Download Accumulation of Ractopamine Residues in Hair

Journal of Analytical Toxicology 2013;37:117 –121
doi:10.1093/jat/bks092 Advance Access publication January 9, 2013
Article
Accumulation of Ractopamine Residues in Hair and Ocular Tissues of Animals during
and after Treatment
Jelka Pleadin1, Ana Vulić1*, Nina Perši1, Svjetlana Terzić2, Miroslav Andrišić2, Irena Žarković2, Ksenija Šandor2, Eleonora Perak2 and
Željko Mihaljević3
1
Laboratory for Analytical Chemistry, Croatian Veterinary Institute, Savska cesta 143, 10000 Zagreb, Croatia, 2Laboratory for
Analyses of Veterinary Medicinal Products, Croatian Veterinary Institute, Savska cesta 143, 10000 Zagreb, Croatia, and 3Deparment
for Pathology, Croatian Veterinary Institute, Savska cesta 143, 10000 Zagreb, Croatia
*Author to whom correspondence should be addressed. Email: [email protected]
The aim of the present study was to assess the accumulation of
ractopamine residues in the hair and ocular tissues of guinea pigs
during repeated ractopamine administration and after treatment.
The experiment was conducted in 38 guinea pigs (30 treated and
eight controls). Treated animals were orally administered ractopamine hydrochloride in a dose of 3.5 mg/kg body mass per day
using probes for seven consecutive days. Ractopamine concentration was determined in hair during the treatment (Days 1, 3 and 7)
with ractopamine hydrochloride and in ocular tissues and hair on
defined days after exposure (Days 1, 10, 20 and 30). Residues were
present in hair in high concentrations as early as Day 3 (86.15 +
87.71 ng/g) and Day 7 (85.25 + 56.97 ng/g). After exposure, residues were found to persist, having depleted from 68.06 +
30.54 ng/g on Day 1 to 8.01 + 2.22 ng/g on Day 30, with a significantly higher concentration in hair in contrast to low residue levels in
ocular tissues (1.20–0.34 ng/g). The results of the study pointed to
high ractopamine accumulation, even in non-pigmented hair, suggesting hair to be used as a matrix in the control of ractopamine abuse in
farm animals because of its many advantages over ocular tissues.
Introduction
Ractopamine is a phenethanolamine (Figure 1) from the group of
b-adrenergic agonists that improves the carcass composition
of animals for meat production by decreasing fat to the benefit
of muscle mass, gaining higher economic benefit to producers
(1–2). It increases the amount of lean meat and decreases the
amount of carcass fat, and its supplementation during the last
24–42 days of feeding improves live weight performance parameters by as much as 26% (3). The biochemical basis of ractopamine effects implies increasing nitrogen retention and protein
synthesis, enhancing lipolysis and suppressing lipogenesis (4–7).
Ractopamine has been approved as a feed additive for swine
and cattle in many countries, including the United States (1),
but its use is prohibited in the European Union (8). Although
all incidents of poisoning have been caused by clenbuterol as a
representative of the group of b-agonists, the European Union
has placed a ban upon the use of these substances and requires
strict monitoring for their illegal use. Because ractopamine is a
b-agonist classified into group A, i.e., unauthorized substances
and substances that have an anabolic effect, its determination is
necessary according to the analytical performance criteria of
Commission Decision 2002/657/EC (9). The selection of a
suitable matrix is necessary for ractopamine determination,
which could indicate its abuse for a long period of time during
fattening and after slaughtering in meat production.
Several analytical methods for the detection of ractopamine
residues in animal tissues have been reported, including
liquid chromatography –mass spectrometry (LC – MS) (10),
gas chromatography –mass spectrometry (GC –MS) (11), LC –
tandem MS (MS-MS) (12 –14), and immunoassays (15 –17).
Immunoassays are usually utilized as a rapid and high-capacity
screening method for monitoring purposes. Enzyme-linked immunosorbent assays (ELISAs) are the most common approach
for screening ractopamine in animal tissues (18).
Earlier investigations by the authors have shown that ocular
tissues are appropriate for the control of the illegal use of the
b-agonist clenbuterol because of its accumulation in retinas
and the possibility of residue determination for a long period
after the cessation of treatment (19 –20). Also, studies have
revealed many advantages of b-agonist determination in animal
hair of different colors (21 –24). Literature data are very limited
on the accumulation of ractopamine in pigmented tissues such
as hair and ocular tissues during treatment and after exposure.
Some studies have presented analyses of ractopamine residues,
mostly performed in body fluid samples taken from living animals
(25–28) and from edible tissues such as liver, kidney and muscle
(17, 29–32). Studies of ractopamine residues have demonstrated
its accumulation in pig hair (13) and have revealed that ractopamine does not accumulate in ocular tissues of some animal
species. Hence, if ocular tissues are used to determine ractopamine exposure, the analytical method employed should have a
sensitivity normally provided by immunoassay or MS (33).
The aim of this study was to evaluate the accumulation potential of residual ractopamine in hair and ocular tissues of
treated guinea pigs by the determination of ractopamine residues on defined days during treatment and for 30 days after exposure. For the quantitative determination of ractopamine
residue in both matrices, a sensitive ELISA was developed as a
screening method.
Materials and Methods
Chemicals and apparatus
A Ridascreen ractopamine kit for ELISA was provided by
R-Biopharm (Darmstadt, Germany). Each kit contains a microtiter plate with 96 wells coated with antibodies to rabbit IgG,
ractopamine standard solution concentrations of 0, 100, 300,
900, 2,700 and 8,100 ng/mL, peroxidase conjugated ractopamine, anti-ractopamine antibody, substrate/chromogen (tetramethylbenzidine), stop reagent (1 M sulfuric acid), sample
dilution buffer and washing buffer (10 mM phosphate buffer,
pH 7.4). Ractopamine hydrochloride standard was provided by
# The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Chemical structure of ractopamine.
Sigma-Aldrich-Chemie (Steinheim, Germany). Bond Elute
Certify solid-phase extraction (SPE) columns (500 mg, 6 mL)
were provided by Varian (Harbor City, CA). All other chemicals
used in the analysis were of analytical grade. ELISA was performed by the use of a ChemWell 2910 (Awareness
Technology Inc., Palm City, FL). Statistical data analysis was performed by use of Statistica software, version 6.1 (StatSoft Inc.,
Tulsa, OK) with statistical significance set at the level of 95%
(P ¼ 0.05).
Animals and sampling procedure
The experiment was conducted in 38 male Weiser-Maples
guinea pigs (non-pigmented hair) with body masses of 500 to
700 g. All animals were kept under controlled conditions
(temperature at 20 –228C and relative air humidity at 50 +
5%) and divided into two groups (treated and control). Thirty
treated animals were orally administered ractopamine hydrochloride by probes, 3.5 mg/kg body mass per day for seven
days, in the form of a solution in water. The applied volume
of ractopamine hydrochloride solution was adjusted to body
mass per animal throughout the study. Eight animals were left
untreated and served as a control group. All animals were
fed ad libitum and had free access to water. Hair samples
(non-pigmented hair) were collected by shaving animals with
a razor blade on Days 1 (after first application), 3 and 7
during the treatment with ractopamine hydrochloride and on
Days 1, 10, 20 and 30 after exposure. On the same days after
exposure, animals were randomly sacrificed in groups of eight
(Days 1 and 10) or seven (Days 10 and 20) and eyes from
each animal were collected. Control animals were sacrificed
on Day 1 after the exposure of the treated group. Hair
samples were stored at room temperature, whereas eyes
were frozen at –208C until analysis of residual ractopamine.
The experimental protocol was designed in accordance
with current regulations and standards issued by the Ministry
of Agriculture, Fishery and Rural Development. The research
protocol was approved by the Ethics Committee of the
Croatian Veterinary Institute.
Preparation and extraction procedure for hair samples
Hair samples were washed with 2 50 mL of distilled water,
dried overnight and stored at room temperature until analysis
of residual ractopamine. After washing and drying, 250 mg of
hair samples were put in test tubes with the addition of 2 mL
of 1 M NaOH. The mixture was gently shaken on a mini-shaker
and heated in a water bath at 658C for 2 h. After cooling at
room temperature, 1.75 mL of 1M HCl and 2.5 mL of 0.25 M
sodium acetate buffer were added. The tubes were then centrifuged for 10 min at 2,000 g. The supernatants were loaded to
SPE columns and cleaned up as described by Nielen et al. (12).
Residues were dissolved in appropriate volumes of distilled
water and applied to the wells of the ELISA kit.
118 Pleadin et al.
Preparation and extraction procedure for eye samples
Whole eyes of animals were weighed into ground centrifuge
tubes and used for the analysis of ractopamine residues. Then,
10 times the sample weight of tris buffer ( pH 8) was added to
the tube and homogenized with the potter. After homogenization, 4 g of suspension was weighed, 40 mL of tris buffer was
added and the mixture was homogenized again in an ultrasonic
bath. Subsequently, 100 mL of the protease solution (50 mg/mL
in water) was added; the samples were vortexed and incubated
overnight at 50– 608C. The next day, the samples were centrifuged for 10 min at 48C and 3,000– 4,000 rpm. The upper layer
was added to a new tube and the centrifugation residue was
extracted twice with 1 mL phosphate buffer ( pH 6), with the
centrifugation repeated each time. The supernatants were combined and the pH was adjusted at 6. The samples were then
loaded to SPE cartridges conditioned with 2 mL of methanol,
2 mL of water and 2 mL of phosphate buffer at pH 6, respectively. The cartridges were washed with 1 mL of 1 M acetic acid and
evaporated to dryness, followed by washing with 2 mL of methanol and evaporating to dryness. The elution was performed with
6 mL of a mixture consisting of ethyl acetate and ammonia at a
ratio of 97:3. The samples were evaporated to dryness under a
stream of nitrogen at 358C. The residues were dissolved in 1 mL
of water and applied to the wells of the ELISA kit.
Analysis of ractopamine
Competitive ELISA was performed as described in the package
insert provided by the manufacturer. Method specificity, determined by analyzing the cross-reactivity to substances similar to
ractopamine, is also provided in the ELISA kit (dobutamine
4.7%, mabuterol 4.4%, ritodrine 1.4%, clenpropanol and cimaterol ,0.1%, and all others ,0.01%). Microtiter strips coated
with sheep antibodies directed against anti-ractopamine were
inserted into the microwell holder for the standards and
samples to be analyzed in duplicate. Using the device, 100 mL of
the diluted antibody solution was added to the microwells and
mixed gently, and the plate was incubated at room temperature
for 15 min. The wells were emptied completely and washed
three times with 250 mL of washing buffer. Then, 20 mL of ractopamine standards (0, 100, 300, 900, 2,700 and 8,100 ng/L)
and prepared samples were added to the microwells. To each
microwell, 100 mL of diluted enzyme conjugate were added,
mixed gently and incubated for 60 min at room temperature.
After washing, 100 mL of the substrate/chromogen solution
were added to all wells and incubated in the dark for 15 min at
room temperature. The reaction was stopped by adding 100 mL
of stop reagent and the absorbance was measured on a microplate reader at 450 nm. The limit of detection (LOD) and limit of
quantification (LOQ) of the method were obtained by adding 3
and 10 times the standard deviations (SDs) of 10 blank samples
to the mean blank value. When calculating the concentration of
ractopamine, the results obtained from the calibration curve
were multiplied with the corresponding dilution factor.
Results and Discussion
Previous studies have indicated the accumulation of
b-adrenergic agonists in pigmented tissues (21, 34), with a considerably lower level of residues in white than in dark hair (22,
35). Their binding in non-colored hair has been suggested to be
possibly attributable to the matrix component or residual
melanin (21). Earlier investigations have shown that pigmented
tissues such as ocular tissue (retina) and hair (also white hair)
have a high accumulation potential for clenbuterol as the
primary representative of b-adrenergic agonists (19, 20, 23, 24).
The advantages of hair as a novel matrix in the control of the
illegal use of b-agonists include simple and rapid sampling
during animal fattening (live animals) and residue persistence
in this matrix for a significantly longer time after treatment cessation (36). Pharmacokinetic studies have shown the retina to
accumulate up to 35-fold levels of b-agonists found in other
tissues, confirming the eye to be a very good target organ to estimate residual clenbuterol. The high level of residual clenbuterol in retinal tissue is attributed to the high clenbuterol
binding affinity for melanin found in the pigmented segment of
the eye (21, 37).
Very few data are currently available on the accumulation of
ractopamine residue in hair and ocular tissues as target matrices during and after animal exposure to an anabolic dosage of
ractopamine (13, 25, 29). In a previous study in pigs administered a significantly lower than anabolic dose of ractopamine
(0.51 mg/kg by weight), the accumulation of ractopamine residues in hair (white) was assessed over a short period of time
after exposure (eight days). The mean concentration of ractopamine determined in treated pig hair samples was 12.12 +
2.42 ng/g on Day 1, 11.52 + 1.99 ng/g on Day 3 and 8.77 +
1.13 ng/g on Day 8 of withdrawal, pointing to ractopamine accumulation in this matrix (13).
Hence, in the present study, ractopamine accumulation was
investigated in hair samples (also non-pigmented) of guinea
pigs during the treatment, on days after exposure for a longer
period (30 days) and in ocular tissues (whole eyes) on the
same days after exposure. The LOD and LOQ values of the
method were 0.4 and 0.8 ng/g for hair and 0.3 and 0.6 ng/g for
eyes, respectively. A typical ELISA standard curve for the determination of ractopamine concentrations in hair and eye
samples is presented in Figure 2.
The concentrations of ractopamine residues determined in
hair samples on days during the treatment and after exposure
are shown in Figure 3.
The analysis performed on hair samples revealed them to
contain residual ractopamine during treatment and for 30 days
after exposure. On Day 1 of treatment, the hair sampled immediately after administration of the first ractopamine dose contained a ractopamine concentration approximately at the LOD
of the method (0.4 ng/g). On Day 3 of treatment, a high ractopamine concentration (86.15 + 87.71 ng/g) was recorded,
with wide value variation ranging from a minimal value of
10.6 ng/g to a maximal value of 243.5 ng/g, pointing to a significant accumulation of ractopamine as early as Day 3 of treatment. Other reports, mostly relating to the study of clenbuterol
residues, show that clenbuterol could be detected in hair from
Day 4 of treatment in male veal calves of different coat color;
in addition, the level of clenbuterol accumulation was found to
rise with the duration of treatment and to persist for a long
period of time after withdrawal (22).
In the present study, on Day 7 of treatment, the mean ractopamine concentration (85.25 + 56.97 ng/g) corresponded
closely to the mean concentration determined on Day 3 of
treatment. All determined concentrations were significantly
higher than those recorded in the control group, and in
Figure 2. ELISA standard curve for ractopamine.
Accumulation of Ractopamine Residues in Hair and Ocular Tissues of Animals during and after Treatment 119
Table I
Concentrations of Ractopamine Residues in Eyes on Days after Exposure
Day after
exposure
Number of determined
concentrations
Ractopamine concentration + SD
(ng/g)
1
10
20
30
8*
5†
3‡
3‡
1.20 + 0.92
0.61 + 0.55
0.37 + 0.05
0.34 + 0.07
*Residues were detectable in all animals (samples).
†
Three of eight animals had no detectable residues (,LOD).
‡
Four of seven animals had no detectable residues (,LOD).
Figure 3. Mean (+ SD) ractopamine concentrations in hair samples on days during
treatment and after exposure.
comparison to the LOD of the method. On Days 1, 10, 20 and
30 after exposure, ractopamine concentration was 68.06 +
30.54, 13.22 + 13.15, 11.11 + 3.69 and 8.01 + 2.22 ng/g, respectively, pointing to slow depletion from Day 10 to Day 30
after exposure. The significant depletion recorded in the
period from Day 1 to Day 10 after exposure may have been
because of the respective stage of hair growth (negative hair
growth), suggesting that the residual level of ractopamine in
hair sample extracts depends on the growth stage at ractopamine administration.
Furthermore, no distinction was made regarding hair length,
and animal hair for residue analysis was whole length hair
(normal hair), so it could be used in animal production during
fattening or after slaughtering. The determination of high concentrations of ractopamine residues in the hair of treated
animals demonstrated hair (even if non-pigmented) to be a
useful matrix for monitoring the abuse of ractopamine in meat
production, even 30 days after the last exposure.
The concentrations of ractopamine residues determined in
eyes (whole-eye homogenates) on days after exposure are
shown in Table I.
The ractopamine residues recorded on Day 1 after exposure
in the eyes yielded a mean concentration of 1.20 + 0.92 ng/g.
On Days 10, 20 and 30, ractopamine concentrations had still
decreased and were only determined in some study samples
with values slightly over (Day 10) or approximately at the LOD
of the method (Days 20 and 30).
The results of this study are consistent with those reported
by Churchwell et al. (29), in which low levels of ractopamine
(0.5 –3 ng/g) were determined after a seven-day treatment
with 20 mg/kg ractopamine in the diet, with concentrations of
unconjugated ractopamine in ovine and bovine retina slightly
increasing over the seven-day withdrawal period. Furthermore,
literature data on the accumulation of ractopamine in ocular
tissues vary greatly depending on many parameters, such as
animal species, pigmentation, ractopamine dose applied, duration of treatment and eye segment analyzed (whole eye, retina
or choroids). A disadvantage of ocular tissues as a matrix in the
control of anabolic misuse is the very small amount of sample
available for analysis, which also depends on animal species,
i.e., body weight.
120 Pleadin et al.
The treatment of pigs with 10 mg of ractopamine per day for
10 days resulted in a high amount of ractopamine residues
detected in retinas (19,480 ng/g) and demonstrated the high affinity for accumulation due to the large number of b-agonist
receptors in this tissue (25). A study evaluating ractopamine accumulation in ocular tissue was conducted on cattle and turkeys
by Smith et al. (33). After a seven-day treatment with [14C] ractopamine of turkeys, fed with a diet containing 7.5, 22.5 and
30 mg ractopamine per kg of feed, ractopamine residues could
be detected in retina/choroids/sclera and cornea/iris tissues,
but not in aqueous humor. The highest mean concentrations of
ractopamine were found in retina/choroids/sclera tissues from
animals fed with a diet containing 30 mg/kg of [14C] ractopamine. After seven days of ruminant treatment with [14C] ractopamine in a dose of 0.90 mg/kg, there were no detectable residues
of ractopamine in whole eye homogenates obtained on Days 2,
4 and 6 of withdrawal. The authors concluded that the residues
of ractopamine did not accumulate in pigmented ocular tissues
of cattle, to the extent to which the residues of other b-agonists
accumulate in ocular tissues of live-stock species (33). Data
obtained in this study confirmed that ractopamine accumulation
in ocular tissues could not be compared to b-agonist clenbuterol, so ocular tissues may not be a safe and useful matrix for
monitoring ractopamine illegal use.
Conclusion
The present study results pointed to a significantly higher accumulation of ractopamine in hair than in ocular tissues. The
results suggested a low accumulation of ractopamine in ocular
tissues and recommended to their limited use in ractopamine
residue determination. Taking into account the many advantages of hair as a matrix in the control of illegal use, the high
accumulation of ractopamine in hair, even non-pigmented hair,
over a short period of ractopamine administration and for a
long period after exposure, animal hair can be proposed as a
useful matrix in the regulatory monitoring of the misuse of the
b-adrenergic agonist ractopamine during fattening and after
slaughtering of farm animals.
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