Detection
of
Designer
Anabolic
Steroids
by
LC‐MS/MS
and
Commercial
Synthesis
of
Oxandrolone
Kristin
Minkowski
Carthage
College
Chemistry
400:
Senior
Seminar
Detection
of
Designer
Anabolic
Steroids
by
LC‐MS/MS
and
Commercial
Synthesis
of
Oxandrolone
11/18/10
Abstract
As
of
late,
the
abuse
of
previously
unknown
steroids
by
professional
and
amateur
athletes
has
become
a
major
concern
for
athletic
governing
bodies.
In
order
to
detect
designer
steroids,
which
have
been
synthesized
specifically
to
evade
current
analytical
techniques,
new
analytical
screening
methods
must
be
devised.
For
liquid
chromatography‐tandem
mass
spectrometry
at
high
collision
energy,
a
precursor
ion
scan
method
was
proposed,
which
allows
for
the
detection
of
potential
steroid
analytes.
The
simultaneous
presence
of
three
specific
fragmentation
ions
at
m/z
105,
91,
and
77
on
an
MS
spectrum
indicates
a
possible
steroid
molecule
is
present
in
the
sample
matrix.
In
addition
to
detection,
a
more
efficient
commercial‐grade
synthesis
was
investigated
regarding
the
anabolic
steroid,
oxandrolone.
The
Searle
procedure
was
used
as
a
starting
point,
for
which
new
reagents
were
proposed
to
generate
specific
reaction
intermediates:
α‐bromoketone,
enone,
and
hydroperoxide.
Perbromides
were
used
in
place
of
molecular
bromine
to
brominate
the
A
ring,
and
an
ozonolysis
process
was
used
in
place
of
toxic
reagents
to
oxidize
the
enone.
Together,
these
mechanistic
steps
significantly
increased
the
initial
yield
from
8%
to
45%.
At
present,
there
is
a
constant
battle
between
those
synthesizing
designer
steroids
to
evade
detection
and
those
developing
analytic
techniques
able
to
identify
previously
unknown
steroids.
Introduction
In recent years, designer steroid abuse has been at the forefront of numerous athletic
scandals, from Major League Baseball to Olympic track and field. In 2004, the UCLA Olympic
Analytical Laboratory identified the compound 17α-methyl-5α-androst-2-en-17β-ol, more
commonly known as the anabolic steroid madol, after receiving an oily substance not currently
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Detection
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Designer
Anabolic
Steroids
by
LC‐MS/MS
and
Commercial
Synthesis
of
Oxandrolone
11/18/10
tested for in urine analysis.1 Madol was only the third performance-enhancer, never
commercially developed, to be identified as a designer steroid. While the methods for
determining known steroid analytes by target detection are fairly straightforward, analytical
methods for detecting new unknown designer steroids (madol) are much more problematic.2
Traditional steroid detection methods involve coupling mass spectroscopy with either gas
chromatography (GC) or liquid chromatography (LC). These techniques allow the sample to be
separated into its individual components before being introduced to the ionization source,
important when dealing with complicated sample matrices. While GC-MS has been the industry
standard since the 1980s, it has its limitations, including complicated sample pretreatment.3
Since steroids and their metabolites can be excreted as both a conjugated (sulfate or glucuronide
derivatives) and unconjugated (free) fraction, sample matrices often require hydrolysis of the
conjugated analytes and a derivatisation step before GC-MS analysis can be run at a suitable
level of sensitivity.3 In an effort to simplify sample pretreatment, analyst’s are turning toward
LC-MS/MS as the chosen instrumentation for detection of steroid compounds.
Figure 1: General Steroid Structure
Steroid molecules share a common four-ringed structure, composed of three cyclohexane
rings (A, B, and C) and one cyclopentane ring (D). The general steroid structure and standard
numbering nomenclatural is shown in figure 1. Due to additional functional groups and double
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Detection
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Anabolic
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LC‐MS/MS
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Synthesis
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Oxandrolone
11/18/10
bonds, which can be added to this basic structure, the number of potential designer steroids is
almost endless. In order to stay ahead of athlete’s abusing performance-enhancing steroids, new
analytical techniques must be developed that not only detect all of the known steroids and their
metabolites, but also detect all of the potential steroid analytes present in urine samples.
Figure 2: Mass spectroscopy components adapted from Harris4
Pozo et al.2 utilized many distinct LC-MS/MS scan modes in an effort to design a method
of steroid detection capable of identify unknown steroids. Figure 2 illustrates the specific
analytical components that make up LC-MS/MS.4 To begin, a sample is separated into a series of
individual components via liquid chromatography. These isolated components are then
introduced to an ion source, electrospray ionization, where the eluent is sprayed through an
electrically charged capillary tube. The charged solvent is sprayed from the tube and allowed to
interact with nitrogen gas. The solvent eventually evaporates, leaving only the positively charged
sample ions. These positively charged ions are attracted to a negative voltage potential allowing
them to travel toward the mass separator.4 In tandem mass spectrometry, two quadrupoles,
separated by a collision cell, are used to isolate and observe specifically selected ions based on
their mass-to-charge ratios (m/z)4. In steroids, a single protonated molecule is generated via ESILC-MS/MS, meaning the m/z is equivalent to the mass of the protonated molecule [M + H]+.
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Oxandrolone
11/18/10
Figure 3: Tandem Mass Spec Scan Techniques (a) Product ion scan, and (b) Precursor ion scan adapted from
Haris.4
Pozo et al.2 used a product ion scan to establish fragmentation patterns for a diverse
group of model steroid compounds (figure 3a). In this procedure, the initial quadrupole is set to
select for a parent or precursor ion from a specific analyte of interest, typically the molecular ion
[M + H]+. Once the [M + H]+ ion is isolated, it passes through the collision cell, where it
collides with nitrogen gas, causing the ion to fragment. These fragmented ions are then analyzed
and separated according to their mass-to-charge ratio by the second quadrupole. The resulting
spectrum is unique for each steroid analyte under investigate.
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Detection
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Oxandrolone
11/18/10
The goal of Pozo et al.2 was to determine specific fragmentation ions common to known
steroid molecules using LC-MS/MS, in order to more accurately predict the presence of
unknown steroid molecules. Figure 3b depicts the proposed precursor ion scan used to detect the
presence of a specific product ion after fragmentation. In this technique, quadrupole 1 allows all
of the sample ions to be transmitted to the collision cell, and quadrupole 2 is selected to monitor
only one specific fragment ion. By analyzing the various fragmentation pathways of a widevariety of steroid compounds at high collision energy, Pozo et al. were able to isolate three
common fragmentation ions. Their proposed technique provides researchers with a
straightforward method to determine previously unidentified steroid compounds in a sample
matrix.
Figure 4: Structures of methylandrostanolone and oxandrolone
As new analytical techniques are being created to aid in steroid detection, new synthetic
pathways are also being devised in an effort to create either new undetectable compounds, i.e.
designer steroids, or to improve overall synthetic yield in commercially produced steroid
compounds.5 In order to produce commercial quantities of the anabolic steroid oxandrolone
(figure 4), Cabej et al. used the initial Counsell and Pappo synthesis6 as a starting point (scheme
1).
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Detection
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Designer
Anabolic
Steroids
by
LC‐MS/MS
and
Commercial
Synthesis
of
Oxandrolone
11/18/10
Scheme 1: Counsell / Pappo sythesis of Oxandrolone (a) Br2, NaOAc, HOAc, RT (b) LiCl, Li2CO3, DMF, (c)
OsO4, Pb(OAc)4, HOAc, H2O, RT (d) NaBH4, NaOH, RT adapted from Cabej et al.5 & Counsell et al.6
Cabej et al. focused on three specific areas upon which the original synthesis needed to
be improved. First, they needed to address the low overall percent yield for oxandrolone, which
was only 8% for the Counsell / Pappo synthesis.6 Second, they needed to find alternatives for
toxic reagents used in the initial synthesis, such as molecular bromine.7 Finally, they needed to
elimination purification via column chromatography8, which is not feasible for a commercial
synthesis. After numerous mechanistic modifications, Cabej et al.5 were able to increase the
overall yield of oxandrolone to 45% and make commercial production a more viable option.
Results
The purpose of Cabej et al.5 was to modify the original synthesis of the anabolic steroid
oxandrolone, in order to make it more suitable for commercial-scale production. The initial
Searle synthesis, developed by Counsell and Pappo6 (scheme 1), was used as the starting point
for the newly proposed up scaling of the oxandrolone synthesis; however, alterations were made
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Detection
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Anabolic
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LC‐MS/MS
and
Commercial
Synthesis
of
Oxandrolone
11/18/10
at numerous steps of the synthetic pathway. Adjustments were necessary in the oxidation step,
due to the toxicity of osmium tetroxide and lead acetate reagents. In addition, the initial
synthesis required purification via column chromatography of the bromoketone and enone
intermediates, which is not feasible for large-scale production. Finally, Cabej et al. addressed the
relative low yields for specific reaction intermediates. While the Searle synthesis was an
excellent guide, the newly proposed synthesis of oxandrolone from methylandrostanolone was
more direct and efficient.
Beginning with methylandrostanolone, Counsell and Pappo brominated C(2) using
bromine in a sodium acetate/acetic acid solution to produce the bromoketone intermediate. Due
to competing reactions, this bromination process only gave the bromoketone intermediate in a
43% yield. Hydrobromic acid (HBr) was a byproduct of the bromination, and its acidic
properties led to competing dehydration reactions on the C(17) alcohol group, lowering the
overall bromoketone intermediate yield. Cabej et al. addressed this issue by brominating
methylandrostanolone with perbromides, first phenyltrimethyl ammonium bromide (PTAB) and
then due to its cost efficiency, pyridinium tribromide (PyHBr3). While PTAB in the aprotic
solvent tetrahydrofuran (THF) gave an 85% yield during the initial small-scale procedure,
similar results were not observed once performed on the commercial level. THF, a cyclic ether
(figure 6), interacts with HBr yielding 4-bromo-1-butanol, a compound capable of remaining in
the product after bromination of C(2) takes place. During the drying process, 4-bromo-1butanol transforms back into THF, releasing HBr, which dehydrates the C(17) alcohol. Since the
THF solvent was responsible for the acid trapping and subsequent dehydration, Cabej et al.
removed THF as a solvent and performed the PTAB bromination in a variety of solvents. Table
1 highlights the reaction conditions, percent yields, and percent purities for each solvent. Percent
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Detection
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Anabolic
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Synthesis
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Oxandrolone
11/18/10
yield reflects the amount of product obtained verse the expected or calculated amount of product;
whereas, the percent purity only takes into account the bromoketone present in the product
collected (bromoketone + impurities) determined via HPLC analysis.
entry
1
2
3
4
5
6
7
8
Table 1: Bromination of methylandrostanolone with PTAB in different solvents
conditions
percent yield (%)
percent purity (%)
comments
THF/0°C EtAOc extraction
75
92-94
HBr trap/dehydration
DME/0°C
64
not determined
bromoketone insoluble
EtOAc/0°C
73
97
bromoketone insoluble
CH2Cl2/0°C
not determined
not determined
dehydration
EtOH/RT
88
90
ketal formed
EtOH/8% H2O/RT
84
89
rx time 5.5 h
EtOH/14% H2O/RT
82
91
rx time 24 h (slow)
MeOH/RT
not determined
not determined
ketal formed
Figure 6: Bromination solvent structures (a) Tetrahydrofuran (THF), (b) Dimethoxyethane (DME), (c) Ethyl
Acetate (EtOAc)
The bromination was performed in both protic (EtOH, H20, MeOH) and aprotic (THF,
DME, EtOAc, CH2Cl) solvents. Figure 6 illustrates the structure of three of the polar aprotic
solvents used in the bromination: THF, DME, and EtOAc. As mentioned above, THF as a
solvent on the commercial level led to HBr trapping and resulted in late-stage dehydration,
despite the small-scale success evident by percent yield and percent purity in Table 1. Entry 2
using DME and Entry 3 using EtOAc not only had low percent yields, 64% and 73%, but also
made isolating the bromoketone intermediate difficult due to its insolubility in both solvents.
Entry 4 was performed in dichloromethane; however, due to dehydration of the C(17) alcohol,
the percent yield and corresponding bromoketone purity were deemed to low to analyze. Entry 5
and 8 were run in ethanol and methanol only (in the absence of water) and resulted in the
formation of a ketal at C(3). The presence of the ketal was not observed in entries 6 and 7, and it
is hypothesized that the addition of water to the ethanol solvent medium (at eight and fourteen
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Oxandrolone
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percent respectively) hydrolyzes the ketal back to into a ketone. In the presence of too much
water (entry 7), the reaction proceeds at a much slower rate (24 hours vs. 5.5 hours), and with
similar percent yields and percent purities, the commercial scale process would be most efficient
in the ethanol / 8% water solvent.
Scheme 2: Reaction intermediates (A ring) during commercial synthesis of oxandrolone (a) PyHBR3, ETOH,
H20, Na2S2O35H20 (aq), Na2CO3 (aq) (b) LiBr, Li2CO3, DMF, Acetic Acid, H20, EtOAC, Heptane (c) O3,
MeOH, NaOH, HCl, MeOH/H2O (d) NaOH, EtOH, H2O, NaBH4, HCl, Filter, MeOH, H2O
Due to its lower cost and its solubility in ethanol, PyHBr3 was used as the brominating
agent in place of PTAB. Sodium thiosulfate pentahydrate and sodium carbonate were added
stepwise to the slurry to quench or neutralize the perbromide and adjust the pH. Scheme 2
illustrates the newly proposed commercial-grade synthesis of Oxandrolone, focusing on the A
ring of the steroid. Reacting PyHBr3 with methylandrostanolone in an ethanol/8% water solvent
produces the bromoketone intermediate in the first step of the synthesis at an 82% yield (92%
purity).
Scheme 3 illustrates the reaction mechanism resulting in the α-bromoketone intermediate.
The key to this reaction proceeding is the formation of the enol isomer from the original ketone
functional group. In the presence of an acid (PyH+), the carboxyl group is protonated, followed
by the removal of an α hydrogen at C(2), the formation of a C(2)-C(3) double bond, and the
creation of a C(3) alcohol. The presence of the alkene (en) and alcohol (ol) give rise to the name
of the isomer (enol). Following the electron arrows, the enol acts as a nucleophile and attacks
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Oxandrolone
11/18/10
the molecular bromine (electrophile). The carbocation is converted to its alternate resonance
form, or the protonated ketone. Finally, the ketone is deprotonated via a bromide ion, and the αbromoketone intermediate is produced.
Scheme 3: Bromination of methylandrostanolone (A ring structure)5
In step two of the synthesis, the newly formed bromoketone undergoes elimination to yield the
enone intermediate shown in scheme 2. Counsell and Pappo reacted the bromoketone
intermediate with LiCl/Li2CO3 in dimethylformamide solvent (figure 8), yielding a crude 93%
mixture of two major enone products (figure 7). To isolate enone A, column chromatograph and
recrystallization were performed, resulting in only a 39% yield of enone A intermediate. Cabej
et al. were unable to use column chromatograph in their commercial-scale synthesis, and
therefore, looked for alternative reagents with which to perform the elimination of the
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bromoketone. Table 2 highlights the different elimination reaction conditions and corresponding
enone A to enone B ratios.
Figure 7: Structure of major elimination productions (enone A and enone B)
Table 2: Bromoketone elimination reaction conditions to yield enone A and enone B
entry
conditions
enone production ratio A/B
1
CaCO3, DMA, reflux
2/1 (100% crude yield)
2
KOH, MeOH, H20 80°C
multiple products
3
Li2CO3, DMF, 100-115°C
multiple products
4
LiCl, DMF, reflux
~ 2/1
5
LiBr, Li2CO3, DMF, 110-120°C
15/1 (91% crude yield)
6
LiBr, Li2CO3, DMA, 110-120°C
46/1 (97% crude yield)
7
LiBr, Li2CO3, DMSO, 110-120°C
multiple products
8
LiBr, DMF, 110-120°C
dehydration
Entry 1 reacts the bromoketone with the base, calcium carbonate in dimethylacetamide
(DMA), yielding a 2:1 ratio of enone A to enone B. Entry 2 uses potassium hydroxide,
methanol, and water for the elimination but resulted in numerous product formations. Entry 3
used the lewis base Li2CO3 in the presence of DMF, but also resulted in a variety of products. In
turn, Entry 4 used only LiCl from the Counsell/Pappo synthesis in a DMF solvent and yielded a
2:1 ratio of enone A to enone B. Cabej et al. had greater success using LiBr as the Lewis acid as
demonstrated by entries 5 – 8. Entry 5 used LiBr in combination with the base Li2CO3 in a DMF
solvent (figure 8) and resulted in a 15:1 ratio of enone A to enone B. Entry 6 uses the same acid
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and base as 5 but uses DMA as a solvent (figure 8), increasing the product ratio to 46:1. Entry 7
again uses LiBr/Li2CO3 but in a dimethyl sulfoxide (DMSO) solvent (figure 8); however,
multiple product formations were reported. Finally, the elimination reaction of the bromoketone
was run in LiBr and DMF, without the base Li2CO3 where the major product was the result of
dehydration at C(17) and a methyl substituent shift (carbocation rearrangement). Scheme 2
highlights the reagents chosen for the elimination of HBr from the bromoketone to produce the
enone A intermediate at a 60 – 80% yield (93 – 97% purity).
Figure 8: Elimination solvent structures (a) DMF, (b) DMA, (c) DMSO
Scheme 4: Formation of enone intermediate via E1 elimination5
Scheme 4 highlights the E1 mechanism involved in the formation of the enone
intermediate. The first step involves the loss of the bromide leaving group, generating a
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Oxandrolone
11/18/10
secondary carbocation. The halide leaving group is favorable because bromide ions are weak
bases, i.e., they are the conjugate base of a strong acid, HBr. With the formation of the
carbocation intermediate, the carbonate ion (CO32-) acts as a moderately strong base and removes
a proton, allowing the C(1)-C(2) double bond to form.
In step three of the Oxandrolone synthesis, Counsell and Pappo oxidized the enone to a
carboxylic acid using osmium tetroxide and lead acetate (scheme 1), compounds that were
deemed too toxic for a commercial-grade procedure. Cabej et al. explored different ozonolysis
methods to address both the toxicity issue and attempt to increase the low 7% yield initially
reported. Ozonolysis trials were performed in various solvents (MeOH, EtOH, EtOAc, CH2Cl2),
but methanol was the most successful, since it lacked the solubility issues of EtOAc and CH2Cl2
and the multiple product formations of ethanol. Aqueous sodium hydroxide and hydrochloric
acid were added stepwise to quench hydroperoxide and adjust the pH, leaving the carboxylic
acid in a 56 – 66% yield (94 – 97% purity). Ozonolysis reaction temperature was an important
factor, and numerous trials were performed to determine the most efficient temperature range.
Although -50 to -30°C was determined to be the optimum temperature range based on product
purity (Table 3), for the commercial synthesis, the minimal temperature their plant could
maintain was -15°C. Percent purity was slightly lower at this temperature, but more than
adequate for their large-scale synthesis. Recrystallization of the crude product was also
performed to increase the overall carboxylic acid yield. Scheme 2 highlights the reagents used to
oxidize the enone via ozonolysis.
Table 3: Various temperature ranges for ozonolysis of the enone intermediate
entry
temperature (°C)
percent yield (%)
percent purity (%)
1
-50 to -40
83
95
2
-40 to -30
90
99
3
-20 to -10
92
90
4
-10 to 0
88
89
5
ambient
not determined
multiple products
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Scheme 5: Ozonolysis of enone intermediate to generate the hydroperoxide via the Criegee Mechanism5
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Scheme 5 uses the Criegge mechanism to transform the enone into the hydroperoxide
intermediate. In the first step, the ozone molecule is added to the carbon-carbon double bond by
electrophilic addition. The arrow starts at the nucleophilic pi bond, opens up to the C(2), and
ends at the double bonded oxygen (electrophile). The second electron arrow begins at the pi
bond and ends at the positively charged oxygen, and the third arrow begins at the negatively
charged oxide electron pair and ends at the C(1) carbon. The newly created functional group is
referred to as a molozonide; however, due to instability, it breaks apart quickly to yield an
aldehyde fragment and a carbonyl oxide fragment. Methanol attacks the carbonyl carbon and the
resulting hydroperoxide intermediate is formed.
In the second half of step three, the hydroperoxide intermediate was neutralized with
aqueous sodium hydroxide (NaOH), and hydrochloric acid (HCl) was added to adjust the pH,
resulting in the formation of the acid intermediate. The final step takes this acid intermediate and
forms the cyclic ester, oxandrolone. As described in scheme 2, Cabej et al. produced the sodium
carboxylate salt from the carboxylic acid and NaBH4 reduced the aldehyde functional group to
its primary alcohol. After the addition of hydrochloric acid, the cyclic ester was generated.
Once the crude product is recrystallized, the percent yield was calculated at 81 – 97% (99 –
100% purity). The complete commercial synthesis of oxandrolone from methylandrostanolone
(scheme 2) resulted in a 45% yield compared to Counsell and Pappo’s 8%.
Pozo et al. sought to devise an analytical method capable of detecting unknown anabolic
steroids and their metabolites. To begin, five different steroid compounds were selected as
‘models’ based on their specific functional groups and both the position and quantity of double
bonds. These five compounds (figure 9) were selected in an effort to encompass a wide variety
of steroids, with their combined analytical results being representative of the ‘steroid’ class of
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molecules as a whole. Compared to the general steroid structure (figure 1), Testosterone has a
C4 – C5 double bond on the A ring and a C17 alcohol group on the D ring. Oxandrolone is
composed of a cyclic ester and has both an alcohol and methyl group on C17. Boldenone has
two double bonds at C1 – C2 and C4 – C5 and a C17 alcohol group. 17α-trenbolone has a
network of conjugated double bonds: C4 – C5, C9 – C10, and C11 – C12. Lastly, 6β-hydroxymetandienone has two double bonds at C1 – C2 and C4 – C5, two alcohol groups at C6 and C17,
and a methyl group at C17. It is important to recognize that all five of the steroids selected
possess a keto group at C3, as this will be the protonated site during electrospray ionization
(ESI).
Figure 9: Structures of five ‘model’ steroid compounds used as standards
The five model compounds were analyzed via tandem mass spectroscopy at six different
collision energies (15, 20, 25, 30, 40, 50 eV). These collision energies correlate to the energy
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applied to the ions in the collision cell, located between quadrupole 1 and quadrupole 3 (figure
2). Pozo et al. utilized a product ion scan (figure 3a), where the first quadrupole selected [M +
H]+ as the precursor ion, the second quadrupole acted as the collision cell, and the third
quadrupole separated the fragmented product ions according to the m/z. It is the additional
energy from the interactions in the collision cell that cause precursor ions to fragment into their
corresponding product ions. Figure 10 focuses on the product ion spectra from three of these
compounds and the unique fragmentation observed at high collision energy (50 eV), medium
collision energy (25 eV), and low collision energy (10 eV). As the collision energies were
increased for oxandrolone, 17α-trenbolone, and 6β-hydroxy-metandienone, the fragmentation
patterns were drastically altered. At low collision energy, the product ions formed by way of
collision-induced dissociation (CID) are typically the result of small bond cleavages, which can
be attributed to the loss of one or more water molecules. The mass spectrum of 6β-hydroxymetanienone at low collision energy (Figure 10c) assigns the product ions at m/z 299 and 281 to
[M + H - H2O]+ and [M + H - 2H2O]+ respectively, meaning the protonated ion has lost 1 or 2
neutral water molecules. For all three compounds, fragmentation is minor at low collision
energy, which is demonstrated by the limited number of ions (peaks) observed on the three
spectra. Since the most abundant ion often corresponds to the initial precursor ion [M + H]+, at
low collision energies, residual energy would be too low for any additional fragmentation to take
place. As the collision energy increases, the amount of residual energy possessed by the
protonated ion also increases, giving rise to a greater amount of fragmentation. Comparing the
low collision energy spectra to the medium collision energy spectra, two behavioral patterns are
reported. First, the medium collision energy spectra for all three compounds have a greater
number of product ions, due to the larger amount of excess energy. Second, the mass-to-charge
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values are smaller for the medium collision energy spectra. These smaller product ion values
correspond to the loss of much larger neutral fragments, than were observed with low collision
energy (i.e. the loss of water).
Figure 10: MS/MS spectra of (a) Oxandrolone, (b) 17α-trembolone, and (c) 6β-hydroxy-metanienone2
While it was important to analyze each steroid molecule at various collision energies, Pozo et al.
believed that the most promising data could be extrapolated from the high collision energy
spectra, which revealed a definite fragmentation pattern among all of the ‘model’ compounds
analyzed. Specifically, three common product ion fragments are present at m/z 105, 91, and 77 in
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the high collision energy spectra for all of the steroid compounds. Figure 11 highlights the
structure of these three common ions: methyl tropylium (m/z 105), tropylium (m/z 91) and
phenyl (m/z 77). Each of these mass-to-charge ratios corresponds to a viable fragmentation of
the steroid (ring B or C), and it is their mutual presence in each of the collected spectra that Pozo
et al. attempt to apply to the determination of unknown steroids and their metabolites.
Figure 11: Common product ions, which appear in spectra, collected at high collision energy
Table 4 summarizes the results obtained from the product ion scan of the 5 ‘model’
steroid compounds selected. For all five compounds, common ions were observed at m/z 105,
91, and 77. In addition, the precursor ions selected by the first quadrupole were identified.
While all five had a [M + H]+ precursor ion, corresponding to the protonation of the C3 keto
group, 6β-hydroxy-metanienone and oxandrolone form additional adducts that can be used as
precursor ions in a product ion scan to yield the three common product ions.
Table 4: Presence of product ions for ‘model’ steroids and their precursor ions
compound
m/z 77
m/z 91
m/z 105
precursor ion
boldenone
YES
YES
YES
[M + H]+
+
6β-hydroxy-metanienone
YES
YES
YES
[M + H] , [M + H - H2O]+, [M + H -2H2O]+
17α-trembolone
testosterone
Oxandrolone
YES
YES
YES
YES
YES
YES
YES
YES
YES
[M + H]+
[M + H]+
+
[M + H] , [M + NH4]+, [M + H + MeOH]+
Collectively, it is important to note that while each of the steroid molecules are
structurally similar, even minor differences, such as the addition of a double bound or an alcohol
group, drastically change the analyte’s fragmentation and result in the unique spectra observed in
figure 10. Using the data from their analysis of ‘model’ steroid compounds, Pozo et al. theorized
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that the simultaneous presence of three common product ions (m/z 105, 91 and 77) would be a
starting point for identifying unknown steroids and their metabolites. With the advent of
designer steroids, new analytical techniques are necessary to detect compounds specifically
engineered to evade current screening protocols. Pozo et al. went on to test their newly proposed
method in two alternate situations. First, they applied the technique to additional non-steroidal
doping agents banned by World Anti-Doping Agency (WADA), in order to test the specificity of
their method. Second, they used their method to detect fluoxymesterone metabolites in positive
urine matrixes, a necessary feature for accurate steroid detection, since most steroid compounds
are highly metabolized in the body. These additional trials were used to determine how
successful their analytical technique would be as an alternative to the standard target detection
approach commonly used in analytical analysis.
Figure 12: Structure of Additional Doping Agents Containing a Six-Membered Ring
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Figure 12 illustrates eight non-steroid doping agents banned by WADA, which were
analyzed for the presence of the product ions at m/z 105, 91, and 77. These compounds were
selected based on their relative size (molecular weight) and the presence of either an aromatic or
six-member ring. While their structures vary quite a bit from the common anabolic steroid
structure (figure 1), the above-mentioned similarities will help test the limitations of the common
ion approach, as these doping agents tend to fragment in a similar behavior.
Table 5: Presence of product ions for additional doping agents
Compound
Doping Agent
MW
m/z 77
m/z 91
Clenbuterol
β-Agonist
276
YES
NO
Formoterol
β-Agonist
344
YES
YES
Methylprednisolone
Corticosteroid
374
YES
YES
Atenolol
β-Blocker
266
YES
YES
Bisoprolol
β-Blocker
325
YES
YES
Propranolol
β-Blocker
259
YES
YES
Clopamide
Diuretic
345
NO
NO
Canrenone
Diuretic
340
YES
YES
m/z 105
YES
NO
YES
YES
YES
NO
YES
YES
As the cornerstone to their theory, the presence of all three common product ions (m/z
105, 91, and 77) is required for a compound to be considered an anabolic steroid. Table 5 shows
that all of the doping agents have at least one of the three necessary ions, and four of the eight
have all three of the common ions. Both canrenone (diuretic) and methylprednisolone
(corticosteroid) have peaks at all three product ions, which Pozo et al. attribute to their steroidlike structures. Figure 11 clearly shows both compounds containing four fused rings (three sixmember rings and one five-member ring) and a keto group at C3. While not classified as
steroids, both canrenone and methylprednisone have structures similar to steroids, hence the
presence of all three common product ions. In addition, two β-blockers (atenolol and bisoprolol)
also had fragmentation corresponding to all three common ions. However, these compounds
were easily ruled out as potential steroids after running scans at low collision energy. Neither
atenolol nor bisoprolol exhibited any traditional steroid behaviors, such as ions resulting from the
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loss of water at low collision energies, which were the case for the ‘model’ steroids (figure 10).
While these doping agents interfered with the proposed method, Pozo et al. easily justified the
simultaneous presence of all three common product ions, and were therefore pleased with their
technique’s ability to distinguish steroid compounds from both aromatic compounds and
compounds containing six-member rings.
Figure 13: Fluoxymesterone metabolism analysis via LC-MS/MS adapted from Pozo et al.2
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Due to extensive metabolism, initially ingested steroid compounds are often not present
in urine samples, and therefore the study and detection of urinary metabolites is of great interest
to analytical chemists. Pozo et al. acquired urine samples that had tested positive for the steroid,
fluoxymesterone (figure 14) and used a precursor ion scan technique (figure 3b) in order to
detect both the parent compound, fluoxymesterone, and its metabolites. To begin, the sample is
separated into a series of individual components by way of liquid chromatography. Then, the
eluent is ionized via ESI and a precursor ion scan was performed for each individual analyte. In
this study, the chromatogram from a negative (blank) urine sample was compared to the
chromatogram of a sample, which previously tested positive for fluoxymesterone. Figure 13
contains the LC chromatogram for each of the pre-selected product ion scans (m/z 105, 91, 77).
Figure 14: Structure of Fluoxymesterone and its metabolites adapted from Pozo et al.2
Any peak that was present in both the negative urine sample and the fluoxymesterone
urine sample could be attributed to a compound in the sample matrix and therefore was not
assigned to a steroid metabolite. In total, six peaks present on the chromatogram were
recognized as fluoxymesterone metabolites. The retention time for each analyte was based on
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the amount of interaction with the non-polar stationary phase of the C18 column as the polar
mobile phase eludes through the column. The more polar the analyte, the less interaction it has
with the non-polar stationary phase and consequently, the quicker it moves through the column.
The structures of the six suspected metabolites (except M3) are proposed in figure 14. As
expected, the more alcohol functional groups the metabolite possesses, the smaller its LC
retention time. Metabolite 4 (M4) has four alcohol groups, is the most polar of the compounds,
has the least interaction with the stationary phase, and has the shortest retention time at 1.7
minutes. A peak can be attributed to M4 at all three of the product ion scans in the
chromatogram in figure 13. In addition, the mass spectrum for metabolite 4 reports a precursor
ion at m/z 337. Of the six metabolites, M4 is the only analyte with a precursor ion not assigned
to [M + H]+. In order to determine the molecular weight and chemical formula of the metabolite,
a full scan of the analyte needs to be performed. From the full scan spectrum, various adducts,
each corresponding to specific m/z values, were recorded for M4. Two common ESI adducts,
[M + NH4]+ and [M + Na]+ were observed at m/z 372 and 377, in addition to the precursor ion at
m/z 337. Using the relationship (M + 18) for the ammonium adduct and (M + 23) for the
sodium adduct, the molecular weight of the metabolite was determined to be 354 Da. Since the
[M + H]+ ion for M4 would have to be located at m/z 355 (a peak not present on the spectrum),
the precursor ion was determined to be [M + H – H2O]+, 18 Da units less or the equivalent of the
loss of one water molecule. Table 6 outlines all of the metabolites detected via the precursor ion
scan method, while figure 14 shows the proposed structures of the six metabolites.
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Metabolite
1
2
3
MW
336
318
318
Table 6: Metabolite Detection via Precursor ion Scan
Molecular
LC retention time
Formula
(min)
precursor ion (m/z)
C20H29O3F
13.7
337
C20H27O2F
22.2
319
C20H27O2F
24.6
319
4
354
C20H31O4F
1.7a
5
350
C20H27O4F
4.0a
6
552
C20H29O4F
5.0a
a
retention times are approximations based on figure
337
351
353
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precursor ion
[M + H]+
[M + H]+
[M + H]+
[M + H H2O]+
[M + H]+
[M + H]+
Looking at the proposed structures for the fluoxymesterone metabolites, it is reasonable
that those with keto groups at C3, would undergo protonation at this site to yield the precursor
ion [M + H]+. This was the same behavior observed for the ‘model’ steroid compounds and for
the non-steroid doping agents. Metabolites 1 – 3 are the least polar and have LC peaks at higher
retention times (13.7, 22.2, and 24.6 minutes). Retention time plays an important role in the
ability to detect analytes. Due to potential interference from compounds within the sample
matrix, two analytes can elude at almost identical times, despite vastly different structural
characteristics. It is the analyst’s responsibility to separate the endogenic compounds (coming
from within) from the exogenic compounds (originating outside the body), which can all be
simultaneously present in a urine sample. The common ion method accurately identified six
fluoxymesterone metabolites, and appears to be a viable procedure to apply to metabolic studies.
Despite the inherent challenges in steroid detection, Pozo et al. managed to devise a
method that is able to not only identify a majority of known steroids and their metabolites, but
also can be used as a starting point to identify previously unknown anabolic steroids. Once a
potential steroid analyte is detected, additional examination is required to determine the actual
structure of the compound.
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Conclusion
Cabej et al. succeeded in constructing a straightforward, high-yield synthesis of
oxandrolone from methylandrostanolone starting material. While using the Searle procedure as a
starting point, they were able eliminate many of the initial issues associated with the oxandrolone
synthesis. They demonstrated an efficient method of bromination, while eliminating (toxic)
molecular bromine as the brominating agent. They also succeeded in eliminating the need for
purification via column chromatography at numerous stages in the synthesis, a technique not
suitable on the commercial scale. Finally by using an ozonolysis procedure, they were able to
again avoid using toxic oxidizing reagents osmium tetroxide (OsO4) and lead acetate
(Pb(OAc)4). With the growing need for large quantities of the anabolic steroid oxandrolone,
Cabej et al. were able to devise a safe and more efficient commercial-scale alternative to the
original Searle synthesis. Oxandrolone is currently being used to aid in weight gain for
individuals with weight-loss related illness9,10, and due to its anabolic properties, it is a tempting
ergogenic aid for many athletes looking for a competitive edge. Oxandrolone is just one of
numerous anabolic androgenic steroids currently on World Anti-Doping Agency (WADA) list of
prohibited substances, and with newly proposed commercial-grade processes being developed,
the ability for athletes to obtain these illicit substances becomes even greater11. To combat the
abuse of known anabolic steroids, as well as remain ahead of any ‘designer’ steroids, analytical
techniques must constantly be improved and adjusted in order for chemists and athletic
governing bodies to stay ahead of dishonest athletes.
In an effort better detect both known and unknown steroid compounds, Pozo et al.
devised a LC-MS/MS technique that allows steroid-like compounds to be readily identified. The
combined presence of methyl tropylium (m/z 105), tropylium (m/z 91), and phenyl (m/z 77) ion
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fragments were shown, to accurately identify steroids and steroid metabolites within a sample
matrix. Although this technique has limitations, overall, it is an excellent method for detecting
potential steroid components. The proposed technique helps to narrow down unknown analytes
of interest in a complex sample, which can then undergo additional separation and full MS
spectral analysis can be performed.
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