Pharmacokinetics of Ractopamine and Its Organ Distribution in Rats

Aug 29, 2014 - Department of Education and Research, Taipei City Hospital, Taipei, Taiwan. ⊥ Department of Pharmacy, Keelung Hospital, Ministry of H...
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Pharmacokinetics of Ractopamine and Its Organ Distribution in Rats Jing-Kai Ho,†,⊥ Teh-Ia Huo,† Lie-Chwen Lin,‡ and Tung-Hu Tsai*,†,¶,§ †

Institute of Pharmacology, and ¶Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan National Research Institute of Chinese Medicine, Taipei, Taiwan § Department of Education and Research, Taipei City Hospital, Taipei, Taiwan ⊥ Department of Pharmacy, Keelung Hospital, Ministry of Health and Welfare, Keelung, Taiwan ‡

S Supporting Information *

ABSTRACT: Ractopamine, a β-agonist, is used to increase the proportion of lean meat in livestock. However, due to potential cardiovascular risks, ractopamine has been banned for use in food-producing animals in many countries. Nevertheless, pharmacokinetic studies of ractopamine have not been completed. The aim of this study was to develop a high-performance liquid chromatography−tandem mass spectrometry (HPLC-MS/MS) method for the determination of ractopamine. This validated method was used to investigate the pharmacokinetics and organ distribution of ractopamine in rats. The validation results complied with the U.S. Food and Drug Administration’s standards. The oral bioavailability of ractopamine was 2.99%. After intravenous administration, ractopamine concentrations varied as follows: kidney > lung > spleen > heart > liver > muscle > plasma > brain. Nonlinear pharmacokinetics and strong partitioning into tissues were observed after intravenous administration of ractopamine. These effects may be due to nonlinear elimination via the kidney. KEYWORDS: bioavailability, HPLC-MS/MS, pharmacokinetics, ractopamine, tissue distribution

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INTRODUCTION In clinical application, β-adrenergic agonists (β-agonists) are widely used as bronchodilators to treat asthma and other pulmonary diseases. Repartitioning effects of β-agonists have been found in rats,1 lambs,2 and steers.3 Some studies have indicated that β-agonists may reduce lipid synthesis and increase lipolysis via an insulin-related mechanism.4 Ractopamine, a β-agonist, decreases sensitivity and responsiveness to insulin-stimulated glucose metabolism in adipose tissue. These effects may be mediated by β-adrenergic receptors.5 Ractopamine is therefore applied to increase the proportion of lean meat in livestock.6 Ingesting meat contaminated with ractopamine may cause cardiovascular risks. The European Food Safety Authority (EFSA) notes that ractopamine induced tachycardia in an animal study.7 The use of ractopamine in food-producing animals is therefore banned in many countries, including the European Union, China, and Russia (although not in the United States). Importing ractopamine-containing meat from the United States has become a political issue in Taiwan. Owing to these safety concerns, use of ractopamine in livestock is prohibited by the Taiwan Food and Drug Administration. Furthermore, meat imported into Taiwan must contain less than a specified level of ractopamine. Previous studies used radiolabeling to investigate the metabolism and disposition of ractopamine in turkey poults8 and the urinary excretion in rats after oral administration of [14C] ractopamine.9 LC-MS/MS has been reported to determine ractopamine residues in livestock tissues (in ppb).10 Microdialysis is another option for sampling unbound drug from multiple sites in a single animal. This technique can be coupled with liquid chromatography to determine the concentration versus time (pharmacokinetic) profiles in various © 2014 American Chemical Society

tissues. In a previous study, the blood and muscle distribution profiles of clenbuterol, a β2 agonist, were evaluated via the combination of microdialysis and HPLC.11 However, detailed pharmacokinetic studies of ractopamine have not been reported. Due to safety concerns over ractopamine, we hypothesized that the potential risks of ingesting meat containing ractopamine would be associated with bioavailability and tissue residues. The aim of this study was to develop a validated HPLC-MS/MS method for the determination of ractopamine in biological samples. This method was applied to investigate the pharmacokinetics, oral bioavailability, and organ distribution of ractopamine in rats.

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MATERIALS AND METHODS

Chemicals and Reagents. Ractopamine hydrochloride was purchased from Fluka (Steinheim, Germany). The internal standard, nylidrin hydrochloride, was provided by RBI (Natick, MA, USA). Pentobarbital sodium, urethane, α-chloralose, and heparin sodium were obtained from Sigma-Aldrich (St. Louis, MO, USA). Triply deionized water from Millipore (Bedford, MA, USA) was used in all experiments. Acetonitrile, methanol, and formic acid were obtained from E. Merck (Dermstadt, Germany). Ractopamine was dissolved in methanol to give a standard solution (1 mg/mL) and then diluted into individual eppendorf vials covered with a paraffin film as a stock solution (10 μg/mL). Working solutions were prepared by dilution of stock solution in 0.1% formic acid/ methanol (60:40, v/v). Nylidrin hydrochloride was dissolved in acetonitrile and stored in individual eppendorf vials covered with a Received: Revised: Accepted: Published: 9273

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Figure 1. Structures and product ion mass spectra of (a) ractopamine (m/z 302.10) and (b) nylidrin (m/z 300.15) in electrospray positive-ion mode. paraffin film as internal standard (30 ng/mL). All stock solutions were stored in darkness at −20 °C. Experimental Animals and Drug Administration. Experimental Animals. Male Sprague−Dawley rats (230 ± 20 g) were purchased from the National Yang-Ming University Animal Center, Taipei, Taiwan. Rats were housed with a 12 h light/dark cycle. Laboratory rodent diet 5001 (PMI Feeds, Richmond, IN, USA) and water were provided freely at all times. The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of National Yang-Ming University (IACUC no. 1011216) and was consistent with the guidelines of the National Research Council, USA.12 Intravenous Administration of Ractopamine. Rats were anesthetized with a mixture of urethane (1 g/kg) and α-chloralose (0.1 g/ kg) by intraperitoneal injection. Fur at the surgical site was shaved, and the skin was disinfected with 70% ethanol. The right jugular vein and the left femoral vein were catheterized with polyethylene tubing for blood sampling and drug administration, respectively. After surgery, ractopamine was administered in normal saline at 1 or 10 mg/kg, intravenously (iv) (n = 6 for each group). A 300 μL blood sample was collected from the jugular vein at 0, 15, 30, 45, 60, 90, 120, 180, 240, and 360 min after drug administration. Blood samples were placed in heparin-rinsed eppendorf vials and centrifuged at 6000g for 10 min at 4 °C to obtain plasma. Plasma was stored at −20 °C until analysis. If the concentration of ractopamine was above 50 ng/mL, the plasma sample was diluted with blank plasma, 5-fold (1 mg/kg, iv) or 50-fold (10 mg/kg, iv). Oral Administration of Ractopamine. Pentobarbital sodium (50 mg/kg) was used to anesthetize the rats via intraperitoneal administration. Surgical sites were shaved and cleaned with 70% ethanol. The right jugular vein was catheterized with polyethylene tubing for blood sampling. The catheter was guided under the skin to the dorsal neck region and fixed.13 After surgery, the rats were allowed to recover in a clean cage for at least 24 h before drug administration. After the stabilization period, ractopamine in water (10 mg/kg, per os (po)) was administered to the rats (n = 6). A 300 μL blood sample was collected from the jugular vein at 0, 5, 15, 30, 45, 60, 90, 120, 180, 240, and 360 min after drug administration. Blood samples were centrifuged at 6000g for 10 min at 4 °C to obtain plasma. Plasma was stored at −20 °C until analysis. Organ Distribution. Forty-five minutes after ractopamine administration (1 mg/kg, iv), blood samples were collected via cardiac puncture, and the rat was sacrificed by decapitation. The brain, liver, heart, spleen, lung, kidney, and gracilis muscle were collected and weighed. These organ samples were stored at −20 °C until analysis.

Sample Preparation. Blood Samples. Plasma samples (100 μL) were mixed with 10 μL of internal standard (nylidrin hydrochloride 30 ng/mL in acetonitrile) and 200 μL acetonitrile for protein precipitation. The mixture was centrifuged at 16000g for 10 min at 4 °C. The supernatant was evaporated to dryness at 40 °C in a centrifuge evaporator. The dried residue was reconstituted in 100 μL of 0.1% formic acid/methanol (60:40, v/v) and filtered through a 0.22 μm filter. An aliquot (5 μL) of the filtrate was analyzed using HPLCMS/MS. Organ Samples. Organ samples were homogenized in 50% aqueous acetonitrile (1:5, w/v), and the homogenate was centrifuged at 16000g for 15 min at 4 °C. The supernatant was collected and stored at −20 °C until analysis. Organ sample preparation was as described under Blood Samples. In brief, the organ sample (100 μL) was mixed with 10 μL of internal standard, followed by 200 μL of acetonitrile for protein precipitation. Samples were then subjected to centrifugation, evaporation, reconstitution, and filtration. Finally, 5 μL of filtrate was introduced into the HPLC-MS/MS system for analysis. If the concentration of ractopamine exceeded the upper limit of the linearity range (50 ng/mL), these organ homogenate samples were diluted with blank organ homogenate. HPLC-MS/MS. The HPLC-MS/MS system consisted of an LCMS8030 triple-quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization interface, coupled to a Shimadzu LC-20AD HPLC system (Shimadzu). The HPLC system was equipped with two pumps, a system controller, an autosampler, a column oven, and an online degasser. Chromatographic separation was carried out at 35 °C on an Acquity BEH C18 column (100 × 2.1 mm, 1.7 μm) with an in-line filter (Phenomenex AF0-8497, 0.5 μm depth × 0.004 i.d.). Isocratic elution was used for separation. The mobile phase was 0.1% formic acid/ methanol (60:40, v/v). The flow rate was 0.2 mL/min, and the injection volume was 5 μL. The temperature of the autosampler was 8 °C. Before analysis, a standard solution of ractopamine (100 ng/mL) was injected into the tandem mass spectrometer. The precursor ion of ractopamine was found in full-scan positive-ion mode. The mass-tocharge ratio (m/z) of the precursor ion was 302. The product ion m/z 164 was selected for quantification (Figure 1). The mass transition of nylidrin was determined using the same procedure. The transition m/z 300 → 150 was chosen for nylidrin (Figure 1). The optimized parameters for the mass spectrometer were as follows: nebulizing gas (nitrogen) flow, 3.0 L/min; drying gas (nitrogen) flow, 15 L/min; collision gas (argon) pressure, 230 kPa; 9274

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dissolution line temperature, 250 °C; and heat block temperature, 400 °C. Method Validation. Linearity, Accuracy, and Precision. The method validation confirms that the method used for the quantification of analyte in a biological sample is reliable and reproducible.14 Calibration standards were prepared from stock solution of ractopamine and then added to blank plasma or homogenate to spike samples with concentrations of 0.5, 1, 5, 10, and 50 ng/mL. These spiked samples were processed as described under Sample Preparation. The calibration curve was constructed from the ratio of the peak areas of ractopamine and the internal standard (nylidrin) to the nominal concentration of ractopamine. The correlation coefficient (r2) of all calibration curves was at least 0.995. Six replications of the calibration curve were performed on the same day (intraday) and over six consecutive days (interday) to evaluate precision and accuracy. Accuracy describes the proximity of mean results (observed concentration, Cobs) of this method to the true concentration (nominal concentration, Cnom). Accuracy can be calculated as bias (%) = [(Cobs − Cnom)/Cnom] × 100. Precision means the closeness of each individual result to the others. Precision, quantified as relative standard deviation (RSD), was calculated as follows: RSD (%) = [standard deviation (SD)/Cobs] × 100. The bias and coefficient of variation were kept within ±15%, except for the lower limit of quantification (LLOQ), which was not permitted to exceed ±20%. Matrix Effect and Recovery Evaluation. The matrix effects and recovery were calculated using three sets of samples. Ractopamine was evaluated at 0.5, 5, and 50 ng/mL, and nylidrin was evaluated at 3 ng/ mL. The three sets were as follows. Set 1: For standard solutions of ractopamine, the ractopamine stock solution was diluted to 0.5, 5, or 50 ng/mL with 0.1% formic acid/ methanol (60:40, v/v). Set 2: Blank plasma or organ homogenate (100 μL) was processed as described under Sample Preparation and reconstituted to obtain blank matrix. Blank matrix (90 μL) was added to 10 μL of standard solution, giving postextraction spiked ractopamine samples at three different concentrations. Set 3: Standard solution (10 μL) and blank plasma or organ homogenate (90 μL) were mixed and subjected to sample preparation and reconstitution. Then a pre-extraction spiked ractopamine sample was acquired and injected into the HPLC-MS/MS system. The matrix effect is given as the ratio of mean peak area for the postextraction spiked samples (set 2) to that of the standard solution samples (set 1). Recovery was quantified as the ratio of mean peak area of the pre-extraction spiked samples (set 3) to that of the postextraction spiked samples (set 2). Stability. The stability of ractopamine was evaluated by the following methods, as recommended by the FDA: 1, freeze and thaw; 2, short term; 3, long term; 4, postpreparative; and 5, stock solution.14 Samples for freeze and thaw stability were stored at −20 °C for 24 h and thawed at room temperature. This freeze and thaw cycle was repeated a total of three times. The samples were analyzed after the third cycle. Short-term stability was measured by maintaining the samples at room temperature for 4 h and then analyzing them. Longterm stability was assessed by storing the samples at −20 °C for 30 days. The samples for postpreparative stability testing were processed as described under Sample Preparation. These samples were then maintained in an autosampler at 8 °C for 8 h. The stock solution stability was evaluated by keeping the stock solution at room temperature for 6 h. Concentrations of 0.5, 5, and 50 ng/mL were used to measure the stability of ractopamine in plasma or organ homogenate. The stock solution for stability evaluation was prepared in the mobile phase before analysis. Stability was calculated using the relative error between the freshly prepared and stored samples. Sample stability was limited to within ±15%. Pharmacokinetic Analysis. Pharmacokinetic parameters were calculated using WinNonlin Standard Edition 1.0 (Pharsight Corp., Mountain View, CA, USA) with compartmental and noncompartmental models for the intravenous and oral groups, respectively.

Akaike’s information criterion (AIC) was used to choose the pharmacokinetic models (one- or two-compartment).15 The lowest AIC value indicated the best way to describe the pharmacokinetic data. The results are expressed as the mean ± standard deviation. The criterion of statistical significance was Student’s t test, with p < 0.05.

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RESULTS HPLC-MS/MS. In positive-ion mode, the multiple reaction monitoring (MRM) transitions were m/z 302 → 164 for ractopamine and m/z 300 → 150 for nylidrin (internal standard). The fragmentation pathway of m/z 302 → 164 was consistent with previous results for ractopamine.16 The second step was optimizing chromatographic separation. Peak shape, run time, and sensitivity were influenced by the composition of the mobile phase. To reduce tailing, 0.1% formic acid was selected as the aqueous phase. Different mobile phase ratios were examined: 0.1% formic acid/methanol (10:90, 60:40, and 90:10, v/v). The experimental results revealed that 0.1% formic acid/ methanol (60:40, v/v) gave the sharpest peak and the best run time. Thus, 0.1% formic acid/methanol (60:40, v/v) was chosen for the remaining experiments. Under these conditions, the retention time of ractopamine was 2.0 min and that of nylidrin was 5.5 min. The similar structures of ractopamine and nylidrin justified the use of this internal standard. The similar fragmentation patterns and the structural similarity between ractopamine and nylidrin may ensure the specificity of this analytical method.17 Thus, nylidrin was an ideal choice as internal standard. Typical chromatograms are shown in Figures S1−S4 in the Supporting Information for blank plasma, liver, heart, spleen, lung, kidney, brain, and muscle homogenates; blank plasma or organ homogenates spiked with ractopamine; and finally samples collected from experimental animals after ractopamine administration (1 mg/kg, iv). There was no significant endogenous interference under optimized HPLC-MS/MS conditions (panels a and d of Figures S1−S4). These results show acceptable selectivity for the determination of ractopamine in the biological samples. Furthermore, carry-over effects were not observed in any experiment. Sample Preparation. A pilot experiment was undertaken to determine the most appropriate sample preparation method. Liquid−liquid extraction and protein precipitation by methanol or acetonitrile were examined. Liquid−liquid extraction gave the lowest recovery of ractopamine in rat plasma, approximately 30%. The extraction recoveries via methanol and acetonitrile protein precipitation were 75 and 85%, respectively. The low recovery by liquid−liquid extraction may be due to the high polarity of ractopamine; thus, this method is not suitable.18 Protein precipitation by acetonitrile was selected as the sample preparation method in this experiment. In the full study, the extraction recovery of ractopamine in rat plasma via acetonitrile protein precipitation was 86.7 ± 2.8% (Supporting Information Table S2, n = 3). Unfortunately, the chromatographic peak obtained using acetonitrile protein precipitation was split, which made integration difficult. This problem was resolved by evaporating the supernatant after protein precipitation. After supernatant evaporation, the chromatographic peak was narrow and sharp. Method Validation. Linearity, Accuracy, And Precision. To evaluate linearity, a calibration curve was derived from the peak area ratios of ractopamine and the internal standard, nylidrin. The linear range for ractopamine in rat plasma and 9275

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organ homogenates was 0.5−50 ng/mL; all correlation coefficients (r2) were >0.999. The limit of detection (LOD) for ractopamine was 0.01 ng/ mL, with a signal-to-noise ratio of 3. The lower limit of quantification (LLOQ) in plasma and organ homogenate was 0.5 ng/mL. Intraday and interday assays were used to evaluate accuracy (as % bias) and precision (as % RSD) for ractopamine (Supporting Information Table S1, n = 6). All bias and RSD values were below 15%, except for the LLOQ (0.5 ng/mL). At this concentration, 20% is the maximum acceptable value.14 These method validation results indicate that this analytical method is reproducible. Matrix Effect and Recovery. The mean matrix effects for ractopamine in plasma, liver, heart, spleen, lung, kidney, brain, and muscle homogenates were 81.2 ± 4.2, 97.5 ± 2.3, 123.3 ± 2.6, 120.8 ± 3.7, 112.6 ± 2.9, 59.5 ± 2.6, 135.0 ± 4.5, and 113.1 ± 1.5%, respectively (Supporting Information Table S2, n = 3). Compared with the standard solution (set 1) and postextraction spiked solution (set 2), it was very easy to determine ion suppression or enhancement in the plasma and organ samples. Thus, the matrix effect of ractopamine should not be ignored in these experiments. Extraction recovery is defined as the difference between preextraction (set 3) and postextraction spiked (set 2) samples. The mean extraction recoveries for ractopamine varied from 86.7 ± 2.8 to 98.0 ± 0.6% (Supporting Information Table S2, n = 3). Significant matrix effects were observed in rat plasma and organ homogenates. However, within a given matrix, the matrix effects and recoveries were independent of concentration within the linear range. To examine the matrix effect and recovery in the whole experiment, samples collected from animals were diluted to within the linear range using blank plasma or organ homogenate. In addition, all experimental samples were diluted to the same linear range (0.5−50 ng/mL). The matrix effect and recovery, correlated with concentration, were not significantly different. The average matrix effects and recoveries of nylidrin in rat plasma and organ homogenates ranged from 98.6 ± 1.3 to 106.9 ± 0.8 and from 80.8 ± 0.3 to 97.1 ± 0.2, respectively (Supporting Information Table S2, n = 3). On the basis of these results, nylidrin had a small matrix effect and extraction loss after sample preparation. Thus, nylidrin is a suitable internal standard for this study. Stability. Following FDA guidelines, five stability evaluation experiments (freeze−thaw cycle, long-term, short-term, postpreparative, and stock solution) were designed to simulate different conditions: sample collection, storage, preparation, or other handling conditions. Stability was quantified as the relative error in concentration between freshly prepared and stored samples. Stability in the freeze−thaw, short-term, longterm, postpreparative, and stock solution analyses were all within ±15% (Supporting Information Table S3, n = 3). These results indicate that ractopamine was stable during all of these experimental processes. Pharmacokinetics of Ractopamine. To investigate the pharmacokinetics and oral bioavailability of ractopamine, it was administered orally (10 mg/kg, po) and intravenously (1 and 10 mg/kg, iv) (Figure 2, n = 6 for each group). Plasma samples were collected and analyzed by the validated method. To describe the pharmacokinetic profile precisely, it is important to select a proper compartmental model (one- or two-compart-

Figure 2. Concentration versus time profile of ractopamine in rat plasma (10 and 1 mg/kg, iv, and 10 mg/kg, po) Data are expressed as the mean ± SD (n = 6).

ment models). Akaike’s information criterion (AIC) provides a suitable criterion for the selection of compartmental models.15 A smaller AIC value means the model has a better fit to the concentration−time curve calculated using WinNonlin. In this experiment, the average AIC values for the one-compartment model (ractopamine 1 and 10 mg/kg, iv) were −86.82 and −23.36, respectively. The mean AIC values of the twocompartment model were −98.27 and −41.16, respectively. The two-compartment model thus describes the pharmacokinetics of ractopamine better (Table 1). Table 1. Pharmacokinetic Data for Plasma Ractopamine in Ratsa pharmacokinetic parameter

ractopamine 1 mg/kg, iv

ractopamine 10 mg/kg, iv

AIC of one compartment AIC of two compartments AUC (min mg/mL) AUC/dose t1/2, α (min) t1/2, β (min) Tmax (min) C0 (μg/mL) Cl (mL/min/kg) MRT (min) Vss (L/kg) F (%)

−86.82 ± 4.40

−23.36 ± 2.93

−98.27 ± 6.53

−41.16 ± 6.92

5.85 ± 0.70

123.92 ± 40.23b

5.85 ± 0.70 21 ± 7.4 118 ± 25

12.39 ± 4.02b 18 ± 8.0 165 ± 49

0.062 173 146 25.4

± ± ± ±

0.015 22 25 5.7

1.65 87.4 185 14.9

± ± ± ±

0.33b 25.2b 65 2.0b

ractopamine 10 mg/kg, po

1.75 ± 0.24

113 ± 23 15 5800 148 945 2.99

± ± ± ±

735 30 196 0.40

a

Abbreviations: AIC, Akaike’s information criterion; AUC, area under the concentration−time curve; t1/2, α, distribution half-life; t1/2, β, elimination half-life; Tmax, time of peak plasma level; C0, initial drug concentration; Cl, clearance; MRT, mean residence time; Vss, apparent volume of distribution at steady state; F, bioavailability. Data are expressed as the mean ± SD (n = 6). bp < 0.05 compared with 1 mg/ kg, iv.

The equation for the two-compartment model is Cp = A e−αt + B e−βt. This reflects the sum of two different processes in the body: distribution and elimination, presented as A e−αt and B e−βt, respectively. The parameters α and β are the distribution and elimination constants, respectively. The A and B values are the intercepts of the distribution and elimination phases. 9276

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Journal of Agricultural and Food Chemistry Additional pharmacokinetic parameters are as follows. Distribution and elimination half-lives are referred to as t1/2, α and t1/2, β, respectively. AUC, the area under the concentration− time curve, is related to the extent of drug absorption. C0, the initial drug concentration, is calculated by extrapolation using the pharmacokinetic equation. Cl, clearance, is an indicator of drug elimination. MRT, mean residence time, could be considered the average period of residence of individual molecules of the drug in the body. Vss is the apparent volume of distribution at steady state, derived from concentration and total amount. According to the pharmacokinetic parameters calculated using WinNonlin, intravenous ractopamine administration at 1 and 10 mg/kg gave the two-compartment models Cp = 0.033 e−0.037t + 0.029 e−0.006t and Cp = 1.258 e−0.047t + 0.392 e−0.005t, respectively. The average elimination half-lives (t1/2, β) for the two doses of ractopamine were 118 and 165 min, respectively (Table 1, n = 6 for each group). The noncompartmental model was selected to describe the pharmacokinetic profile after oral administration of ractopamine (10 mg/kg, po) (Table 1, n = 6). The pharmacokinetic parameter Tmax indicates the time of peak plasma level. Bioavailability (F) was used to describe systemic availability: F (%) = 100 × (AUCoral/doseoral)/(AUCiv/doseiv). Tmax was 15 min, indicating that ractopamine is rapidly absorbed in rats. Bioavailability was derived from the AUC after intravenous (1 mg/kg) and oral administration (10 mg/kg). The oral bioavailability of ractopamine (F = 100 × (1.75/10)/5.85) was around 2.99%. Organ Distribution of Ractopamine. To investigate the tissue distribution of ractopamine, tissues and organs were collected 45 min after administration (1 mg/kg, iv). The ractopamine levels in liver, heart, spleen, lung, kidney, brain, and muscle tissue were 307.85 ± 54.82, 391.11 ± 22.81, 675.77 ± 49.54, 1058.41 ± 78.44, 1211.17 ± 89.25, 24.35 ± 1.85, and 294.81 ± 28.00 ng/g, respectively (Figure 3, n = 6). Plasma samples for the organ distribution experiment were collected by cardiac puncture. Plasma ractopamine was 25.09 ± 3.51 ng/ mL.

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DISCUSSION

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ASSOCIATED CONTENT

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Nonlinear Pharmacokinetics of Ractopamine. Significant differences were found (p value < 0.05) in the pharmacokinetic parameters of the two intravenous doses: AUC/dose (5.85 ± 0.70 vs 12.39 ± 4.02), clearance (173.20 ± 21.75 vs 87.39 ± 25.21), and Vss (25402 ± 5734 vs 14879 ± 1971). That these pharmacokinetic parameters varied with dose may imply nonlinear pharmacokinetics after intravenous ractopamine administration at between 1 and 10 mg/kg.19 From these results, we hypothesize that excess ractopamine may accumulate due to organ saturation and retarded elimination in the kidney. Oral Bioavailability of Ractopamine. The oral bioavailability of ractopamine was derived from the AUC for intravenous and oral administration. Due to nonlinear pharmacokinetics at high doses, the AUC data for intravenous ractopamine (10 mg/kg) were not utilized. In this experiment, the oral bioavailability of ractopamine was 2.99%. Oral bioavailability can be evaluated by comparing the amount of parent drug in the collected urine with the total amount administered. Smith and Paulson found, via urine collection, that the oral bioavailability of ractopamine in rats was 2%.9 Although in our experiment the bioavailability of ractopamine was determined via plasma collection, the result is consistent with the previous study. However, due to the different methods of evaluation, the experimental results may differ. In addition, oral bioavailability has been studied in turkeys and swine, and the results were 8 and 4−16%, respectively.8,20 Thus, bioavailability may vary between species. Ractopamine Accumulation in Rat Organs. On the basis of the above pharmacokinetic results, a time point of 45 min was selected because that was close to the intersection of the first and second compartments. The organ distribution results suggest that ractopamine is extensively distributed into tissues 45 min after intravenous administration. The lowest concentration was found in the brain, suggesting that the distribution of ractopamine, with its high polarity, may be restricted by the blood−brain barrier. The highest level was observed in the kidney, which may indicate that the kidney is the primary organ of elimination for this drug in rats. This is consistent with the hypothesis given under Nonlinear Pharmacokinetics of Ractopamine. The nonlinear pharmacokinetics of ractopamine in high doses may be due to nonlinear elimination in the kidney. In a previous study, Smith and Paulson reported that after oral administration of ractopamine (9.9 mg/kg), approximately 59% of the drug was eliminated in bile. Thus, they suggested that bile excretion is the most important elimination pathway for ractopamine in rats.9 Our findings differ. This inconsistency may be explained by the first-pass effect. If the rats were given ractopamine orally, most of the drug would be eliminated via bile and would not enter the circulation. Intravenous administration of ractopamine, however, avoids the first-pass effect. Thus, most of the drug may be eliminated by the kidney. This may also explain the low oral bioavailability of ractopamine in rats: most of it may be excreted in bile.

Figure 3. Plasma, liver, heart, spleen, lung, kidney, brain, and muscle levels of ractopamine after administration (1 mg/kg, iv). Data are expressed as the mean ± SD (n = 6). Concentrations in plasma and organs are micrograms per milliliter and micrograms per gram, respectively.

S Supporting Information *

Method validation results and typical HPLC-MS/MS chromatograms. This material is available free of charge via the Internet at http://pubs.acs.org. 9277

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(16) Thevis, M.; Opfermann, G.; Schanzer, W. Liquid chromatography/electrospray ionization tandem mass spectrometric screening and confirmation methods for β2-agonists in human or equine urine. J. Mass Spectrom. 2003, 38, 1197−1206. (17) Antignac, J. P.; Marchand, P.; Le Bizec, B.; Andre, F. Identification of ractopamine residues in tissue and urine samples at ultra-trace level using liquid chromatography-positive electrospray tandem mass spectrometry. J. Chromatogr., B, Anal. Technol. Biomed. Life Sci. 2002, 774, 59−66. (18) McDowall, R. D. Sample preparation for biomedical analysis. J. Chromatogr. 1989, 492, 3−58. (19) Mehvar, R. Principles of nonlinear pharmacokinetics. Am. J. Pharm. Educ. 2001, 65, 178−184. (20) Dalidowicz, J. E.; Thomson, T. D.; Babbitt, G. E. Ractopamine hydrochloride, a phenethanolamine repartitioning agent. In Xenobiotics and Food-Producing Animals; Hutson, D. H., Hawkins, D. R., Paulson, G. D., Struble, C. B., Eds.; American Chemical Society: Washington, DC, USA, 1992; Vol. 503, pp 234−243.

AUTHOR INFORMATION

Corresponding Author

*(T.-H.T.) Mail: Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, 155 Li-Nong Street, Section 2, Taipei 112, Taiwan. Fax: (886-2) 2822 5044. Phone: (886-2) 2826 7115. E-mail: [email protected]. Funding

This work was supported in part by research grants from the National Science Council Taiwan (NSC102-2113-M-010-001MY3) and by TCH 103-02 and TCH 10301-62-021 from Taipei City Hospital, Taiwan. Notes

The authors declare no competing financial interest.

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dx.doi.org/10.1021/jf5026168 | J. Agric. Food Chem. 2014, 62, 9273−9278