Assessment of the withdrawal period for ractopamine hydrochloride in the goat and sheep

Ractopamine hydrochloride often used as a bronchodilator, but its β-adrenergic agonist effects on un-striated muscle and its withdrawal time have not been assessed for Etawah goats and sheep. The aim of this study was to determine the safe time to slaughter goats and sheep post-treatment with ractopamine. Five clinically healthy adult goats and sheep (20 kg body weight) were treated with a single dose of ractopamine (1 mg, intravenously). Whole blood was sampled from the jugular vein at 120 min, 180 min and 300 min post-treatment. Ractopamine as a veterinary drug was analysed using HPLC at wavelength 225 nm. The concentrations at 120 min, 180 min and 300 min were 817.156 ± 13.460 μg.mL, 554.468 ± 50.157 μg.mL, and 294.588 ± 52.845 μg.mL in goats and 706.266 ± 89.856 μg.mL, 579.194 ± 45.664 μg.mL, and 209.36 ± 54.797 μg.mL in sheep, respectively. The withdrawal times in goats and sheep were 1141.710 ± 255.85 h and 989.741 ± 167.633 h, respectively, no drug residues detected. The safe time to slaughter goats after administration of 1 mg of ractopamine was approximately 3 months and 5 days post-treatment at a safety factor of 2, which was later than the sheep at 2 months and 22 days by a safety factor of two.

Introduction β-Agonists exhibit short-acting effects and long-acting effects and are frequently used to promote growth in livestock, but problems arise when the residues of such veterinary drugs have the potential to influence human health after being consumed in animal products (1)(2)(3)(4)(5). Evaluation of β-agonist as a veterinary drug is a new concept to implement the principle using medicines for veterinarian by motto: logic and responsibility. Logic in science has the following characteristics: a methodology, based on evidence and theory update. Responsibility means that a professional veterinarian has a moral responsibility to monitor the effects of administration of a drug that they have recommended (6).
In early 2018, a new concept for calculation of withdrawal time was developed on the basis of pharmacokinetic and bio-analysis data. This improved approach permits the calculation of withdrawal periods for veterinary drugs in any local animal, so it logical to use it to re-assess the current withdrawal period for ractopamine (7).
It was therefore decided to use this updated approach to re-assess the withdrawal period of ractopamine after treatment in goats and sheep, both of which are distributed across Asia, Europe, Australia and Africa, where they are used to produce human food, drugs and cosmetics. They are also closely related to livestock in several countries that are free from adverse drug responses (8). Specifically, we tested whether the withdrawal period of ractopamine, after a single dose was administered intravenously, differs between Indonesian (Etawah) goats and sheep (9). A second goal was to determine the length of time required for β-agonist residues to decrease to safe levels in animals that are to be slaughtered post-treatment.

Experiment animals and animal ethic clearance
This research was conducted at Airlangga University in collaboration with the Indonesia Veterinary Pharmacy and Pharmacology Association under control by the Indonesia Medical Veterinary Association (IMVA) from August 2016 to August 2018, with rainy conditions and subtropical temperatures of approximately 22-25ºC, and was conducted in compliance with the animal ethics clearance from University of Airlangga. Etawah goats and Javanese thintailed sheep, both local Indonesian breeds, were obtained from local farmers in Surabaya, Indonesia. The study was conducted in five of goats and five of sheep. The criteria of experimental animals were from the second generations of the native species of goats and sheep and were healthy adult males aged 1 year (live weight 18-22±1.41kg) raised under controlled veterinarian practices. Before treatment, all animals were adapted in animal housing for approximately 2 weeks.

Research design and experimental protocol
The experimental design consisted of a time series of measurements at 120 min, 180 min and 300 min posttreatment, followed by analysis of the elimination rate constant (K el ) and limit of quantification for method analysis (10).

Sample preparation
Certified reference material of Ractopaminehydrochloride (Vetranal) at HPLC grades was purchased from Sigma-Aldrich in CAS-N0 90274-24-1 at level of purity 95.5%. Pharmaceutical-grade ractopaminehydrochloride was produced by Shanghai Hannochem International Corporation. (www.hannochem.com). The drug was dissolved in aqua pro injection solution (w/v), and drops of 0.1% phosphoric acid for HPLC grade (Merck Catalog No.7664-38-2) were added until a solution formed. pH was adjusted 6.8-7 using with 0.1 M NaOH. After filtration through a 0.20 µm filter, the solution of ractopamine was ready to use for injection (11). One milligram of the drug was intravenously injected into the jugular veins of goats and sheep. Jugular blood was sampled with a disposable syringe (5 mL) at 120 min, 180 min and 300 min post-treatment. All samples were placed in 10 mL heparinized tubes and centrifuged at 3500 rpm for 30 min. The plasma samples were placed in Nunc tubes (5 mL) and stored at -20ºC until all samples were prepared.

Analysis of ractopamine
Analysis of the drug and drug residues in the liver was performed using a Shimadzu HPLC LC-6AD pump with a PDA detector. Optimization and validation of the HPLC method were modified with the mobile phase consisted of acetonitrile: aqua pro chromatography reagent (30:70) containing 0.1% phosphoric acid at pH 3.8 (11)(12)(13). A reverse-phase ODS C 18 column was used, and the detector was set at 225 nm. Rheodyne ® sample loops were configured to perform 2 µL injections (14). Analysis was carried out using isocratic elution without internal standards. Analysis of the method parameters resulted in an optimal flow rate of 0.5 mL.min -1 and an analysis time of 20 min. The method was validated for quality assurance at high impact levels according to the new research report (7,8,10).

Analysis of withdrawal time
The withdrawal time was calculated by using new method namely "Lazuardi Theory" base on K el data for ractopamine to determine the elimination half-live (T 1/2β ) and the limit of quantification (LOQ), as described by equation at below (7,9,14). The drug residues in goat and sheep in liver were monitored one-month post treatment.

Statistical analysis
The relationship between the ractopamine concentration and HPLC detector response and the differences in the kinetics profiles based on goat versus sheep data were assessed with SPSS 24.0 at 5% significance. The research hypothesis was tested at significance 5% with SPSS 24.0

Validation method and concentration of ractopamine in matrix biology
The optimal UV-Vis wavelength for ractopamine detection was 225 nm, as indicated in Fig. 1. The HPLC retention time was 3.890 min for the ractopamine standard ( Fig. 2) but 4.082 min for ractopamine extracted plasma by SPE (Fig. 3).
The peak area of ractopamine increased linearly with the concentration over the evaluated range of 0.242 to 1.601 µg.mL -1 , with a regression equation of Y = -0.205 + 33925175 X and an R-squared value of 0.99. The coefficients of variation (CVs) for the intraday precision and accuracy at concentrations ranging from 0.18 µg.mL -1 to 0.956 µg.mL -1 were less than 8% (Table 1).
Regarding method sensitivity, the limit of detection (LOD) was 3.5 ×10 -6 µg.mL -1 , and the LOQ was 1.06 ×10 -5 µg.mL -1 . The precision and accuracy had CVs of less than 20%, and the percent recovery in artificial plasma was more than 80% ( Table 2). The plasma concentrations of the drug in goats and sheep are detailed in Table 3.

Kinetic parameters of ractopamine
The exponent equation (K el ) and T ½β determined on the basis of the data from goat and sheep between 5-300 min post-treatment are presented in Table 4. These data show that the drug was eliminated more slowly in the goat than in the sheep (P>0.05).

Discussion
To determine the ractopamine levels in plasma collected from healthy goats and sheep treated with this agent, we initially utilized the HPLC assay described by other researchers without modification (9,15,16). However, we were unable to reproduce the reported assay so we developed a simpler assay using an isocratic mobile phase. Our protocol was validated and the CV from linearity analysis was no more than 10%. For intra-day precision and accuracy, CVs were less than 20%. We needed to analyse plasma samples containing low concentrations of ractopamine, below 1.00 μg.mL -1 , because such concentrations are expected in animals receiving a typical dose regimen but measurements at this level are known to be un certainty, primary due to instrument error and human error during sample preparation. The LOQ we achieved (1.06 x 10 -5 μg.mL -1 ) was lower than the mean ractopamine concentration at 300 min posttreatment in goats and sheep. To produce an accurate, simple assay with high recoveries, sample components were separated on two adsorption columns: an HPLC column and SPE column (17,18). The recovery of ractopamine from plasma was 88-99% (19,20), consistent with reported recoveries of the half-life in these two species (21,22). and supporting suggestions that it is more extensively distributed in goat tissues than in sheep tissues (23,24). Therefore, in goats, ractopamine is not easily metabolized and must be processed more than 1.15 times as much as in sheep. Overall. It is clear that the risk of exposure to ractopamine residues is greater in goat tissues than in sheep tissues (4,25). The residues in liver and became undetectable after 95 h in goat and 82 h in the sheep, similar to delays reported for clenbuterol, a β 2 -agonist, in goat tissues (8,26). No ractopamine residues were detected in liver samples at a safety factor of 2 and 3, comparable to the factors reported by Ho and Colleagues the rat (27).
Ractopamine is known for its potential to be distributed into deep tissues in organs such the spleen, liver and kidney, increasing the risk of cancer (28). It is therefore important to acknowledge the risk of residues in animal slaughtered before the end of the withdrawal period. It is known that ractopamine residues do not melt at temperatures 0f 100-120 0 C so imperfectly cooked meat will lead to the consumption of residues so persistent consumption is likely to increase the risk of cancer.

Conclusions
The withdrawal time of 1 mg of ractopamine administered intravenously is longer in goats than that in sheep. In goats, the withdrawal time is 3 months and 5 days post-treatment at a safety factor of two, and 4 months and 8 days post-treatment at a safety factor of three. In sheep, the withdrawal time is 2 months and 22 days post-treatment at a safety factor of two, and 4 months and 4 days post-treatment at a safety factor of three. It is clear that, more research is still needed to improve the detection of ractopamine residues in various tissues. A safety factor of more than 3 will be increase confidence that all residues had disappeared.