Journal of Analytical Toxicology Advance Access published April 23, 2015 Journal of Analytical Toxicology 2015;1 – 7 doi:10.1093/jat/bkv038

Article

Clenbuterol Distribution and Residues in Goat Tissues After the Repeated Administration of a Growth-Promoting Dose Zhen Zhao1, Ting Yao1, Yuchang Qin2, Xiaowei Yang3, Jun Li1, Junguo Li1 and Xu Gu1* 1

Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 10081, PR China, 2Institute of Food and Nutrition Development, Ministry of Agriculture, Beijing 10081, PR China, and 3 Supervision Institute of Veterinary Medicine and Feed of Tianjin, Tianjin 300402, PR China *Author to whom correspondence should be addressed. Email: [email protected]

This study was conducted to investigate the deposition and depletion process of clenbuterol (CL) in goat tissues, plasma and urine after the repeated administration of a growth-promoting dose. The experiment was conducted in 24 goats (21 treated and 3 controls). Treated animals were administered orally in a dose of 16 mg/kg body mass once daily for 21 consecutive days and randomly sacrificed on days 0.25, 1, 3, 7, 14, 21 and 28 of the withdrawal period. CL in goat tissues was extracted with organic solvents and determined using liquid chromatography tandem mass spectrometry. The depletion rates of tissue differed significantly. The highest concentrations of CL in all tissues are detected on day 0.25 of treatment discontinuation. After administration had been discontinued for 28 days, CL still residues in all tissues, especially, in whole eye, where the concentrations reach 363.29 + 31.60 mg/kg. These findings confirmed that the whole eye, which are rich in pigment, showed a much higher concentration than any other studied tissue during the withdrawal period.

Introduction b2-adrenergic agonists can be used as growth promoters in economically important animals. Clenbuterol (CL), 4-amino3,5-dichloro-alpha-[[(1,1-dimethylethyl)amino]methyl]benzenemethanol, is an efficient b2-adrenoceptor agonist; it was originally developed for the clinical treatment of bronchial obstructions (1). If administered at a concentration 5 – 10 times higher than the therapeutic dose, it can promote protein deposition and increase fat biolysis in various vertebrates and, thus, increase muscle growth (2, 3). The 3,5-dichlone substitution pattern gives CL a higher lipid solubility and longer half-life than other b-agonists. It also prevents CL from metabolizing, causing its accumulation and the deposition of its residues in livestock products (4). Due to its high lipid solubility (log P ¼ 2.47), CL readily crosses membranes and easily passes the blood –brain barrier (5). In addition to its illegal use as a stimulant to improve performance in human athletes, CL has been widely abused to increase lean meat production and promote the growth of farm animals in certain Asian countries (6). Although CL has been banned for more than 10 years, the illicit use of CL has caused a number of food poisoning events that have occurred following the consumption of CL-contaminated liver or meat (7 –10). Most Asian countries and the Commissions of European Communities (11) have banned the use of all these additives in animal feed. While in recent years, a tendency for the illegal use of b-adrenergic agonists in ruminants has been reported (12). To our knowledge, there are several studies reported the residues in the tissues, the elimination in urine and the bio-availability in plasma in bovine and equidae (13 – 16), but the studies in ovine, especially in goats, are few. Hence, it is necessary to investigate the CL residues in

the tissues of livestock and to compare residual concentrations between various tissues to facilitate the prevention of the illegal use of this xenobiotic in goats. The aim of this study was to clarify the deposition and depletion rules of CL in different tissues of the goat to provide an appropriate regulatory matrix for the control of the abuse of this compound. Liquid chromatography tandem mass spectrometry (LC–MS-MS) was used to determine the content of CL in plasma, urine and tissues after repeated exposure to an anabolic dosage of CL. Materials and methods Chemicals CL with a purity of 98.5% was obtained from Dr Ehrenstorfer GmbH (Augsburg, Germany). A stable-isotope-labeled internal standard was used in the LC – MS-MS experiments; [2H9]-CL (99.4% isotopic purity) was purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany). Automatic solid-phase extraction apparatus (Fotector-06C) was provided by Reeko Technologies (XiaMen, China). The methanol used for the analysis was HPLC grade, as were all other chemicals and solvents used in the analysis. The solvents used for LC–MS-MS analysis were of HPLC grade. Animals and sampling procedure The research on animals performed in this study was conducted under the guidelines for animal experiments and standards of the Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China. The experiment was performed on 24 male goats (21 treated and 3 controls). The subjects were crossbred Boer and Haimen goats with an average body weight of 30 kg. The animals were farm-bred and were maintained under the same conditions. The 21 treated goats received the same treatment, the administration of oral irrigation CL at 16 mg/kg body mass once daily for 21 days. The goats were fed a standard diet ad libitum and had free access to fresh water for 21 consecutive days. Three medicated animals were randomly slaughtered on each sampling day, namely, days 0.25, 1, 3, 7, 14, 21 and 28 of the withdrawal period. All control animals were sacrificed on day 0 after the discontinuation of CL in the treated group. Tissue samples, including muscle, liver, kidney, heart, lung, whole eye, fat, plasma, urine and bile, were collected and kept at 2408C prior to analysis of the CL residues. Preparation of standard solutions A stock solution of 1,000 mg/L of CL was prepared as follows: 100 mg of CL was transferred into 100 mL volumetric flasks and diluted with methanol. The solution was stored at 48C (at

# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

this temperature, it remains stable for 6 months). Calibration standards of CL at 0.5, 5, 10, 20, 50, 100, 200 and 500 mg/L were prepared by combining 2 mM of an aqueous solution of aqueous ammonium acetate with 0.1% formic acid in water. The injection volume was 100 mL.

Sample preparation All samples preparation methods were developed according to the previous report by Tang et al. (17) with some changes. Briefly, all samples were hydrolyzed with 50 mL of b-glucuronidase/arylsulfatase at 378C for 16 h. After centrifugation at 8,000g for 10 min, for the fat and eye samples, the supernatant was transferred into a new centrifuge tube. A total of 3 mL n-hexane was added to the tube. The tube was then vortex mixed for 2 min to extract the fat or pigment. The n-hexane phase was abandoned. A total of 3 mL n-hexane was then added for a second extraction. Lastly, the subnatant was loaded onto the SPE columns (3 mL, 60 mg; Waters). For other samples, the supernatant was loaded onto the SPE columns, directly.

Apparatus and chromatographic conditions The extracts were analyzed using LC–MS-MS on an Agilent 1200 series HPLC and an Agilent 6460 triple quadrupole mass spectrometer. The column was an Atlantis T3 column (2.1 mm  150 mm, 3 mm particle size; Waters, Milford, MA, USA). Chromatographic separation was performed with the multiple reaction monitoring (MRM) mode in the positive ESI mode. Analytical data were obtained from the area ratios of the m/z 277/203 and m/z 286/ 204, respectively, for CL and for [2H9]-CL. The gradient elution used two solutions: solution A was prepared by combining 2 mM aqueous ammonium acetate aqueous solution and 0.1% formic acid in water, and solution B was constituted with acetonitrile. The gradient elution program was as follows: solution A was changed from 75% at 0 min to 20% at 5 min; changed from 20% at 5 min to 10% at 5.5 min; held 0.5 min; changed from 10% at 6 min to 75% at 6.1 min; held 4.9 min; the total run time was 11 min. A flow rate of 0.3 mL/min was used in all steps.

Calibration curves and assay validation The blank tissues used for method validation were first analyzed by the above-described method, and no CL residue was detected. The standard deviation (SD) and the relative standard deviation (RSD) (RSD ¼ SD/mean  100%) were calculated over the entire calibration range. Recovery estimation was performed over three concentrations (0.5, 5 and 10 mg/kg for muscle and fat; 0.5, 5 and 20 mg/kg for plasma; 0.5, 10 and 50 mg/kg for lung, heart and brain; 0.5, 50 and 100 mg/L for bile and urine; 0.5, 50 and 200 mg/kg for liver and kidney and 0.5, 100 and 1,000 mg/kg for eye). These samples were analyzed by LC – MS-MS, and the signal-to-nose (S/N) ratio was recorded. The limit of detection (LOD) and limit of quantification (LOQ) for each analyte were defined as the concentrations in the tissue, plasma, urine or bile samples that produced a S/N ratio of 3 and 10, respectively. Curves of the concentration in tissue materials (Y, mg/kg or mg/L) vs. the time elapsed since the discontinuation of the treatment (X, days) were obtained with a nonlinear regression analysis (GraphPad Prism 6.0). 2 Zhao et al.

Table I The Concentration (mg/L) Range and Equation of Linear Calibration Curves Tissue

Concentration

Equation

R2

Liver Lung Muscle Kidney Heart Fat Eyes Plasma Urine Bile

0.5 –200 0.5 –50 0.5 –10 0.5 –200 0.5 –50 0.5 –10 0.5 –1,000 0.5 –20 0.5 –100 0.5 –100

Y ¼ 0.6434x 2 0.1073 Y ¼ 1.6169x þ 0.2278 Y ¼ 04780x þ 0.1035 Y ¼ 0.3705x þ 0.1169 Y ¼ 1.2385x þ 0.0141 Y ¼ 1.5491x 2 0.0025 Y ¼ 0.1302x þ 0.0089 Y ¼ 0.7138x þ 0.0318 Y ¼ 1.3597x þ 0.0613 Y ¼ 1.1944x þ 0.0045

0.9989 0.9941 0.9994 0.9990 0.9993 0.9960 0.9994 0.9949 0.9920 0.9938

Table II Mean Recoveries (%) (+SE) (n ¼ 3) of CL from Fortified Samples (n ¼ 3) Samples

Fortified concentration (mg/kg or mg/L)

Mean recovery + RSD (%)

Liver

0.5 50 200 0.5 10 50 0.5 5 10 0.5 50 200 0.5 10 50 0.5 5 10 0.5 100 1,000 0.5 50 100 0.5 50 100 0.5 5 20

78.3 + 5.2 73.2 + 4.7 70.7 + 4.1 82.1 + 4.8 85.4 + 6.5 74.2 + 4.2 87.4 + 6.1 104.5 + 5.7 93.6 + 4.8 89.2 + 7.3 91.1 + 5.0 83.9 + 4.5 74.6 + 5.1 72.8 + 4.7 69.3 + 3.4 89.6 + 4.9 104.5 + 5.3 96.2 + 3.9 85.4 + 4.3 90.0 + 4.0 82.5 + 5.2 92.3 + 4.5 97.5 + 9.2 84.6 + 3.9 80.3 + 5.4 79.1 + 6.9 78.4 + 8.3 98.7 + 5.4 106.1 + 3.4 96.5 + 4.1

Lung

Muscle

Kidney

Heart

Fat

Eyes

Bile

Urine

Plasma

Results and discussion Validation of method The concentration range and equation of the linear calibration curves are summarized in Table I. The calibration curves for CL typically gave R 2 values .0.9920 for each matrix. The mean recoveries of CL from the tissue samples were determined at three fortification concentrations, as shown in Table II. The LOQ, defined as the concentration that produced a S/N of 10, was 0.50 mg/kg or 0.50 mg/L. The LOD for CL was 0.15 mg/kg or 0.15 mg/L. Single-ion chromatograms (m/z) of CL and CL-D9 are shown in Figure 1. The results indicated that the methods were efficient and reliable for the analysis of CL. Deposition of CL in goat tissues for repeated treatments in a growth-promoting dose Figure 2 shows the mean concentrations of CL (+SD) measured in various tissues of the treated goats after 21 days of repeated administration. These results shows that the concentration of

Figure 1. Single-ion chromatograms (m/z) of 1 mL plasma fortified with CL (5 mg/L) and CL-D9 (10 mg/L).

CL is the highest in the eye, followed by bile, liver, urine, kidney, lung, fat, heart and muscle. All of these concentrations are higher than the concentration of CL in plasma. Previous reports have shown comparable results for plasma (1.1  1023 mg/mL) and edible tissues in an analogous study with calves (14). These reports indicated that CL accumulated heavily in the eye, liver and kidney, whereas muscle and fat had a low level of accumulation, 0- to 2-fold greater than the plasma level (18). Thus, the distribution of CL in various mammals appears to be similar. The profile of the distribution of CL residues in various tissues was in agreement with other studies in the literature (15). The highest concentration of CL is found in whole eye. This level is 909-fold greater than the plasma level, 28-fold greater than the bile level, 37.8-fold greater than the liver level, 51.6-fold greater than the urine level, 158.5-fold greater than the kidney level, 460-fold greater than the lung level, 700-fold greater than the level in fat and in the heart and 900-fold greater than the level in muscle. The high concentration of CL in the eye may be associated with the presence of pigment. This finding is consistent with the results of previous studies. For example,

unpigmented and pigmented rats were subcutaneously injected twice with 5 mg of CL, and the eyes were analyzed with an enzyme immunoassay after 63 h of withdrawal. Only the eye of the pigmented treated rats showed a clear accumulation of CL (68.1–81.5 mg/kg), whereas the eye of the unpigmented treated rats demonstrated concentrations similar to the negative controls (,0.27 mg/kg) (13). Many previous studies have suggested a correlation between CL deposition and pigment (16, 18). In bile, the content of CL is 32-fold greater than that in plasma. A previously published report showed that biliary elimination of CL and (or) its metabolites in rats was a significant route of elimination because almost 10% of the radioactivity in an intraduodenal [14C] CL dose was excreted via bile (19). This mechanism may explain the high concentration of CL found in bile. The concentrations of CL in liver and kidney are 24-fold and 5.7-fold greater, respectively, than that in plasma. This result may be related to the density of b-receptors in these tissues. b-adrenoceptor density has been determined in rats using a suitable hydrophilic b-adrenoceptor tracer and positron emission tomography (20). A higher in vivo uptake of the tracer was observed in heart, lung, kidney and liver than in muscle and fat. Clenbuterol Distribution Residues of Goats After Treatment 3

Figure 2. Deposition of CL in goat various tissues (orally administered 16 mg/kg body weight CL for 21 consecutive days) on withdrawal day 0.

Figure 3. Mean (+SD, mg/L) CL concentrations in goat urine (orally administered 16 mg/kg body weight CL for 21 consecutive days) on withdrawal days 0.25– 7 (n ¼ 3).

In addition, it is probable that kidney showed a higher CL content than muscle because CL is primarily eliminated by the kidney.

Elimination of CL in plasma and urine after withdrawal The concentrations (mean + SD, mg/L) of CL on days 0.25, 1 and 3 of treatment cessation in urine are 72.05 + 12.63, 52.04 + 4.01 and 0.44 + 0.22, respectively. These values are lower than the LOD over a withdrawal period of 7 days. After a withdrawal period of 21 days, the concentration of CL in plasma had declined from 8.64 to 0.29 mg/L; the concentration of CL in plasma is lower than the LOD after a withdrawal period of 28 days (Figure 3). The results of this study is consistent with the report of Smith, who found that the routes of elimination are via urine 4 Zhao et al.

and feces of calves orally dosed with 3 mg/kg BW [14C]CL-HCl, and that the concentrations of CL in bile, urine and serum are 12.53, 7.75 and 0.45 mg/L, respectively, after a 48-h withdrawal period (15). In this experiment, CL residues in urine are below LOD on withdrawal day 7, while the contents of CL in plasma and bile are still detectable after a withdrawal period of 21 and 28 days, respectively, the results indicate that the major route of the later stage of CL elimination is via bile. The decrease in the concentration of CL in plasma is best described by a one-phase exponential decay model. A nonlinear regression analysis is performed to determine the depletion of CL residues in plasma (GraphPad Prism 6.0) (Figure 4). The regression equations and half-lives of CL are shown in Table II (R 2 ¼ 0.9335).

Figure 4. The clearance of CL in goat plasma (orally administered 16 mg/kg body weight CL for 21 consecutive days, n ¼ 3).

Table III Tissue Pharmacokinetic Parameter Estimates of CL in Goat (Orally Administered 16 mg/kg Body Weight Clenbuterol for 21 Consecutive Days) Tissue

Equation

k (2d)a

t1/2 (2d)b

Liver Lung Muscle Kidney Heart Fat Eyes Plasma Bile

Y ¼ 123.1e 20.3285t þ 9.938 Y ¼ 9.211e 20.4475t þ 0.6414 Y ¼ 5.426e20.3215t þ 0.3073 Y ¼ 48.50e 20.5243t þ 1.071 Y ¼ 8.696e 20.3988t þ 0.1838 Y ¼ 5.817e 20.6487t þ 0.7413 Y ¼ 3285.0e 20.1427t þ 207.90 Y ¼ 9.025e 20.4648t þ 0.5724 Y ¼ 203.3e 20.6330t þ 2.678

0.3285 0.4475 0.3215 0.5243 0.3988 0.6487 0.1427 0.4648 0.6330

2.11 1.55 2.16 1.32 1.74 1.07 4.86 1.49 1.10

a

Elimination rate constant. Elimination half-life.

b

The CL in plasma declined after the final dosage with an elimination half-life (t1/2) of 35.8 h. The data are similar with the t1/2 of 35 h in people (21), but the t1/2 is longer than in cattle, rat, rabbit and horse, which are 18, 30, 9 and 12.9 h, respectively (16, 21, 22). These could be explained by differences among animal species, method of administration and CL dose applied.

Depletion of CL in tissues after discontinuation of administration Curves of the concentration in tissue (Y, mg/kg or mg/L) versus discontinuation times (X, days) were obtained with a nonlinear regression analysis (GraphPad Prism 6.0). The regression equations had R 2  0.9335 for all tissues (Table III). On day 28 of treatment discontinuation, CL can be detected in all tissues except plasma and urine (,0.15 mg/L). Over a withdrawal period of 28 days, liver, lung, muscle, kidney, heart, fat, the whole eye and bile still contained 5.9%, 7.1%, 3.9%, 0.5%, 2.4%, 10.4%, 11.4% and 0.7%, respectively, of the initial residue concentration. CL concentrations decreased rapidly in fat, bile and kidney relative to plasma. In lung, heart, liver and muscle tissues, elimination proceeded more slowly. The rate of decrease is slowest for the whole eye, as shown in Figure 5. In liver, the content of CL (mg/kg) decreased over the withdrawal period from 123.40 + 12.69 on day 0.25 to 7.23 + 0.33 on day 28. Liver, as an alimentary matrix where CL remains in greater amounts and for a longer period, has been associated with the highest number of incidents attributed to CL (13).

Hypothetically, liver can serve as an alimentary matrix and as a matrix for the control of CL abuse. However, CL depletion by day 28 of treatment cessation do not reach the concentration of 0.5 mg/kg. This finding is not consistent with the results of a previous study of pigs, in which the content of CL in liver decreased below 0.5 mg/kg (23). The pigs in this previous study received a dose of 10 mg/kg body mass orally twice daily for 28 days. These differences between the results of this study and the previous study of pigs may have occurred because the goat is a ruminant, whereas the pig is a nonruminant. Additionally, this study and the previous study used different doses and different treatment times. In contrast, the mean concentration of residual CL in the whole eye on day 21 of CL withdrawal is 45.4-fold greater than that measured in the liver, with similar mean concentration ratios on day 28 of treatment withdrawal (49.1:1). These results agree with the reports in choroid/pigmented retinal epithelium and the liver and of Gojmerac et al. (23), which also means the whole eye and the choroid/pigmented retinal epithelium have similar content of CL. These data suggest that the highest accumulation potential of CL is found in the whole eye and that the rate of depletion is very slow between days 21 and 28 after withdrawal. This result supports the view that2 the accumulation of CL in pigmented tissues is associated with the presence of melanin (13). The finding that the slowest rate of depletion occurred in the whole eye is also consistent with the results of Smith (24), who found that CL residues deplete slowly in ocular tissues in cattle and poultry. Indeed, tests for CL in ocular tissues have played an important role in the surveillance of illicit CL use. We find no statistically significant differences between the depletion rate in liver and muscle. Moreover, the depletion rate was the same in bile, fat, kidney and plasma. Furthermore, the concentrations are similar in heart and muscle at 0.25 –1 day of withdrawal; on day 3 of treatment cessation, however, the residue concentration in heart is lower than that in muscle as a result of the more rapid rate of depletion in heart than in muscle. European regulators have set maximum residue limits (MRLs) of 0.5, 0.5 and 0.1 mg/kg CL for liver, kidney and muscle, respectively, in bovine and equidae (25). The MRLs could be used to analyze CL residues in the goat in this study for goats are belonged to bovine. In the context of this study, the following conclusion is evident. On day 28 of treatment discontinuation, the concentration of CL in liver exceeded the MRL by 14-fold. In muscle, fat and heart, the residues of CL are low, while CL residues in muscle still exceeded MRL (0.1 mg/kg) on day 28 of treatment cessation. The content of CL in kidney had fallen below the MRL on day 21 of treatment cessation. Moreover, the CL content exceeded the LOD of the methods in all test tissue samples even on day 28 of the withdrawal period; in plasma and urine, the CL content is less than the LOD on days 7 and 28, respectively, of treatment cessation. Conclusions The depuration of each tissue, urine and plasma followed firstorder kinetics. The absorption of CL is rapidly and widely, and CL residues decreased differently in various tissues, plasma and urine of the goat during the withdrawal period. However, accumulation of CL in the whole eye is high and persistent compared with that in urine, plasma and other tissues; thus, the whole eye might furnish a new sensitive indicator of b2-adrenergic agonist Clenbuterol Distribution Residues of Goats After Treatment 5

Figure 5. The depletion of CL in goat tissues (orally administered 16 mg/kg body weight CL for 21 consecutive days) on withdrawal days 0.25 to 28 (n ¼ 3): (a) lung, (b) liver, (c) muscle, (d) kidney, (e) bile, (f ) eyes, (g) fat and (h) heart.

6 Zhao et al.

residues, serving to detect illegal treatment with CL even after a longer period of withdrawal.

Funding This work was supported by the Special Fund for Agro Scientific Research in the Public Interest (HY201203088).

References 1. Shelver, W.L., Smith, D.J. (2000) Evaluation of commercial immunoassays for cross-reactivity to clenbuterol stereoisomers and bovine metabolites. Food Additives and Contaminants, 17, 837– 845. 2. Deutsch, D.A., Abukhalaf, I.K., Wineski, L.E., Aboul-Enein, H.Y., Pitts, S.A., Parks, B.A. et al. (2000) b-agonist-induced alterations in organ weights and protein content: comparison of racemic clenbuterol and its enantiomers. Chirality, 12, 637– 648. 3. Pleadin, J., Vulic´, A., Persˇi, N., Vahcˇic´, N. (2010) Clenbuterol residues in pig muscle after repeat administration in a growth-promoting dose. Meat Science, 86, 733– 737. 4. Zalko, D., Perdu-Durand, E., Debrauwer, L., Bec-ferte, M.P., Tulliez, J. (1998) Comparative metabolism of clenbuterol by rat and bovine liver microsomes and slices. Drug Metabolism and Disposition, 26, 28 –35. 5. Liu, Q.Y. (2009) Analysis of differentially expressed genes in pig liver and adipose tissue after clenbuterol administration. Graduate Dissertation. China Agricultural University. 6. Delbeke, F.T., Desmet, N., Debackere, M. (1995) The abuse of doping agents in competing body builders in Flanders (1988 – 1993). International Journal of Sports Medicine, 16, 66– 70. 7. Brambilla, G., Loizzo, A., Fontana, L., Strozzi, M., Guarino, A., Soprano, V. (1997) Food poisoning following consumption of clenbuteroltreated veal in Italy. Journal of the American Medical Association, 278, 635– 640. 8. Garay, J.B., Jimenez, J.E.H., Jimenez, M.L., Sebastian, M.V., Matesanz, J.P., Moreno, P.M. et al. (1997) Clenbuterol poisoning—clinical manifestations and analytical findings in an epidemic outbreak in Mostoles, Madrid. Revista Clı´nica Espan˜ola, 197, 92 –95. 9. Pulce, C., Lamaison, D., Keck, G., Bostvironnois, C., Nicolas, J., Descotes, J. (1991) Collective human food poisonings by clenbuterol residues in veal liver. Veterinary and Human Toxicology, 33, 480–481. 10. Gu, Z.H., Zheng, L.J. (2007) Lessons from outbreak of food poisoning caused by clenbuterol in Shanghai in Sept. 2006. Chinese Journal of Food Hygiene, 19, 10 –12.

11. Commission of the European Communities (1996) Council Directive 96 /22 /EC on the Prohibition of the Use of Certain Substances Having a Hormonal and Thyrostatic Action and b-Agonists in Animal Husbandry. 12. Three cases of illegal usage of b-adrenergic agonist on ruminants. (2011) http://www.chinanews.com/fz/2011/10-26/3416474.shtml. 13. Du¨rsch, I., Meyer, H.H., Karg, H. (1995) Accumulation of the beta agonist clenbuterol by pigmented tissues in rat eye and hair of veal calves. Journal of Animal Science, 73, 2050– 2053. 14. Meyer, H.H.D., Rinke, L.M. (1991) The pharmacokinetics and residues of clenbuterol in veal calves. Journal of Animal Science, 69, 4538. 15. Smith, D.J., Paulson, G.D. (1997) Distribution, elimination, and residues of [14C]clenbuterol HCl in Holstein calves. Journal of Animal Science, 75, 454– 461. 16. Sauer, M.J., Pickett, R.J.H., Limer, S. (1995) Distribution and elimination of clenbuterol in tissues and fluids of calves following prolonged oral administration at a growth-promoting dose. Journal of Veterinary Pharmacology and Therapeutics, 8, 81–86. 17. Tang, C., Zhang, J., Li, L., Zhao, Q., Bu, D. (2014) Ractopamine residues in urine, plasma and hair of cattle during and after treatment. Journal of Analytical Toxicology, 38, 149– 154. 18. Malucelli, A., Ellendorff, F., Meyer, H.H. (1994) Tissue distribution and residues of clenbuterol, salbutamol, and terbutaline in tissues of treated broiler chickens. Journal of Animal Science, 72, 1555– 1560. 19. Kopitar, V.Z., Zimmer, A. (1976) Pharmakokinetik and metabolitenmuster von clenbuterol bei der Ratte. ArzneimittleForschung—Drug Research, 26, 1435– 1441. 20. Elsinga, P.H., Waarde, A.V., Vaalburg, W. (2004) Receptor imaging in the thorax with PET. European Journal of Pharmacology, 499, 1– 13. 21. Yamamoto, I., Iwata, K., Nakashima, M. (1985) Pharmacokinetics of serum and urine clenbuterol in man, rat and rabbit. Journal of Pharmacobio-dynamics, 8, 385– 391. 22. Soma, L.R., Uboh, C.E., Guan, F. (2004) Tissue distribution of clenbuterol in the horse. Journal of Veterinary Pharmacology and Therapeutics, 27, 91 –98. 23. Gojmerac, T., Pleadin, J., Bratosˇ , I., Vulic´, A., Vahcˇic´, N. (2008) Xenobiotic clenbuterol in food producing male pigs: various tissue residue accumulation on days after withdrawal. Meat Science, 80, 879–884. 24. Smith, D.J. (2000) Total radioactive residues and clenbuterol residues in swine after dietary administration of [14C] clenbuterol for seven days and preslaughter withdrawal periods of zero, three, or seven days. Journal of Animal Science, 78, 2903–2912. 25. European Commission. (2010) Commission Regulation (EU) No. 37/ 2010 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin.

Clenbuterol Distribution Residues of Goats After Treatment 7

Clenbuterol Distribution and Residues in Goat Tissues After the Repeated Administration of a Growth-Promoting Dose.

This study was conducted to investigate the deposition and depletion process of clenbuterol (CL) in goat tissues, plasma and urine after the repeated ...
454KB Sizes 1 Downloads 10 Views