Disturbance in sex-steroid serum profiles of cattle in response to exogenous estradiol: A screening approach to detect forbidden treatments

Article abstract of DOI:10.1016/j.steroids.2010.12.005

Estradiol benzoate (EB) has been one of the most widely used estrogenic agents in animal husbandry, as a way of exogenously introducing the natural hormone estradiol-17β into the animal organism. Estradiol was previously employed to induce anabolic effects or reproductive improvements in cattle. However, the employment of EB in European countries has been permanently forbidden by Directive 2008/97/EC to guarantee consumers' health. Despite this prohibition, the control of estradiol-17β and its esters continues to be a difficult task for residue-monitoring plans in European Communities because official analyses of natural thresholds for hormones in cattle have not yet been established, leading to a lack of confirmation for any exogenous administration of natural hormones. Several researchers have worked on excretion profiles of metabolites, variation in specific hormonal ratios and metabolomic fingerprints after hormonal treatments. This research focuses on the possible existence of disturbances in the serum profile of animals treated with EB in terms of steroid sex hormones (androgens, oestrogens and progestogens), by investigating the serum levels of several of these hormones. The serum samples were collected from three groups of cows: one treated with an intramuscular injection of EB, one treated with a combination of intravaginal EB and progesterone and a control (non-treated) group. The samples have been analysed by a validated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method, and 17 natural hormones were identified and quantified. Subsequently, data from the serum profiles were submitted for statistic and multivariate analysis, and it was possible to observe a manifest variation between animal groups. The obtained results can help in the development of a viable screening tool for monitoring purposes in cattle.

Full text of DOI:10.1016/j.steroids.2010.12.005

Steroids 76 (2011) 365–375
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Disturbance in sex-steroid serum profiles of cattle in response to exogenous
estradiol: A screening approach to detect forbidden treatments
∗
Patricia Regal , Carolina Nebot, Mónica Díaz-Bao, Rocio Barreiro, Alberto Cepeda, Cristina Fente
Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Veterinary Medicine, University of Santiago de Compostela, Lugo, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2010
Received in revised form
Estradiol benzoate (EB) has been one of the most widely used estrogenic agents in animal husbandry, as a
way of exogenously introducing the natural hormone estradiol-17␤ into the animal organism. Estradiol
was previously employed to induce anabolic effects or reproductive improvements in cattle. However,
the employment of EB in European countries has been permanently forbidden by Directive 2008/97/EC to
guarantee consumers’ health. Despite this prohibition, the control of estradiol-17␤ and its esters contin-
ues to be a difficult task for residue-monitoring plans in European Communities because official analyses
of natural thresholds for hormones in cattle have not yet been established, leading to a lack of con-
firmation for any exogenous administration of natural hormones. Several researchers have worked on
excretion profiles of metabolites, variation in specific hormonal ratios and metabolomic fingerprints after
hormonal treatments. This research focuses on the possible existence of disturbances in the serum profile
of animals treated with EB in terms of steroid sex hormones (androgens, oestrogens and progestogens),
by investigating the serum levels of several of these hormones. The serum samples were collected from
three groups of cows: one treated with an intramuscular injection of EB, one treated with a combination
of intravaginal EB and progesterone and a control (non-treated) group. The samples have been analysed
by a validated high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS)
method, and 17 natural hormones were identified and quantified. Subsequently, data from the serum
profiles were submitted for statistic and multivariate analysis, and it was possible to observe a manifest
variation between animal groups. The obtained results can help in the development of a viable screening
tool for monitoring purposes in cattle.
1
0 November 2010
Accepted 11 December 2010
Available online 21 December 2010
Keywords:
HPLC–MS/MS
Bovine
Serum
Estradiol benzoate
Sex hormone
©
2010 Elsevier Inc. All rights reserved.
Throughout history, humans have bred animals to accommo-
date increasing human requirements in terms of food and other
products, such as leather and wool, the demand for which is par-
ticularly important in developed countries. A large number of
substances, both natural and synthetic, have been applied in stock
farming to speed up and improve animal growth, and to decrease
feed costs [1,2]. Among these veterinary drugs, there are some nat-
ural hormones and substances with hormonal effects that have
been applied to animals for different purposes, especially as growth
promoters and as fertility regulators in cattle. The steroid hor-
mones, which are steroids acting as hormones, contain the sex
hormones oestrogens, gestagens and androgens (EGAs), and the
corticosteroids. Although these hormones have a wide variety of
applications within the veterinary field, they have been used in
animal fattening due to the anabolic effect that increases weight
gain in treated animals, and induces changes that are generally
characterised by lower fat content and more lean mass [3–5]. The
zootechnical use of some sex hormones, such as estradiol or its
esters (i.e., estradiol benzoate (EB)), which successfully regulate
oestrus in cattle, has also led to important improvements and finan-
cial gain in stock farming [6].
There have been several European regulations regarding the
use of EGAs as animal growth promoters because of their possible
toxic effect on public health. In the Council Directive 96/22/EC [7],
the European Union prohibited the administration of substances
having thyreostatic, oestrogenic, androgenic or gestagenic effects
and of beta agonists in animal husbandry, while certain therapeu-
tic applications of these drugs were still allowed. These anabolic
steroids are included in group A substances according to Annex
I of Directive 96/23/EC [8], which pertains to growth-promoting
agents abused in animal fattening and unauthorised substances
with no maximum residue limit (MRL). A zero-tolerance policy
has been adopted, and especial analytical requirements have been
stated in regard to these hormones [9]. In particular, estradiol-
∗ Corresponding author at: Laboratorio de Higiene e Inspección de Alimentos,
Departamento de Química Analítica, Nutrición y Bromatología, Facultad de Veteri-
naria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
Tel.: +34 982285900; fax: +34 982254592.
E-mail addresses: patricia.regal@rai.usc.es, patricia regalopez@hotmail.com
(
P. Regal).
0
039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.12.005

3
66
P. Regal et al. / Steroids 76 (2011) 365–375
ment of 13C/12C ratios by gas chromatography-combustion-isotope
1
7␤, used with the aim of promoting animal growth, was deemed
as a complete carcinogen by the Scientific Committee on Veteri-
nary Measuresrelatingto Public Health(SCVPH) [10]. Estradiol-17␤
exerts both tumour-initiating and tumour-promoting effects, and
the data currently available do not make it possible to obtain a
quantitative estimate of the risk. Although Directive 2003/74/EC,
amending Directive 96/22/EC, permanently prohibited the use of
estradiol-17␤ and its ester-like derivatives as growth promoters,
a temporary exemption was given until 14 October 2006 for their
use as an oestrous-induction tool in cows, horses, sheep or goats.
As alternative effective products exist and are implemented in the
market [11], the European Parliament banned estradiol-17␤ and
its ester-like derivatives, including those with a therapeutic pur-
pose in farm animals, in 2008, to ensure human health protection
within the European Community [12]. However, the possibility of
widespread abuse of hormonal substances by unscrupulous farm-
ers and veterinary professionals in some parts of Europe still exists,
mainly due to the economic benefits these substances provide in
animal husbandry [13].
The control of growth promoters in meat-producing animals is
probably one of the most challenging tasks in the field of Euro-
pean residue-monitoring plans, as it involves a wide number of
target substances, the variability of their chemical structures and
their concentration levels and biological matrices used in residue
surveillance [1,4]. With regard to the confirmation of use of xenobi-
otic analogues of natural sex steroids and non-steroidal compounds
such as stilbenes and zeranol, there is an extensive range of success-
ful methods that has been performed on different analytic matrices
thathave made theconfirmationof illicit administrations ofanabol-
ics in cattle feasible [2,14–17]. However, hormones of natural
origin, such as estradiol-17␤, testosterone (T) or progesterone, are
still a weak area in residue-monitoring plans due to their endoge-
nous origin, as the target compound is always present. In such a
case, the confirmation of an exogenous administration involves
logical difficulties associated with distinguishing an exogenous ori-
gin from an endogenous (naturally occurring) presence of these
hormones. In fact, it has been found that treatments with T or
estradiol in bovines lead to equal or lower plasma concentrations
of these compounds [18,19]. On the other hand, exogenous nat-
ural hormones are usually administered as simple semi-synthetic
esters (i.e., 17␤-estradiol benzoate and T decanoate), and a subse-
quent rapid hydrolysis of these compounds takes place as soon as
they reach the bloodstream, where they generate non-esterified
forms that are indistinguishable from naturally occurring forms
ratio mass spectrometry (GC-C-IRMS) can be a powerful tool to
trace the true origin of steroids, and is one of the most promis-
ing approaches for the control of exogenous administration of
natural hormones, as it has already been applied for anti-doping
in sports [23,24]. In recent years, the potential of ‘-omic’ tech-
nologies (metabolomics, proteomics with transcriptomics) coupled
with bioinformatics has been investigated for the development
of reliable molecular biomarkers, and to obtain discrimination
based on targeted profiling of metabolites [25–27]. Other research
has focussed on the variation of the excretion profile of phase II
metabolites asaconsequenceofexogenously administered steroids
[28,29], and in variation of some urinary or plasmatic metabolites
from the biosynthetic pathway of sex hormones [18,30], or, even
in blood chemistry [31].
Summing up, more information concerning steroid levels in
animals treated with natural hormones seems necessary to estab-
lish acceptable thresholds of natural hormones or for use as a
screening approach in residue-monitoring plans. In the present
study, an analytical evaluation of serum profiles of several nat-
ural hormones from the biosynthetic pathway of sex hormones
has been performed to prove the existence of any disturbance
in the serum profile in response to exogenous estradiol admin-
istration in cattle. Bovine serum samples were analysed using
a method based on liquid chromatography–tandem mass spec-
trometry (high-performance liquid chromatography tandem mass
spectrometry, HPLC–MS/MS) [32], previously validated according
to Decision 657/2002/EC criteria [9]. The samples were obtained
from cows treated intramuscularly with the main ester of natu-
ral estradiol, 17␤-estradiol benzoate, and animals treated with a
common intravaginal combination of 17␤-estradiol benzoate and
progesterone. Serum from non-treated animals, which were used
as control animals, was also collected. Free plasma concentra-
tions of 17 natural steroids belonging to the three existing groups
of sex hormones (EGAs) were submitted for further statistical
analysis. From the data analysis, useful and valuable descrip-
tive information about the natural steroid levels in bovines was
obtained. In addition, an overview of the disturbance in plasma
profiles of cattle treated with the oestrogenic compound was
gathered.
1. Experimental
[
20]. These exogenous compounds follow the same pathways as
1.1. Samples
the natural compounds biosynthesised by the animal, making the
detection and confirmation of their exogenous administration dif-
ficult, if not impossible. These circumstances have led to the lack
of success in detecting EB in serum or plasma, which has only
been confirmed in hair from animals treated with this ester [14,21].
In addition, the demonstration of an exogenous administration of
natural steroids, for instance, T, estradiol or cortisol, remains prob-
lematic, as no official threshold has been stated for natural hormone
concentrations, mainly due to the fact that concentrations of nat-
urally occurring hormones depend on the type of animal product,
breed, gender, age, disease, medication and physiological condition
Serum samples were obtained from 72 Holstein cows that were
between 24 months and 5 years in age, all from the same intensive
dairy farm. Twenty-four cows were treated with an intramuscular
injection of EB, consisting of 5 ml of a veterinary drug (Neonida N
from Pfizer S.A., Madrid, Spain) containing 1250 I.U. of chorionic
gonadotropin and 5 mg of EB, while 13 other cows were treated
with an intravaginal device composed of a progesterone-releasing
spiral (1.55 g of progesterone dispersed in an elastomeric silicone
matrix) and a capsule containing 10 mg of EB (PRID® from CEVA
Salud Animal, Barcelona, Spain). The experimental cows were fed
a diet typically used in animal husbandry practices, and provided
ad libitum access to water.
The administration of these hormonal preparations took place
within a typical and real bovine reproductive control programme
under the supervision of a veterinary surgeon. The estrogenic
compounds were administrated before their total prohibition in
October 2006. Blood samples from the animals treated with EB and
EB combined with progesterone were collected on days 3 and 6,
respectively, after a single-dose treatment. Thirty-five untreated
animals were used as a control group (so-called non-treated or C).
[
22]. Furthermore, no list of discriminative marker metabolites has
ever been stated, accepted or published by the community refer-
ence laboratories (CRLs) or by the European Commission, regarding
the misuse of natural hormones in stock farming.
The development of methods to provide unequivocal discrim-
ination between the natural presence of an endogenous hormone
and its presence as a consequence of an illegal exogenous adminis-
tration remains a challenge. Some promising analytical approaches
have been published in the past few years regarding this critical
point of controlling residues in food of animal origin. The measure-

P. Regal et al. / Steroids 76 (2011) 365–375
367
Table 1
HPLC–MS/MS transition used for the measurement of the assayed natural hormones, obtained CC␣ and CC␤ when relevant [29]. (IS: internal standard; RT: retention time;
CC␣: decision limit; CC␤: detection capability).
CC␣ (ng dL−1)
−1
CC␤ (ng dL )
Steroid
RT (min)
MRM (Q1/Q3)
P4
24.5
24.3
18.9
23.4
23.3
18.5
18.3
18.3
18.3
16.6
18.3
17.2
16.4
18.1
17.1
17.5
19.4
19.2
19.8
19.7
17.4
17.2
18.6
18.1
17.8
345.093/124.100
354.124/128.200
361.068/124.200
332.065/86.200
336.121/90.100
348.081/330.300
350.97/333.200
286.064/253.000
287.965/255.100
302.041/269.100
305.951/273.100
302.041/269.100
316.035/283.100
319.95/287.000
316.044/283.200
301.912/251.100
304.105/253.200
306.005/213.200
304.084/124.100
306.994/124.100
320.086/112.000
320.071/143.100
317.086/112.200
333.05/112.100
333.034/124.100
18.68
31.83
P4-d9 (IS)
1
7OHP4
13.06
10.92
22.25
18.60
P5
P5-d4 (IS)
1
7OHP5
7.33
7.27
6.95
12.49
12.39
11.84
1
7OHP5-d3 (IS)
E1
E1-d2 (IS)
2
2
4
2
2
4
1
OHE1
OHE1-d4 (IS)
OHE1
MeE1
MeE1-d4 (IS)
MeE1
6.00
12.98
12.10
22.12
14.31
8.99
14.99
24.21
15.33
25.54
6OHE1
DHEA
DHEA-d2 (IS)
T
19.46
33.16
T-d3 (IS)
7
1
OHT
9OHT
19.38
7.48
9.16
13.33
10.65
33.02
12.75
15.60
22.71
18.14
A
7
1
OHA
9OHA
In all cases, the samples were collected from the tail by a veterinary
surgeon using vacuum-extraction tubes. After blood clotting into
the extraction tubes and 10 min of centrifugation at 814 × g, the
with 0.1% formic acid, mixed in a binary gradient from a previously
validated method [32].
◦
serum was removed from all samples and stored at −20 C, until
1.4. Serum analysis by LC–MS/MS
further analysis in 2009.
The serum samples (0.5 ml) were processed following an
analytical method previously validated according to the European
Commission Decision 2002/657/EC criteria [9] and described by
Regal et al. [32]. This method permitted reliable detection and
quantification of hormones from the three groups of natural sex
1
.2. Reagents and chemicals
Experimental materials included the following deuterated
analogues of steroids: deuterium-labelled progesterone (P -d9),
4
pregnenolone (P5-d4), 17-hydroxy pregnenolone (17OHP5-d3),
estrone (E -d2), 2-hydroxy oestrone (2OHE -d4), 2-methoxy
hormones (EGAs): pregnenolone (P ) [5-pregnen-3ß-ol-20-one],
5
1
1
progesterone
progesterone
17-hydroxy-pregnenolone
(P4)
(17OHP4)
[4-pregnen-3,20-dione],
[4-pregnen-17-ol-3,20-dione],
(17OHP5) [5-pregnen-3ß,17-diol-
20-one], DHEA [5-androsten-3␤-ol-17-one], androstenedione
(A) [4-androsten-3,17-dione], 7␣-hydroxyandrostenedione
(7OHA) [4-androsten-7␣-ol-3,17-dione], 7␣-hydroxytestosterone
17-hydroxy-
estrone (2MeOE -d4), dehydroepiandrosterone (DHEA-d2) and T-
1
d3, purchased from CDN Isotopes (Quebec, Canada). The formic
acid was obtained from AcrosOrganics (Geel, Belgium). The
compounds methyl tert-butylether (MTBE), hexane, HPLC-grade
methanol and acetonitrile were supplied by Merck (Darm-
stadt, Germany). The hydroxylamine solution (99.999%, 50 wt. %
(7OHT)
17␤-ol-3-one],
[4-androsten-7␣,17␤-diol-3-one],
T
[4-androsten-
(19OHA)
in H O) and ascorbic acid were obtained from Sigma–Aldrich
19-hydroxyandrostendione
17-dione],
[4-androsten-17␤,19-diol-3-one],
2
(
(
St Louis, MO, USA). Milli-Q organic-free water from Millipore
Bedford, MA, USA) was used. All reagents were of analytical
[4-androsten-19-ol-3,
19-hydroxytestosterone
estrone (E1)
[1,3,5(10)-estratien-3-ol-17-one], 2-methoxyestrone (2MeOE1)
[1,3,5(10)-estratien-2,3-diol-17-one 2-methyl ether], 4-
methoxyestrone (4MeOE ) [1,3,5(10)-estratrien-3, 4-diol-17-one
(19OHT)
grade.
The blood extraction materials consisted of single-use blood
collection tubes, needles and needle holders from Vacutainer (Ply-
mouth, UK).
1
4-methyl
ether],
2-hydroxyestrone
(2OHE1)
[1,3,5(10)-
estratrien-2, 3-diol-17-one], 16␣-hydroxyestrone (16OHE1)
[1,3,5(10)-estratrien-3,16␣-diol-17-one] and 4-hydroxyestrone
1.3. Materials and apparatus
(
4OHE ) [1,3,5(10)-estratrien-3,4-diol-17-one].
1
The HPLC system consisted of a quaternary pump, degasser and
The serum samples (500 ␮l) were placed in a 2-ml Eppendorf
autosampler (Agilent Technologies model 1100; Minnesota, USA).
A Q-Trap 2000 mass spectrometer with ion source turbo spray
from Applied Biosystems MSD Sciex (Toronto, Canada) was used.
Nitrogen was produced by a high-purity nitrogen generator (PEAK
Scientific Instruments Ltd, Chicago, IL, USA). Nitrogen was used for
the curtain, nebuliser and collision gases.
Aliquots of sample extracts were separated by HPLC using a
Synergi 2.5 ␮m Fusion-RP 100A (100 mm × 2 mm) column from
Phenomenex (Torrance, CA, USA) with a guard column of the same
filling material. The mobile phase was water and methanol, both
tube containing 1000 ␮l of the internal standard mixture in acetoni-
−
1
trile (100 ng dL ) and 250 ␮l of hexane. The tubes were capped,
vortexed for 1 min, sonicated for 30 min and centrifuged at 16
100 × g for 15 min using an Eppendorf Centrifuge 5415D (Ham-
◦
burg, Germany). The assembly was cooled for 15 min at −20 C.
The hexane supernatant and the precipitate were discarded, and
the acetonitrile layer was evaporated under a nitrogen gas stream
◦
at 37 C. For steroid derivatisation, the residue was re-dissolved
in 300 ␮l of a 1.5 M hydroxylamine solution (pH 10) for 30 min
◦
at 90 C. After this derivatisation procedure, 700 ␮l of water was

3
68
P. Regal et al. / Steroids 76 (2011) 365–375
Table 2
Median, mean, minimum (Min) and maximum (Max) values of hormonal levels in bovine serum (ng dL−1) and the percentage of samples below the CC␣ level in each group
of animals; statistically significant differences are also shown (SD: standard deviation).
Natural hormones
Statistics
Animal group
Estradiol benzoate (EB)
EB and progesterone (EB + P4)
Control
DHEA*
,†
Median
25.6
34.0
13.1
<25.5
<25.5
5.0
<25.5
<25.5
5.9
Mean (ng dL−1)
SD
Min
Max
<25.5
61.3
<25.5
32.2
<14.9
40.8
%
<CC␣
0.0
0.0
11.4
T
Median
<19.5
<33.2
23.9
41.9
49.3
34.6
<19.5
48.1
60.6
Mean (ng dL−1)
SD
Min
Max
<33.2
120.1
0.0
<33.2
144.4
0.0
<19.5
214.6
17.1
%
<CC␣
A*
,†
Median
<15.6
20.5
15.5
<9.2
<15.6
1.8
<9.2
<15.6
2.8
Mean (ng dL−1)
SD
Min
Max
<15.6
70.0
0.0
<9.2
<15.6
92.3
<9.2
21.1
8.6
%
<CC␣
7
OHT
Median
<33.0
33.4
<33.0
35.4
<19.4
28.9
Mean (ng dL−1)
SD
13.6
7.8
12.7
Min
Max
<19.4
65.2
<33.0
51.1
<33.0
71.5
%
<CC␣
0.0
<22.7
37.1
0.0
0.0
42.2
45.4
28.3
<22.7
109.7
0.0
OHA*
,‡
Median
135.5
161.5
136.0
36.9
475.2
0.0
7
Mean (ng dL−1)
SD
Min
Max
40.8
<22.7
188.1
0.0
%
<CC␣
1
9OHT
Median
319.1
450.6
347.2
51.4
1198.6
0.0
582.5
624.8
292.4
243.2
1152.1
0.0
455.5
561.7
538.4
61.8
1804.0
0.0
52.0
69.9
47.0
28.5
194.3
0.0
483.4
506.3
125.8
305.6
676.0
0.0
42.8
48.8
23.5
<22.1
98.1
374.5
423.0
321.2
32.7
1264.6
0.0
141.4
161.1
142.3
<18.1
755.8
0.0
Mean (ng dL−1)
SD
Min
Max
%
<CC␣
9OHA*
,‡
Median
47.1
1
Mean (ng dL−1)
108.7
158.2
18.2
676.5
0.0
185.5
191.1
100.5
55.9
378.4
0.0
703.8
892.3
532.8
416.8
2718.6
0.0
162.9
163.2
94.7
34.1
401.2
0.0
735.3
1123.9
938.9
416.8
3940.8
0.0
SD
Min
Max
%
<CC␣
E1*
,†
Median
40.2
48.6
33.5
Mean (ng dL−1)
SD
Min
Max
<12.4
204.5
0.0
377.9
396.3
163.9
85.5
913.1
0.0
22.4
%
<CC␣
OHE1*
,†
Median
2
2
4
4
Mean (ng dL−1)
SD
Min
Max
%
<CC␣
MeOE1*
,†
Median
Mean (ng dL−1)
50.3
46.4
SD
Min
Max
<22.1
162.9
0.0
378.8
396.3
163.9
85.5
913.1
0.0
<14.3
<22.1
2.4
%
<CC␣
0.0
OHE1*
,†
Median
492.7
530.7
149.3
305.6
824.2
0.0
<14.3
23.1
19.2
Mean (ng dL−1)
SD
Min
Max
%
<CC␣
MeOE1*
,†,‡
Median
96.7
114.1
94.7
Mean (ng dL−1)
SD
Min
Max
<14.3
354.6
16.7
<14.3
81.9
61.5
<14.3
<24.2
94.3
%
<CC␣

P. Regal et al. / Steroids 76 (2011) 365–375
369
Table 2 (Continued)
Natural hormones
Statistics
Animal group
Estradiol benzoate (EB)
EB and progesterone (EB + P4)
Control
33.9
6OHE1*
,†
Median
107.1
140.8
114.7
22.7
35.0
38.8
30.0
1
Mean (ng dL−1)
43.1
42.8
<8.9
SD
Min
Max
<8.9
450.2
0.0
109.9
15.4
218.6
17.1
%
<CC␣
P5
Median
252.4
287.1
123.9
110.0
586.3
0.0
113.8
262.5
354.8
<18.6
1531.0
8.3
221.8
230.1
79.2
128.3
382.4
0.0
1302.0
1460.4
779.6
26.0
2669.6
0.0
196.7
243.2
139.8
58.8
553.4
0.0
2556.1
2899.4
2774.3
<18.6
12,364.3
17.1
Mean (ng dL−1)
SD
Min
Max
%
<CC␣
P4*
,†
Median
Mean (ng dL−1)
SD
Min
Max
%
<CC␣
1
7OHP5*
7OHP4*
,†
,†
Median
112.4
127.2
44.7
71.1
249.4
0.0
22.3
33.0
26.9
42.4
56.9
36.7
<12.5
112.0
0.0
<13.1
<22.3
2.5
65.4
67.8
22.8
33.3
157.6
0.0
<13.1
<22.3
21.0
Mean (ng dL−1)
SD
Min
Max
%
<CC␣
1
Median
Mean (ng dL−1)
SD
Min
Max
<13.1
126.4
4.2
<13.1
22.3
53.8
<13.1
127.8
65.7
%
<CC␣
Ratio T/P4*
,†
Median
Mean
SD
Min
Max
Median
Mean
SD
Min
Max
0.179
0.464
0.532
0.013
1.826
2.599
4.751
6.796
0.166
13.690
0.027
0.135
0.356
0.009
1.316
0.139
0.478
1.073
0.083
4.031
0.013
0.217
0.396
0.004
1.042
0.095
2.050
4.535
0.024
20.675
Ratio P5/P4*
,†
†
‡
Mann–Whitney U test for EB and C p < 0.01.
Mann–Whitney U test for CEB + P4 and C p < 0.01.
KRUSKAL–WALLIS test between 3 groups p < 0.01.
*
added, and the oxime steroid derivatives were extracted twice with
ml of MTBE. After evaporation under a nitrogen gas stream, the
terone (EB + P , intravaginal) and 35 non-treated animals as
group C.
4
2
residue in 100 ␮l of methanol:water (10:90 (v/v)) was used for the
chromatographic procedure.
Adequate measures were taken to minimise pain or discomfort
to the animals, and the experiments were conducted in accordance
with international standards on animal welfare, and were compli-
ant with local and national regulations.
1.5. Identification and quantification of steroids
The transitions monitored for each of the assayed hormones
1.6. Data analysis
and for their internal standards and main validation parame-
−
1
ters, CC␣ and CC␤ (in ng dL ) [32], are shown in Table 1. The
automatic quantification tool of Analyst 1.4.1 software (MDS
SCIEX) was used to integrate the area corresponding to the
chromatographic peaks of each hormone and internal stan-
dard. The serum levels of each hormone in their unconjugated
forms were interpolated from calibration curves constructed by
calculating the area ratios of the analyte peak area/internal stan-
dard (IS) peak area versus the analyte concentration (calibration
with IS).
Univariate statistics were performed using PASW Statistics 18.0
(SPSS Ibérica, Madrid, Spain). Breakdown analyses are represented
−
1
by the mean (ng dL ), median, standard deviation, ranges (max-
−
1
imum and minimum quantified level in ng dL ) and percentage
of samples that were below the decision limit (CC␣) in each
group of animals. When an analyte was not detected (below the
CC␣), its value was expressed as the CC␣ level; when an analyte
was detected but not accurately quantifiable (below CC␤), it was
expressed as the CC␤ level. The values of these performance limits,
CC␣ and CC␤, are shown in Table 1 for each hormone. In addition,
discrimination tests were applied to search for any possible
statistically significant differences in hormonal levels, according
to animal group. Differences between groups were evaluated
by means of the Mann–Whitney U test and Kruskal–Wallis
test, as the distribution of variables was proven to be
non-Gaussian.
The data obtained on the level of free, unconjugated steroid
hormones in animal serum samples were subsequently submit-
ted to statistical analysis, to obtain descriptive results related
to animal group and to observe discriminative and multivari-
ate features. The results obtained from the serum samples were
organised and evaluated in three groups: 25 animals treated
with EB (EB, intramuscular), 13 treated with EB plus proges-

3
70
P. Regal et al. / Steroids 76 (2011) 365–375
Fig. 1. Box plots showing serum levels of the steroid hormones identified by HPLC–MS/MS that were found to be significantly different (p < 0.01) between cows treated with
estradiol benzoate (intramuscular, EB), treated with estradiol benzoate plus progesterone (intravaginal, EB + P4) and the control group (C). The box limits are in the 25th and
7
5th percentile, and the band in the middle of the box is the median; the whiskers are in the 1.5 interquartile range. Data not included between the whiskers are plotted as
an outlier with a small circle (mild) or star (extreme outlier).

P. Regal et al. / Steroids 76 (2011) 365–375
371
Fig. 2. Box plots showing the hormonal ratios that were found to be significantly different (P5/P4 and T/P4) between cows treated with estradiol benzoate (intramuscular,
EB), treated with estradiol benzoate plus progesterone (intravaginal, EB + P4) and the control group (C), when p < 0.01. The box limits are in the 25th and 75th percentile, and
the band in the middle of the box is the median; the whiskers are in the 1.5 interquartile range. Data not included between the whiskers are plotted as an outlier with a small
circle (mild) or star (extreme outlier).
Multivariate analysis was performed by means of SIMCA-P+12.0
Umetrics AB, Sweden) software. As the name indicates, multivari-
treated cows. The sampling times of day 3 for EB and day 6 for
(
EB + P were established by the veterinary surgeon as the time fixed
4
ate analysis groups a set of techniques dedicated to the analysis of
data sets with more than one variable. To perform multiple sam-
ple comparison by multivariate modelling, all samples must be
described by a common set of variables, which, in this case, is the
concentration of free natural hormones present in bovine serum.
All variables were log transformed to become uniform and suitable
for being submitted to multivariate analysis.
for artificial insemination when using each treatment, based on the
purpose of these drugs.
The possible occurrence of problems in the hormonal stability
due to long-term freezing storage has also been taken into account;
therefore, control samples from the same farm were also collected
at the same time. It is expected that possible variations in hormonal
levels would affect both treated and non-treated samples, and dif-
ferences in hormonal levels between groups would be maintained
despite storage.
Unsupervised principal components analysis (PCA) is a method
that reduces data dimensionality through a covariance analysis
between factors without referring to class labels, which, in this
case, are animal groups [33]. PCA was run to obtain a general
overview of the variance in hormones throughout samples. Mul-
tivariate regression analysis in terms of orthogonal partial least
squares discriminant analysis (OPSLS-DA) [34,35] was applied to
extract the systematic variation in the quantified serum profiles
related to the animal groups. OPLS-DA is a supervised method that
uses a multiple linear regression technique to find the maximum
covariance between a data set and the sample class. By analysing
the S-plot, hormones that provide a greater ability to discriminate
between animals treated with EB and the control animals were
identified. The robustness of the OPLS-DA model was verified by
a predictive model, in which a random selection of two-thirds of
the samples (of known class) was used to predict the rest of the
samples (of unknown class). The samples used as predictors were
randomly selected with respect to a two-third-rate in each animal
group.
2.2. Analytical methodology
Oximation with hydroxylamine provided a method for the
simultaneous analysis of 17 steroid compounds, including pro-
gestagens, androgens and estrogens, all of which have reactive
keto groups. Monitored hormones were selected with the inten-
tion of including precursors and metabolites from the three existing
groups of sex hormones (PGAs), and accounting for the possibility
of oximation with hydroxylamine. Despite inhibiting the analysis
of estradiol (E ), hydroxylamine was selected based on previous
2
research that was focussed on detecting the parent compound [36]
without obtaining promising results in terms of discrimination of
animals treated with EB. Moreover, deproteinisation and extraction
with acetonitrile, without later steps, permitted the HPLC–MS/MS
analysis of the obtained steroid–oxime derivatives. As the existing
validation demonstrates [32], this LC–MS/MS methodology enables
an efficient measurement of steroids from bovine serum with min-
imal background interferences. Good efficiency and peak shape
were achieved with the proposed gradient conditions, and it was
possible to separate closely related steroids when monitoring the
2
. Results and discussion
2.1. Selected drugs and sampling
same transitions (e.g., 2OHE and 4OHE ). The obtained derivatives
1
1
Veterinary commercial products were selected based on their
widespread employment in husbandry before the prohibition of
7␤-estradiol and its esters by the European Union. The selected
were easily ionisable with an electrospray source in positive mode.
Two multiple reaction monitoring (MRM) transitions (Table 1)
were monitored for each analyte, and the most intense transition
was monitored for quantification. Adequate chromatographic sep-
aration and evaluation of the two transitions enabled compound
confirmation.
The serum concentrations of some analysed hormones were too
low to be confirmed (below CC␣) and/or quantified (below CC␤),
especially in the case of some androgens and, particularly, in ani-
mals treated with EB combined with P4 and in group C. To avoid
eliminating these samples and some experimental animals with
very low hormonal levels from the research, a fixed value was given
1
veterinary drugs were employed in the past for zootechnical pur-
poses, such as controlling oestrus and synchronising pregnancy in
cows. Due to the high effectiveness of estradiol in controlling and
inducing oestrus in cattle [6], it can be illegally used nowadays, even
though a permanent prohibition has been stated for it. It should
be noted that these hormonal treatments were real, and the sam-
ples were collected from non-experimental dairy cows at a Spanish
dairy farm, before the permanent prohibition of estradiol in Octo-
ber 2006, aiming to induce oestrus and, subsequently, inseminate

3
72
P. Regal et al. / Steroids 76 (2011) 365–375
5
4
3
2
1
0
2
2
1
2
3
3
2
1
3
3
2
2
2
1
3
3
2
3
33
1
3
3
2
1
1
1
3
2
3
1
2
2
1
2
3
1
1
3
3
1
1
3
3
3
1
-
1
2
3
4
5
3
3
1
1
1
1
1
1
3
3
1
1
3
3
-
-
-
-
3
3
3
3
1
3
3
3
3
1
3
-
6
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
t[1]
Fig. 3. Principal components analysis (PCA) score plots derived from the HPLC–MS/MS data set consisting of hormonal profiles from animals treated with estradiol benzoate
1, red box), estradiol benzoate and progesterone (2, green open triangle) and control animals (3, blue dot), showing that the profile is different between groups 1 and 3, but
not between 2 and 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(
to the hormonal levels when they could not be quantified, as men-
tioned in Materials and Methods section, and this step must be
taken into account for consideration throughout our research. Fur-
thermore, as the relation between variables depends on the size
of the sample as well, the results of discrimination regarding ani-
mals treated with EB + P4 (n = 13) should not be considered to be as
reliable as the results from the EB-treated animals (n = 24), from a
statistical point of view.
When analysing the obtained results, the mode of application
of EB should be considered, as it is known that the rate of absorp-
tion of a steroid will differ significantly depending on the means of
administration and formula [29,30]. When EB is injected intramus-
cularly, the absorption will be higher than for an intravaginal pill
because the latter was previously a veterinary reproductive tool,
which was not aimed at reaching high rates in blood, but only in
reproductive organs. The above statement is in agreement with the
results obtained for animals treated with the EB + P4 intravaginal
device, as few differences were found after comparison with the
control cows.
the non-parametric alternative for this test. In this study, the
Mann–Whitney U test was performed to compare EB samples with
C samples and EB + P samples with C samples. Differences between
4
the groups were considered statistically significant when p < 0.01.
Both descriptive and discriminative results obtained from univari-
ate statistical analyses are shown in Table 2.
When differences between the three groups were checked
statistically by means of the Kruskall–Wallis test (see Table 2),
it was concluded that the variations were significant in almost
every assayed hormone, except for pregnenolone, T and its
hydroxides (7OHT and 19OHT), as p > 0.01. When performing the
Mann–Whitney U test for comparison of the hormonal profiles
of the EB and C groups, steroids that were significantly different
in the Kruskall–Wallis test were also selected (p < 0.01), except
for the hydroxides of androstenedione (7OHA and 19OHA), which
appeared to be non-altered. Interestingly, for animals treated with
EB and P , variations in serum profiles were not evident in any hor-
4
mone (p > 0.01), except for the hydroxides of androstenedione and
4MeOHE . These statistically proven differences can be clearly seen
1
in Fig. 1, which shows box plots for each hormone that was found
significantly modified relative to group C (p < 0.01).
2
.3. Univariate analysis
Hormonal ratios were also evaluated, but only T/P4 and P5/P4
were found to be different among the three groups. These hypo-
thetically significant ratios are also described in Table 2. Fig. 1
summarises the results of the concentrations of hormones in the
free form of the three different groups: the C group, the EB-treated
As for distribution of variables (hormonal levels), precise infor-
mation was obtained by performing one of the tests of normality to
determine the probability that the sample originated from a nor-
mally distributed population. The Kolmogorov–Smirnov test was
performed on available quantitative variables corresponding to the
hormonal level of each hormone in serum samples, and it was con-
cluded that the hormonal levels did not follow a normal distribution
group and the EB + P -treated group. Here, statistically significant
4
difference among the groups could be visualised when comparing
the analysed hormones of animals and the significant ratios. Fig. 2
shows a comparison among the selected ratios through all the sam-
ples; it should be noted that an obvious variation of these ratios,
(
p > 0.05). In addition, corresponding histograms of variables were
built to visualise the distribution of the data, confirming the results
of this test. Subsequent discrimination tests were applied according
to these results.
T/P4 and P5/P , could be observed for the EB group (p < 0.01), but
4
not for cows treated with EB + P , and the variation was particularly
4
evident in the P5/P ratio. This finding shows that a single hormone
4
Non-parametric methods were chosen to evaluate the exis-
tence of statistically significant variations between animal groups
in terms of serum profiles. When multiple groups were compared,
then the Kruskall–Wallis analysis, which is the non-parametric
equivalent of analysis of variance/multivariate analysis of variance
could appear non-disturbed, as in the case of pregnenolone and T,
although a significant change is observed in terms of the ratio of
this hormone to related substances.
(
ANOVA/MANOVA) analysis, was used. If there are two inde-
2.4. Multivariate analysis
pendent groups of samples which could be compared by their
mean values for some variable of interest, the t-test for inde-
pendent samples is usually used; the Mann–Whitney U test is
Statistical analyses were performed on all continuous variables,
representing the levels of several unconjugated EGAs, using SIMCA-

P. Regal et al. / Steroids 76 (2011) 365–375
373
A
3
2
1
0
.0
.0
.0
.0
1
1
1
1
1
3
1
3
1
1
1
1
1
1
3
1
3
3
1
3
1
3
3
3
3
3
11
1
1
1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1
1
3
2
2
1
2
3
3
2
3
3
-
-
-
1.0
2.0
3.0
1
2
2
2
2
3
2
2
2
2
2
-5
-4
-3
-2
-1
0
1
2
3
4
5
t[1]
B
3
2
1
0
.0
.0
.0
.0
1
1
1
1
1
1
1
3
1
1
3
1
3
3
11
1
3
1
1
1
3
1
3
3 3
1
1
3
3
3
3
3
3 3
3
1
1
3
3
3
1
3
3
2
3
33
3
1
1
3
2
3
3
2
3
-
-
-
1.0
2.0
3.0
3
3
23
2
2
2
2
2
2
2
2
2
-5
-4
-3
-2
-1
0
1
2
3
4
5
t[1]
Fig. 4. OPLS-DA score plot derived from the HPLC–MS/MS data set consisting of hormonal profiles from animals treated with estradiol benzoate (1, red box), estradiol
benzoate and progesterone (2, green open triangle) and control animals (3, blue dot). The first score plot (A) shows OPLS-DA derived from the complete data set, and the
second (B) is derived from the data set without hormones that were not statistically different between groups. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
P + version 12.0 (Umetrics AB, Sweden). Unsupervised principal
component analysis (PCA) was run to obtain a general overview of
the variance in serum profiles, and supervised OPLS-DA was per-
formed to obtain information on the differences in the hormonal
profiles of samples.
ent between samples (T and its hydroxides, 7OHT and 19OHT,
and pregnenolone) were eliminated from the data set of OPLS-
2
2
DA (R (Y) = 0.70; Q (Y) = 0.61), and the three groups can clearly
be distinguished (Fig. 4). The S-plot from these two OPLS-DA
models showed a high sharing of oestrogens in discriminatory
power, androgens and especially T and its hydroxides being the
less powerful components in terms of discrimination. The conclu-
sions extracted from the S-plot were in agreement with the results
obtained from the statistical tests from the previous univariate
analysis.
2.5. PCA and OPLS-DA
As shown in the PCA score plot (Fig. 3), significant differences
were detected between animals treated intramuscularly with EB
and the control animals, but no difference was detected in the
animals treated with progesterone combined with intravaginal
EB, which were grouped with the control samples in the PCA
score plot. The OPLS-DA model showed a good discrimination
between the control and EB-treated animals, but the EB + P4-
treated animals were not clearly distinguished from the other
2.6. Predictive model
The prediction ability of the previous OPLS model was assessed
by a new OPLS built with two-thirds of the previous data set.
Forty-eight samples (two-thirds of the data) were used to build the
predictor set, and the other 24 (one-third) were used as the inde-
pendent validation set to be predicted. The first model was built
using the three groups of animals, and a second model included
only the animals treated intramuscularly and group C. The resulting
characteristics of the second model were better than in the first, in
which animals treated intravaginally appeared slightly grouped but
2
2
two groups (R (Y) = 0.63; Q (Y) = 0.50), as can be seen in Fig. 4.
When eliminating these 13 samples from the data set, the charac-
2
teristics the OPLS-DA model improved significantly (R (Y) = 0.76;
2
Q (Y) = 0.75), resulting in a better discrimination between the
EB group and the C group. A clear improvement could also be
observed when the hormones that were not statistically differ-

3
74
P. Regal et al. / Steroids 76 (2011) 365–375
A
4
3
2
1
0
2
5
2
4
1
4
2
2
1
4
1
2
5
3
26
3
4
4
4
6
3
1
3
6
6
2
2
1
3
3
5
1
3
6
4
3
1
5
3
1 1
5
1
1
6
11
3
6
3
-
1
2
3
4
3
3
3
3
3
3
14
-
-
-
6
3
3
1
3
3
1
3
6
3
6
6
-7
-6
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
7
tPS[1]
B
3.0
2.0
1.0
0.0
1
1
1
1
1
4
1
4
6
3
6
1
6
3
6
3
4
1
1
1
1
3
3
3
1
4
1
1
3
3
3
5
6 3
4
4
33 3
6
4
1
3
3
3
3
3
4
1
3
6
6
6
3
6
5
6
3
2
5
-
-
-
1.0
2.0
3.0
5
3
2
2
3
2
2
5
2
2
2
-5
-4
-3
-2
-1
0
1
2
3
4
5
tPS[1]
Fig. 5. Assessment of the predictive ability of the OPLS models built in Fig. 4, derived from the HPLC–MS/MS data set consisting of hormonal profiles from animals treated
with estradiol benzoate (1, red box), estradiol benzoate and progesterone (2, green open triangle) and the control animals (3, blue dot). Here, two-thirds of the data were
used for building the predictive model, and one-third of the data was used for the prediction, represented in the figure as follows: estradiol benzoate (4, orange box), estradiol
benzoate and progesterone (5, green triangle) and control (6, purple dot). (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
were mixed with group C (Fig. 5). The independent set was success-
Acknowledgements
2
fully predicted in the second predictive OPLS model (R (Y) = 0.77;
2
Q (Y) = 0.73).
We express our thanks for financial support received from
Xunta de Galicia Programa Sectorial de Tecnoloxías da Ali-
mentación, PGIDIT07TAL025261PR. Ms. Regal acknowledges a
fellowship from Ministerio de Educación, Formación de Profe-
sorado Universitario (FPU) del Programa Nacional de Formación
de Recursos Humanos de Investigación, Plan Nacional de Investi-
gación Científica, Desarrollo e Innovación Tecnológica 2008–2011
(Spain).
3
. Conclusions
In general, the obtained results showed that the main ester of
estradiol-17␤, EB, leads to changes in serum profiles of cattle with
regard to sex steroid hormones. Variations in the serum profile after
the administration of EB were not as evident in animals treated
intravaginally as they were in animals treated intramuscularly with
EB. The hormones that were not found to be evidently modified
according to a first evaluation produced significant hormonal ratios.
Multivariate analysis has hinted at the possibility of developing a
future analysis using steroid serum profiles with screening pur-
poses, as a contribution to the fight of the European Union against
the prevalent use of illegal treatments with hormones in stock
farming. However, further research in terms of age, breed, sex,
nutritional status and/or animal location must be developed as
well, as it is highly necessary to assess long-term administrations
of EB and the possible existence or the absence of long-term serum
disturbances associated with these treatments.
References
[
1] Kinsella B, O’Mahony J, Malone E, Moloney M, Cantwell H, Furey A, et al. Current
trends in sample preparation for growth promoter and veterinary drug residue
analysis. J Chromatogr A 2009;1216:7977–8015.
[
2] De Brabander HF, Le Bizec B, Pinel G, Antignac J-P, Verheyden K, Mortier
V, et al. Past, present and future of mass spectrometry in the analysis of
residues of banned substances in meat-producing animals. J Mass Spectrom
2007;42(8):983–98.
[
3] Hancock DL, Wagner JF, Anderson DB. Effects of estrogens and androgens on
animal growth. In: Pearson AM, Dutson TR, editors. Growth regulation in farm
animals: advances in meat research. London: Elsevier Applied Science; 1991.
p. 255–97.

P. Regal et al. / Steroids 76 (2011) 365–375
375
[
[
4] Courtheyn D, Le Bizec B, Brambilla G, De Brabander HF, Cobbaert E, Van De Wiele
M, et al. Recent developments in the use and abuse of growth promoters. Anal
Chim Acta 2002;473(1–2):71–82.
5] Lone KP. Natural sex steroids and their xenobiotic analogs in animal
production: growth, carcass quality, pharmacokinetics, metabolism, mode
of action, residues, methods, and epidemiology. Crit Rev Food Sci Nutr
[22] Hartmann S, Lacorn M, Steinhart H. Natural occurrence of steroid hormones in
food. Food Chem 1998;62(1):7–20.
[23] Bichon E, Kieken F, Cesbron N, Monteau F, Prévost S, André F, et al. Devel-
opment and application of stable carbon isotope analysis to the detection
of cortisol administration in cattle. Rapid Commun Mass Spectrom 2007;21:
2613–20.
1
997;37(2):93–209.
[24] Cawley AT, Trout GJ, Kazlauskas R, Howe CJ, George AV. Carbon isotope
ratio (␦13C) values of urinary steroids for doping control in sport. Steroids
2009;74:379–92.
[25] Gardini G, Del Boccio P, Colombatto S, Testore G, Corpillo D, Di Ilio C, et al.
Proteomic investigation in the detection of the illicit treatment of calves with
growth-promoting agents. Proteomics 2006;6(9):2813–22.
[
6] Cavalieri J, Rabiee AR, Hepworth G, Macmillan KL. Effect of artificial insem-
ination on submission rates of lactating dairy cows synchronised and
resynchronised with intravaginal progesterone releasing devices and oestra-
diol benzoate. Anim Reprod Sci 2005;90(1–2):39–55.
[
[
[
7] Council Directive 96/22/EC. Off J Eur Comm 1996;L125:3–9.
8] Council Directive 96/23/EC. Off J Eur Comm 1996;L125:10–32.
9] Commission Decision 2002/657/EC. Off J Eur Comm 2002;L221:8–36.
[26] Mooney MH, Situ C, Cacciatore G, Hutchinson T, Elliott C, Bergwerff AA. Plasma
biomarker profiling in the detection of growth promoter use in calves. Biomark-
ers 2008;13(3):246–56.
[
[
10] Directive 2003/74/EC. Off J Eur Comm L262:17–21.
11] Lane EA, Austin EJ, Crowe MA. Oestrous synchronisation in cattle-current
options following the EU regulations restricting use of oestrogenic compounds
in food-producing animals: a review. Anim Reprod Sci 2008;109(1–4):1–16.
12] Directive 2008/97/EC. Off J European Union 2008; L318:9–11.
13] Stephany R. Hormones in meat: different approaches in the EU and in the USA.
APMIS 2001;109:S357–63.
[27] Della Donna L, Ronci M, Sacchetta P, Di Ilio C, Biolatti B, Federici G, et al. A
food safety control low mass-range proteomics platform for the detection of
illicit treatments in veal calves by MALDI-TOF-MS serum profiling. Biotechnol
J 2009;4:1596–609.
[28] Antignac J-P, Brosseaud A, Gaudin-Hirret I, Andre ˇı F, Bizec BL. Analytical strate-
gies for the direct mass spectrometric analysis of steroid and corticosteroid
phase II metabolites. Steroids 2005;70(3):205–16.
[29] Maume D, Le Bizec B, Pouponneau K, Deceuninck Y, Solere V, Paris A,
et al. Modification of 17␤-estradiol metabolite profile in steer edible tis-
sues after estradiol implant administration. Anal Chim Acta 2003;(483):
289–97.
[
[
[
14] Duffy E, Rambaud L, Le Bizec B, O’Keeffe M. Determination of hormonal
growth promoters in bovine hair: comparison of liquid chromatography-
mass spectrometry and gas chromatography–mass spectrometry methods
for estradiol benzoate and nortestosterone decanoate. Anal Chim Acta
2
009;637(1–2):165–72.
[
[
[
[
15] Kaklamanos G, Theodoridis G, Papadoyannis IN, Dabalis T. Determination of
anabolic steroids in muscle tissue by liquid chromatography–tandem mass
spectrometry. J Agric Food Chem 2007;55(21):8325–30.
16] Noppe H, Le Bizec B, Verheyden K, De Brabander HF. Novel analytical methods
for the determination of steroid hormones in edible matrices. Anal Chim Acta
[30] Becue I, Van Poucke C, Rijk JCW, Bovee TFH, Nielen M, Van Peteghem C. Investi-
gation of urinary steroid metabolites in calf urine after oral and intramuscular
administration of DHEA. Anal Bioanal Chem 2010;396:799–808.
[31] Cunningham RT, Mooney MH, Xia X-, Crooks S, Matthews D, O’Keeffe
M, et al. Feasibility of
a clinical chemical analysis approach to predict
2
008;611(1):1–16.
misuse of growth promoting hormones in cattle. Anal Chem 2009;81(3):
977–83.
17] Regal P, Nebot C, Vázquez BI, Cepeda A, Fente CA. Determination of 17␣-
methyltestosterone in bovine serum using liquid chromatography tandem
mass spectrometry. J Food Drug Anal 2010;18(1):51–7.
18] Simontacchi C, Perez De Altamirano T, Marinelli L, Angeletti R, Gabai G.
Plasma steroid variations in bull calves repeatedly treated with testosterone,
nortestosterone and oestradiol administered alone or in combination. Vet Res
Commun 2004;28(6):467–77.
19] Scippo M-, 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 1994;119(12):2639–44.
20] Stolker A, Groot M, Lasaroms J, Nijrolder A, Blokland M, Riedmaier I. Detectabil-
ity of testosterone esters and estradiol benzoate in bovine hair and plasma
following pour-on treatment. Anal Bioanal Chem 2009;395(4):1075–87.
21] Regal P, Vázquez BI, Franco CM, Cepeda A, Fente CA. Development of a rapid
and confirmatory procedure to detect 17␤-estradiol 3-benzoate treatments in
bovine hair. J Agric Food Chem 2008;56(24):11607–11.
[32] Regal P, Vázquez BI, Franco CM, Cepeda A, Fente C. Quantitative LC–MS/MS
method for the sensitive and simultaneous determination of natural hormones
in bovine serum. J Chromatogr B 2009;877(24):2457–64, 8/15.
[33] Eriksson L, Antti H, Gottfries J, Holmes H, Johansson E, Lindgren F, et al. Using
chemometrics for navigating in the large data sets of genomics, proteomics and
metabolomics (gpm). Anal Bioanal Chem 2004;380:419–29.
[34] Trygg J, Wold S. Orthogonal projections to latent structures (O-PLS). J Chemom
2002;16:119–28.
[35] Bylesjö M, Rantalainen M, Cloarec O, Nicholson JK, Holmes E, Trygg J. OPLS
discriminant analysis: combining the strenghts of PLS-DA and SIMCA classifi-
cation. J Chemometr 2006;20:341–51.
[36] Fritsche S, Rumsey T, Meyer H, Schmidt G, Steinhart H. Profiles of steroid hor-
mones in beef from steers implanted with synovex-S (estradiol benzoate and
progesterone) in comparison to control steers. Z Lebensm Unters Forsch A
1999;208(5–6):328–31.
[
[
[