Using complementary mass spectrometric approaches for the determination of methylprednisolone metabolites in human urine

Article abstract of DOI:10.1002/rcm.6129

RATIONALE The metabolism of methylprednisolone is revisited in order to find new metabolites that could be important for distinguishing between different routes of administration. Recently developed liquid chromatography/tandem mass spectrometry (LC/MS/MS) strategies for the detection of corticosteroid metabolites have been applied to the study of methylprednisolone metabolism. METHODS The structures of these metabolites were studied using two complementary mass spectrometric techniques: LC/MS/MS in product ion scan mode with electrospray ionization and gas chromatography/mass spectrometry (GC/MS) in full scan mode with electron ionization. Metabolites were also isolated by semipreparative liquid chromatography fractionation. Each fraction was divided into two aliquots; one was studied by LC/MS/MS and the other by GC/MS after methoxyamine-trimethylsilyl derivatization. RESULTS The combination of all the structural information allowed us to propose a comprehensive picture of methylprednisolone metabolism in humans. Overall, 15 metabolites including five previously unreported compounds have been detected. Specifically, 16β,17α,21-trihydroxy-6α-methylpregna-1,4-diene- 3,11,20-trione, 17α,20β,21-trihydroxy-6α-methylpregna-1,4- diene-3, 11-dione, 11β,17α,21-trihydroxy-6α- hydroxymethylpregna-1,4-diene-3,20-dione, 11β,17α,20,21-tetrahydroxy- 6α-hydroxymethylpregna-1,4-diene-3-one, and 17α,21-dihydroxy- 6α-hydroxymethylpregna-1,4-diene-3,11,20-trione are proposed as feasible structures for the novel metabolites. In addition to the expected biotransformations: reduction of the C20 carbonyl, oxidation of the C11 hydroxy group, and further 6β-hydroxylation, we propose that hydroxylation of the 6α-methyl group can also take place. CONCLUSIONS New metabolites have been identified in urine samples collected after oral administration of 40mg of methylprednisolone. All identified metabolites were found in all samples collected up to 36h after oral administration. However, after topical administration of 5g of methylprednisolone aceponate, neither the parent compound nor any of the metabolites were detected. Copyright

Full text of DOI:10.1002/rcm.6129

Research Article
Received: 6 October 2011
Revised: 2 December 2011
Accepted: 12 December 2011
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2012, 26, 541–553
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6129
Using complementary mass spectrometric approaches for
the determination of methylprednisolone metabolites in
human urine
1
1,2
1
1,2
*
Oscar J. Pozo , Josep Marcos , Xavier Matabosch , Rosa Ventura and
Jordi Segura1,2
1Bioanalysis Research Group, IMIM, Institut de Recerca Hospital del Mar, Doctor Aiguader 88, 08003 Barcelona, Spain
2Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003 Barcelona, Spain
RATIONALE: The metabolism of methylprednisolone is revisited in order to find new metabolites that could be important
for distinguishing between different routes of administration. Recently developed liquid chromatography/tandem mass
spectrometry (LC/MS/MS) strategies for the detection of corticosteroid metabolites have been applied to the study of
methylprednisolone metabolism.
METHODS: The structures of these metabolites were studied using two complementary mass spectrometric techniques:
LC/MS/MS in product ion scan mode with electrospray ionization and gas chromatography/mass spectrometry (GC/
MS) in full scan mode with electron ionization. Metabolites were also isolated by semipreparative liquid chromatography
fractionation. Each fraction was divided into two aliquots; one was studied by LC/MS/MS and the other by GC/MS
after methoxyamine-trimethylsilyl derivatization.
RESULTS: The combination of all the structural information allowed us to propose a comprehensive picture of methylprednis-
olone metabolism in humans. Overall, 15 metabolites including five previously unreported compounds have been detected.
Specifically, 16b, 17a,21- trihydroxy- 6a- methylpregna-1, 4 -diene-3,11,20-trione, 17a,20b,21-trihydroxy-6a-methylpregna-1,
4-diene-3, 11- dione, 11b, 17a, 21-trihydroxy-6a-hydroxymethylpregna-1, 4-diene-3,20-dione, 11b,17a, 20x,21-tetrahydroxy-6a-
hydroxymethylpregna-1,4-diene-3-one, and 17a,21-dihydroxy-6a-hydroxymethylpregna-1,4-diene-3,11,20-trione are
proposed as feasible structures for the novel metabolites. In addition to the expected biotransformations: reduction
of the C20 carbonyl, oxidation of the C11 hydroxy group, and further 6b-hydroxylation, we propose that hydroxylation
of the 6a-methyl group can also take place.
CONCLUSIONS: New metabolites have been identified in urine samples collected after oral administration of 40 mg of
methylprednisolone. All identified metabolites were found in all samples collected up to 36 h after oral administration.
However, after topical administration of 5 g of methylprednisolone aceponate, neither the parent compound nor any of
the metabolites were detected. Copyright © 2012 John Wiley & Sons, Ltd.
Methylprednisolone (11b,17a,21-trihydroxy-6a-methylpregna-
1,4-diene-3,20-dione, MP) is a moderately potent glucocorticoid
with anti-inflammatory and immunosuppressive effects.[1] It
has been shown to be effective in a wide range of conditions
such as asthma, systemic lupus, rheumatoid arthritis, and renal
transplant rejection.[2,3] Its use in sport is prohibited by the
World Anti-Doping Agency (WADA). All glucocorticosteroids
are prohibited in-competition when administered orally, rectally,
intravenously or intramuscularly,[4] and its use requires a thera-
peutic use exemption approval. However, topical preparations
when used for dermatological, auricular, nasal, ophthalmic,
buccal, gingival and perianal disorders are not prohibited and
do not require any form of therapeutic use exemption.
In humans, MP undergoes phase I and II metabolism. By
using gas chromatography/mass spectrometry (GC/MS),
Rodchenkov et al. found 11-keto and 20-hydroxy, and 6,7-
dehydro as the main metabolites in human urine after oral
administration of 20 mg of the drug.[5] A different study,
based on liquid chromatography/tandem mass spectrometry
(LC/MS/MS) and nuclear magnetic resonance (NMR), con-
cluded that the metabolic pathway of MP after intravenous
administration was simple: reduction of the C20 carbonyl
group, further oxidation of the C20-C21 side chain, and C6
oxidation.[6] In a recent study performed by LC/MS/MS,
metabolites detected after intramuscular and intra-articular
administrations were described.[7] Metabolites obtained after
11-oxidation, reduction of C20 and oxidation of the C20-C21
side chain were also detected. Some metabolites conjugated
with glucuronic acid were also identified.
In spite of the knowledge that we have about glucocorticoid
metabolism, detection of the use of MP in sport is routinely
performed by methods aimed at detecting the presence of
the parent drug in urine.[8–10] The characterization of minor
* Correspondence to: X. Matabosch, Bioanalysis Research
Group, IMIM, Institut de Recerca Hospital del Mar, Doctor
Aiguader 88, 08003 Barcelona, Spain.
E-mail: xmatabosch@imim.es
Rapid Commun. Mass Spectrom. 2012, 26, 541–553
Copyright © 2012 John Wiley & Sons, Ltd.

O. J. Pozo et al.
corticosteroid metabolites can be of great importance in the
doping control field as it may improve the detection of drug
misuse, or allow for differentiation between different routes
of administration.
Z-spray electrospray ionization (ESI) interface (Waters) inter-
faced to an Acquity ultra-performance liquid chromatography
(UPLC) system (Waters) for the chromatographic separation.
Nitrogen (N2) was used as the drying gas, nebulizing gas, cone
gas and desolvation gas. The desolvation gas flow rate was
approximately 1200 L/h and the cone gas flow rate was
50 L/h. A cone voltage of 25 V and a capillary voltage of
3.0 kV were used in positive ionization mode. The nitrogen
desolvation temperature was set to 450 ꢀC and the source
Recently, different non-targeted LC/MS/MS methods able
to detect anabolic steroids, glucocorticosteroids and their
metabolites, based on a specific structural characteristics,
have been studied.[7,11,12] LC/MS/MS in triple quadrupole
instruments can be applied in different acquisition modes
such as neutral loss (NL) or precursor ion (PI) scan modes.
The use of these modes allows for the detection of untargeted
compounds containing a specific structure which is very
useful for metabolic studies. Several methods based on these
scan modes have been developed and successfully applied
for the detection of steroid metabolites.[11,13,14] Based on these
results, different approaches for the non-targeted detection of
corticosteroids in urine were evaluated in this study. These
screening methodologies may provide a list of potential
metabolites of MP with unknown structures. A more compre-
hensive study should then allow us to propose feasible
structures for those candidate metabolites. The combination
of classical GC/MS strategies in full scan mode[15] and LC/
MS/MS strategies based on the integration of different
mass spectrometric scan modes[16] can provide the most
comprehensive picture of the metabolism of corticosteroids
in general, and MP in particular.
The goals of the present work were: first to enhance our
knowledge of the oral metabolism of MP by using comple-
mentary mass spectrometric techniques, and second to
develop specific and sensitive LC/MS/MS qualitative meth-
ods in selected reaction monitoring (SRM) mode for the
detection of these metabolites in human urine. These methods
were then applied to the detection of potential metabolites of
MP after topical use, in order to estimate the differences in
metabolism between routes of administration.
ꢀ
temperature to 120 C. In the PI scan methods three different
collision energies were used (50, 55 and 60 eV) and a precursor
ion window from m/z 300 to 600 was monitored. In the PI scan
methods two different collisions energies were used (20 and
30 eV) and a window from m/z 50 to 400 was monitored. In
negative ionization mode, a cone voltage of 25 Vand a capillary
voltage of 2.5 kV were used. The nitrogen desolvation tem-
perature was set to 400 ꢀC and the source temperature to
120 ꢀC. In the NL scan methods, a collision energy (CE) of
15 eV was used and a precursor ion window from m/z 300
to 600 was monitored.
The LC separation was performed using an Acquity BEH
C18 column (100 mm  2.1 mm i.d., 1.7 mm particle size) at a
flow rate of 400 mL/min. Water (solvent A) and acetonitrile
(solvent B), both with formic acid (0.01%), were selected as
the mobile phase solvents. A gradient program was used:
the percentage of organic solvent was linearly changed as
follows: 0 min, 10% B; 0.6 min, 10% B; 5 min, 20% B;
12 min, 30% B; 16.5 min, 90% B; 17 min, 90% B; 17.5 min,
10% B; 20 min, 10%B.
GC/MS instrumentation
GC/MS analysis was carried out on a 6890 N gas chromato-
graph coupled with a 5975 quadrupole mass spectrometer with
a triple-axis detector (Agilent Technologies, Palo Alto, CA,
USA). The steroids were separated on a HP-Ultra1 cross-linked
methyl-silicone column (16.5 m 0.2 mm i.d., 0.11 mm film
thickness; J&W Scientific, Folsom, CA, USA). Helium was used
as the carrier gas at a constant pressure of 5 psi. A 2 mL aliquot
of the final derivatized extract was injected into the system
operated in splitless mode (valve opened at 2 min). The GC
column temperature was ramped as follows: initial 50 ꢀC, held
for 3 min, increased to 230 ꢀC at 30 ꢀC min–1, thereafter
increased to 285 ꢀC at 2 ꢀC min–1. The injector and transfer line
were kept at 280 ꢀC. In full scan mode, the mass range scanned
was from m/z 70 to 850.
EXPERIMENTAL
Chemicals and reagents
Methylprednisolone, sulfatase from Helix pomatia type H-1, b-
glucuronidase/arylsulfatase from Helix pomatia type H-2,
sodium borohydride, and methoxyamine hydrochloride were
obtained from Sigma (St. Louis, MO, USA). The b-glucuronidase
preparation (type E. coli K12) was purchased from Roche
Diagnostics GmbH (Mannheim, Germany). Analytical grade
disodium hydrogen phosphate, sodium hydrogen phosphate,
ethyl acetate, cyclohexane, and pyridine were obtained from
Merck (Darmstadt, Germany). Trimethylsilylimidazole (TMSI)
was from Macherey-Nagel (Düren, Germany).
Acetonitrile and methanol (LC gradient grade) and formic
acid (LC/MS grade) were purchased from Merck. Milli-Q
water was obtained using a Milli-Q purification system
(Millipore Ibérica, Barcelona, Spain). Sep-PakW C18 cartridges
were obtained from Waters (Milford, MA, USA).
Semipreparative HPLC
In order to isolate the metabolites, the urine sample (15 mL)
was extracted as described previously for the LC/MS/MS
analysis. The extract was reconstituted in 250 mL of a water/
methanol mixture (90:10, v/v) and 200 mL were injected into
the HPLC system. An HP1090 liquid chromatograph with
automatic injection system and diode-array detector (Hewlett-
Packard, Waldbronn, Germany) was used. The LC column
was a Hypersil C18 BDS (100 mm 4.6 mm i.d., 3 mm particle
size). The mobile phase was water/methanol (90:10, v/v) at a
flow rate of 1 mL/min. The gradient elution program was as
follows (where B is methanol): 0 min, 10% B; 0.5 min, 10% B;
15 min, 50% B; 18 min, 100% B; 20 min, 100% B; 20.5 min,
LC/MS/MS instrumentation
SRM, precursor ion (PI), neutral loss (NL) and product ion
scans were carried out using a triple quadrupole (Quattro
Premier XE) mass spectrometer provided with an orthogonal
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Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2012, 26, 541–553

Determination of methylprednisolone metabolites in urine
10% B; 22 min, 10% B. Along the whole chromatogram time
frame, fractions of 30 s were collected in separate glass tubes.
corticosteroid, were used for evaluation of the selectivity of
the method.
Ethical approval for the study was granted by Comité Ètic
d’Investigació Clínica of our institute (CEIC-IMAS no.94/
467) and the Spanish Health Ministry (DGFPS no.95/75).
All the subjects participating in the study gave their written
informed consent.
Sample preparation
Urine samples (5 mL) were passed through a C18 solid-phase
extraction (SPE) cartridge, previously conditioned with
4 mL methanol and 4 mL water. The column was then
washed with 4 mL water and finally the steroids were
recovered with 4 mL methanol.
Structural elucidation
The methanolic extract was evaporated under a stream of
nitrogen and 3 mL of sodium acetate buffer (0.1 M, pH 4.5)
were added. After adding 10 mg of sulfatase type H-1 and
12 mL of b-glucuronidaseꢀtype H-2, hydrolysis was allowed
to proceed for 16 h at 55 C. After the sample had cooled to
room temperature, liquid-liquid extraction was performed
by the addition of 5 mL ethyl acetate. After centrifugation,
the organic layer was separated and evaporated to dryness.
For LC/MS/MS analysis, the residue was dissolved in 150
mL of a mixture of water/acetonitrile (50:50, v/v) and 10 mL
were directly injected into the system.
For GC/MS analyses, the residue was dissolved in 100 mL
of a 2% (w/v) methoxyamine hydrochloride solution in
pyridine, and derivatization allowed to proceed for 60 min
at 60 ꢀC. The pyridine was blown off and 50 mL of TMSI were
added. The silylation proceeded for 16 h at 80 ꢀC. The invola-
tile reagents were removed prior to GC/MS analysis by a
cyclohexane/water extraction. The organic layer was trans-
ferred to an injection vial taking care not to transfer any of
the aqueous layer.
Data from several mass spectrometric experiments were
evaluated in order to propose putative metabolite structures.
By GC/MS, electron ionization mass spectra of the
methyloxime-trimethylsilyl (MO-TMS) derivatives of steroids
typically give the following fragment ions: [M–31]+ (loss of
-OCH3 from the oxime group), sequential losses of 90 Da
(HOTMS group), loss of 103 Da (ÀCH2-OTMS group), and
combinations thereof (Fig. 2(D)).[18] Two features of steroid
oximes have to be taken into account; first, an 11-carbonyl
group is sterically hindered and is thus not derivatized.
Second, each carbonyl can generate both the syn and the anti
isomers when derivatized. These isomers may or may not be
resolved by GC.[18]
By LC/MS/MS, the use of ESI in both positive and
negative mode was evaluated. The occurrence of a [M+H]+
ion in positive ion mode was indicative of the presence of a
conjugated 3-keto function[19] while the presence of abundant
[M+H–nH2O]+ ions was considered as symptomatic of
reduction in the A ring.[16] Corticosteroids are mainly ionized
in negative ion mode by the formation of [M+HCOO]– ions.
This adduct is formed by interaction between the formate
ion and the carbonyl and hydroxyl groups at C20 and C21,
respectively. Therefore, only corticosteroids containing a keto
group in the C20 position and two oxygen atoms at C17 and
C21 will ionize in negative ion mode.[16]
In addition, the ions produced in the product ion spectra at
collision energies of 20 and 30 eV were studied. At 20 eV, the
ions appearing between m/z 275 and the [M+H]+ ion were
evaluated. The number of losses of water from a protonated
steroid is related to the number of hydroxyl groups in the
steroid molecule and to the conjugation of the keto group.[20]
A loss of CO to form ([M+H–28–nH2O]+) product ions is
related to the presence of a keto function at C11[16] and other
losses (e.g. of 26 Da, 30 Da, and 58 Da) can provide further
structural information on the steroid.[20] At a CE of 30 eV,
the most abundant product ions are derived from the
steroidal skeleton.[20] The ions produced for one metabolite
were compared with those obtained for the parent drug in
order to yield valuable structural information.
Chemical reduction of methylprednisolone
Chemical reduction of the C20 carbonyl group was performed
in order to compare authentic materials with the putative
20-hydroxy metabolites extracted from the urine. Reduction
was performed by adding 100 mg of NaBH4 to a solution of a
MP standard (50 mg) in 6 mL of methanol and 1 mL of water.
After the mixture had been stirred at room temperature for
5 h, a few drops of acetic acid were added. The mixture was
then diluted with ethyl acetate, washed with saturated
NaHCO3 solution and water, and dried with anhydrous
Na2SO4. The organic layer was evaporated to dryness, and
the mixture of products obtained analyzed by LC/MS/MS
and GC/MS. It is known that the C20 carbonyl group present
in side chains of the pregnane and 21-hydroxypregnanes series
is reduced preferentially to the 20b configuration.[17]
Administration study samples
One oral dose of 40 mg of methylprednisolone (Urbason,
Sanofi-Aventis, Paris, France) was administered to two healthy
male volunteers. Blank samples were collected twice before
administration. Urine samples were collected from 0–8 h,
8–24 h, 24–36 h, and 36–48 h, 48–60 h, and 60–72 h after drug
intake. All samples were stored at À20 ꢀC until analysis.
Methylprednisolone aceponate (5 g; AdventanW, Schering,
Berlin, Germany) were administered to two volunteers (one
male, one female) as a cream (2.5 g in each arm). Urine
samples were collected from 0–4 h, 4–8 h, 8–24 h, 24–32 h,
RESULTS AND DISCUSION
Screening of possible MP metabolites by LC/MS/MS
Several methods based on both PI and NL scans were applied
(NL of 76 Da in negative ion mode; PI scan of m/z 77, 91 and
105 and PI scan of m/z 237 in positive ion mode).[16] In a
previous, preliminary approach four major metabolites were
found, and feasible structures were proposed,[16] and in this
present work a more in-depth study was carried out in order
to identify more metabolites.
ꢀ
and 32–48 h, and stored at À20 C until analysis. Six blank
samples, collected from volunteers who did not take any
Rapid Commun. Mass Spectrom. 2012, 26, 541–553
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O. J. Pozo et al.
Using these approaches, more than 30 peaks were detected
in MP post-administration urines. After combining the
information from all the PI and NL methods, and contrasting
this with the results from pre-administration samples, the list
of possible metabolites was reduced to 15 potential MP
metabolites candidates (Table 1). In positive ion mode, the
occurrence of [M+H]+ and [M+H-nH2O]+ ions is possible
for corticosteroids while in negative ion mode, [M–H]– and
[M+HCOO]– ions are possible. Combining the information
obtained from both positive and negative ion techniques
molecular mass for each metabolite could be calculated (Table 1).
(m/z 171, 147 and 121) but with a difference of 14 m/z units
due to the presence of the methyl group at C6 in MP.[14,17,21]
In addition, the product ion at m/z 211 can be explained as
containing rings C and D together with carbons C20 and
C21 (Fig. 2).
When urine samples were fractionated by LC, MP was
collected between 7.5 and 8 min. When this fraction was
analyzed, the same retention times and equivalent relative
abundances were obtained for MP as from the MP standard.
The isomer in the C11a configuration that was found
previously in rat urine[22] has not been detected in our study.
M1
Proposal of structures for the metabolites
In the LC fractionation experiments this compound was mainly
collected between 7.5 and 8 min. In the LC/MS/MS analyses
this compound eluted at 11.1 min. According to the data
obtained by the precursor ion scan methods, M1 had a molecu-
lar mass of 372 (Table 1). M1 ionized in both positive and
negative ion mode, indicating the presence of a keto function
at C20 and a hydroxyl group at C21. The product ion spectrum
of the [M+H]+ ion at m/z 373 at a CE of 20 eV showed three
losses of water (m/z 355, 337, 319 in Table 2) and several
peaks related to losses of CO (m/z 327 [M+H–CO–H2O]+, 309
[M+H–CO–2H2O]+, and 291 [M+H–CO–3H2O]+) suggesting
the presence of a keto function at C11. At 30 eV (Table 2), the
same product ions as were detected in the parent drug were
obtained (m/z 185, 161 and 135) indicating that both the A and
the B rings remained unaltered. Based on these data, 17a,21-
dihydroxy-6a-methylpregna-1,4-diene-3,11,20-trione is suggested
as the structure of M1.
In the GC/MS analyses this steroid gave two peaks (at 23.5
and 24.1 min), representing the syn and anti steroisomers of
the C3 oxime group. The GC/MS spectrum for the MO-
TMS derivative of M1 showed a molecular ion at m/z 574,
characteristic fragment ions formed by loss of the oxime and
O-TMS groups at m/z 543, 453, 484 and 453 (Table 2) and ions
at m/z 202, and 229 arising from C ring fragmentations. This is
in agreement with published data.[5]
The proposed metabolism for MP is depicted in Fig. 1. In
addition to the unchanged drug, 15 metabolites were
detected in all urine samples collected in the first 36 h after
oral intake, and some of them were detected even up to
72 h after intake. These compounds were labelled M1 to
M15 and tentatively identified as shown below.
Parent compound (MP)
In the GC/MS analyses the MP standard gave two peaks (at 25.1
and 25.8 min), representing the syn and anti steroisomers of
the C3 oxime group. The GC/MS spectrum for the MO-TMS
derivatives showed a molecular ion at m/z 648, and distinctive
fragment ions at m/z 617, 558, 527, 496, 468, and 437 were formed
by losses of the oxime, and silylated groups (Table 2). In addi-
tion, B ring fragment ions at m/z 163 and m/z 189 were also
detected. The proposed fragmentation pathways of the molec-
ular ion of the MO-TMS derivative of MP are shown in Fig. 2.
In the LC/MS/MS runs, the MP standard eluted at
11.3 min and the [M+H]+ ion at m/z 375 showed the expected
fragmentation in a product ion scan at a CE of 20 eV with four
losses of water to form product ions at m/z 357, 339, 321 and
303 (Table 2). At a CE of 30 eV, the most abundant product
ions were found at m/z 185, 161 and 135. These ions were
equivalent to those obtained typically for 1,4-diene steroids
Table 1. Metabolites of methylprednisolone (MP). Retention times obtained by LC/MS/MS, protonated molecules [M+H]+
and formate adduct ions [M+HCOO]- in LC/MS/MS in positive and negative ESI modes, EI molecular ion detected in GC/MS
after MO-TMS derivatization and molecular mass (MW)
Retention time
(min)
m/z
m/z
m/z
MO-TMS
Metabolite
[M+H]+
[M+HCOO]–
MW
MP (parent drug)
11.3
11.1
9.4
10.1
10.5
9.1
9.7
3.5
4.3
2.4
2.9
9.4
9.9
3.9
3.3
4.5
375
373
377
377
373
375
375
391
389
393
393
375
375
391
393
389
419
648
574
693
693
646
691
-
374
372
376
376
372
374
374
390
388
392
392
374
374
390
392
388
M1
417
M2
-
-
M3
M4
417
-
-
435
433
-
-
-
-
M5
M6
M7
736
-
M8
M9
781
781
-
M10
M11
M12
M13
M14
M15
-
435
-
433
-
-
-
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Rapid Commun. Mass Spectrom. 2012, 26, 541–553

Determination of methylprednisolone metabolites in urine
Figure 1. Proposed methylprednisolone metabolism in humans. Metabolic pathways: (i) 11-oxydation,
(ii) 20-reduction, (iii) 6,7-dehydrogenation, (iv) 6-hydroxylation, (v) 16-hydroxylation, and (vi) 22-hydroxylation.
Production of such a metabolite was expected since 11-
oxidoreduction is one of the principal catabolic reactions
undergone by steroid hormones and it was also found after
intra-articular or intramuscular administration;[7] however,
this metabolite was not detected after the administration of
a high intravenous dose for therapeutic treatment in multiple
sclerosis patients.[6]
In the LC/MS/MS run, the retention times for the M2
and M3 metabolites were 9.4 and 10.1 min, respectively.
M2 and M3 exhibited ionization only in positive mode
(Table 1), indicating a change in either C20 or C21. Since
the molecular weight of these metabolites is 2 Da more
than that of the parent compound, reduction of the keto
moiety at C20 seems to be the most feasible alternative. In
addition, the product ion spectra of the [M+H]+ ions at
m/z 377 for both metabolites exhibited four losses of water
at a CE of 20 eV (m/z 359, 341, 323, 305) and similar ions
to those yielded by the parent drug at a CE of 30 eV
(Table 2), supporting the proposed structure. The most
intense isomer in the biological samples was the 20a, as
previously reported;[24] however, in an other study the 20b
structure was proposed for the only isomer reported.[6]
In order to establish a comparison with authentic material,
sodium borohydride reduction was used to produce the
20a- and 20b-hydroxy metabolites from the MP standard.
The mixture of several reaction products was then analyzed
by GC/MS. The most intense pair of peaks, previously
reported as the 20b isomer,[17] had retention times of 25.8
and 26.4 min. The other pair eluted at the same retention time
as M2 (26.8 and 27.5 min). As expected, the spectra obtained
were in good agreement with the spectrum obtained from
the urine (Fig. 3).Thus, the structures for M2 and M3 are
probably 11b,17a,20a,21-tetrahydroxy-6a-methylpregna-1,4-
diene-3-one and 11b,17a,20b,21-tetrahydroxy-6a-methylpregna-
1,4-diene-3-one, respectively.
M2 and M3
In the LC fractionation experiments M2 appeared at
7–8 min and M3 was mainly collected between 7.5
and 8 min.
GC/MS analyses showed that these two steroids
were stereoisomers. Each of them gave two peaks which
correspond to the syn and anti forms. The four peaks
(25.8, 26.4, 26.8 and 27.5 min) exhibited the same electron
ionization (EI) mass spectrum; one of them is shown in
Fig. 3. The molecular ion was at m/z 693, and distinctive
fragment ions were formed at m/z 662 (M–31), m/z 572
(M–31–90), m/z 482 (M–31–90–90), and m/z 392 (M–31–90–
90–90). The base peak is the side-chain-derived ion at
m/z 205, characteristic of steroids containing hydroxyl
groups at C20 and C21.[23] The presence of fragment ions
at m/z 163 and 189 pointed towards the presence of a
methyl group at C6. Previous GC/MS studies obtained
similar results and it was concluded that those peaks corre-
sponded to the 20-hydroxy metabolites.[5]
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O. J. Pozo et al.
Table 2. Main fragment ions (relative abundance in brackets) obtained by GC/MS and main product ions of [M+H]+
obtained by LC/MS/MS for the detected metabolites at the indicated collision energies
Metabolite
MP
GC/MS
LC/MS/MS (20 eV)
LC/MS/MS (30 eV)
648 (10) 617(60), 558(20), 527(30), 496(25), 375 (10), 357 (100), 339 (45), 321 (45),
468(15), 437(25), 366(30), 307(35), 276(60),
211 (30), 185 (55), 173 (20),
161 (100), 135 (80), 121 (45)
303(20), 293 (20), 279 (30), 253 (40)
233(50), 189(40), 163(100), 129(55), 103(50)
M1
M2
574 (15), 543(20), 484(5) 453(10), 414 (20), 373 (55), 355 (100), 337 (65), 327 (50),
211 (25), 185 (70), 173 (50),
161 (100), 135 (40), 121 (35)
185 (50), 173 (20), 161 (45),
135 (100), 121 (50)
323(70), 292(25), 229(100), 202(25), 103(50)
693(2), 662(15), 603(5), 590(5), 572(10), 513
(10), 500(15), 482(15), 423(5), 392(15), 205
(100), 163(30)117(30)
319 (45), 309 (45), 291 (25), 281 (55)
377 (5), 359 (35), 341 (30), 323 (15),
305 (20), 281 (100)
M3
M4
M5
M6
M7
693(2), 662(15), 603(5), 590(5), 572(10), 513
(10), 500(15), 482(15), 423(5), 392(15), 205
(100), 163(30)117(30)
646(85), 615(80), 600(20), 556(10), 525(20),
435(10), 291(35), 272(70), 182(55), 129
(100), 103(10)
377 (10), 359 (55), 341 (30), 323 (25),
185 (80), 173 (30), 161 (60),
305 (25), 281 (100)
135 (100), 121 (50)
373 (15), 355 (100), 337 (5), 319 (5),
185 (100), 173 (25), 161 (80)
159 (75), 135 (25), 121 (20)
309 (10), 291 (5), 280 (60)
691(10), 660(5), 601(10),511(10), 486(30),
396(20), 291(50), 205(100), 129(60)
375 (35), 357 (10), 339 (25), 321 (40),
303 (30), 279 (55),
263 (45), 185 (100), 173 (10),
161 (15), 159 (20), 135 (15),
121 (10)
263 (30), 185 (100), 173 (10),
161 (25), 159 (30), 135 (15),
121 (15)
251 (50), 211 (40), 185 (100),
161 (45), 159 (65), 147 (50),
135 (35), 131 (40), 121 (60)
227 (100), 185 (10), 173 (15),
161 (30), 159 (15), 121 (15)
211 (15), 197 (30), 189 (30),
185 (100), 161 (20), 159 (15),
147 (70), 135 (20), 131 (40),
121 (20)
211 (15), 197 (20), 189 (20),
185 (100), 161 (25), 159 (20),
147 (50), 135 (40), 131 (25),
121 (20)
N.D.a
375 (30), 357 (30), 339 (25), 321 (70),
303 (30), 279 (60),
736(30), 707(5), 646(20), 615(5), 556(5), 525
(5), 470(5), 380(45), 251(45), 219(100), 191
391 (10), 373 (5), 355 (65), 337 (55),
319 (50)301 (15), 279 (90)
(20), 103(25)
M8
M9
N.D.a
389 (10), 371 (20), 353 (25), 335 (10),
331 (25), 325 (20)
393 (10), 375 (5), 357 (15), 339 (30),
321 (35), 303 (20), 297 (20), 279 (100)
781(25), 750(2), 691(15), 601 (5), 511 (5),
425(30), 396(20), 335(85), 251(50), 205(70),
191(35), 147(100), 117(30), 103(25)
M10
781(25), 750(2), 691(10), 601 (7), 595(10),
425(30), 396(20), 335(65), 251(35), 205(90),
191(45), 147(100), 117(40), 103(40)
393 (30), 375 (5), 357 (15), 339 (40),
321 (65), 303 (20), 297 (10), 279 (100)
M11
M12
M13
M14
M15
N.D.a
N.D.a
N.D.a
N.D.a
N.D.a
375 (55), 357 (30), 339 (30), 329 (5),
321 (20), 311 (15), 303 (10), 297 (25),
293 (10), 279 (100)
375 (35), 357 (40), 339 (40), 329 (10),
321 (35), 311 (20), 303 (15), 297 (15),
293 (20), 279 (100)
391 (40), 373 (20), 355 (60), 343 (30),
337 (70), 325 (25), 319 (50), 307(60),
301 (40), 291 (40), 277 (100)
393 (10), 375 (5), 357 (10), 339 (10),
327 (5), 321 (10), 309 (5), 303 (20), 297
(25), 279 (100)
201 (20), 185 (60), 173 (50),
161 (100), 135 (30), 121 (30)
201 (30), 185 (60), 173 (60),
161 (100), 135 (30), 121 (30)
185 (60), 171 (100), 159 (80),
147 (70), 121 (50)
185 (90), 171 (100), 159 (40),
147 (100), 121 (35)
389 (45), 371 (5), 353 (90), 341 (100),
335 (40), 325 (5), 323 (15), 317 (20),
309 (20), 307 (10), 305 (30), 277 (40)
185 (30), 171 (100), 159 (20),
147 (10), 145 (10), 121 (20)
aNot detected.
M4
of steroids and they are present normally in steroids with
high conjugation of the 3-keto function.[20] At CE of 30 eV,
in addition to the ions common to the steroid skeleton of
the [M+H]+ ion of the parent compound (m/z 185, 173, 161, 135
and 121), an ion at m/z 159 was observed (Table 2). This ion
(2 m/z units less than the parent compound ion at m/z 161)
suggests that there is a double bond situated in the B ring.
Thus, the most likely structure for M4 is 11b,17a,21-
trihydroxy-6-methylpregna-1,4,6-triene-3,20-dione. Although
6,7-dehydration is not considered a major steroid metabolic
In the LC fractionation experiments, M4 eluted between 7.5
and 8 min. LC/MS/MS analysis of the LC fraction showed
the presence of a steroid with a protonated mass of m/z 373
(Table 1). Ionization in negative mode suggested the presence
of an unaltered C20-C21 part of the molecule. The product ion
spectrum of m/z 373 at a CE of 20 eV showed, in addition to
three losses of water, an odd-electron ion at m/z 280. The pre-
sence of odd-electron ions is uncommon in the fragmentation
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Determination of methylprednisolone metabolites in urine
A
B
C
D
Figure 2. Proposed fragmentation origin of the most abundant ions for
methylprednisolone. A, C, CID fragmentation of the [M+H]+ ion; in LC/MS/MS;
B, EI fragments of the MO-TMS derivative in GC/MS; D, typical losses 31, 90
and 103 Da of steroids, caused by fragmentation of oxime, HOTMS, and
-CH2OTMS groups in GC/MS analysis.
A
B
D
C
Figure 3. Mass spectra of M2 obtained from urine (A, B) and chemically reduced from MP standard (C, D); (A, C) as
MO-TMS derivatives obtained by EI in GC/MS, and (B, D) CID spectra obtained from [M+H]+ ion at 20 eV collision
energy in LC/MS/MS analysis.
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O. J. Pozo et al.
biotransformation, several 6,7-dehydro metabolites have
been recently reported for some steroids,[21,25,26] but they
have not been reported for MP after intra-articular or
intramuscular administration[7] or after an intravenous
high-dose pulse.[6]
In the GC/MS analysis of the LC fraction, two peaks (25.1
and 25.6 min) were detected. The mass spectrum of the
second peak is shown in Fig. 4. The molecular ion was at
m/z 646, and distinctive fragment ions formed by loss of
the oxime group and silylated hydroxyl groups at m/z 615,
556, 525, 435 were also detected. The base peak was the
ion at m/z 129. This is in accordance with previously
reported data.[5]
Both GC/MS and LC/MS/MS proved that M4 eluted
between 7.5 and 8 min in the LC fractionation. Interestingly,
in the GC/MS analysis of the 4–4.5 min fraction, M4 was also
observed. Thus, it is likely that the same steroid was formed
as a derivatization artifact as suggested by Gallicano et al.[27]
This artifact probably had its origin from the 6b-hydroxy
metabolite (M7, see below), since M7 was detected in the
same 4–4.5 min fraction.
M5 and M6
The M5 and M6 metabolites were collected between 7.5 and
8 min during LC fractionation. By LC/MS/MS, both M5
and M6 ionized only in positive mode (Table 1), suggesting
a modification at either C20 or C21. In agreement with the
GC/MS data, the reduction of C20 seems to be the most likely
option. Fragmentation of the [M+H]+ ion at m/z 375 at a CE of
20 eV (Table 2) showed the expected four losses of water (m/z
357, 339, 321 and 303). At 30 eV CE, in addition to the common
ions for MP (m/z 185, 173, 161, 135 and 121), the ion at m/z 159
also appeared with a similar abundance to the m/z 161 ion.
This situation was analogous to that presented above for
M4, suggesting the presence of an additional double bond
in the B ring. Similarly to M4, a 6,7-dehydro structure was
suggested for M5 and M6.
It was not possible to determine the stereochemistry for M5
and M6. However, if we assume that the chromatographic
behaviour of the C20a/b isomer products is not altered by
the presence of a C6-C7 unsaturation, M6 (shorter retention
time in the LC run) should be identified as 11b,17a,20a,21-
Figure 4. Mass spectra of putative M4 (A, B), M7 (C, D) and M9 (E, F) (A, C, E) as MO-TMS derivatives
obtained by EI in GC/MS analysis and (B, D, F) CID spectra obtained from [M+H]+ ion at 20 eV CE in
LC/MS/MS analysis.
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Determination of methylprednisolone metabolites in urine
tetrahydroxy-6-methylpregna-1,4,6-triene-3-one. Thus, M5
will then be 11b,17a,20b,21-tetrahydroxy-6-methylpregna-1,
4,6-triene-3-one.
product ion spectrum of m/z 389 at 30 eV CE was dominated
by an ion at m/z 227. This ion, 16 m/z units higher than the ion
at m/z 211, suggests the presence of the hydroxyl group in
either the C or D ring. The occurrence of the ions at m/z 185,
173, 161, 135 and 121 indicated an unchanged A-B-C ring
structure. Therefore, the D ring was selected as the most
feasible place for the hydroxylation.
16b-Hydroxylation has not been reported in the meta-
bolism of MP,[6,7] although this is a common metabolic
pathway for several steroids[28] and, therefore, 16b,17a,21-
trihydroxy-6a-methylpregna-1,4-diene-3,11,20-trione is the
most plausible structure for M8. In addition, the presence
of an additional hydroxyl group at C16 can make derivati-
zation difficult, thus explaining the inability to detect M8
by GC/MS.
During GC/MS analysis, only two of the four expected
peaks were detected. In a previous study, Rodchenkov and
co-workers found the four peaks, but the intensities of
two of them were very low.[5] Low intensity signals show-
ing a molecular ion at m/z 691 were present at 26.7 and
27.2 min. The rest of the diagnostic ions are summarized
in Table 2. This mass spectrum resembles those obtained
for M2 and M3 with a 2 m/z units shift. Since the 20b meta-
bolites seems to be excreted in a lesser amount than the 20a
isomers (M2 vs. M3), it is likely that both peaks correspond
to M5.
M5 and M6 have not been reported after intra-articular
or intramuscular administration[7]or after intravenous high-
dose pulse[6] for MP.
M9 and M10
In the LC fractionation experiments M9 appeared between
3.5 and 4 min, whereas M10 was mainly collected between
4 and 4.5 min. Thus, both metabolites seem to be more polar
than MP.
M7
This metabolite was collected between 4 and 4.5 min in
the LC fractionation, symptomatic of some polar groups
being added to the MP structure. M7 ionized in both posi-
tive and negative mode, suggesting that the C20 and C21
positions were unaltered (Table 1). Fragmentation of the
[M+H]+ ion at m/z 391 at a CE of 20 eV showed five losses
of water (m/z 373, 355, 337, 319 and 301, see Table 2), con-
firming the presence of an additional hydroxyl group. At
30 eV CE, in addition to the ions at m/z 185, 161, 135
and 121 common to MP, the ion at m/z 159 suggested a
modification in the B ring. This ion was also present in
M4, M5 and M6 and it was associated with the presence
of a double bond in position 6. Therefore, C6 and C7 are
the most feasible positions for the additional hydroxyl
group in M7.
The GC/MS spectrum for the MO-TMS derivative of M7 is
displayed in Fig. 4, showing the molecular ion at m/z 736. Apart
from the distinctive ions formed by losses of the oxime and
silylated groups, the characteristic ion at m/z 103 for the
primary TMS group at C21 was detected. The ion at m/z 251
corresponds to the B-ring fragmentation at C9-C10 and
C6-C7 (equivalent to the m/z 163 ion seen in the MP
spectrum). Similar to the other metabolites described, two
peaks (27.5 and 28.4 min) were present in the GC run. Thus,
6b-hydroxymethylprednisolone was selected as a feasible
structure for M7.
M9 and M10 were only observed to ionize in positive
mode, indicating a change at either C20 or C21 (Table 1).
Taking into account that the molecular weights of M9 and
M10 are 392, reduction of C20 seems to be the most plausible
option for this change. In the fragmentation of the [M+H]+
ions at m/z 393 at a CE of 20 eV, the five losses of water
corroborated the presence of an extra hydroxyl group. At
30 eV CE, the product ion spectra of protonated M9 and M10
showed the characteristic ions of MP (m/z 185, 161, 135 and
121), but also ions at m/z 159 and 147. These ions were also
present in M7 (proposed as 6b-hydroxy-methylprednisolone)
indicating that there is the same modification in rings A
or B. Peaks corresponding to losses of water, and also a
peak at m/z 297, are in agreement with ions in the product
ion mass spectra described for these compounds at a CE
of 25 eV,[6] although in that report mass spectra were not
obtained at higher CEs so it is not possible to compare
information related to the steroidal skeleton. Therefore,
6b,11b,17a,20a,21-pentahydroxy-6a-methylpregna-1,4-diene-
3-one and 6b,11b,17a,20b,21-pentahydroxy-6a-methylpregna-
1,4-diene-3-one were proposed as feasible structures for M9
and M10, respectively.
Similarly to M5 and M6, and in agreement with previous
results,[6] the proposed stereochemistry of the hydroxyl group
at C20 was based on the elution order. M9 eluted first and
therefore it was selected as the 20a isomer while a b stereo-
chemistry was proposed for M10.
Although 6b-hydroxylation has been described as
a
pathway in MP metabolism,[6] previous attempts to detect
this metabolite by GC/MS in SIM mode have failed.[5]
GC/MS analyses showed that these two steroids were
stereoisomers, and each gave two peaks corresponding to
the syn and anti forms. The four peaks (27.8, 28.7, 29.1 and
30 min) exhibited the same mass spectrum; as an example,
the spectrum of M9 is shown in Fig. 4. The molecular ion
was at m/z 781 and the distinctive fragment ions are listed
in Table 2. The ion at m/z 251 was characteristic of a B ring
containing an extra O-TMS group. The ion at m/z 396
probably originates from the (M–90–90) ion at m/z 601 by
losing 205 Da (side chain). By retention analogy, the peaks
at 27.8 and 28.7 min would correspond to M10, and the peaks
at 29.1 and 30 min to M9. Again, the 20a isomer was of higher
intensity than its 20b isomer. Previous studies by GC/MS
were not able to identify these metabolites.
M8
M8 was not detected by GC/MS. Therefore, a putative
structure was established based only on the LC/MS/MS
data. The molecular weight of M8 was 14 Da higher than that
of the parent drug (Table 1) suggesting a hydroxylation and
an oxidation. M8 ionized in both positive and negative mode
and, therefore, no modification was proposed for C20 and
C21. In addition to several losses of water, the product ion
spectrum of the [M+H]+ ion at m/z 389 at a CE of 20 eV
exhibited a loss of CO (m/z 325, [M+H–CO–2H2O]+) which
can indicate the presence of a carbonyl group at C11. The
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O. J. Pozo et al.
M11 and M12
Although no metabolite of MP with a hydroxyl group in
the C22 position has been reported previously, 11b,17a,21-
trihydroxy-6a-hydroxymethylpregna-1,4-diene-3,20-dione
was selected as a feasible structure for M13.
M11 and M12 eluted in the fractions between 7 and 7.5 min
during LC fractionation and were not detected by GC/MS.
By LC/MS/MS it was found that the molecular weight of
both analytes was the same as the parent drug. This could
be associated to a combination of oxidation and reduction
processes. Both metabolites were only ionized in positive
mode indicative of a change at either C20 or C21. The
reduction of the keto function in C20 seems to be the most
feasible metabolic pathway for this change. The fact that the
[M+H]+ ions of the two metabolites displayed the same
product ion spectra (as described before for M2/M3, M5/
M6 and M9/M10, all of which corresponded to the 20a/20b
metabolites) supports this assignation.
M14
This metabolite was collected between 4.5 and 5 min during LC
fractionation and it was not detected by GC/MS. A molecular
mass of 392 Da was obtained for M14 (Table 1) with a retention
time of 3.3 min. This corresponded to hydroxylation and reduc-
tion of the parent MP molecule. Similarly to other metabolites
(M2, M3, M5, M6, M9, M10, M11 and M12), ionization of M14
was only possible in positive mode suggesting that the
reduction took place at C20. At a CE of 20 eV, the product ion
spectrum of protonated M14 exhibited five losses of water
and two ions related to losses of 30 Da (m/z 327 and 309).
Similar to M13, these losses of 30 Da can be associated with
the presence of a hydroxymethyl group in the molecule. In
addition, several of the most abundant ions at a CE of 30 eV
(m/z 171, 147 and 121) corresponded to the steroidal skeleton
without a methyl group at C6. Therefore, 11b,17a,20x,21-
tetrahydroxy-6a-hydroxymethylpregna-1,4-diene-3-one was
selected as a feasible structure for M14. In this case, it was
not possible to predict the stereochemistry at C20 as only
one metabolite was detected. However, as for the other
metabolites described here with the C20 hydroxyl group,
the a isomer was always detected in higher concentration;
this suggests that M14 is the C20a isomer and that the C20b
isomer is below the detection limit.
The product ion spectra of the [M+H]+ ions at m/z 375 for
M11 and M12 at a CE of 20 eV exhibited four losses of water
together with ions involving a loss of CO (m/z 329, 311 and
293) which can be associated with the presence of a carbonyl
group at C11. The presence at CE of 30 eV of the same ions as
for MP (Table 2) supports the absence of additional changes
in the A, B, C rings.
Therefore, 17a,20a,21-trihydroxy-6a-methylpregna-1,4-diene-
3,11-dione and 17a,20b,21-trihydroxy-6a-methylpregna-1,4-diene-
3,11-dione were proposed as feasible structures for M11
and M12, respectively. Similarly to M5/M6 and M9/M10,
the proposed stereochemistry of the hydroxyl group at
C20 was based on the retention time. Only one of these
two compounds has been reported previously (by Panusa
et al.[7]) probably the C20a isomer due to it being present
at a higher concentration than the C20b isomer which
was below their detection limit.
M15
M15 was collected between 5 and 5.5 min during LC frac-
tionation and it was not detected by GC/MS. M15 has a
molecular weight of 388 (Table 1) which corresponds to one
hydroxylation and one oxidation. Its ionization in both
M13
This metabolite was collected between 5 and 5.5 min during
LC fractionation; and it was not detected by GC/MS.
The molecular weight of M13 (390 Da, see Table 1) corre-
sponded to a hydroxylated metabolite. The ionization of
this metabolite in both positive and negative mode sug-
gested that no modification had taken place at C20 and
C21. The product ion spectrum of the [M+H]+ ion at m/z
391 for M13 at a CE of 20 eV exhibited five losses of water
combined with losses of 30 Da (m/z 343, 325 and 307). This
neutral loss of 30 Da is characteristic of steroids containing
a hydroxymethyl group.[20,29] Thus, a hydroxylation at
C18, C19 or C22 seems to be the most feasible metabolic
pathway for M13.
In the product ion scan of m/z 391 at CE of 30 eV, some
of the most abundant ions (m/z 171, 147 and 121) were
similar to those obtained for steroids and corticosteroids
without the methyl group at C6.[7,12,30,31] These ions can
be produced after the loss of the hydroxymethyl group
in C6. For this reason, hydroxylation at C22 was selected
as the most plausible metabolic pathway for the formation
of M13. In order to corroborate this, a urine sample col-
lected after the administration of prednisone (same struc-
ture as MP, but without the methyl group at C6) was
analyzed. No metabolite having losses of 30 Da in the
product ion scan of the [M+H]+ ion was found, supporting
the hypothesis that the M13 extra hydroxylation takes
place at C22.
Table 3. Transitions and collision energies (CE) of the SRM
method for the detection of MP metabolites by LC/MS/MS.
Cone voltage was set at 25 V for all transitions
Precursor ion
Product ion
Metabolite
(m/z)
(m/z)
CE (eV)
MP
M1
375
373
377
377
373
375
375
391
389
393
393
375
375
391
393
389
161
161
281
281
280
185
185
185
227
189
189
279
279
171
171
341
30
30
15
15
15
25
25
30
20
15
15
15
15
30
30
15
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
M13
M14
M15
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Determination of methylprednisolone metabolites in urine
Figure 5. (a) Excretion rates for methylprednisolone and its metabolites after the oral
intake of 40 mg (peak areas obtained by LC/MS/MS for each metabolite with respect
to time). (b) Detection of metabolites of MP during time course of excretion.
modes indicated the presence of a carbonyl group at C20
a methyl group in the C6 position. Therefore, 17a,21-dihydroxy-
6a-hydroxymethylpregna-1,4-diene-3,11,20-trione was proposed
as a feasible structure for M15.
Some of these metabolites were not detected by GC/MS.
This could be because the addition of more functional groups,
especially alcohol moieties, makes the derivatization step
difficult (as in M8, for example). Low concentrations of the
metabolites and lower sensitivity in GC/MS than in UPLC/
MS/MS (M13 and M14) and a combination of both are other
possible explanations.
and a hydroxyl group at C21. At a CE of 20 eV, the product
ion spectrum of protonated M15 exhibited four losses of
water, losses of CO (m/z 325 and 307) and losses of 30 Da
(m/z 341, 323 and 305). These losses suggested the presence
of a keto group at C11 (similar to M1, M8, M11 and M12)
and a hydroxymethyl group in the C6 position (similar to
M13 and M14). This last consideration was supported
by the fact that at a CE of 30 eV, the most abundant ion
(m/z 171) corresponded to a steroidal skeleton without
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O. J. Pozo et al.
Analysis of excretion studies
Acknowledgements
Once all metabolites had been identified, the whole set of
samples corresponding to the excretion studies was analyzed.
A LC/MS/MS method in SRM mode was developed for this
purpose. The optimized SRM method allowed the detection
of MP at concentrations of around 1 ng/mL. Table 3 sum-
marizes the most critical parameters for the 15 metabolites.
The method was found to be selective for all the selected
analytes as no interferences were found in any of the six blank
samples analyzed.
Grants from Instituto de Salud Carlos III FEDER (CP10/00576),
and financial support from the World Anti-Doping Agency
(WADA research grant 07C12JS), Ministerio de Ciencia e Innova-
ción (Spain) (DEP2009-11454) and Generalitat de Catalunya
(Consell Català de l’Esport and DIUE 2009SGR4929) are grate-
fully acknowledged. The authors would like to thank Dr
Magí Farré and Esther Menoyo for their collaboration in
the excretion studies.
Figure 5 shows the time course of excretion. Although the
amount excreted dramatically decreased after the first 48 h,
it was possible to unequivocally identify six metabolites
72 h after the intake of the drug. When administered
topically, none of the reported metabolites was detected in
any of the samples.
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CONCLUSIONS
A combination of different hyphenated mass spectrometric
approaches has been used to shed new light on methylpred-
nisolone metabolism. By combining all the information col-
lected, the overall metabolism for MP has been re-evaluated.
Plausible structures for 15 metabolites have been proposed,
five of them being novel compounds.
As expected, the activities of the biotransformations of some
of the principal corticosteroids were confirmed. It is known that
11b-hydroxysteroid dehydrogenase type 2 present in the
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enzymatic activity seems to be diminished, if the cortisol struc-
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the excretion of M1 (MW 372) was expected.
Analogously, the unequivocal identification of M2 and M3
(MW 376) confirmed that C20 reduction catalyzed by the two
enzymes 20a- and 20b-hydroxysteroid dehydrogenases takes
place. A combination of these two basic biotransformations,
as occurs in the cortisol metabolism, produced M11 and
M12 (MW 374). 6b-Hydroxylation is the origin of M7 (MW
390) as was described by Vree et al.[6] The combination of
6b-hydroxylation and C20 reduction produces M9 and M10
(MW 392). According to the data obtained, further unreported
hydroxylations may occur at C16a, and C22. We propose that
four novel metabolites, M8 (MW 388), M13 (MW 390), M14
(MW 392) and M15 (MW 388), are excretion products generated
through these extra hydroxylations.
Finally, in the present work three 6,7-dehydro metabolites
(M4 (MW 372), M5 and M6 (MW 374)) have been detected,
thus proving that MP undergoes 6,7-dehydration. These
metabolites were previously detected only by GC/MS.[5]
Their detection by LC/MS/MS leads to the conclusion that
they are authentic metabolites and not simply derivatization
artefacts.
A method for discriminating between different routes of
administration of MP has also been evaluated. However,
none of the metabolites was found after topical application
of a high dose of the drug. Thus, it seems that establishing
cut-off values would suffice for discriminating routes of
administration.
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Rapid Commun. Mass Spectrom. 2012, 26, 541–553

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