DETECTION OF BOLDENONE, METHANDRIOL AND TESTOSTERONE IN
EQUINE URINE BY GC-MS
F. I. Keskin, E. Turhan, N. Yazicioglu and S. Kaya*
Central Veterinary Control and Research Institute Doping Laboratory. Ankara, Turkey.
*Ankara University Veterinary Faculty, Pharmacology-Toxicology Department. Ankara,
Turkey.
ABSTRACT
Anabolic androgenic steroids are synthetic derivatives of testosterone, which is the primary
male sex hormone, or a related compound that is derived from (or similar in structure and
action to) this hormone. They are an important class of performance enhancing drug in horse
racing and other sports. That’s why using of them in racehorses is prohibited in many racing
jurisdictions around the world.
This paper describes an analytical method developed for the detection of anabolic
androgenic steroids boldenone, methandriol and testosterone in equine urine. The equine urine
fortified with the steroid standards. The steroids were extracted from equine urine with
dichloromethane at pH 6.8. The extracts were analysed by Agilent 6890N-5973 Gas
Chromatography coupled to Mass Spectrometry (GC-MS) using 12 m L x 0.20 mm ID, 0.33
µm particle size HP 1 column. The method was validated for methandriol. Extraction
recovery, limit of detection and limit of quantification were 90%, 3.54 ng/ml and 11.8 ng/ml,
respectively.
This results point out that the method’s recovery is extremely high, reproducible and
sensitive.
INTRODUCTION
Traditional attempts to increase sports performance have been done by using
pharmacologically active substances external to the body (Kintz et al. 2001). The use of
performance enhancing drugs is unethical and against all principles of fair competition
(MacAuley 1996; Holt et al. 2009).
Anabolic androgenic steroids are synthetic derivatives of testosterone with related
structure and activity (Lenehan 2004; Ho et al. 2007b). They are an important class of
performance enhancing drug with potential for misuse in horse racing and other sports
because of their capability to increase muscle mass and strength, and to promote
aggressiveness (Ho et al. 2005; Hungerford et al. 2005). The use of them is prohibited by the
International Olympic Committee via its anti-doping arm, the World Anti-Doping Agency, in
human sports (Guan et al. 2005). The International Olympic Committee has banned the use of
anabolic steroids since 1976 (Yu et al. 2005). They are also prohibited substances according
to the Article 6 of the International Agreement on Breeding, Racing and Wagering by the
International Federation of Horseracing Authorities and rules of Federation Equestre
Internationale in the horseracing industry as well as in food producing animals in the
European Union (Guan et al. 2005; Ho et al. 2007a; Roig et al. 2007). As a result, the integrity
of the horse racing industry relies on stringent doping control measures targeting these agents
(Hungerford et al. 2005).
Anabolic steroids have been used for a variety of therapeutic and non-therapeutic
purposes (Lenehan 2004). They are used in treating delayed puberty, select impotence, and
wasting of the body caused by some diseases (Guan et al. 2005). These agents promote the
growth of skeletal muscle, take nitrogen in the albuminous tissues, accelerate the synthesis of
albumin and decrease the breaking up of amino acids and proteins (anabolic effects) and the
development of male sexual characteristics (androgenic effects), and also have some other
effects (Kaya and Bilgili 2002; Kaya 2007; Parker and Parker 2002).
Testosterone (17-hydroxyandrost-4-en-3-one) is the primary male sex hormone and the
main anabolic androgen (Bahrke and Yesalis 2004; Dehennin et al. 2007). The first recorded
case of the use of testosterone as a means of improving performance was in 1941 in an 18year-old horse named Holloway, who won many races with improved performance following
treatment (Holt et al. 2009).
Boldenone (1,4-androstadien-17-ol-3-one or 1,2-dehydrotestosterone) is an oil based
common anabolic steroid used in veterinary practice. Its structure is highly similar to
testosterone with dehydrogenation at the C-1,2 position. It is highly anabolic and moderately
androgenic (Ho et al. 2004; Lenehan 2004).
Methandriol (17α-methylandrost-5-ene-3,17-diol) is 5-androstenediol that has had its
chemical structure modified by adding a methyl group. It is slightly anabolic and androgenic
(Steroid 2009).
This study describes a simple extraction procedure for boldenone, methandriol and
testosterone using enzyme hydrolysis and liquid-liquid extraction and detection by GC-MS.
MATERIALS AND METHODS
Chemicals
The reference standards boldenone was obtained from Sigma (USA), methandriol was
obtained from Steraloids Inc. (Newport, USA) and testosterone was obtained from Riedel-de
Haen (Germany). .glucuronidase helix pomadia was obtained from Merck (Darmstadt,
Germany, 100.000 IU/ml). N-Methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) was
obtained from Merck (Darmstadt, Germany). Extract clean 8 ml filter was obtained from
Alltech (Illinois, USA). Hydrochloric acid was obtained from Carlo Erba (Rodano). Sodium
hydroxide was obtained from Riedel-de Haen (Sigma-Aldrich, Seelze). Dichloromethane was
obtained from Riedel-de Haen (Sigma-Aldrich, Seelze) and ethyl acetate was obtained from
Scharlau Chemie S. A. (Barcelona, Spain). All solvents were of analytical grade. Milli-Q
water was used were required.
Extraction procedure
Urine (5 ml) was spiked with boldenone, methandriol and testosterone standards at a final
concentration of 200 ng/ml. It’s pH was adjusted to 6.8 with 1 N HCl and/or 1 M NaOH,
hydrolysed with .glucuronidase helix pomadia (50 µl) and incubated at 55 ºC for 2 h. After
cooling to room temperature, the enzyme treated urine was extracted with dichloromethane (4
ml) and centrifuged (3000 rpm, 5 min). NaOH (3 ml, 0.5 M) was added on the organic layer
for clean-up. After centrifugation (3000 rpm, 5 min) the organic layer was filtered from
extract clean filter and transferred to a clean tube. The eluate was evaporated to dryness under
a stream of nitrogen at 50 ºC, the residue was dissolved in ethyl acetate (100 µl). The dried
residue of methandriol analysed by GC-MS. Boldenone and testosterone spiked urine samples
were derivatizated with MSTFA (100 µl) at 80 ºC for 30 min and analysed by GC-MS.
Instrumentation
GC/EI/MS analysis was performed on an Agilent 6890 GC-5973 MSD equipped with a 7683
autoinjector. Separations were performed on 12 m L x 0.20 mm ID, 0.33 µm particle size HP
1 column with helium as the carrier gas at a constant flow rate of 0.6 ml/min. The oven
temperature was held initially at 90 ºC for 1 min, then increased to 320 ºC at 12 ºC/min with a
final hold time of 4 min. Injections of 2 µl sample were made in splitless mode with an
injector temperature of 250 ºC. Run time of the analyse was 24.17 min. Qualitative data were
collected with full scan mode from m/z 50-550.
Method validation
The method validation was performed on an Agilent 6890 GC-5973 MSD. Based on GC-MS
method data the following parameters were evaluated in the validation study: selectivity,
specificity, sensitivity, limit of detection (LOD), limit of quantification (LOQ), extraction
recovery, precision and linearity in accordance with ISO EN 17025:2005 guidelines.
Blank and spiked equine urine samples were extracted and analysed by the procedures
outlined above. Different equine blank urine samples (n=6) were analysed for selectivity and
specificity. The presence of any interfering substance at the retention time methandriol
standard was verified.
The sensitivity of the method was evaluated by spiking different amounts of methandriol
standards (50, 100, 200 ng/ml) to equine blank urine sample.
Quality control samples were prepared by spiking 50 ng/ml methandriol standard to
equine blank urine sample and injected to GC-MS 20 times.
For the validation, 4 point calibration curves (50, 100, 200, 500 ng/ml) were prepared for
methandriol with ethyl acetate standard solutions.
Equine blank urine sample was spiked with 50 ng/ml methandriol standard and injected to
GC-MS 20 times in two different days, one day injected 10 times and the other day injected
10 times. The standard deviation of the extraction recovery was calculated. LOD and LOQ
were defined as 3 and 10 times the value of standard deviation, respectively.
To determine the extraction recovery, equine blank urine samples (n=6) spiked with
methandriol standard (50, 100, 200 ng/ml), analysed by the procedures outlined above and
injected to GC-MS 6 times. Recovery was measured by comparing the peak areas with
calibration curves.
Precision was expressed as the relative standard deviation (RSD) of the methandriol
standard spiked (50, 100 and 200 ng/ml) equine blank urine samples.
RESULTS
In this study, the results obtained from spiked urine samples. The retention times and mass
spectrums of boldenone, methandriol and testosterone were determined by SCAN mode and
analysed by SIM mode. The retention times and monitored ions are shown in Table 1.
Extracted ion mass chromatograms of boldenone, methandriol and testosterone and extracted
ion mass chromatograms and ion chromatograms of boldenone, methandriol and testosterone
are shown separately in Figure 1, 2, 3 and 4, respectively.
The selectivity and specificity were evaluated after analysis of 6 different equine blank
urine samples. The developed method is selective and concluded satisfactory, because no
matrix interferences were detected at the targeted ion masses and retention time of
methandriol.
Good linearity of the calibration curve was observed. For methandriol, calibration curve
was linear from 50 to 500 ng/ml, response linearity was found adapted to the needs
(R2=0.998). Calibration curve of methandriol is shown in Figure 5.
In the present study, LOD and LOQ of methandriol were 3.54 and 11.8 ng/ml,
respectively.
The validated method gave extraction recovery of %90, which was considered enough for
qualitative testing of methandriol misuse in horse.
Precision was %0.269, %0.94 and %1.01 for 50, 100 and 200 ng/ml, respectively.
In summary, these validation results showed that the developed method is fit, reproducible
and reliable for detection of methandriol abuse in equine urine by GC-MS.
DISCUSSION
GC-MS is an important analytic technique in doping control of anabolic steroids. Several
extraction procedures for detection of anabolic steroids in equine urine by GC-MS have been
reported (Dumansia et al. 1986; Singh et al. 1989; Houghton et al. 1990; Ho et al. 2004). No
reports of the detection of methandriol in equine urine by GC-MS have been detected.
Liquid-liquid extraction procedure at pH 6.8 using dichloromethane was chosen as the
most suitable method for the detection of analytes of interest in equine urine with satisfactory
results. The developed method is simple, rapid, effective.
CONCLUSION
At present, for screening and confirmation of anabolic steroids, GC-MS is the effective
technique commonly used for the analysis of urine samples by anti-doping or analytical
toxicology laboratories.
In this study simple, sensitive and reproducible method for determination of boldenone,
methandriol and testosterone in equine urine have been developed and validated for
methandriol according to the ISO EN 17025:2005 by using GC-MS. The method was
validated for selectivity, specificity, sensitivity, LOD, LOQ, extraction recovery, precision
and linearity. Validation of this quantitative method is presented. The method has proven to
be suitable for the quantitative determination of methandriol.
ACKNOWLEDGEMENTS
We would like to thank to Etlik Central Veterinary Control and Research Institute for their
support.
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c
e
4
8
0
0
0
4
6
0
0
0
4
4
0
0
0
4
2
0
0
0
4
0
0
0
0
3
8
0
0
0
3
6
0
0
0
3
4
0
0
0
3
2
0
0
0
3
0
0
0
0
2
8
0
0
0
2
6
0
0
0
2
4
0
0
0
2
2
0
0
0
2
0
0
0
0
1
8
0
0
0
1
6
0
0
0
1
4
0
0
0
1
2
0
0
0
1
0
0
0
0
8
0
0
0
6
0
0
0
4
0
0
0
2
0
0
0
1
I o
I o
I o
n
n
n
2
2
3
M
7
5
0
1
3
4
. 0
. 0
. 0
E
T
H
0
0
0
A
( 2
( 2
( 3
N
D
7
5
0
0
2
3
R
1
4
. 4
. 7
. 7
. 7
I O
4
0
0
0
t o
t o
t o
2
2
3
7
5
0
1
3
4
. 7
. 7
. 7
0
0
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) :
) :
) :
6
6
6
0
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1
1
1
0 0 5 . D
01 05 5. 2. D6
0 0 5 . D
L
. 9
5
1
4
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5
1
4
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1
5
1
5
. 2
1
5
7
. 3
1
0
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5
. 4
4
5
. 4
9
3
1
5
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8
0
1
T
im
e
- - >
4
. 5
0
1
4
. 6
0
1
4
. 7
0
1
4
. 8
0
1
4
. 9
0
. 0
0
1
5
. 1
0
1
5
. 2
0
1
5
. 3
0
1
5
. 4
0
1
5
. 5
0
b)
A b u n d a n c e
S c a n
1 9 0 1
( 1 4 . 9 5 3 m in ) :
2 5 3
6 0 0 1 0 0 5 .D
1 3 0 0 0
1 2 0 0 0
1 1 0 0 0
2 1 3
1 0 0 0 0
9 0 0 0
8 0 0 0
9 1
2 7 1
7 0 0 0
6 0 0 0
1 4 5
5 5
5 0 0 0
3 0 4
1 1 9
4 0 0 0
2 3 1
1 7 3
3 0 0 0
2 0 0 0
1 0 0 0
7 2
1 9 5
3 2 4
3 4 6
4 4 6
0
4 0
6 0
8 0
1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0
m / z -->
Fig 3: Extracted ion mass chromatogram (a) and ion chromatogram (b) of methandriol.
a)
A
b u n d a n c e
I o n
I o n
I o n
1 3 0 0 0 0
1 2 4 . 0 0
2 8 8 . 0 0
2 4 6 . 0 0
(1 2 3 . 7 0
(2 8 7 . 7 0
(2 4 5 . 7 0
t o
t o
t o
1 2 4 . 7 0 ):
2 8 8 . 7 0 ):
2 4 6 . 7 0 ):
6 0 0 1 0 0 5 . D
6 0 0 1 0 0 5 . D
6 0 0 1 0 0 5 . D
1 2 0 0 0 0
T
E
S
T
O
S
T
E
R
O
N
E
1 1 0 0 0 0
1 5 . 4 9
1 0 0 0 0 0
9 0 0 0 0
1 5 . 4 9
8 0 0 0 0
7 0 0 0 0
1 5 . 4 9
6 0 0 0 0
5 0 0 0 0
4 0 0 0 0
3 0 0 0 0
2 0 0 0 0
1 0 0 0 0
T
im
11 55 . . 22 68
1 4 . 3 7
1 31 . 138 3.19. 90 6
0
1 3 . 5 0
1 4 . 0 0
1 15 5. 6. 76 1
1 15 5. 6. 75 0
1 14 4. 9. 91 5
1 4 . 5 0
1 5 . 0 0
1 5 . 5 0
1 6 . 2 0
1 6 . 0 0
1 6 . 5 0
1 7 . 0 0
1 7 . 5 0
e -->
b)
A
b
u
n
d
a
n
c
e
1
0
0
0
0
0
9
5
0
0
0
9
0
0
0
0
8
5
0
0
0
8
0
0
0
0
7
5
0
0
0
7
0
0
0
0
6
5
0
0
0
6
0
0
0
0
5
5
0
0
0
5
0
0
0
0
4
5
0
0
0
4
0
0
0
0
3
5
0
0
0
3
0
0
0
0
2
5
0
0
0
2
0
0
0
0
1
5
0
0
0
1
0
0
0
0
5
0
0
0
S
1
2
c
a
n
1
9
9
5
( 1
5
. 4
9
1
2
9
i n
) :
4
0
0
1
2
8
8
0
0
5
3
0
6
0
0
. D
4
6
7
2
5
1
0
0
3
7
2
2
8
2
1
1
7
6
1
1
5
m
4
6
8
7
0
5
5
3
3
0
4
m
/
z
- - >
0
6
0
8
0
1
0
0
1
2
0
1
4
0
1
6
0
1
8
0
2
0
0
2
2
0
2
4
0
2
6
0
2
8
0
3
3
2
3
0
3
0
3
4
0
5
3
5
6
4
0
3
8
0
4
0
0
4
2
2
0
9
c)
A
b u n d a n c
e
I
I
I
I
I
I
3 2 0 0 0 0
3 0 0 0 0 0
2 8 0 0 0 0
o
o
o
o
o
o
n
n
n
n
n
n
1
3
2
4
2
4
2
6
7
3
0
1
9
0
0
2
9
7
. 0
. 0
. 0
. 10
. 0
. 0
0
( 1
0
( 3
0
( 2
50 . 8( 47
0
( 2
0
( 4
2
5
6
3
0
1
8
9
9
1
8
6
1 5 . 8 7
T E S T
O
.
.
.
.
.
.
7
7
7
7
7
7
0
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0
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0
0
t
t
t
t
t
t
o
o
o
o
o
o
1
3
2
4
2
4
2
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.
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7
7
7
7
7
7
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)
)
)
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)
:
:
:
:
:
:
6
6
6
6
6
6
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
.
.
.
.
.
.
D
D
D
D
D
D
2 6 0 0 0 0
2 4 0 0 0 0
2 2 0 0 0 0
S
T
E
R
O
N
T
M
S
2 0 0 0 0 0
1 8 0 0 0 0
1 6 0 0 0 0
1 5 . 8 7
1 4 0 0 0 0
1 6 . 0 7
1 2 0 0 0 0
1 6 . 0 1
1 0 0 0 0 0
1 5 . 6 7
8 0 0 0 0
6 0 0 0 0
1 5 . 6 6
1 5 . 9 2
4 0 0 0 0
1 5 . 9 1
1 5 . 8 7
1 5 . 81 9511. 595 .2.99 33
2 0 0 0 0
1 5 . 6 7 1 5 .17 50 . 7 2 1 5 . 17 56 . 7 8
1 5 . 6 5
0
1 5 . 6 5
1 5 . 7 0
1 5 . 7 5
1 5 . 8 0
T
im
1 5 . 8 5
1 5 . 9 0
1 6 . 1 0
1 6 . 0 7
1 6
6 .1
.0
067
7. 0 9
1
6 .0
. 00 11
1 6 11 . 6
0
1 5 . 9 9
1 5 . 9 5
1 6 . 0 0
1 6 . 0 5
1 6 . 1 0
1 6 . 1 9
1 6 1. 116 68. 1. 29 0
1 6 . 1 5
1 6 . 2 0
e - - >
d)
A b u n d a n c e
S c a n
3 2 0 0 0 0
2 0 6 0
(1 5 . 8 6 3
m
in ) :
6 0 0 1 0 0 1 . D
7 3
3 0 0 0 0 0
1 2 9
2 8 0 0 0 0
2 6 0 0 0 0
2 4 0 0 0 0
3 6 0
2 2 0 0 0 0
2 0 0 0 0 0
1 8 0 0 0 0
1 6 0 0 0 0
1 4 0 0 0 0
2 7 0
1 2 0 0 0 0
1 0 0 0 0 0
2 2 6
8 0 0 0 0
1 0 5
6 0 0 0 0
3 0 4
1 8 5
4 0 0 0 0
1 5 9
2 0 0 0 0
3 3 1
2 4 7
5 1
3 8 7
0
5 0
m
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 1 5 4 3 84 5 94 8 0
4 0 0
4 5 0
5 0 6
5 3 5
5 0 0
/ z -->
Fig 4: Extracted ion mass chromatogram (a) and ion chromatogram (b) of testosterone; extracted ion
mass chromatogram (c) and ion chromatogram (d) of testosterone TMS derivative.
Response
R2=0.998
4 x 105---
3.5 x 105-
3 x 105---
2.5 x 105-
2 x 105---
1.5 x 105-
1 x 105---
0.5 x 105-
0
50
100
Fig 5: Calibration curve of methandriol.
200
300
400
500
Amount (ng/ml)
TABLE 1: Retention times and monitored ions of boldenone, methandriol and testosterone
Compound
Retention Time
Main Ion and Product Ions
Boldenone
15.70
286, 122, 147
Boldenone TMS derivative
16.07
358, 122, 147
Methandriol
14.95
304, 271, 253
Testosterone
15.49
288, 124, 147
Testosterone TMS derivative
15.86
360, 129, 270