
345
329
—
332
319
255
—
—
—
—
255
247
234
—
217
204
187
260
247
234
—
217
204
187
255
265
252
—
235
222
196
—
—
—
233
217
204
233
217
204
251
235
222
a A D 2,2,3,4,4-d5-androsterone glucuronide-per-TMS; B D androsterone glucuronide-17-enol-
TMS-tetrakis-d9-TMS; C D 2,2,3,4,4-d5-androsterone glucuronide methyl ester tetrakis-TMS; D D
androsterone glucuronide methyl ester 17-enol-TMS-tris-d9-TMS.
b Dashes indicate fragment peak intensity 5% of base peak or not detected.
c Originating from the glycosidic moiety.
d Originating from the steroid moiety.
Copyright  2001 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2001; 36: 159–168

Mass spectrometry of steroid glucuronide conjugates
165
moiety but also fragments of steroid-specific mass/charge
values.
androsterone glucuronide which also contains the fragment
of m/z 504 because of the removal of the substituted molecule
part (ester group). Further, the spectra of d5-androsterone
glucuronide show the counterpart with m/z 509, and one
deuterated TMS group shifts the fragment of m/z 504 to m/z
513 (Table 1). The main product ions of m/z 504 and 509 are
m/z 489 and 494, respectively. The loss of a methyl group
responsible for the difference of 15 u can not be located at
the sugar residue because a subsequent loss of the remaining
glycosidic moiety is observed. Second, the loss of the methyl
group cannot originate from C-19 because measurements
of 19-norandrosterone glucuronide per-TMS show highly
comparable spectra in agreement with the mass difference
(Fig. 4) of 14 u. In the literature the removal of a methyl
group from norandrosterone-bis-TMS7 and from estrone20
was proved to originate from C-18, which is proposed in this
case also as shown in Fig. 12.
The loss of the remaining sugar moiety accompanied
by a hydrogen transfer is observed from m/z 489 and
504, generating the ions of m/z 329 and 344 in the case
of unlabelled androsterone glucuronide. These are also
observed with TMS-labelled derivatives (Table 1) because
in this step the last deuterated TMS group is removed. The
transferred hydrogen must be located at different carbons
of the steroid because the product ion spectra of d5-labelled
androsterone glucuronide show fragments of m/z 333 and
334 (Table 1) originating from a loss of 160 and 161 u,
respectively, from m/z 494 (Fig. 12).
m/z 577
The cleavage of the glycosidic ring structure is the starting
point for several fragmentation steps initialized by the
ionization of the O-glycosidic bond between the steroid
and the glucuronic acid. The loss of 249 u from the molecular
ion (Mr D 826) leads to m/z 577 as presented in Fig. 10. In
case of 2,2,3,4,4-d5-androsterone glucuronide per-TMS, this
fragment shifts to m/z 582, and deuterated TMS groups
increment m/z 577 of androsterone glucuronide per-TMS to
m/z 595, due to two labelled and one unlabelled (enol ether)
TMS groups (Table 1).
m z 504 ! 489 ! 329
The base peak of this spectrum is m/z 504, the generation
of which is proposed to start identically with m/z 577 by
the fission of the ring structure, but followed by a transfer
of the TMS group from C-3 to the obtained oxygen radical
(Fig. 11). Owing to the cleavage of a ring bond, the molecular
conformation can be changed, enabling a transfer of the
6-positioned TMS group and a subsequent loss of 322 u
comprising the ester group. Support for this proposal is
given by the spectrum of the methyl ester derivative of
OTMS
COOTMS
O
m z 375 ! 345 ! 255 (m z 380 ! 350 ! 260)
H
TMSO
TMSO
O
The generation of m/z 375 was initially described from the
glycosidic moiety, but the appearance of this ion in spectra
of the methyl ester derivatives of androsterone glucuronide
and its stereoisomers epiandrosterone glucuronide and
etiocholanolone glucuronide besides m/z 317 suggested
another source for this ion in the case of this steroid structure.
This was proved with d5-androsterone glucuronide, the
spectra of which show m/z 375 and 380 as the per-TMS
H
OTMS
−249
OTMS
C
CH
CH
O
TMSO
H
m/z 577
Figure 10. Possible mechanism of the loss of 249 u from the
molecular ion of androsterone glucuronide.
OTMS
COOTMS
O
TMSO
TMSO
O
H
OTMS
COOTMS
OTMS
COOTMS
O
H
TMSO
OTMS
TMSO
TMS–O
O
O
O
H
H
OTMS
OTMS
O
H
HC
CHO
m/z 504
C
OTMS
Figure 11. Proposed generation of m/z 504 of androsterone glucuronide per-TMS.
Copyright  2001 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2001; 36: 159–168

166
M. Thevis et al.
OTMS
OTMS
2,2,4,4-d5-19-Norandrosterone glucuronide
per-TMS (Mr = 816)
The spectrum of deuterium-labelled 19-norandrosterone
glucuronide (Fig. 5) contains many fragment ions incre-
mented by 4 u in comparison with the unlabelled nandrolone
metabolite. The ion of m/z 563 is shifted to m/z 567, m/z
490 to 494, m/z 475 to 479, m/z 361 to 365, the aglycone
m/z 331 to 335 and the product ion, generated by a neutral
loss of TMS-OH (ꢀ90), is present with m/z 245. Further,
the above-proposed loss of 160 and 161 u in case of d5-
androsterone glucuronide (Fig. 12) is also observed with
d4-norandrosterone glucuronide per-TMS, resulting in the
fragment ions of m/z 318 (m/z 479 ꢀ 161) and m/z 319 (m/z
479 ꢀ 160). This indicates that the deuterium, transferred to
the leaving group, must not originate from C-3. The frag-
ments originating from the glycosidic moiety (m/z 375, 305,
292, 217, 204 and 169) are not influenced by the deuteration.
O
O
H
H
O
H
C
H
H
HC
HC
CHO
C
C
m/z 504 (m/z 509)
OTMS
OTMS
OTMS
OTMS
−15
H
O
CH3
CH2
O
HC
CH–OH
C
HC
CH–OH
CH
m/z 489 (m/z 494)
OTMS
OTMS
−160 (−161)
11b-Hydroxyetiocholanolone glucuronide
per-TMS (Mr = 914)
OTMS
The fragmentation of the 11 -hydroxylated etiocholanolone
glucuronide as the per-TMS derivative (Fig. 6) and methyl
ester pentakis-TMS derivative conforms to the described
fragmentations in compliance with the increment of 88 u
or the subtraction of 2 u which result from the additional
11-O-TMS group or its loss as TMS-OH (ꢀ90), respectively.
CH3
CH2
m/z 329 (m/z 333, 334)
Figure 12. Possible mechanism of the elimination of C-18 and
the consecutive loss of the glycosidic moiety after hydrogen
(deuterium) transfer generating the fragment ions of m/z 489
(494) and m/z 329 (333, 334), respectively.
m z 914 ! 899 ! 809, m z 914 ! 824 ! 809
The molecular ion of the per-TMS derivative is observed
with m/z 914 and also the consecutive losses of 15 and 90 u
generating the ions m/z 899, 824 and 809.
derivative and m/z 317 and 380 as the methyl ester product
(Table 1). Further, TMS labelling experiments yielded m/z
375 (aglycone) and m/z 402 (glycosidic residue), and MS/MS
experiments on m/z 375 and 380 of androsterone glucuronide
and d5-androsterone glucuronide generated m/z 345 and 350,
respectively, by the loss of 30 u. The subsequent loss of TMS-
OH from the remaining steroid fragments (m/z 345 and 350)
led to m/z 255 and 260.
m z 592 ! 577, m z 502
The fragments of m/z 592 and 502 correspond to m/z 504 of
androsterone glucuronide (incremented by 88 u and reduced
by 2 u) and m/z 577 is the counterpart to m/z 489 resulting
from a loss of a methyl group from m/z 592.
These basic fragmentation patterns can be applied to
the other steroid glucuronides derivatized as described
with respect to their individual structural difference. The
spectra of androsterone glucuronide and its stereoiso-
mers (epiandrosterone glucuronide and etiocholanolone
glucuronide) are identical with only minor differences in
ion abundance ratios so their mass spectrometric behaviour
is not described separately.
m z 433 ! 343 ! 253
An abundant aglycone fragment is present with m/z 433,
which subsequently loses TMS-OH (ꢀ90) twice to give
m/z 343 and 253. This is also observed with TMS-labelled
11 -hydroxyetiocholanolone glucuronide, where m/z 433 is
shifted to 442, due to one d9-TMS and one unlabelled TMS
group (enol ether).
The glucuronide specific ions (m/z 375, 305, 292, 217,
204 and 169) resulting from the glycosidic moiety are also
observed with high abundances.
19-Norandrosterone glucuronide per-TMS
(Mr = 812)
Comparable to androsterone glucuronide per-TMS (Fig. 2),
all fragment ions of 19-norandrosterone glucuronide per-
TMS (Fig. 4) comprising the steroid moiety are detected with
respect to a difference of 14 u due to the missing methyl
group with C-19. The ion of m/z 563 corresponds to m/z
577, m/z 490 to 504, m/z 475 to 489, m/z 361 to 375 (steroid
origin), m/z 331 to 345 (aglycone), m/z 315 to 329 and m/z
241 to 255. The glucuronic acid-specific fragment ions (m/z
375, 305, 292, 217, 204 and 169) are present with unchanged
mass/charge values.
1a-Methyl-5a-androstan-3a-ol-17-one glucuronide
per-TMS (Mr = 840)
The per-TMS and methyl ester tetrakis-TMS derivatives
of the phase II metabolite of mesterolone (1 -methyl-5 -
androstan-17 -ol-3-one) fragments very much like andros-
terone glucuronide, bearing an extra methyl group at C-1
resulting in a difference of 14 u. The molecular ion is observed
weakly with m/z 840 and the ꢀ15 u product ion of m/z 825
(Fig. 7).
Copyright  2001 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2001; 36: 159–168

Mass spectrometry of steroid glucuronide conjugates
167
Table 2. Chemical shifts of carbons and selected hydrogens of the androsterone glucuronide and
19-norandrosterone glucuronide (19-NorAG) in 13C and 1H NMR experiments
Steroid carbons
AG chemical shift (ppm)
19-NorAG
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
33.64
26.46a
75.62a
35.30a
40.44
29.33
32.07
36.37
55.70
37.04
21.16
32.87
49.17
52.92
22.71
36.69
224.21
14.20
11.85
25.11
30.51a
75.35
40.28a
37.40
34.55
31.02
42.07
49.60
48.17
26.03
32.83
49.23
52.09
22.59
36.70
224.29
14.23
C-9
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
Glucuronic acid carbons
C-10
C-20
C-30
C-40
C-50
C-60
103.03
74.83
77.64
73.21
76.57
172.72
103.07
74.83
77.62
73.19
76.57
172.68
Glucuronic acid hydrogens
ꢀ
H-10
H-20
H-30
H-40
H-50
4.36
3.28
3.37
3.52
3.75
4.37
3J D 7 8 Hz
3.23
3.37
3.52
3.76
a Labelled positions in deuterated counterparts.
bond to the glycosidic oxygen. The chemical shifts of carbons
of the deuterated and the unlabelled conjugates in 13C
experiments were identical except those bearing the 2H
atoms, which appeared as quaternary carbons and were
not detected in the DEPT spectrum. A comparison of these
data with 13C data for unconjugated androsterone found
in the literature21 indicates the position of glucuronidation
by deviation of the chemical shifts mainly of the carbons
C-2, C-3 and C-4. The influence of the C-19 methyl group is
obviously present on carbons C-1 to C-11, where the chemical
shifts differ significantly between androsterone glucuronide
and 19-norandrosterone glucuronide, but C-12 to C-18 have
nearly identical values.
m/z 591, m z 518 ! 503 ! 343
The fragment of m/z 591 corresponds to m/z 577, m/z 518
to 504, m/z 503 to 489 and m/z 343 to 329 of androsterone
glucuronide (Fig. 2).
m z 359 ! 269, m/z 389
The base peak is performed by the aglycone with m/z 359,
which generates m/z 269 by the neutral loss of TMS-OH
(ꢀ90) and m/z 389 represents the aglycone fragment plus 30
mass units comparable to m/z 375 in the case of androsterone
glucuronide per-TMS. The glucuronic acid fragment ions
(m/z 375, 305, 292, 217, 204 and 169) are also present with
equivalent abundances.
The NMR data for labelled and unlabelled androsterone
glucuronide and 19-norandrosterone glucuronide, obtained
by different experiments, as described above, are listed
in Table 2. The coupling constant 3J D 7 8 Hz proved an
axial–axialconfiguration of the hydrogens located at C-10 and
C-20 of the glucuronide moiety and thus the -configuration
of the analysed steroid glucuronides because of an equatorial
CONCLUSION
The GC/MS data obtained from 5 -/5 -androstan-/estran-
17-one glucuronide conjugates present common fragments
and their individual changes are due to structural peculiari-
ties of the steroids. With the combination of two derivatives
Copyright  2001 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2001; 36: 159–168

168
M. Thevis et al.
3. Sanaullah, Bowers LD. J. Steroid Biochem. Mol. Biol. 1996; 58:
225.
4. Bort DJ, Bowers LD. J. Mass Spectrom. 2000; 35: 50.
5. Budzikiewicz H, Djerassi C, Williams DH. Structure Elucidation
of Natural Products by Mass Spectrometry. Holden-Day: San
Francisco, 1964.
6. Scha¨nzer W, Horning S, Opfermann G, Donike M. J. Steroid
Biochem. Molc. Biol. 1996; 57: 363.
7. Masse´ R, Laliberte´ C, Tremblay L. J. Chromatogr. 1985; 339:
11.
8. Harvey DJ, Vouros P. Biomed. Mass. Spectrom. 1979; 4: 135.
9. Du¨rbeck HW, Bu¨ker I. Biomed. Mass. Spectrom. 1980; 10: 437.
10. Masse´ R, Bi H, Ayotte C, Dugal R. Biomed. Environ. Mass. Spec-
trom. 1989; 18: 429.
11. Paulson GD, Zaylskie RG, Dockter MM. Anal. Chem. 1973; 45:
21.
(methyl ester per-TMS, per-TMS), structurally related com-
pounds and deuterated analogues, information concerning
the origin of distinct fragment ions, and possible ways of
their generation are provided. This allows the completion
of characteristic studies on synthesized reference materials.
The generally present fragment ions of the glycosidic moiety
(m/z 375, 305, 292, 217, 204 and 169) facilitate the identifi-
cation of steroids bearing glucuronic acid with a saturated
A-ring structure in combination with expected aglycone frag-
ments, and those resulting from a common fission through
the glycosidic part of the molecule (neutral loss of 322 u).
The consecutive loss of C-18 (ꢀ15) and the remaining sugar
residue (ꢀ160) produces a typical fragmentation pattern indi-
cating partly the structure of the conjugates. The benefit of
derivatization and GC/MS analysis is the presence of abun-
dant fragment ions of both parts of the molecule, the steroid
and glycosidic moiety which may be missing with other mass
spectrometric and ionization techniques, e.g. LC/MS, where
mainly fragments of the steroidal moiety are generated.
12. Bollenback GN, Long JW, Benjamin DG, Lindquist JA. J. Am.
Chem. Soc. 1955; 77: 3310.
13. Scha¨nzer W, Donike M. Anal. Chim. Acta 1993; 275: 23.
14. Scha¨nzer W, Donike M. In Proceedings of the 12th Cologne
Workshop on Dope Analysis, Donike M, Geyer H, Gotzmann A,
Mareck-Engelke U (eds). Sport & Buch Strauss, Edition Sport:
Cologne, 1995; 93–112.
15. Koenigs W, Knorr E. Chem. Ber. 1901; 34: 957.
16. Goebel WF, Babers FH. J. Biol. Chem. 1935; 110: 707.
17. Woitz HH, Smakula E, Lichtin NN, Leftin JH. J. Am. Chem. Soc.
1959; 81: 1704.
Acknowledgement
We thank the Bundesinstitut fu¨r Sportwissenschaft, Cologne, for
financial support.
18. Elce JS, Carpenter JGD, Kellie AE. J. Chem. Soc. 1967; 542.
19. Thevis M, Opfermann G, Scha¨nzer W. J Chromatogr. 2000; 744:
345.
REFERENCES
20. Djerassi C, Wilson JM, Budzikiewicz H, Chamberlin JW. J. Am.
Chem. Soc. 1962; 84: 4544.
21. Blunt JW, Stothers JB. Org. Magn. Reson. 1977; 9: 439.
1. Scha¨nzer W. Clin. Chem. 1996; 42: 1001.
2. Donike M, Zimmermann J, Ba¨rwald KR, Scha¨nzer W, Christ V,
Klostermann K, Opfermann G. Dtsch. Z. Sportmed. 1984; 1: 14.
Copyright  2001 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2001; 36: 159–168