Detection of Designer Anabolic Steroids by LC‐MS/MS and Commercial Synthesis of Oxandrolone

29 Pages·2010·2.4 MB·English

by  Kristin Minkowski

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
of
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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Designer
Anabolic
Steroids
by
LC‐MS/MS
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Commercial
Synthesis
of
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 ESI-
LC-MS/MS, meaning the m/z is equivalent to the mass of the protonated molecule [M + H]+.

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Detection
of
Designer
Anabolic
Steroids
by
LC‐MS/MS
and
Commercial
Synthesis
of
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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Designer
Anabolic
Steroids
by
LC‐MS/MS
and
Commercial
Synthesis
of
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 wide-
variety 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
of
Designer
Anabolic
Steroids
by
LC‐MS/MS
and
Commercial
Synthesis
of
Oxandrolone
 11/18/10


Scheme 1: Counsell / Pappo sythesis of Oxandrolone (a) Br , NaOAc, HOAc, RT (b) LiCl, Li CO , DMF, (c)
2 2 3
OsO , Pb(OAc) , HOAc, H O, RT (d) NaBH , NaOH, RT adapted from Cabej et al.5 & Counsell et al.6
4 4 2 4
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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Designer
Anabolic
Steroids
by
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 (PyHBr ). While PTAB in the aprotic
3
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-1-
butanol 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
of
Designer
Anabolic
Steroids
by
LC‐MS/MS
and
Commercial
Synthesis
of
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.
Table 1: Bromination of methylandrostanolone with PTAB in different solvents
entry conditions percent yield (%) percent purity (%) comments
1 THF/0°C EtAOc extraction 75 92-94 HBr trap/dehydration
2 DME/0°C 64 not determined bromoketone insoluble
3 EtOAc/0°C 73 97 bromoketone insoluble
4 CH Cl /0°C not determined not determined dehydration
2 2
5 EtOH/RT 88 90 ketal formed
6 EtOH/8% H O/RT 84 89 rx time 5.5 h
2
7 EtOH/14% H O/RT 82 91 rx time 24 h (slow)
2
8 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, H 0, MeOH) and aprotic (THF,
2
DME, EtOAc, CH Cl) solvents. Figure 6 illustrates the structure of three of the polar aprotic
2
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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Detection
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Designer
Anabolic
Steroids
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LC‐MS/MS
and
Commercial
Synthesis
of
Oxandrolone
 11/18/10

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) PyHBR , ETOH,
3
H 0, Na S O 5H 0 (aq), Na CO (aq) (b) LiBr, Li CO , DMF, Acetic Acid, H 0, EtOAC, Heptane (c) O ,
2 2 2 3 2 2 3 2 3 2 3
MeOH, NaOH, HCl, MeOH/H O (d) NaOH, EtOH, H O, NaBH , HCl, Filter, MeOH, H O
2 2 4 2
Due to its lower cost and its solubility in ethanol, PyHBr was used as the brominating
3
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 PyHBr with methylandrostanolone in an ethanol/8% water solvent
3
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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