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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. 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