Elimination and Concentration Correlations between Edible Tissues

Article pubs.acs.org/JAFC

Elimination and Concentration Correlations between Edible Tissues and Biological Fluids and Hair of Ractopamine in Pigs and Goats Fed with Ractopamine-Medicated Feed Lingli Huang,†,§ Jingfei Shi,† Yuanhu Pan,‡,§ Liye Wang,†,§ Dongmei Chen,‡ Shuyu Xie,†,§ Zhenli Liu,‡,§ and Zonghui Yuan*,†,‡,§ †

MOA Laboratory of Risk Assessment for Quality and Safety of Livestock and Poultry Products, ‡National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, and §Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Huazhong Agricultural University, Wuhan, Hubei 430070, China ABSTRACT: Ractopamine (RAC), a β-adrenergic leanness-enhancing agent, endangers the food safety of animal products because of overdosing and illegal use in food animals. Excretion and residue depletion of RAC in pigs and goats were investigated to determine a representative biological fluid or surface tissue for preslaughter monitoring. After a single oral gavage of RAC, 64− 67% of the dose was excreted from the urine of pigs and goats within 12−24 h. RAC persisted the longest in the hair of pigs and goats but depleted rapidly in the plasma, muscle, and fat. Urine and hair were excellent for predicting RAC residues in edible tissues of pigs, whereas plasma and urine were satisfactory body fluids for the prediction of RAC concentrations in edible tissues of goats. These data provided a simple and economical preslaughter living monitoring method for the illegal use and violative residue of RAC in food animals. KEYWORDS: ractopamine, pig, goat, residue depletion, correlation, preslaughter monitoring

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and biological fluids are significantly correlated.11−14 Furthermore, the concentrations of clenbuterol, which is representative of β-adrenoceptor agonist, in urine are significantly correlated with the concentrations in edible tissues.15 Therefore, biological fluids can be used to monitor the illegal use and violative residues of RAC in edible tissues of food animals. Numerous papers reported the determination methods of RAC in plasma, urine, and hair of pigs, sheep, and cattle.16−19 Several papers reported the depletion of RAC in edible tissues and urine of pigs.20−22 However, the depletion of RAC in goats remains unclear. A recent paper described the depletion of RAC in the hair of pigs, sheep, and cattle.23 These studies focused mainly on the determination of RAC in different matrices of food animals, and no data indicated the correlations among edible tissues, biological fluids, and surface tissues. The present study investigated the excretion and depletion of RAC in pigs and goats to characterize the kinetics of RAC in biological fluids (plasma and urine), surface tissues (hair), and edible tissues (muscle, liver, kidneys, fat, lung, and intestine). The correlations of RAC among edible tissues, biological fluids, and surface tissues were investigated for the first time. The optimal biological fluid (plasma or urine) or surface tissue was identified as a representative matrix for monitoring the illegal use and violative residue of RAC in food animals. These results may provide a suggestive preslaughter living monitoring method for estimating the concentration of RAC in edible

INTRODUCTION Ractopamine (RAC), a phenolethanolamine-adrenergic agonist, has been used to improve weight gain, carcass leanness, and feed efficiency in livestock by diverting nutrients from fat deposition to muscle tissue production.1,2 RAC was approved by the U.S. Food and Drug Administration for use in finishing swine in 1999 and is now cleared in 21 other countries.3 However, this additive has been banned in China and Europe because of the potential risks to the cardiovascular and central nervous systems of humans via transfer from RAC-treated animals to human diet.4,5 In practice, RAC is still overdosed or illegally used to improve carcass composition and gain high economic benefits.6 β-Adrenoceptor agonists are potentially harmful to human health and of great concern worldwide for its safety; thus, the overdose or illegal use of RAC in food animals must be monitored, and its residues in animal production should be predicted. Many determination methods have been developed to detect the residues of RAC in edible tissues.7−10 These methods are effective tools for the monitoring of RAC residues in animal products because they focus directly on the detection of real edible tissues, which would be consumed by humans. However, animal slaughter and tedious sample processing and detection cost too much based on these direct determinations of RAC in real edible tissues. Instead, a preslaughter living monitoring strategy by determining the correlations of drug residues between edible tissues, biological fluids, and surface tissues would be an efficient and low-cost method to prevent animal waste and economy loss. Plasma, urine, and hair are popular samples for the living monitoring of drug residues because they are practical and convenient for sampling. Several papers demonstrated that drug concentrations between edible tissues © XXXX American Chemical Society

Received: January 28, 2016 Revised: February 15, 2016 Accepted: February 17, 2016

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DOI: 10.1021/acs.jafc.6b00456 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

merged, and 4 mL of 2% formic acid was added into the mixed extracts. The extract was evaporated to dryness under nitrogen stream at 45 °C. Approximately 2 mL of n-hexane was added into the tube, and the mixture was vortexed for 2 min. The solution was centrifuged at 8000 rpm for 10 min. The lower solution was finally subjected to SPE. Hair. Approximately 1.0 g of hair was soaked in 40 mL of 0.2% Tween 80 aqueous solution for 30 min and shaken in an ultrasonic bath at room temperature for 20 min. The hair was washed three times with distilled water and dried in a dry oven at 60 °C overnight. About 500 mg of dried hair was cut into pieces and placed into a test tube, and 4 mL of 1 M NaOH was added. The mixture was gently shaken on a vortex finder and heated in a water bath at 65 °C for 2.5 h. When the mixture was cooled to room temperature, 3.5 mL of 1 M HCl was added, and the pH was adjusted to 9−10. About 5 mL of ethyl acetate was added into the mixture, which was vortexed for 2 min. The extraction solution was removed into a separate tube. Another three repeated extractions with ethyl acetate were conducted. The mixed extraction solutions were centrifuged at 8000 rpm for 10 min. The supernatant was transferred to another tube, and 4 mL of 2% formic acid was added. The mixture was evaporated to dryness under nitrogen stream at 45 °C. About 2 mL of n-hexane was added into the tube and then vortexed for 2 min. The solution was centrifuged at 8000 rpm for 10 min. The lower solution was finally subjected to SPE. Tissues and Feces. Aliquots of 2.0 ± 0.01 g of homogenized tissues (or feces) were placed into a 50 mL disposable plastic centrifugal tube. Approximately 3 mL of 0.2 mol/L acetate buffer (pH 5.2) was added into the tube. When the samples needed to be hydrolyzed, another 40 μL of β-glucuronidase was added into the tube and the mixture was incubated at 37 °C for 12 h. About 6 mL of 5% ammonia−ethyl acetate was added into the mixture, followed by shaking in an ultrasonic bath for 15 min at room temperature. The mixture was centrifuged at 8000 rpm for 10 min. The supernatant was transferred to another tube, and the residue was extracted with another 6 mL of 5% ammonia−ethyl acetate twice again. The extraction was combined, and 4 mL of 2% formic acid was added into the mixed extraction. The mixed extraction was evaporated to dryness under nitrogen stream at 45 °C. Subsequently, 2 mL of n-hexane was added, followed by centrifugation at 8000 rpm for 10 min. The lower solution was subjected to SPE. Sample Purification. The MCX cartridge column (60 mg, 3 mL) (Waters Corp., Milford, MA, USA) was conditioned sequentially with 3 mL each of methanol, water, and 2% formic acid. The extracted solution was loaded onto the SPE column at a flow rate of 1 mL/min. The column was washed with 3 mL each of 2% formic acid and methanol and then dried by purging air at a rate of 10 mL/min for 5 min. The analytes were eluted with 5 mL of a mixture consisting of methanol and ammonia at a ratio of 20:1 at a flow rate of 1.0 mL/min. The eluent was evaporated to dryness under a gentle stream of nitrogen at 40 °C and dissolved in 1 mL of HPLC mobile phase consisting of 0.1% formic acid and 5 mM ammonium acetate in the mixture of water/methanol (90:10, v/v). LC-MS/MS Analysis. Analyses were performed with a LC-MS/MS system (Thermo Electron Corp., Wyman, Waltham, MA, USA) consisting of a Finnigan Surveyor Plus system with an online degasser, a Surveyor autosampler, and a TSQ Quantum triple-stage quadrupole mass spectrometer equipped with an electrospray interface operating in the positive mode (ESI+). Separation was achieved on a Hypersil ODS C18 column (150 mm × 2.1 mm, 5 μm) at 40 °C. The mobile phase A was 0.1% formic acid solvent with 5 mmol/L ammonium acetate in water, whereas the mobile phase B was methanol. The gradients were as follows: 0.0−1.0 min, A/B (10/90); 9.0−15 min, A/ B (90/10); 16.0−22.0 min, A/B (10/95). Flow rates were set at 250 μL/min. The Surveyor autosampler was performed in partial loop injection mode. The injection volume was 10 μL, and the needle was rinsed with H2O/methanol (50/50, v/v) between injections. To optimize the tuning parameters for each compound, a standard solution containing 1 μg/mL RAC was infused into the ESI source at 25 μL/min with 200 μL/min H2O/methanol mobile phase (50/50, v/ v). The spray voltage was held at 4600 V. The capillary temperature

tissues of pigs and goats before animal products are exposed to consumers.

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MATERIALS AND METHODS

Drugs and Chemicals. The standard of RAC hydrochloride (≥99.7%) was purchased from Sigma-Aldrich Chemie b.v. (Zwijndrecht, The Netherlands). The standard stock solution (1.0 mg/mL) was prepared by dissolving 10.0 mg of RAC in methanol. The standard working solution (100 μg/mL) was prepared by dilution of the stock standard in methanol. β-Glucuronidase was purchased from Sigma Chemical Co. (Helix pomatia, Type H-2, aqueous solution, ≥85 000 units/mL). High-performance liquid chromatography (HPLC)-grade methanol was purchased from Merck Chemicals Co. (Darmstadt, Germany). Oasis MCX solid-phase extraction cartridges were purchased from Waters Co. (Milford, MA, USA). Deionized water (Milli-Q; Millipore, Bedford, MA, USA) was used throughout the study. All other chemicals were of analytical grade. Animals. The use of animals in this study was performed in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals of China (permit SYXK 2007-0044). A total of 28 healthy Landrace-Large white crossbred castrated male pigs (weight, 25−30 kg) were purchased from the China Breeding Swine Testing Center (Wuhan, China). A total of 28 healthy Boer goats (weight, 20−25 kg) were purchased from Huazhong Agricultural University Veterinary Hospital (Wuhan, China). All of the animals were allowed a 7 day acclimation period before the experiments were conducted. A standard ration based on corn and soybean was fed twice a day, and tap water was available ad libitum. Dosing and Sampling. Pigs and goats were randomly divided into groups A, B, and C. Group A (n = 5) was fed with standard ration without RAC. Group B (n = 4) was given a single oral administration of RAC at 1.2 mg/kg bw. Group C (n = 20) was provided with medicated feed containing RAC (20 mg/kg) for 14 consecutive days. For group B, urine and feces were collected at 0−6, 6−12, and 12−24 h and every 24 h thereafter. All of the urinary and feces samples were weighed and stored frozen at −20 °C. For groups A and C on days 0.25, 1, 3, 7, and 14, one control and four medicated pigs were anesthetized and sacrificed after the last dose. The pigs and goats were slaughtered using a captive bolt stunner and exsanguinated in accordance with the guidelines provided by the American Veterinary Medical Association for euthanasia.24 At each slaughter time point, blood samples (10 mL) were collected from the right or left jugular vein using disposable heparinized vacutainer tubes. Plasma was prepared by placing the blood sample in an ice bath for 2 h and centrifuged at 25 °C for 10 min at 1100 g (r = 107 mm). Edible tissue samples (liver, kidney, muscle, fat, lung, large intestine, and small intestine) and hair were collected and placed in plastic bags on an ice bath. Urine samples were collected daily after the last dose. All samples were assayed immediately or were frozen at −20 °C until analysis. Sample Extraction. Plasma. An aliquot of thawed plasma (2 mL) was placed into a 50 mL disposable plastic centrifugal tube. About 3 mL of 0.2 mol/L acetate buffer (pH 5.2) was added into the tube, and the mixture was vortexed for 2 min. When the samples had to be hydrolyzed, another 40 μL of β-glucuronidase was added into the tube and the mixture was incubated at 37 °C for 12 h. Approximately 4 mL of methanol was added into the mixture to precipitate the protein. Subsequently, the sample was centrifuged at 8000 rpm for 10 min. The supernatant was subjected to solid-phase extraction (SPE). Urine. Approximately 2 mL of urine was poured into a 50 mL disposable plastic centrifugal tube. About 3 mL of 0.2 mol/L acetate buffer (pH 5.2) was added into the tube, and the mixture was vortexed for 2 min. When the samples needed to be hydrolyzed, another 40 μL of β-glucuronidase was added into the tube and the mixture was incubated at 37 °C for 12 h. When the mixture was cooled to room temperature, 6 mL of 5% ammonia−ethyl acetate was added. The samples were shaken in an ultrasonic bath at room temperature for 15 min and centrifuged at 8000 rpm for 10 min. The supernatants were transferred to another tube, and the residue was extracted with 6 mL of 5% ammonia−ethyl acetate twice again. The extracted solutions were B


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Journal of Agricultural and Food Chemistry was 350 °C, and the tube lens offset was set at 104 V. The sheath and aux gas pressures were 30 and 10 arb, respectively. The parent ion of RAC was 302.00 m/z. The quantitative daughter ions of RAC were 106.998, 121.167, and 284.101 m/z, and the collision energies (Ev) of the daughter ions were 33, 24, and 9 eV, respectively. Method Validation. The method was validated with reference to the implemented validation procedure for residues in food animal products as described in EU Commission Decision 2002/657/EC.25. The validation of specificity, matrix effects, linearity, CCα, CCβ, accuracy, and precision of the method were determined by spiking blank matrixes with RAC standard solution. The specificity of the method was evaluated by the presence of interfering substances around the RAC retention time. Matrix effects on the ionization of analytes were evaluated by comparing the peak area of the standard solution with those of the matrix extract solution. Blank samples of pigs and goats were used as matrices for calibration curve study. The matrix−match calibration curves were constructed using fortified blank tissues with RAC at six levels as follows: 0.5, 1.0, 2.0, 4.0, 8.0, and 16 μg/kg (n = 3). The calibration curve was y = 119 540x + 145 853 (r = 0.999). CCα values were defined as three times the signal−noise ratio (S/N) and established by the following steps: 20 blank samples of pigs and goats were analyzed, and the S/N was calculated at the time window in which the analyte was expected. CCβ was calculated by analyzing 20 blank samples spiked with the concentration at CCα, and the CCα value plus 1.64 times the corresponding standard deviation (SD) was equal to CCβ (β = 5%). The CCα and CCβ values of the method were within the range of 0.57−0.65 and 0.64−0.80 μg/kg in tissues of pigs and goats, respectively, but 1.1 and 1.2 μg/kg in hair of pigs and goats, respectively. Accuracy and precisions (intraday, interday, and within laboratory) were calculated from the determination of five aliquots of each tissue fortified at 0.5, 1.0, and 2.0 μg/kg. The recovery of RAC in plasma, urine, and hair of pigs and goats ranged within 71.8−83.0%, with the intraday relative SD less than 10.8%. The recovery of RAC in hair of pigs and goats ranged within 70.8−84.3%, with the intraday relative SD less than 12.6%. Data Analysis. Descriptive statistical parameters, such as mean, SD, and CV, were calculated. Statistical analysis of data and correlation analysis among plasma, urine, hair, and tissues were performed using SigmaPlot 11.0 (SPSS, Inc.). The residue depletion profile of RAC in tissues of pigs and goats following withdrawal from the medicated diet was estimated by linear regression. The half-life (t1/2) of RAC in plasma and tissues during the elimination phase was calculated graphically by fitting linear regression. Linear regression, exponential function, and power function analysis were performed by analyzing RAC concentrations in plasma, urine, hair, and edible tissues to describe the concentration correlations of RAC between body fluid/ surface tissues and edible tissues.

Figure 1. Excretion of ractopamine in pigs and goats after a single oral administration at 1.2 mg/kg bw (mean ± SD).

Table 1. Comparison of Free and Bound Ractopamine (RAC) Concentrations in Urine of Pig and Goat (μg/kg) (n = 4) time (days)

concentrations of free RAC (μg/kg)

concentrations of total RAC (μg/kg)

free/ bound

pigs

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0.25 0.5 1 2 3 4 5 6

775 834 1195 143 86 42 29 24

± ± ± ± ± ± ± ±

202 232 248 18 11 7 4 3

0.25 0.5 1 2 3 4 5 6

1364 2291 2375 4122 2564 2553 854 456

± ± ± ± ± ± ± ±

383 570 591 814 472 672 157 69

8660 7732 1829 175 87 42 30 21

± ± ± ± ± ± ± ±

1033 779 664 20 10 8 4 1

9/91 11/89 65/35 82/18 100/0 100/0 100/0 100/0

11 021 17 218 12 941 5782 3146 1908 923 461

± ± ± ± ± ± ± ±

1809 4984 3041 985 728 501 127 68

12/88 13/87 18/82 71/29 82/18 100/0 100/0 100/0

goats

RESULTS Excretion of RAC in Pigs and Goats. The urine and fecal samples were hydrolyzed with β-glucuronidase and analyzed by HPLC-MS/MS. Within 7 days after a single oral dosage of 1.2 mg/kg bw, approximately 72.2% and 74.5% of the dose was excreted by the pigs and goats, respectively, of which 64−67% of the dose was excreted from urine and only 7% was excreted from feces. RAC was excreted more rapidly in pigs than in goats as more than 60% of the dose was excreted in pigs, whereas only 38% was excreted in goats within 24 h (Figure 1). Nearly 90% of the excreted drug was bound RAC in urine of pigs and goats within 12−24 h; subsequently, 80−100% of the excreted drug was free RAC (Table 1). Tissue Depletion of RAC in Pigs and Goats. The RAC concentrations in tissues, plasma, and excreta after different sample treatments are listed in Tables 2 and 3. In pigs, the highest concentration of RAC was observed in hair followed by urine and lung at all sampling times. No statistically significant differences were observed between the concentrations of RAC

with hydrolysis and nonhydrolysis treatments for most tissues, but significant differences were observed in urine, plasma, and kidney at withdrawal times of 6 and 24 h (Table 2). The concentration of RAC with hydrolysis treatment was 10 and 20 times the concentration of RAC without hydrolysis treatment in urine and plasma, respectively. RAC could be detected in most tissues and excreta until 7 days, but it was only detected in muscle, fat, and plasma at withdrawal times of 6 and 24 h. The longest persistence was observed in lung and hair because RAC was still detectable at a withdrawal time of 14 days. C


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Table 2. Concentration of Ractopamine in Pig Tissues, Plasma, and Excreta with Different Treatments (Hydrolysis and Nonhydrolysis) after Being Dosed with 20 mg/kg RAC in Feed for 14 Consecutive Days (μg/kg) (n = 4)a samples liver kidney muscle fat lung large intestine small intestine plasma urine plasma urine hair feces

treatment nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis

49.4 52.1 76.9 102.2 3.2 3.5 4.7 4.5 163.3 166.4 47.6 49.6 58.0 55.9 5.8 55.2 52.1 958.2 5.8 55.2 52.1 957.8 1196.4 1155.3 160.4 154.2

6h

1 day

3 days

7 days

14 days

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

9.2 ± 2.3 9.1 ± 2.2 12.2 ± 3.2 14.1 ± 2.8 0.6 ± 0.3 0.6 ± 0.1 ND ND 130.6 ± 24.2 133.4 ± 21.3 17.3 ± 4.2 18.3 ± 4.0 11.7 ± 4.9 11.7 ± 4.1 1.3 ± 0.5 2.8 ± 1.0* 7.3 ± 0.9 74.9 ± 20.1** 1.3 ± 0.5 2.8 ± 1.0 7.3 ± 0.9 74.9 ± 20.1 759.2 ± 191.3 788.4 ± 205.2 84.1 ± 47.6 86.0 ± 37.4

3.8 ± 0.8 3.6 ± 1.2 5.1 ± 1.1 5.7 ± 1.2 ND ND ND ND 97.6 ± 24.1 96.8 ± 20.1 10.0 ± 2.1 9.8 ± 2.0 5.7 ± 3.8 5.7 ± 3.6 ND ND 8.2 ± 3.3 9.1 ± 3.7 ND ND 8.2 ± 3.3 9.1 ± 3.7 447.3 ± 64.2 476.4 ± 70.1 12.1 ± 2.2 14.3 ± 2.0

0.72 ± 0.04 0.83 ± 0.07 0.87 ± 0.55 0.96 ± 0.75 ND ND ND ND 13.7 ± 1.8 15.2 ± 1.7 1.7 ± 0.2 1.8 ± 0.3 1.4 ± 0.3 1.6 ± 0.4 ND ND 1.5 ± 0.5 1.6 ± 0.6 ND ND 1.5 ± 0.5 1.6 ± 0.6 286.3 ± 39.8 297.8 ± 44.2 0.3 ± 0.1 0.4 ± 0.2

ND ND ND ND ND ND ND ND 2.6 ± 0. 6 2.6 ± 0.4 ND ND ND ND ND ND ND ND ND ND ND ND 131.3 ± 21.4 142.3 ± 25.2 ND ND

8.1 9.1 19.6 22.1* 1.2 0.9 3.0 2.7 36.2 34.2 5.9 6.6 13.0 19.6 3.4 14.2** 24.3 203.1** 3.4 14.2 24.3 203.1 337.2 323.1 48.2 37.2

ND = not detected. *Significant difference between nonhydrolysis and hydrolysis treatments (P < 0.05). between nonhydrolysis and hydrolysis treatments (P < 0.01). a

**

An extremely significant difference

function equations were used to describe the relationship of RAC concentration between all assayed edible tissues and urine and hair in pigs. The correlation coefficients ranged from 0.94 to 0.98 between the liver, kidney, intestine, and urine (Figure 3) and from 0.92 to 0.93 between the liver, kidney, lung, intestine, and hair (Figure 4). On the basis of the correlation equation, when the concentration of RAC in the liver and kidney decreased to around that of the MRL (40 and 90 μg/kg for liver and kidney, respectively), the corresponding concentration of RAC in the urine was around 720 and 890 μg/kg, which means that if the concentration of RAC in the urine was higher than 723 μg/kg the residue amount of RAC in the liver could exceed the MRL and if the concentration of RAC in the urine was higher than 890 μg/kg the residue amount of RAC in the kidney could exceed the MRL. No correlations were developed for muscle and fat, because RAC depleted rapidly in the muscle and fat of pigs, and it was detectable in the early two sampling times. In goats, excellent correlations were developed when power function equations were used to describe the relationship of RAC concentration between most edible tissues (liver, kidney, lung, large intestine, and small intestine) and plasma, urine, and hair. The correlation coefficients were 0.96, 0.97, and 0.98 between the liver, kidney, and lung and plasma, respectively. Both power function and linear regression equations showed unsatisfactory correlations between fat and plasma, but satisfactory correlation with a correlation coefficient of 0.95 was developed when the exponential equation was used (Figure 5). Power function equations showed satisfactory correlations between the liver, kidney, and lung and urine with correlation

Furthermore, the RAC concentration in hair was more than 100 μg/kg at 14 days. The RAC concentrations in all tissues and plasma in goats were significantly lower than those in pigs, but those in urine (4215 μg/kg) and feces (606 μg/kg) were significantly higher than those in pigs. High RAC concentration was also observed in hair and urine at all sampling times. Notably, the RAC concentration in hair in goats (54.2 μg/kg) was extremely lower than that in pigs (1155 μg/kg). Similar to pigs, significant differences between the concentrations of RAC with hydrolysis and nonhydrolysis treatments were observed in urine, plasma, and kidney in goats at withdrawal times of 6 and 24 h. RAC persisted in most tissues for 7 days after the last dose, but it could not be detectable in liver, muscle, fat, and plasma at a withdrawal time of 3 days; it was still detected in hair at a low level at 14 days after the last dose (Table 3). In general, RAC persisted for the longest time in hair of pigs and goats but depleted rapidly in muscle, fat, and plasma (Figure 2). The depletion half-life (t1/2) of RAC in hair of pigs and goats was 6.22 and 7.29 days, respectively, whereas that in muscle, fat, and plasma was less than 0.5 days. RAC depleted more rapidly in lung, liver, kidney, and intestine of pigs with t1/2 ranging within 0.53−1.19 days compared with that of goats with t1/2 ranging within 1.5−2.17 days. Correlation of RAC Concentration between Edible Tissues and Biological Fluids/Surface Tissues in Pigs and Goats. In pigs, the correlations of RAC concentration between edible tissues and urine and hair were analyzed using linear regression, exponential function, and power function method, respectively. Excellent correlations were developed when power D


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Table 3. Concentration of Ractopamine in Goat Tissues, Plasma, and Excreta with Different Treatments (Hydrolysis and Nonhydrolysis) after Being Dosed with 20 mg/kg RAC in Feed for 14 Consecutive Days (μg/kg) (n = 4)a samples liver kidney kidney muscle fat lung large intestine small intestine plasma urine plasma urine hair feces

treatment nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis nonhydrolysis hydrolysis

6h 20.7 16.8 80.6 125.7 80.6 125.7 1.8 3.8 16.3 19.2 23.5 34.5 48.9 47.6 86.0 86.4 2.8 10.9 711.4 4215.3 2.8 10.9 711.2 4215.0 56.2 54.2 614.2 606.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.2 4.1 13.4 14.5* 13.4 14.5 0.7 1.2 3.9 7.5 4.7 8.0 13.2 14.2 16.4 12.2 0.8 3.4** 111.2 312.2** 0.8 3.4 111.3 312.2 12.8 11.4 92.3 98.1

1 day 14.9 13.0 48.3 70.7 48.3 70.7 0.7 1.0 3.0 4.9 15.9 18.2 17.2 16.1 26.0 26.7 1.3 7.1 253.4 1746.3 1.3 7.1 253.2 1746.0 36.9 38.7 232.3 246.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.3 2.5 11.0 16.6* 11.0 16.6 0.4 0.3 0.9 1.0 4.1 4.7 4.0 3.5 5.7 3.8 0.3 1.8** 66.2 353.2** 0.3 1.8 66.1 353.2 7.5 8.4 55.2 65.3

3 days

7 days

14 days

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

ND ND ND 0.3 ± 0.1 ND 0.3 ± 0.1 ND ND ND ND 0.4 ± 0.1 0.4 ± 0.1 0.6 ± 0.1 0.5 ± 0.1 0.8 ± 0.2 0.8 ± 0.2 ND ND 1.5 ± 0.2 3.5 ± 0.9 ND ND 1.5 ± 0.2 3.5 ± 0.9 12.5 ± 3.4 13.3 ± 4.4 0.6 ± 0.2 0.7 ± 0.2

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 8.9 ± 1.9 9.7 ± 2.1 ND ND

1.3 1.2 2.3 3.8 2.3 3.8 0.4 0.4 0.5 0.8 1.7 2.1 7.1 6.9 6.6 6.4 0.3 0.9 10.7 171.3 0.3 0.9 10.7 171.2 27.0 26.2 26.8 27.4

0.2 0.1 0.8 1.3 0.8 1.3 0.1 0.1 0.2 0.3 0.4 0.6 2.1 2.2 1.6 1.6 0.2 0.3* 2.9 23.1** 0.2 0.3 2.9 23.1 7.8 8.6 5.6 4.6

a ND = not detected. *Significant difference between nonhydrolysis and hydrolysis treatments (P < 0.05). between nonhydrolysis and hydrolysis treatments (P < 0.01).

**

An extremely significant difference

coefficients of 0.97, 0.98, and 0.98, respectively, and 0.94 and 0.93 between the large and small intestines and urine, respectively. Good correlations were developed when linear regression equations were used to fit the relationship of RAC concentration between the muscle, large intestine, and small intestine and urine with correlation coefficients of 0.96, 0.95, and 0.98, respectively (Figure 6). Satisfactory correlation between large intestine and small intestine and the hair with correlation coefficients of 0.94 and 0.93 was developed when the power equation was used (Figure 7).

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DISCUSSION RAC could be excreted rapidly from the animals because a total of 79% and 70% of the administered single oral dose of 14CRAC was recovered from the dog and monkey, respectively, during a collection period of 72 h. Moreover, 88% and 9% of the dose was excreted in urine and feces, respectively, when pigs were dosed with RAC,26 thereby indicating that urinary excretion was the primary route of RAC in pigs. Our study demonstrated that nearly 70% of the dose was excreted from urine, and only less than 10% was excreted from feces when pigs and goats were administered with a single oral dose of RAC; these results further confirmed that RAC was excreted mainly via urine when dosed orally to animals. Monoglucuronides are the main forms of RAC in the urine of pigs, dogs, and rats,27−29 so all samples were hydrolyzed with βglucuronidase in this study. Nearly 90% of the excreted drugs

Figure 2. Depletion of ractopamine in pigs and goats after 14 days of consecutive administration in feed at 20 mg/kg (mean ± SD).

E


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Figure 3. Correlation of ractopamine concentrations in urine and liver (A), kidney (B), large intestine (C), and small intestine (D) of pigs.

Figure 4. Correlation of ractopamine concentrations in hair and liver (A), kidney (B), lung (C), and large intestine (D) of pigs.

higher than those in other edible tissues but extremely low in muscle and fat at less than 5.0 μg/kg. These observations indicated that the lung and kidney could be selected as the target tissues for residue monitoring of RAC. RAC is extensively metabolized to glucuronide conjugates in animals. Consequently, a comparison of hydrolysis with β-glucuronidase and without hydrolysis was investigated for all edible tissues in the present study. Significant differences were observed for the

in urine of pigs and goats within 24 h comprised the glucuronides of RAC. The highest residue amount of RAC in edible tissues of pigs was found in the retina, lung, and kidney of pigs.6,21,22 In the present study, body hair, instead of the retina, was examined because removing the eyes for drug residue monitoring during the growth period of animals is unacceptable. The RAC concentrations in the lung and kidney of pigs were significantly F


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Figure 5. Correlation of ractopamine concentrations in plasma and liver (A), plasma and kidney (B), plasma and fat (C), and plasma and lung (D) of goats.

Figure 6. Correlation of ractopamine concentrations in urine and liver (A), kidney (B), muscle (C), lung (D), large intestine (E), and small intestine (F) of goats.

RAC concentrations in urine, plasma, and kidney. At a withdrawal time of 6 h, the levels of RAC and its conjugates in plasma and urine of pigs processed with enzyme hydrolysis were 10 and 20 times that of plasma and urine samples analyzed without enzyme hydrolysis, respectively. However, the levels of RAC and its conjugates in plasma and urine of goats processed with enzyme hydrolysis were only five and six times that of plasma and urine samples analyzed without enzyme hydrolysis, respectively. This difference indicated that RAC could conjugate with glucuronide more easily in pigs than in goats. Another significant difference is that the residue amount

of RAC in hair and lung of pigs was extremely higher than that of goats, which might be due to the difference between the disposition of RAC in goats and pigs. These data indicated that RAC could accumulate in pig’s hair more easily than in goat’s hair. Although the amount of RAC in hair of goats was extremely lower than that of pigs, RAC persisted in hair of goats as long as in hair of pigs. Plasma and urine can be used to monitor drug residues in edible tissues.11,13−15,30 Plasma is considered the most appropriate biological fluid for in vivo drug monitoring because the drug concentration in plasma represents an instantaneous G


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Figure 7. Correlation of ractopamine concentrations in hair and large intestine (A) and small intestine (B) of goats.

Funding

concentration (not an average concentration over a period of time), which makes the estimated tissue concentration more meaningful for routine residue monitoring. However, RAC was depleted rapidly in plasma of pigs, so no drug could be detected after 24 h, whereas RAC could persist for 7 days in most edible tissues. Hence, plasma was unsuitable for residue monitoring of RAC in pigs. Good correlations of RAC concentrations between plasma and most tissues of goats were observed, thereby indicating that plasma could be used to monitor the drug residues of RAC in edible tissues of goats. The clenbutero concentrations in pigs between tissues and urine were significantly correlated.15 Excellent correlations of RAC concentrations between urine and all of the tissues of pigs and goats were observed in this study, which demonstrated that urine was a satisfactory sample for RAC residue monitoring. These developed correlation equations could be used for routine monitoring to judge a suitable time for animal slaughter. If the estimated RAC concentrations in the tissues using the developed correlation equations are above the MRL, additional time for RAC elimination needs to be provided, and further urine testing at a later time should be performed until the estimated RAC levels in the tissues are below the MRL. Research on agonists found in cattle and human hair has shown that hair can be considered a good matrix to determine the presence of β-agonists in living animals.31,32 Suo et al.23 reported that hair is a suitable medium to monitor the illegal use of RAC in livestock production. In the present study, the RAC concentration in hair was significantly correlated with the concentrations in liver, kidney, lung, and intestine of pigs. Remarkable correlations of RAC concentrations between the hair and the kidney and the intestine were observed in goats. However, the correlations of RAC concentrations between the hair and the liver, muscle, fat, and lung were unsatisfactory, with correlation coefficients less than 0.8. On the basis of the results of correlation analysis, invasive determination in edible tissues at the cost of animal slaughter was avoided to monitor the overdosing or illegal use of ractopamine in pigs and goats. Both urine and hair could be used to monitor RAC residues in edible tissues of pigs. Hair could also be used to predict the RAC concentrations in the kidney and intestine of goats. Both plasma and urine could predict the RAC concentrations in all edible tissues of goats, but urine might be preferred over plasma for preslaughter testing of drug abuse or residue monitoring because sample collection is convenient and harmless to animals.

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This work was supported by grants from the Special Fund for Agroscientific Research in the Public Interest (No. 201203040) Notes

The authors declare no competing financial interest.

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Elimination and Concentration Correlations between Edible Tissues

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