GROWTH – DEDICATED CALL – 10/00
TOPIC IV.16
Determination of the origin of hormones in cattle
1. CONFORMITY WITH THE WORK PROGRAMME
This topic falls under the Competitive and Sustainable Growth Programme, generic
activity Measurement and Testing. Specifically, it is related to Objective GROW-20006.2.2 Measurement and Testing anti-fraud methodologies for which expressions of
interest have been called.
2. KEYWORDS
Anti-fraud-regulations, anabolic steroids, cattle, GC-C-IRMS, abuse, growth promoters
3. SUMMARY OF OBJECTIVES AND JUSTIFICATION
The fight against abuse of anabolic substances for growth promotion in cattle is of main
importance in the EU, but the illegal administration of endogeneous hormones is
difficult to control. The established methods are based on an indirect detection of
administration e.g. on calculation of a statistically signi-ficant decision limit above
which a natural occurence is highly improbable. A new approach is the use of Gas
Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS). This
method is based on the premise that the stable isotope composition of the natural
endogeneous hormones is di-stinct from the synthetically produced ones. The
administration of endogeneous hormones to animals affects the isotopic abundance and
offers thereby the advantage of a direct proof of illegal used anabo-lic substances in
livestock farming. The possibility to detect the abuse of endogeneous hormones for
growth promotion helps to ensure the quality of food for human consumption.
4. BACKGROUND
The use of the steroid hormones to enhance growth in animal production is common
practice for more than 30 years. According to their chemical structure a classification
can be made between endogeneous steroids (e.g. testosterone, progesterone, estradiol17), exogenous steroids (e.g. 19-nortesto-sterone) and non-steroidal extraneous
(diethylstilbestrol) compounds. These substances are particularly effective in ruminants,
especially in cattle fattening.
Hormones, including those used to promote growth in livestock farming, are known to
be related to cancer in laboratory animals and humans. In addition it is assumed that
hormones may magnify the effects of known carcinogens.
Because residues of these substances may be dangerous to the consumers and may also
affect the quality of foodstuffs of animal origin, the use of all substances having a
hormonal action for fattening purposes is banned in the EU since 1988 (88/146/EEC
DC 10/00/Topic IV.16/ Pg 2
replaced by 96/22/EC). In contrast, in the United States the FDA has approved the three
naturally occuring hormones, progesterone, testosterone and estradiol-17 and the three
synthetic hormones trenbolone acetate, zeranol and melengestrol acetate for use in
cattle. From countries where the administration of these substances is allowed the
importation of farm animals, meat or meat products into the EU is prohibited or under
strict control.
The Council Directive 96/23/EC regulates the surveillance of these substances and
residues in the animals and animal products.
One of the implications of the ban is the necessity of developing a system including
analytical methods for monitoring the presence or the concentrations of anabolic
steroids in offal and excrementa of animals reared for meat production.
The location of sampling is on the farm, at the slaughterhouse, at retail level and at the
borders. The nature of the taken samples depends on the place of sampling.
For the detection of exogeneous steroids in the different tissues sensitive and specific
methods and for screening purposes multi-residue methods are well established.
As the endogeneous hormones are naturally present in animals the detection of an
illegal administration presents a more difficult challenge, because no absolute and
definitive method to distinguish between the natural and administered hormone is
available.
The physiological levels and proportions of the hormones, their precursors and
metabolites depend on the gender, the reproductive status, age, breed, nutrition, housing
and season. The data obtained so far for endogeneous anabolic steroids clearly indicate
that under physiological conditions there are high variations between animals. Above
all, they also vary within each animal. Highest residue concentrations could be
measured in urine, fat and kidney. Steroid hormones are naturally present in the tissues
of meat producing animals in the lower µg/kg to ng/kg range.
Hormones are also conjugated to glucuronic and sulfuric acid, bound to plasma proteins
and esterified with fatty acids.
Administration of endogeneous hormones to animals leads either to an increase, to a
decrease or to no significant difference in the concentrations of the adminstered
hormone, the metabolites or the precursors.
To monitor the abuse of endogeneous hormones different approaches are described:

Establishing of a physiological baseline level of the hormone in the different tissues
of the different animals and definition of a decision limit, beyond which the animal
is considered to have been illegally treated (9).
By reason of the above described variability of the hormones in the tissues of
animals the applicability of this method is restricted.

Establishing of steroid profiles and ratios. For this the concentration ratio for the
substance in question or a metabolite relative to an endogeneous reference
compound (ERC) is determined. As administered natural steroid hormones have to
be metabolized in the same way as the endogeneous compounds, the ratios of
hormones to their precursors and/or metabolites may be distorted by treatment. This
method is used for detection of anabolic abuse in doping analysis in human and
equine sports (3,6,11). In cattle different steroid profiles are examined (5).
The disadvantages of such a test are:
DC 10/00/Topic IV.16/ Pg 3
The hormone pattern and also the concentration ratio is influenced by different
factors such as age, the examined tissue and by the breed.
The test can be defeated by simultaneous administration of the endogeneous
reference compound as known from doping control in athletes.

Use of IRMS. A new approach is the determination of the 13C/12C ratio of the
hormones, precursors and metabolites with IRMS.
Depending on the photosynthetic pathways of CO2 incorporation into plant
material, namely C3 and C4, the 13C/12C ratio differs significantly: The C4 plants
are distinguished from C3 plants by a higher 13C abundance. Within each
photosynthetic type only slight variations are observed.
Endogeneous hormones in animals are biosynthesised from precursor molecules
derived from a wide variety of vegetable materials, whereas synthetic hormones are
made from a single plant species (mostly soy) which have a lower 13C abundance.
The method to detect the abuse of steroids is based on this premise.
Administration with an endogenous hormone will alter the 13C/12C ratio of the
incurred hormone and of the metabolites, whereas the 13C/12C ratio of the
precursors remain unchanged.
The method is used for the detection of steroid abuse in human and equine sports
(1,2,10) and work has already been started with cattle (4,8). After testosterone
administration a significant difference was observed between the 13C/12C ratio of a
precursor and the metabolites of testosterone in urine of cattle. In bile only small
differences were found between the compounds but the ratio of an ERC and the
metabolite was affected significantly. The reason for these variations may be
explained by the different feeding regimen of the animals.
The advantage of this method is a direct proof of illegal administration of
endogeneous hormones.
Another advantage of this method is that as far known the 13C/12C-ratio of the
endogeneous hormones is only dependent on the diet of the animals. The method of
isotope ratio mass spectrometry has not yet been applied to other tissues than urine
and bile in cattle
5. ECONOMIC AND SOCIAL BENEFITS
The implementation of the Council Directives 96/22/EC and 96/23/EC demands the
availability of reliable analytical methods to monitor the observance of the ban for these
substances. The results produced by these methods must often fulfil criteria valid in
juridical cases. The occurrence of false positive or false negative results has to be
excluded.
Existing methods to prove the abuse of endogeneous hormones in fattening cattle are
based on indirect determinations. The introduction of IRMS methods into the analysis
of hormones offers the ability to distinguish directly between natural endogeneous and
administered endogeneous hormones. The use of GC-C-IRMS in detection of illegally
administered endogeneous hormones is described so far only for urine and bile in cattle.
Through an extension of these methods and the determination of the influence of
nutrition the proof of illegal administration will be facilitated and thereby it improves
the efficiency with which the EU ban can be enforced. It is important to maintain the
high sanitary level of protection for the consumers chosen by the EU.
From the development of effective control methods the following advantages are
expected:
DC 10/00/Topic IV.16/ Pg 4

Because all member states are obliged to monitor food producing animals, food and
food products for the presence of banned compounds a reliable tool to secure the
unambiguous detection of illegal administered hormones for the control authorities
is necessary.

The method may not only assist in the on-farm and slaughterhouse inspections but
also in the monitoring of live animals and food imports from third countries into the
EU.

Through the productive collaboration between laboratories from different EU
Member States a further harmonisation of the control is expected.
An overall aim is the protection of the consumers in regard to undesired residues in food
and an in-crease of the confidence of the consumers into the quality of food and by it
will help to reduce the concern in this area.
On the other side the use of effective control methods will protect the honest farmer
from farmers who use the banned substances to enhance their profit and distort
competition.
Through the installation and harmonisation of an effective control of food producing
animals and foods, trade disputes both between the member states and between the EU
and third countries will be reduced.
6. SCIENTIFIC AND TECHNOLOGICAL OBJECTIVES
For the purposes of establishing a GC-C-IRMS procedure to identify meat from cattle
administered with endogeneous hormones and an extension of the procedures for
control of illegal treatment at on-farm and slaughterhouse inspections the following
specific objectives are essential:

Development of an efficient extraction method
For analysis by GC-C-IRMS, new extraction methods for the hormones, their
precursors and their metabolites from muscle tissue, faeces, fat, liver and kidney
shall be applied to improve the yield and reproducibility of the extraction method
and compared with customary extraction methods.

Hydrolysis of conjugated and bound steroids
An investigation of different hydrolysis methods for the conjugated and bound
steroids in the various matrices is necessary.

Development of a purification procedure
The clean-up procedure for analysis by GC-C-IRMS is of critical importance (very
low level of use range (ng/kg), losses, interferences,…). For this reason, several
effective methods for the clean-up of the different matrices have to be tested.

Optimisation of the GC-C-IRMS procedure
The GC analysis including derivatisation procedures and inlet systems has to be
optimized according to the specific requirements of IRMS.

Establishing a range of 13C values of steroids in cattle
A standardized and controlled feeding study shall be the essential part to determine
the biological variability of isotopic patterns serving as scientifically accepted
database for official control purposes. Cattle reared for meat production shall be
DC 10/00/Topic IV.16/ Pg 5
raised under defined environmental conditions in special regard to the nutrition and
further shall be treated with the respective endogenous hormones. During rearing
of the animals urine, faeces and blood, and after slaughtering muscle tissue, fat,
liver, kidney and also urine, faeces and blood shall be taken.
From the samples obtained in the above experiments the profiles of the hormones,
metabolites and precursors in the different tissues shall be determined by methods
with sufficient specificity and detection capability.
The 13C/12C-ratio of the analytes (hormones, metabolites and precursors) occured in
satisfactory concentrations for the GC-C-IRMS shall be determined.

Validation of the method
The method for the detection of endogeneous hormones in urine will be validated by an
intercomparison study.
7. TIME SCALE
Although no rigid time scale requirements apply to this project, a maximum of 4 years
is considerated as being realistic to conduct such a research including the validation step
8. IMPORTANT ADDITIONAL INFORMATION
The main stakeholders (control laboratories, national authorities, …) should be deeply
involved in the whole process (development, implementation, validation, etc).
References
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Hatton C.K.,
Screening urine for exogeneous testosterone by isotope ratio mass spectrometric analysis of one
pregnanediol and two androstanediols
Journal of Chromatography B 727, (1999), 95-105
2. Becchi M., Aguilera R., Farizon Y., Flament M.-M., Casabianca H., James P.,
Gas Chromatography/Combustion/Isotope-ratio mass spectrometry analysis of urinary steroids
to detect misuse of testosterone in sport
Rapid Communications in Mass Spectrometry 8, (1994), 304-308
3. Bonnaire Y., Dehennin L., Plou P., Toutain P.L.
Testosterone administration to mares: Criteria for detection of testosterone abuse by analysis of
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Journal of Analytical Toxicology 19, (1995), 175-181
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Determination of the Exogenous Character of Testosterone in Bovine Urine by Gas
Chromatography-Combustion-Isotope Ratio Mass Spectrometry
Analyst 123, (1998), 2617-2620
DC 10/00/Topic IV.16/ Pg 6
5. Fritsche S., Schmidt G., Steinhart H.
Gas chromatographic-mass spectrometric determination of natural profiles of androgens,
progestogens, and glucucorticoids in muscle tissue of male cattle
European Food Research and Technology 209, (1999), 393-399
6. Horning S., Geyer H., Flenker U., Schänzer W.
Detection of exogeneous Steroids by 13C/12C Analysis
In Recent Advances in Doping Analysis (5):
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Gotzmann, A.; Mareck-Engeleke) Sport und Buch Strauss, Cologne 1998
7. Houghton E., Grainger L., Dumasia M.C., Teale P.
Application of Gas Chromatography/Mass Spectrometry to Steroid Analysis in Equine Sports:
Problems with Enzyme Hydrolysis
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The Use of Stable Carbon Isotop Analysis to Detect the Abuse of Testosterone in Cattle
Analyst 123, (1998), 2405-2408
9. Scippo M.L., Degand G., Duyckaerts A., Maghuin-Rogister G., Delahaut P.
Control of the illegal administration of natural steroid hormones in the plasma of bulls and
heifers
Analyst 119, (1994), 2639-2644
10. Shackleton C.H.L., Phillips A., Chang T., Li Y.
Confirming testosterone administration by isotope ratio mass spectrometric analysis of urinary
androstanediols
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11. Teale P., Houghton E.
The development of a gas chromatography/mass spectrometric screening procedure to detect the
administration of anabolic steroids to the horse
Biological Mass Spectrometry 20, (1991), 109-114