CARDIOPULMONARY/EXERCISE
Review of Anabolic Steroid Testing in a Racing
Environment
Keith H. Soring, DVM; and Walter G. Hyde, PhD, FAORC
Routine urine testing for common anabolic steroids can be accomplished in horse racing and can serve as a
deterrent for use or overuse of this class of substances. Authors’ addresses: Iowa Racing & Gaming
Commission, 717 East Court Avenue, Des Moines, IA 50309 (Soring); and Racing Chemistry, VDL-VDPAM,
Iowa State University Diagnostic Lab, Ames, IA 50011 (Hyde); e-mail: [email protected]. © 2008 AAEP.
1.
Introduction
Recent controversy surrounding the use of anabolic
steroids in human and animal athletes has led to
widespread investigations and a call for implementing more stringent rules, penalties, and widespread
testing procedures in an effort to curtail or eliminate
their use.1– 4
In the past, equine athletes have routinely been
given androgenic and anabolic steroids to promote
muscle development, appetite, aggressiveness, or
other desirable effects.5 Agents such as testosterone, nandrolone, and boldenone are endogenous to
the equine. Additionally, anabolic/androgenic steroids based on testosterone have been synthesized to
increase the anabolic pharmacology, such as protein
synthesis and accelerated muscle development.
These steroids also attempt to decrease the androgenic sexual and aggressive side effects.6
Europe, Asia, and many international regulatory
authorities have a ⬎30-yr history of recognizing this
class of steroids as agents of significant concern and
possible abuse in racing. The International Federation of Horseracing Authorities (IFHA) has long
recognized and adopted international thresholds for
endogenous versus exogenous steroid presence
NOTES
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2008 Ⲑ Vol. 54 Ⲑ AAEP PROCEEDINGS
based on the scientific work of several internationally recognized labs.7–12 These same thresholds
have been peer reviewed and tested legally many
times over the same time period.
Because of the possible integrity and welfare issues regarding the misuse or overuse of anabolic/
androgenic agents, the Iowa regulatory authority
established deterrence polices in 1991 to monitor for
the presence of exogenous levels of these agents in
racing horses. Until recently, Iowa has been the
only racing jurisdiction in the United States to implement an anabolic/androgenic steroid testing and
penalty policy. The Iowa regulatory agency has
used the internationally established urinary thresholds as the base for the determination of negative or
positive sample status.
In 2007, the Racing Medication and Testing Consortium in Lexington, Kentucky proposed national
model rules for testing and penalties of the four
common anabolic steroids. They used urinary
threshold concentrations very similar to the internationally accepted IFHA Article 6 levels. However, over the past 18 mo, these proposed policies
have faced opposition from some trainers, owners,
horse associations, veterinarians, and chemists,
CARDIOPULMONARY/EXERCISE
which has resulted in many jurisdictions’ reluctance
to start testing.
The purpose of this review is to show that testing
for anabolic steroids can be accomplished in the
horse racing world and that, at least in one jurisdiction, has proven to be effective in curtailing the use
of these products.
2.
Materials and Methods
Selection of Test Subjects
All first- and second-place finishers plus other random finishers selected by the stewards or state veterinarian were sent to the detention barn for urine
and blood collection after each racing performance
at Prairie Meadows Racetrack located in Altoona,
Iowa. The samples were identified by bar code and
sex, and they were sent by courier in sealed containers to the Racing Chemistry Lab located at Iowa
State University after each performance. The subjects tested were racing Thoroughbreds, Quarter
Horses, and Standardbreds.
Testing Methods
Limits of detection were based on the IFHA Article 6
recommendations.
Boldenone: 0.015 ␮g free and conjugated boldenone/ml in urine from male horses (other than
geldings).
Estranediol: the mass of free and conjugated 5alpha-estrane-3beta, 17alpha-diol in male horses
to the mass of free and conjugated 5 10-estrene-3 beta, 17 alpha (non-gelding) diol in
urine from male horses (other than geldings) at
a ratio of 1.
Testosterone: 0.02 ␮g free and conjugated testosterone/ml in urine from geldings or 0.055 ␮g
free and conjugated testosterone/ml in urine
from fillies and mares (unless in foal).
Drug testing programs routinely use screening tests
to determine which samples submitted by the regulatory authority are suspicious; if this happens, all
other samples are declared negative, and further
work is stopped. The suspicious samples undergo
additional investigation using more rigorous analysis to meet legal requirements for forensic evidence.
Anabolic/androgenic steroid screening has been
successfully conducted using immunoassay enzymelinked immunosorbent assay (ELISA) methods.5,13
This testing is based on kits that use antibody-antigen specific reactions to detect and estimate concentrations of analyte in the biological sample. Two
commercially available test kits are routinely used
in the screen-testing detection for nandrolone, boldenone, and testosterone in equine urine and blood
serum or plasma: the Boldenone ELISA kita and
the Nandrolone ELISA kit.a Limits of detection at
⬍1 ␮g/ml for these anabolic steroids. ELISA
screening allows a relatively inexpensive and rapid
Fig. 1.
Immunoassay nandrolone calibration curve data.
screening means of differentiating samples that may
be positive from samples that are negative and need
no further investigation. One disadvantage of such
ELISA testing is that the antibody-antigen reaction
is not perfectly specific, meaning that the Boldenone
ELISA will react with and therefore “hit” on samples
containing nandrolone and testosterone also. Similarly, the Nandrolone ELISA kit will detect samples
containing boldenone and testosterone. Therefore,
the estimated concentration of these agents in the
sample will add up and cannot be individually differentiated. Using pure nandrolone to create authentic calibration response samples, Figs. 1 and 2
illustrate the sensitive nature of immunoassay
ELISA screening capabilities.
Note that the response curve is a reverse correlation polynomial fit. This means that the lower the
analyte concentration, shown here on the x axis, the
higher the kit reading will be, shown here on the y
axis. Kit testing limit of sensitivity is ⬃150 –200
pg/ml, but this is dependent on sample non-specific
background that can contribute to the reading.
Forensic confirmation is accomplished using analyte
extracted from the urine sample followed by gas chromatography/mass spectrometry of the extract by one of two
distinct processes, either the Hydroxylamine-HCl (in
pyridine)-(N-methyl-N(t-butyldiemethylsiliyl trifouroacetamide) (OX-TBDMS) process or the MethoxyamineHCl solution (in pyridine)-(N-methyl-N-(trimethylsiliyl
trifluoroacetamide) (MOX-TMS) process.
Extraction involves enzymatic hydrolysis and/or
solvolysis of the urine to convert all anabolic agents
in the sample from conjugated metabolites to the
free agents. These are then extracted from the
mixture using solid-phase extraction (SPE) with
C18 Sep Pak Waters SPE extraction cartridges.b
This yields a concentrated and cleaner extract ready
for mass-spectral instrumental analysis.
Figure 3 summarizes the extraction process.
Derivatization
The OX-TBDMS gas chromatography/mass spectrometric process involves forming an oxime-silylated
AAEP PROCEEDINGS Ⲑ Vol. 54 Ⲑ 2008
39
CARDIOPULMONARY/EXERCISE
Fig. 2.
Immunoassay nandrolone calibration curve.
derivative of the anabolic steroids in the sample
with hydroxyamine hydrochloride and N-methyl-Nt-butyldimethylsiliyl
trifluoroacetamide. These
OX-TBDMS derivatives of the anabolic steroids in
the sample yield very sensitive detection limits for
the various individual anabolic steroids down to 1
␮g/ml and less and also allows monitoring of other
naturally occurring hormones and related compounds of interest. Figures 4 and 5 illustrate the
sensitivity of gas chromatography/mass spectrometry for the forensic detection and identification of
Nandrolone in urine as the OX-TBDMS derivative.
Nandrolone in Equine Urine by Gas Chromatography/Mass
Spectrometry (GCMS)
The MOX-TMS gas chromatography/mass spectrometric process involves forming an oxime-silylated
derivative of the anabolic steroids in the sample
with methoxyamine hydrochloride and N-methyl-Ntrimethylsiliyl trifluoroacetamide. These MOXTMS derivatives of the anabolic steroids in the
sample yield slightly less sensitive detection limits
than does the OX-TBDMS process, but the mass
spectral data are much more specific and allow
much more confident identification of the various
anabolic steroids for legal purposes.
3.
Results
In 1991, the Iowa commission instituted an anabolic
steroid deterrence policy, and the stated intent was
to give 3-mo notice of the upcoming anabolic steroid
policy. No withdrawal times were recommended before implementing this policy (Tables 1 and 2; Fig. 6).
40
2008 Ⲑ Vol. 54 Ⲑ AAEP PROCEEDINGS
4.
Discussion
The data presented shows a long-standing and effective monitoring and deterrence policy based on
well-established international standards. This is
especially apparent compared with recently collected data from two other U.S. jurisdictions sampled using these same screening and confirmatory
methods where no deterrence policy has been
adopted.
Table 1 reveals a static number of positive tests
over the 16-yr period. Certain year spikes could be
attributed to an influx of new veterinarians and
trainers who perhaps tested the recommended withdrawal times. The testing procedures were explained to all veterinarians and trainers in preseason meetings. The penalties issued were as
follows:
First Offense ⫽ $300 fine to trainer
Second Offense ⫽ $500 fine to trainer
Third Offense ⫽ $1000 fine to trainer/loss of
purse/suspension; only one trainer ever met the
conditions of the third offense.
It was realized by the Iowa Racing and Gaming
Commission and Board of Stewards that by
adopting the above listed penalties, horses with
uncertain medication status from other jurisdictions could still race at Prairie Meadows without fear of purse loss or suspension. Although
perhaps giving an advantage to those individuals that were only shipping in for select races,
there were relatively few positive results from
those horses. Many out-of-state trainers con-
CARDIOPULMONARY/EXERCISE
Anabolic Steroid Analysis in Equine Urine
Dispense blank, sample(s), or spike(s) urine
Adjust each to pH 6.8 with 1M phosphate buffer
:
:
Enzymatic Hydrolysis
_-Glucuronidase (E. coli) in Phosphate buffer pH 6.8
Hydrolyze overnight at 37°C or for 2 hours at 50°C overnight
:
:
Add D-3 androstanediol (internal standard)
:
:
Steroid Extraction using Solid Phase Extraction
C18 Sep Pak cartridges
Add sample and:
-wash with hexane
-dry thoroughly
:
:
:
:
Elute the Glucuronide fraction
Elute the SO4 fraction
with diethyl ether.
With EtAc : MeOH : H 2SO4
Solvolyze at 37°C or
for 2 hours at 50°C overnight
:
:
:
Add 2M NaOH and mix
Discard the aqueous phase
Reserve the Organic fraction
Repeat
:
Add sat’d NaCl and mix
Discard the aqueous phase
Reserve the organic fraction
:
Dry over anhydrous Na2SO4
Conc. to dryness with OFN and heat
Ready for derivatization to either OX-TBDSM or the MOX-TMS
Derivatization
The OX-TBDMS gas chromatography/mass spectrometric process involves forming an
oxime-silylated derivative of the anabolic steroids in the sample with hydroxyamine
Fig. 3.
Anabolic standard analysis method outline.
tacted the state veterinarians to inquire about
withdrawal times before shipping in to race. It
was also noted that the penalties for a positive
test should be set in a realistic manner because
conc
D3
epinandrol nandrolone nand/D3 nand
258
460
460
ng/ml
gluc-tbdms 12.58 13.35,13.68 14.48,14.77
SPK 1
761169
535747
0.703848 100
SPK 1a
758408
536419
0.707296 100
SPK 2
618844
939616
1.518341 200
SPK 2a
604715
952889
1.575765 200
Fig. 4. Gas chromatography/mass spectrometry nandrolone in
vitro spiked calibration in urine data.
it was the only jurisdiction in the United States
that was currently testing for anabolic steroids.
Although no withdrawal time studies were ever
conducted, “trial and error” by the practicing
veterinarians determined the withdrawal for
the smallest one-time administration of boldenone to be ⬃17 days, for nandrolone and testosterone to be ⬃14 days in geldings and 21
days for fillies, and for stanozolol to be ⬃14 days
(all dose-dependent estimates). Multiple doses
or higher concentrations could likely increase or
double all withdrawal times and would reflect
Racing Medication and Testing Consortium
(RMTC) current recommendations. It is felt by
the authors that the threshold and penalty
phase set in place in Iowa has effectively curAAEP PROCEEDINGS Ⲑ Vol. 54 Ⲑ 2008
41
Ratio
CARDIOPULMONARY/EXERCISE
Fig. 5.
1.8
y = 0.0078x - 0.0388
1.6
R2 = 0.996
1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2 0
50
100
Nandrolone/D3
Linear
(Nandrolone/D3)
150
ng/ml
200
250
Gas chromatography/mass spectrometry nandolone in vitro spiked calibration in urine curve.
tailed the overuse of anabolic steroids; additionally, it still allows some room for legitimate use
by practicing veterinarians.
Over the past 16 yr, the Iowa Racing and Gaming
Commission policy has proven an effective deterrent to anabolic steroid abuse by testing
equine urine samples using international practices and standards of testing.
There has been a suggestion in the United States
that anabolic steroid testing should or must be
done in blood. Our experiences illustrate that
urine-based testing for the anabolic steroids
pursuant to international practice has resulted
in effective deterrent compared with jurisdictions that have no testing or deterrence policy in
place. Certainly, anabolic steroids can be
tested in blood as well. Although both urine
and blood offer analytical and policy advantages
and disadvantages, these advantages and disadvantages should be considered when a jurisdiction is deciding when and how to adopt
testing and deterrent policies for anabolic ste-
Table 1. Test Results
Year
Urines
Tested
Nandrolone
Testosterone
Boldenone
Stanozolol
Total
1991
1992
1993
1994
1995
1996
1997
1998
1999*
2000
2001
2002
2003
2004
2005
2006†
2007
Total
2,317
No Racing
1,067
1,062
1,059
1,058
1,652
1,856
2,256
2,542
2,035
2,029
1,964
2,047
1,808
1,828
1,833
28,413
0
No racing
0
0
0
0
0
0
0
0
1
0
0
0
1
0
1
3
7
No racing
0
0
0
1
0
1
10
7
0
0
1
3
3
8
2
43
2
No racing
0
0
0
0
0
7
8
4
1
0
0
0
2
4
0
28
0
No racing
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
9
No racing
0
0
0
1
0
8
18
11
2
0
1
3
6
13
5
77
*One trainer with four testosterone positives.
†Feed additive attributed to five testosterone positives.
Table 2.
Jurisdiction
Agency A
3 mo
Agency B
1.5 mo
42
Urines
Tested
Nandrolone
Testosterone
Boldenone
Other
Total
925
23
44
116
3
186 (20%)
350
0
3
20
0
23 (7%)
2008 Ⲑ Vol. 54 Ⲑ AAEP PROCEEDINGS
CARDIOPULMONARY/EXERCISE
Anabolic Steroid Positive Tests
20
15
10
5
Fig. 6.
2007
2006
2005
2004
2003
2002
2001
2000
1999
1998
1997
1996
1995
1994
1993
1992
1991
0
Total number of steroid positives per year.
roids. It should be noted, however, that no international thresholds, no national thresholds,
and no agency-approved and peer-reviewed
thresholds currently exist for blood-serum testing at the time of this review.
The authors wish to acknowledge and thank the
staff of the Iowa Racing and Gaming Commission
veterinary team and the analysts and staff in the
Iowa State Racing Chemistry Laboratory for the
many years of work that has yielded the basis for
this presentation. Also a huge thanks to the HFL,
LTD (formerly the Horseracing Forensic Laboratory, Newmarket, United Kingdom), Dr. Edward
Houghton, Dr. Minoo Dumasia, and the HFL research group for the many years of collaboration in
this area of testing.
References and Footnotes
1. National Thoroughbred Racing Association. NTRA CEO
Alex Waldrop’s testimony on drugs in sports. NTRA Available online at http://www.NTRA.com. 2008;2/27.
2. Brown K. The steroid debate. The Horse Available online
at http://www.TheHorse.com. 2008;5/1.
3. Finley B. Horse racing officials move toward steroid ban.
The New York Times 2007;2/28.
4. Bahrke M, Yesalis C. Abuse of anabolic androgenic steroids
and related substances in sport and exercise. Curr Opin
Pharmacol 2004;6:614 – 620.
5. Snow D. Anabolic steroids. Vet Clin North Am [Equine
Pract] 1993;9:563–576.
6. Tobin T. The anabolic steroid androgen group of drugs.
J Equine Med Surg 1978;2:163–165.
7. International Federation of Horseracing Authorities. International agreement on breeding, racing and wagering. Article 6.
Prohibited substances—thresholds. Available online at http://
www.ifhaonline.org. Accessed on Feb 10, 2008.
8. Houghton E, Dumasia M. Studies related to the metabolism
of anabolic steroids in the horse: testosterone. Xenobiotica
1979;9:269 –279.
9. Dumasia M, Houghton E. Development of a gas chromatographic—mass spectrometric method using multiple analytes
for the confirmatory analysis of anabolic steroids in horse
urine. J Chromatogr Journal version A 1986;377:23–33.
10. Teale P, Houghton E. The development of a gc-ms-screening
procedure for endogenous and exogenous steroids in the
horse urine, in Proceedings. 7th International Conference of
Racing Analysts and Veterinarians 1988;25– 43.
11. Teale P, Houghton E. The development of a gas chromatographic/mass spectrometric screening procedure to detect the
administration of anabolic steroids to the horse. Biological
Mass Spectrometry 1991;20:109 –114.
12. Houghton E. Steroid analysis in the horse, in Proceedings.
Pari-Mutual Anabolic Steroid Workshop 1991.
13. Hale N, Tobin T, Tai HH, et al. Elisa assay for stanozolol, in
Proceedings. 13th International Conference of Racing Analysts and Veterinarians 2000;341–344.
a
Neogen Corporation, 620 Lesher Place, Lansing MI 48912.
Waters Corporation, 34 Maple St., Milford, MA 01757.
b
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