Tissue Depletion of Quinocetone and Its Five Major Metabolites in

Oct 3, 2014 - Xu Wang , María-Aránzazu Martínez , Guyue Cheng , Zhaoying Liu , Lingli ... María-Rosa Martínez-Larrañaga , Arturo Anadón , Zongh...
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Tissue Depletion of Quinocetone and Its Five Major Metabolites in Pigs, Broilers, and Carp Fed Quinocetone Premix Juan Li,†,‡,§ Lingli Huang,†,‡ Yuanhu Pan,† Dongmei Chen,† Xu Wang,† Ijaz Ahmad,† Yanfei Tao,† Zhenli Liu,† and Zonghui Yuan*,†,‡ †

National Reference Laboratory of Veterinary Drug Residues (HZAU) and MOA Key Laboratory for Detection of Veterinary Drug Residues and ‡MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei 430070, People’s Republic of China § College of Chemistry, Xiangtan University, Xiangtan 411105, People’s Republic of China ABSTRACT: A residue depletion study was performed to investigate the tissue kinetics of quinocetone (1) and its major metabolites. Quinocetone and its major metabolites were simultaneously quantitated with a high-performance liquid chromatography−ultraviolet (HPLC-UV) method. A total of 25 pigs, 30 broilers, and 50 carp were fed 100 mg/kg quinocetone for 90, 42, and 60 days, respectively. Liver, kidney, muscle, and fat (skin) tissues were collected at five different withdrawal times for analysis. Results revealed that quinocetone, 1-desoxyquinocetone (2), carbonyl-reduced 4-desoxyquinocetone (4), 3methylquinoxaline-2-carboxylic acid (5), and carbonyl-reduced dideoxyquinocetone (6) could be depleted quickly in tissues; by contrast, dideoxyquinocetone, 3, persisted for a long time in the liver. Therefore, the liver is possibly the target tissue of quinocetone, and 3 is the residual marker; the recommended withdrawal times (WDTs) are 0 days in pigs and carp and 3 days in broilers. These results provided clear monitoring tools and technical standards to evaluate the food safety of quinocetone. KEYWORDS: quinocetone, metabolites, tissue depletion, withdrawal time, estimates of dietary intake, HPLC

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INTRODUCTION Quinocetone [1, 3-methyl-2-quinoxalinbenzenevinylketo-1,4dioxide (CAS Registry No. 81810-66-4, C18Hl4N2O3)] is an antibacterial agent that belongs to the quinoxaline-1,4-dioxide family (Figure 1). Given its beneficial effects in animal husbandry, quinocetone was approved as a medication premix at 50−75 mg/kg level in the diet and regarded as a replacement for carbadox and olaquindox in China in 2003.1 However, studies have observed that quinocetone elicits adverse effects, such as genotoxicity, hepatotoxicity, and nephrotoxicity, in vitro and in vivo.2−8 Therefore, food safety of quinocetone in foodproducing animals should be evaluated because quinocetone residues and metabolites in animal tissues may endanger consumer health. Veterinary drugs for food-producing animals should be assigned a scientific withdrawal time (WDT) based on acceptable daily intake and residue depletion study to ensure food safety. However, no WDT of quinocetone has been established yet. Hence, residue studies should be performed to address issues related to practical use and food safety. In vitro and in vivo metabolisms of quinocetone have been investigated, and findings have shown that quinocetone can be easily metabolized to produce a number of compounds.9−11 Moreover, isotopic tracing distribution and metabolism studies have shown that 1-desoxyquinocetone (2), dideoxyquinocetone (3), carbonyl-reduced 4-desoxyquinocetone (4), and carbonylreduced dideoxyquinocetone (6) are the major metabolites in food-producing animals,12 and 3-methyl-quinoxaline-2-carboxylic acid (5) is an important metabolite of quinocetone in a previous study.13 The studied compound structures are shown in Figure 1. Studies have been conducted to examine the residual depletion of quinocetone in pigs, chickens, and carp.14−16 However, these studies have primarily focused on © XXXX American Chemical Society

quinocetone and a few of its known metabolites (2, 3, and 5), but new and major metabolites (4 and 6) have not been described. To obtain basic data for food safety evaluation of quinocetone in pigs, broilers, and carp, researchers should conduct further studies. Sensitive and accurate bioanalytical methods should be developed to quantitate quinocetone and its metabolites; a residue marker and monitoring standard of these compounds in food-producing animals should also be proposed. To the best of our knowledge, limited information is available regarding the simultaneous quantitation of these six compounds; previous studies mainly investigated 1, 3, and 5 by HPLC-UV.13,17−20 Although LC-MS/MS methods have been developed to analyze 1 and 3−6,21,22 LC-MS is not readily available for routine monitoring. In the current study, residue depletion of quinocetone in pigs, broilers, and carp was investigated to characterize the kinetics of quinocetone and its metabolites in the muscle, liver, kidney, and fat (skin) tissues. Subsequently, the WDT of quinocetone in food-producing animals was calculated on the basis of residue depletion data. A sensitive and accurate HPLCUV method was investigated for the first time to simultaneously and quantitatively determine quinocetone and its five main metabolites in edible tissues of pigs, broilers, and carp. Our results provided a scientific food safety standard to monitor quinocetone residues; an efficient method was also proposed to estimate the concentration of quinocetone and its metabolites in edible tissues. Received: June 27, 2014 Revised: October 2, 2014 Accepted: October 3, 2014

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Figure 1. Molecular structures of quinocetone, 1, and its main metabolites, 2−6.

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into a control group (n = 5, 6, and 10 for pigs, broilers, and carp, respectively) and a test group (n = 25, 30, and 50 for pigs, broilers, and carp, respectively). The control groups were fed the standard ration without quinoxaline compounds. The test groups were provided with medicated feed for 90, 42, and 60 consecutive days for pigs, broilers, and carp, respectively. The medicated feed contained a standard ration premixed with quinocetone at a level of 100 mg/kg diet, which was slightly higher than the recommended level of quinocetone preparations.1 At different time points [6 h, 1, 3, 7, and 14 days (6 h, 1, 3, 5, and 7 days for broilers, and 6 h, 1, 3, 5, and 10 days for carp)], 1 control and 5 medicated pigs (6 medicated broilers and 10 medicated carp) were anesthetized and sacrificed. The pigs and broilers were slaughtered using captive bolt stunning equipment and exsanguinated in accordance with the guidelines provided by the American Veterinary Medical Association for euthanasia,23 the carp were euthanized in a MS-222/water (1:10000) bath. Liver, kidney, muscle, and fat (skin) specimens were collected. All of the samples were placed in labeled plastic bags in an ice bath, immediately homogenized, and frozen at −20 °C until these samples were analyzed. Analysis of Analytes. Sample Preparation. Samples (2 ± 0.1 g) were transferred into a 50 mL centrifuge tube, and 8 mL of 1% metaphosphoric acid in MeOH/MeCN/water (50:20:30, v/v/v) was added to the sample. The mixtures were shaken using a vortex system for 5 min, ultrasonicated for 10 min at room temperature, and centrifuged at 2160g for 10 min. The supernatant was then transferred into a 50 mL centrifuge tube. The same extraction procedure was repeated. The two supernatants were combined, and 1 mL of 10% aqueous zinc acetate was added to the extracts. These extracts were mixed by vortexing for 2 min and centrifuged at 13500g for 10 min; afterward, the supernatant was transferred and diluted with 1% metaphosphoric acid to obtain a final volume of 64 mL, which was ready for the cleanup procedure. The HLB cartridge (60 mg, 3 mL) (Waters Corp., Milford, MA, USA) was preconditioned with 3 mL of MeOH and 3 mL of water. Flow rates for conditioning and washing were set at 3 mL/min. The entire extracts were loaded onto the SPE column at a flow rate of 1 mL/min. The column was washed with 3 mL of water and 3 mL of 10% MeOH and dried by purging air at a rate of 10 mL/min for 5 min. The analytes were eluted with 3 mL of MeCN at a flow rate of 1.0 mL/min into a 10 mL tube and evaporated to dryness under a stream of nitrogen at 45 °C. The dry residue was dissolved in 1 mL of 20% MeCN, vortexed for 1 min, and centrifuged at 16600g for 10 min. The resulting solution was then filtered using a 0.22 μm nylon Millipore chromatographic filter, and a 20 μL aliquot was analyzed by HPLC.

MATERIALS AND METHODS

Chemicals and Reagents. The analytical standards of 1−6 (98% purity) were obtained from the Institute of Veterinary Pharmaceuticals (Huazhong Agricultural University, Wuhan, People’s Republic of China). Individual stock standard solutions (1000 μg/mL) of all analytes were prepared by dissolving each pure standard in methanol (MeOH). A mixed standard fortification solution [20 μg/mL (10 μg/ mL for 1 and 2)] was prepared by combining 2.0 mL (1.0 mL for 1 and 2) of each stock standard and diluting with MeOH to obtain a final volume of 100 mL. The stock solutions were stored in amber vials at −20 °C and stabilized for 3 months. The mixed fortification solution was also stored in an amber vial at −20 °C and then stabilized for 1 month. Distilled water was further purified by passing it through a Milli-Q Plus apparatus (Millipore, Bedford, MA, USA). HPLC grade MeOH and acetonitrile (MeCN) were purchased from Tedia (Fairfield, OH, USA). Other chemicals, including formic acid, metaphosphoric acid, and zinc acetate, were of analytical reagent grade. Feed. The feed used in this study was purchased from Wuhan Lvhong Bioscience and Technology Co., Ltd. (Wuhan, People’s Republic of China). The feed formula was strictly in accordance with the recommended formula of the U.S. National Research Council. Premix (4%; 2.5 g/kg quinocetone concentration) and complete feed (100 mg/kg quinocetone concentration) were initially prepared. The blank feed and the medicated feed were analyzed using the HPLC method described by Wu et al.17 to determine drug content. The results indicated that no quinoxaline compound was detected in the blank feed, and only quinocetone (98 ± 6 mg/kg feed, n = 9) was found in the medicated feed. Animals and Sampling. The 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). Thirty healthy castrated crossbred (large white × landrace) pigs (50−65 kg) were purchased from the Breeding Pig Testing Center (Wuhan, People’s Republic of China); these pigs were housed in six 8 m × 10 m pigpens, which were cleaned daily. Thirty-six 1-day-old healthy Cobb500 broilers were purchased from Charoen Pokphand Group (Wuhan, People’s Republic of China); these broilers were kept in stainless steel cages. Sixty healthy carp (400−600 g) were purchased from the Wuhan Fish Breeding Farm (Wuhan, People’s Republic of China); these carp were placed in a fish tank with circulating water. The animal houses were maintained at 25 ± 2 °C room temperature with 45−65% relative humidity. All of the animals were allowed a 7 day acclimation period before our experiments were conducted; a standard ration based on corn and soybean was fed twice a day and tap water was available ad libitum. The animals were randomly divided B

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Figure 2. Chromatograms of (A) blank pig liver, (B) kidney, (C) broiler muscle, (D) sebum, and (E) carp skin sample, (F) blank pig liver, (G) kidney, (H) broiler muscle, (I) sebum, and (J) carp skin sample spiked with quinocetone and its main metabolites. separation was conducted using a ZORBAX SB-C18 column (250 mm × 4.6 mm i.d., 5 μm; Agilent Technology, USA) coupled with a 2 mm C18 guard column at a flow rate of 1.0 mL/min at 30 °C in a column oven. Mobile phase component A was 1% formic acid, and component B was MeCN. The mobile phase gradient profile was as

The other tested cartridges were an Oasis MAX (60 mg, 3 mL) (Waters Corp.) and an SCX (500 mg, 3 mL) (Agilent, Santa Clara, CA, USA). HPLC Analysis. HPLC analysis was performed using a Waters 2695 HPLC system coupled with a UV detector. Chromatographic C

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follows: 0 min, 80% A; 8 min, 80% A; 15 min, 35% A; 20 min, 10% A; 20.1 min, 80% A; 25 min, 80% A. The UV detector was set at a wavelength of 320 nm for all of the compounds. The developed 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 under Council Directive 96/23/ EC.24 Calibration Curve and Linearity. The standard mixture calibration curves were generated on five different days at seven concentration levels from 2 to 500 μg/L for 1 and 2 and from 4 to 500 μg/L for 3−6. The analyses were performed in triplicate. The method was further tested by matrix-matched calibration curves, which were obtained by fortification with the six compounds at each of the six concentrations from 2 to 200 μg/kg. The calibration curves constructed on five separate days were analyzed to evaluate the linearity of each curve. Slope, intercept, and correlation coefficient were calculated for each standard curve. Unknown concentrations were calculated from the equation of the calibration curve. Limits of Detection (LOD) and Limits of Quantification (LOQ). LOD was calculated by comparing the 3-fold variation of signal-tonoise ratio (S/N = 3:1) obtained from the extract analysis of the blank samples; LOQ was calculated using S/N = 10:1. Accuracy and Precision. Blank samples were fortified at three different levels (1, 2, and 4 times the LOQ). For the intraday experiment, six sets of each concentration were run. Six replicates of the three concentrations were analyzed on five different days for the interday experiment. Accuracy was defined by the mean absolute recovery, which was calculated by comparing the peak area of the extracted sample with that of the standard working solution. Precision was defined by relative standard deviation (RSD). Specificity. Method specificity was verified by analyzing blank samples and observing residue-interfering peaks. The results were evaluated by the presence of interfering substances at the specified retention time. Stability. Stability experiments were conducted to investigate the stability of quinocetone and its metabolites in incurred samples stored at −20 °C within 6 months. The incurred samples were extracted, thawed, and analyzed every week. The measured values were compared with those in freshly incurred samples in triplicate. The results indicated that quinocetone and its metabolites were stable for 4 months in the incurred samples during storage at −20 °C. Data Analysis. To determine the tissue depletion profile, we analyzed the concentrations of quinocetone and its metabolites by using a linear regression model of natural log-transformed average concentration of quinocetone and its metabolites (ln C) against time. The last three time-point data were fit to the first-order rate equation C = C0 e−kt, where C is the concentration of quinocetone and its metabolites on day t, C0 is the initial concentration, elimination rate constant (k) is the slope of the linear regression equation for the logtransformed residue concentration (ln C) against time, and the half-life of elimination (t1/2k) is calculated from the equation t1/2k = ln 2/k for each tissue. The withdrawal time (WDT) was estimated by the Statistical Tool for Data Analysis of Draft Maximum Residue Limits for Veterinary Drugs (MRLVDs) in Edible Tissues.25 The approach is primarily based on linear regression analysis and statistical estimation of onesided upper tolerance limits for the marker residue depletion in the individual target tissues. An iterative procedure is then used to calculate for different time points on the depletion curve the intake of residues of concern in the food basket. The calculated intake of residues is compared with the acceptable daily intake (ADI), and the time point of depletion below the ADI is selected to determine the MRLs. The estimated daily intake (EDI) was adopted to calculate the quantity of residues of toxic concern for a food basket that includes 300 g of muscle, 100 g of liver, 50 g of kidney, and 50 g of fat. The concentration of residues was calculated using the median of the residue distribution, expressed as the marker residue 3, with inclusion of factors (when required) to convert the marker residue concentration to total residues. For each food commodity, the highest

species-specific median residue value is used in the calculation. The global estimated chronic dietary exposure (GECDE) to quinocetone residue for the population group of interest is “the highest exposure calculated using the 97.5th percentile consumption figure for a single food selected from all the foods plus the mean dietary exposure from all the other relevant foods” and is calculated as

GECDE = highest exposure from one animal product + total mean exposure from all other products Descriptive statistical parameters, such as mean, standard deviation (SD), and coefficient of variation (CV), were calculated. Data were statistically analyzed using Microsoft Excel 2007.

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RESULTS AND DISCUSSION Sample Pretreatment. Low recoveries of all six analytes were obtained when MeOH or MeCN was used alone as an extraction solvent. However, almost all of the analytes were extracted when 30% water was added to MeOH and MeCN, but the recovery of 5 was low. All of the compounds were extracted with recoveries of >80% when 1% metaphosphoric was added to MeOH/MeCN/water (50:20:30, v/v/v). On the basis of the properties of the six compounds and the sample/ matrix composition, we used the following test cartridges: Oasis HLB, Oasis MAX, and SCX cartridges. With SCX, the recovery was low, and interferences were observed at the same retention time as 5. Sin et al.26 recommended the SPE cleanup on Oasis MAX for 5, but the method was not suitable for other compounds. Comparative studies indicated that Oasis HLB was superior to the other cartridges in terms of good recovery (>90%) and low matrix interference. During the elution steps of Oasis HLB, MeCN and MeOH were selected because of their good extraction efficiencies. MeCN showed high elution power to acquire high recoveries and low interference. The techniques were improved, thereby reducing the total time of sample pretreatment to 0.9999 for all of the curves. Matrix-matched calibration standard curves were used to quantitate the target analytes in animal tissues and to ensure that the method was as accurate as possible. The correlation coefficient values of the analytes within the range from 2 to 200 μg/kg were >0.99. Specificity. On the basis of the comparison of background noise with various matrices, our results showed that no interference peak was detected at the retention time of the tested compounds (Figure 2). LOD and LOQ. The LODs of 1 and 2 were 5 μg/kg in liver and kidney tissues and 3 μg/kg in muscle and fat tissues. The LODs of 3−6 were 10 μg/kg in liver and kidney tissues and 6 μg/kg in muscle and fat tissues. The LOQs of 1 and 2 were 20 μg/kg in liver and kidney tissues and 10 μg/kg in muscle and fat tissues. The LOQs of 3−6 were 40 μg/kg in liver and kidney tissues and 20 μg/kg in muscle and fat tissues. Accuracy and Precision. The results of recovery and reproducibility of the method at the specified concentration range on five separate days were calculated. The mean recoveries ranged from 61.5 to 81.5%. The interday values were muscle > kidney > liver. This result indicated that the liver is the key organ of quinocetone residues. According to the principle of pharmacokinetics, the distributions of compounds in the body depend on organ blood flow and compound affinity to tissues. Blood supply to the liver and kidney is higher than that to muscle and fat. Therefore, a comparatively higher level of 3 was found in the liver and kidney than in other organs or tissues. The depletion rate of the six compounds in the liver was observed in the order 6 > 2 > 1 > 5 > 4 > 3. This result F

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therefore, it is recommended that the WDT of quinocetone is 0 days in pigs and carp and 3 days in broilers.

indicated that 3 is the main compound that produces residues in pigs, broilers, and carp for the longest time when quinocetone was administered consecutively to these animals. Therefore, 3 could be the key compound for quinocetone residue monitoring compared with other compounds. The long half-lives of 3 in tissues may be attributed to the following reasons. 3 (didesoxyquinocetone) is one of the reduced metabolites of quinocetone. The polarity of 3 is much lower than that of other metabolites. As a result, 3 may be easily retained in organs and tissues. Although the residue marker of quinocetone in the three animals was possibly 3, certain differences were found in the residue characteristics of different animals. 6 could be detected only in pig liver. 5 could not be detected in carp tissues. Our results showed that the differences in metabolic processes could be accounted for the differences in distribution. In addition, t1/2 ke‑3 in the liver was detected in the following order: carp > pigs > broilers. This result could be attributed to the differences in the metabolic absorption and metabolic enzymes of different animals. Acceptable Daily Intake (ADI). On the basis of the toxicology results, the no observed effect level (NOEL) of quinocetone is 5 mg/kg bw/day.27 The safety factor usually chosen is 100 in the situation where a NOEL is derived from a long-term animal study on the assumption that humans are 10 times as sensitive as the test animals used in such studies and that a 10-fold range of sensitivity within the human population may exist, but genotoxicity of quinocetone was noted in previous studies,2 so a higher safety factor of 1000 was employed.28 Then the ADI is 5 μg/kg bw/day. Dietary Exposure Assessment. Estimated Daily Intake (EDI). Results below the LOQ are assigned a value of half of the LOQ when the median residue concentration is calculated. On the basis of the established model diet, the exposure to quinocetone expressed as the EDI, and calculated from the M:T and analytical method recovery adjusted median residue, was 208 μg/person/day. Dietary exposure was estimated to be 69% of the ADI (Table 2). Assumptions for Dietary Exposure Assessment. The following assumptions were used in dietary exposure estimates: residues are found in pork, poultry, and fish; residues are found in muscle, liver, kidney, skin, and fat; all poultry offal is assumed to be kidney (worst-case scenario). Global Estimated Chronic Dietary Exposure (GECDE). For the general population group, mammalian liver was the major contributor to estimated exposure from quinocetone residue (Table 2). Mammalian fat and kidney and poultry fat and skin contributed only negligible amounts to overall exposure estimates. Using the median residue as input, GECDE was 4.7 μg/kg bw/day, 94% of the ADI of 5 μg/kg bw/day (Table 2), and did not exceed the ADI. Comparison to EDI Estimates. The calculations based on the model diet found that the median residue resulted in exposure estimated below the ADI for the general population. Similarly, the GECDE calculations found that exposure estimates were below the ADI for the general population, but they were higher than the EDI. Mammalian liver was the major contributor to chronic exposure estimates. In summary, none of the chronic exposure estimates resulted in exceeding the ADI. Withdrawal Time (WDT). The EDI chart reveals that the EDI was below the ADI at zero withdrawal intervals in pigs and carp and at the withdrawal time of 72 h in broilers (Figure 3);

Figure 3. Estimated intakes (μg/person/day) versus time in pigs, broilers, and carp.

The prolonged presence of quinocetone-related residues in edible tissues is a potential risk for consumers. Considering that the established WDT was based on dietary exposure assessment, we could use the proposed WDT for residue monitoring of quinocetone in food-producing animals. In contrast to LCMS/MS, HPLC-UV is probably more suitable for routine quinocetone residue monitoring analyses. HPLC-UV could be efficiently applied in a quinocetone monitoring program. The application of this technology could significantly improve the monitoring procedures for the illegal use of quinocetone to ensure food safety. Our results provided the residue profile of quinocetone and its metabolites in edible tissues. On the basis of these residue data in edible tissues, our conclusion is that the liver is the target tissue and 3 is the residual marker of quinocetone in food animals. A withdrawal period of 0 days was estimated for quinocetone in pigs and carp, and 3 days was the estimated WDT in broilers. In addition, an HPLC-UV method was established to simultaneously and quantitatively determine quinocetone and its five major metabolites in edible tissues of pig, broiler, and carp. The recoveries in edible tissues spiked at G

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10−160 μg/kg ranged from 61.5 to 81.5% with interday RSD < 11.7%. These results also provided scientific evidence for the routine residue monitoring of quinocetone in food-producing animals.

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(10) Shen, J.; Yang, C.; Wu, C.; Feng, P.; Wang, Z.; Li, Y.; Li, Y.; Zhang, S. Identification of the major metabolites of quinocetone in swine urine using ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2010, 24, 375−383. (11) Wu, H.; Yang, C.; Wang, Z.; Shen, J.; Zhang, S.; Feng, P.; Li, L.; Cheng, L. Metabolism profile of quinocetone in pig by ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry. Eur. J. Drug Metab. Pharmacokinet. 2012, 37 (2), 141− 154. (12) Li, J.; Huang, L.; Wang, X.; Pan, Y.; Liu, Z.; Chen, D.; Tao, Y.; Yuan, Z. Metabolic disposition and excretion of quinocetone in rats, pigs, broilers, and carp. Food Chem. Toxicol. 2014, 69, 109−119. (13) Huang, L.; Xiao, A.; Fan, S.; Yin, J.; Liu, D.; Qiu, Y.; Yuan, Z. Development of liquid chromatographic methods for determination quinocetone and its main metabolites in edible tissues of swine and chicken. J. AOAC Int. 2005, 88 (2), 472−478. (14) Hu, G. M.; Huang, L. L.; Yuan, Z. H. Pharmacokineties and residue depletion of quinocetone in carp. Master’s dissertation, Huazhong Agricultural University, Hubei, China, 2008 (in Chinese). (15) Li, J. Y.; Li, J. S.; Xu, Z. Z.; Zhao, R. C.; Miao, X. L.; Zhang, J. Y.; Lu, R. H. The determination and residues of quinocetone in swines. Prog. Vet. Med. 2004, 25 (4), 117−120. (16) Li, J. Y.; Li, J. S.; Zhao, R. C.; Miao, X. L.; Zhang, J. Y.; Zhou, X. Z. Study on residues of quinocetone in edible chicken tissues. Prog. Vet. Med. 2008, 29 (4), 34−37. (17) Wu, Y.; Wang, Y.; Huang, L.; Tao, Y.; Yuan, Z.; Chen, D. Simultaneous determination of five quinoxaline-1,4-dioxides in animal feeds using ultrasonic solvent extraction and high-performance liquid chromatography. Anal. Chim. Acta 2006, 569, 97−102. (18) Wu, Y.; Yu, H.; Wang, Y.; Huang, L.; Tao, Y.; Chen, D.; Peng, D.; Liu, Z.; Yuan, Z. Development of a high-performance liquid chromatography method for the simultaneous quantification of quinoxline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid in animal tissues. J. Chromatogr., A 2007, 1146, 1−7. (19) Zhang, J.; Li, M.; Li, L.; Li, Y.; Peng, B.; Zhang, S.; Gao, H.; Zhou, W. Investigation of the ultrasound effect and target analyte selectivity of dispersive liquid−liquid microextraction and its application to a quinocetone pharmacokinetic study. J. Chromatogr., A 2012, 1268, 1−8. (20) Fang, J.; Li, Y.; Wu, S.; Ma, K.; Li, H.; Gao, Z.; Dong, F. Determination of quinocetone and its two major metabolites in chicken liver and muscle tissues by liquid chromatography-tandem mass spectrometry. Anal. Methods 2012, 4, 1149−1154. (21) Yong, Y.; Liu, Y.; He, L.; Xu, L.; Zhang, Y.; Fang, B. Simultaneous determination of quinocetone and its major metabolites in chicken tissues by high-performance liquid chromatography tandem mass spectrometry. J. Chromatogr., B 2013, 919−920, 30−37. (22) Li, Y.; Liu, K.; Beier, R. C.; Cao, X.; Shen, J.; Zhang, S. Simultaneous determination of mequindox, quinocetone and their major metabolites in chicken and pork by UPLC−MS/MS. Food Chem. 2014, 160, 171−179. (23) AVMA. Panel on Euthanasia 2000 Report of the AVMA panel on euthanasia. J. Am. Vet. Med. Assoc. 2001, 218, 669. (24) Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (2002/657/EC). Off. J. Eur. Communities 2002, L221, 8. (25) 62nd meeting of FAO/WHO Joint Expert Committee on Food Additives, 2003; http://www.fao.org/fileadmin/user_upload/agns/ zip/tool_1.1551.zip (accessed June 2, 2013). (26) Sin, D. W. M.; Chung, L. P. K.; Lai, M. M. C.; Siu, S. M. P.; Tang, H. P. O. Determination of quinoxaline-2-carboxylic acid, the major metabolite of carbadox, in porcine liver by isotope dilution gas chromatography-electron capture negative ionization mass spectrometry. Anal. Chim. Acta 2004, 508 (2), 147−158. (27) Zhang, W.; Huang, L. L.; Wang, Y. L.; Yuan, Z. H. Preclinical toxicity of quinocetone. Master’s dissertation, Huazhong Agricultural University, Hubei, China, 2007 (in Chinese).

AUTHOR INFORMATION

Corresponding Author

*(Z.Y.) Phone: 0086-27-87287186. Fax: 0086-27-87672232. Email: [email protected]. Funding

This work was supported by grants from the National Natural Science Foundation of China (31272614), the National Basic Research Program of China (2009CB118800), the Special Fund for Agro-scientific Research in the Public Interest (201203040), and the Recommend international advanced agricultural science and technology plan (2011-G4). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ADI, acceptable daily intake; WDT, withdrawal time; MRL, maximum residue limit; EMEA, European Medical Evaluation Agency; AVMA, American Veterinary Medical Association; EDI, estimates of dietary intake; GECDE, global estimated chronic dietary exposure

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REFERENCES

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dx.doi.org/10.1021/jf5042867 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX