Rapid Screening of Toxic Salbutamol, Ractopamine, and Clenbuterol In

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Available online at www.sciencedirect.com 65 66 1 ScienceDirect 67 2 68 3 69 4 70 5 journal homepage: www.jfda-online.com 71 6 72 7 73 8 74 9 Original Article 75 10 76 11 Rapid screening of toxic salbutamol, ractopamine, 77 12 78 13 and clenbuterol in pork sample by high- 79 14 80 15 d 81 16 Q1 performance liquid chromatography UV method 82 17 83 18 a,b,*,1 a,1 a a,b 84 19 Q18 Kunping Yan , Huiqun Zhang , Wen Li Hui , Hongli Zhu , 85 20 a a c a,d,* Q2 Xinbo Li , Fangyi Zhong , Xiu'e Tong , Chao Chen 86 21 87 a 22 College of Life Science, Northwest University, Xi'an, PR China 88 23 b Shaanxi Lifegen Co., Ltd, Xi'an, PR China 89 24 c Weinan Products Quality Supervision and Inspection Institute, Weinan, PR China 90 25 d National Engineering Research Center for Miniaturized Detection System, Xi'an, PR China 91 26 92 27 93 28 article info abstract 94 29 95 30 96 Q4 Article history: A rapid and simple high-performance liquid chromatographyeUV method was developed 31 97 Received 16 December 2014 for the separation and quantification of salbutamol, ractopamine, and clenbuterol in pork. 32 98 Received in revised form A mixture of acetonitrileeformic acideammonium acetate was used as the mobile phase to 33 99 b 34 15 June 2015 separate three -agonists on a C18 column with gradient. The effects of the addition of 100 b 35 Accepted 4 December 2015 formic acid and ammonium acetate to mobile phases on the separation of -agonists were 101 36 Available online xxx investigated. These additives can greatly improve the resolution and sensitivity. Under the 102 37 optimized chromatographic condition, this separation does not need extra sample prep- 103 38 Keywords: aration. Complete baseline separation of three b-agonists was achieved in < 20 minutes; 104 39 b-agonist the linear range is 0.2e50 mg/L with a correlation coefficient R2 value of > 0.99. Excellent 105 40 method reproducibility was found by intra- and interday precisions with a relative stan- 106 41 clenbuterol high-performance liquid dard deviation of < 3%. The detection limit (S/N ¼ 3) was found to be <0.05 mg/L; this 107 42 108 chromatographyeUV method can be used for routine screening of the b-agonist residues in foods of animal 43 109 pork origin before being identified by confirmatory methods. 44 110 © 45 ractopamine Copyright 2016, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan 111 46 salbutamol LLC. This is an open access article under the CC BY-NC-ND license (http:// 112 47 creativecommons.org/licenses/by-nc-nd/3.0/). 113 48 114 49 115 50 116 51 117 52 118 activity [1]. The main applications in human and veterinary 53 119 1. Introduction medicine are as tocolytic and bronchodilator agents [2]. 54 120 55 However, these compounds are also used as growth pro- Вntr-agonists are phenyl ethanolamines with different sub- 121 56 moters in livestock to increase the lean meat-to-fat ratio and 122 stituents on the aromatic ring and on the terminal amino 57 improve feed conversion efficiency [3]. Numerous literatures 123 group. These molecules have a powerful pharmacological 58 124 59 125 60 * Corresponding authors. Q3 126 61 E-mail addresses: [email protected] (K. Yan), [email protected] (C. Chen). 127 62 1 These two authors contributed equally to this study. 128 63 http://dx.doi.org/10.1016/j.jfda.2015.12.002 129 64 1021-9498/Copyright © 2016, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Please cite this article in press as: Yan K, et al., Rapid screening of toxic salbutamol, ractopamine, and clenbuterol in pork sample by high-performance liquid chromatographydUV method, Journal of Food and Drug Analysis (2016), http://dx.doi.org/10.1016/ j.jfda.2015.12.002 JFDA295_proof ■ 22 February 2016 ■ 2/7

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1 have reported various poisoning effects and potential hazards water was prepared using a Milli-Q water filtration system 66 2 of b-agonists, including cardiac palpitation, tachycardia, (Millipore Corp.). All solvents for HPLC were filtered through Q7 67 3 68 nervousness, muscle tremors, and confusion [4]. Therefore, 0.45 mm filters (Millipore Corp.) and degassed in an ultrasonic 4 69 no b-agonists have been permitted in most countries for bath. 5 70 growth-promoting purposes in farm animals. Therefore, in- Stock solutions of the three b-agonists were prepared by 6 71 b 7 spection of -agonists is very important in the food industry. dissolving 5 mg of each compound in 50 mL methanol. The 72 b 8 A survey found that the levels of residues of -agonists standard solutions were prepared by diluting the stock solu- 73 9 have decreased over the years due to optimization of admin- tions to 1 mg/mL. As sample matrixes, pork were obtained 74 10 istration schemes and application of comedication to avoid from supermarkets, free markets, and street vendors, and 75 11 detection. Many laboratories now use a combination of preserved at 20C until use. 76 12 screening and confirmatory methods to cope with a relatively 77 13 78 large number of samples and to increase the reliability of the 2.2. Sample preparation from tissue 14 final result [5]. Confirmatory methods include liquid chro- 79 15 80 matography/tandem mass spectrometry [6e8] and gas chro- b 16 In pork, -agonists were extracted using a previously reported 81 matographyemass spectrometry [9,10]. These confirmatory 17 method with minor modification [10]. Briefly, aliquots of 2 g 82 18 methods based on chromatographic approaches can provide finely homogenized pork were transferred into 50 mL conical 83 19 both qualitative and quantitative detection results with glass flasks. Eight milliliters of 0.2 mol/L sodium acetate and 84 20 satisfactory sensitivity and reproducibility. However, they 50 mLofb-glucuronidase/aryl sulfatase were added and mixed. 85 21 require expensive instrumentation, and thus are not suitable It was incubated at 37C for 12 hours. The homogenate was 86 22 for routine inspection. then centrifuged at 5000 rpm for 10 minutes, and the super- Q8 87 23 As alternatives of confirmatory methods, various routine natant was recovered and transferred to a 50 mL flask. The 88 24 b 89 screening methods for the determination of -agonist resi- tissue residue was washed with 5 mL sodium acetate, 25 90 dues in animal tissue and body fluids have been developed. repeated twice. Then 5 mL 0.1 mol/L perchloric acid was added 26 91 These methods use immunoassay [11e14], capillary electro- 27 into 4 mL supernatant, and the pH was adjusted to 1.0 with 92 phoresis [15], electrochemical sensors [11,16,17], surface 28 1.0 mol/L perchloric acid. The mixture was centrifuged at 93 plasmon resonance [18], supercritical fluid chromatography 29 5000 rpm for 10 minutes again. The supernatant was trans- 94 30 [19], chemiluminescence [20], molecularly imprinted poly- ferred into another 50 mL centrifuge tube, and the pH was 95 e 31 mers [21 23], and high-performance liquid chromatography adjusted to 11 with 10 mol/L sodium hydroxide. The mixture 96 e e 32 (HPLC) UV methods [24]. Among them, HPLC UV is one of the was completely extracted with a mixed solution of 10 mL 97 33 most widely used methods in all the inspection centers and saturated sodium chloride solution and isopropanoleethyl 98 34 laboratories, but the current established methods need sam- acetate (6:4, v/v). Then the supernatant was centrifuged at 99 35 b 100 ple clean-up steps or are able to determine only one -agonist 5000 rpm for 10 minutes. The organic phase was transferred 36 e 101 in one chromatographic run [24 27]. into another centrifuge tube, dried under a gentle nitrogen 37 e 102 This work was to develop a simple HPLC UV analytical stream, and reconstituted to 1.0 mL with methanol/water 38 103 method for simultaneous determination of several b-agonists 39 containing 0.1% formic acid (1/9, v/v) followed by filtering 104 without extra sample clean-up steps for routine analysis, m 40 through 0.22 m organic filters for HPLC analysis. 105 41 which can be used as a screening method for the detection of 106 b-agonists before being identified by confirmatory methods. 42 2.3. Analysis of b-agonists by HPLC 107 43 The linearity of calibration curve, recovery, precision, and 108 lower limit of detection were studied to evaluate the devel- 44 The analyses were carried using an Agilent Technologies 109 45 oped method. The method was successfully used to deter- 110 HP1200 series HPLC system equipped with a quaternary 46 mine the content of b-agonist residues in pork. 111 gradient pump, a diode array detector, and a Chemstation 47 112 48 data-analysis system (Agilent, Palo Alto, CA, USA). Chro- 113 matographic separation was performed at 25C on Agilent 49 2. Methods 114 50 Eclipse C18 (250 mm 4.6 mm i.d., 5 mm particle) analytical 115 51 columns (Agilent) at a flow rate of 0.8 mL/min via a ternary 116 2.1. Chemicals, standard solutions, and materials 52 gradient. Eluent A consisted of 0.1% formic acid, eluent B is 117 53 0.1% formic acid in acetonitrile solution, and eluent C is 118 Three b-agonist standards, including salbutamol (SAL), rac- 54 50 mmol/L ammonium acetate aqueous solution. Separation 119 topamine (RAC), and clenbuterol (CLB), were purchased from 55 was performed with a gradient, as shown in Table 1. The in- 120 56 the laboratories of Dr. Ehrenstorfer (Augsburg, Germany). 121 jection volume was 20 mL, and the detection wavelength of the 57 Methanol and acetonitrile (HPLC grade) were purchased from 122 detector was set at 225 nm [28]. 58 Q5 the Fisher Chemicals (Fairlawn, NJ, USA); b-glucuronidase/aryl 123 59 sulfatase was purchased from Roche Diagnostica GmbH 124 60 125 (Mannhein, Germany). Formic acid (HPLC grade) was obtained e 61 Table 1 Gradients used for liquid chromatography. 126 Q6 from Tedia Company Inc. (Fairfield, OH, USA). Hydrochloric 62 Time (min) Eluent A (%) Eluent B (%) Eluent C (%) 127 acid and sodium hydroxide were obtained from Sinopharm 63 128 0.01 71 4 25 64 Chemical Reagents Co., Ltd (Shanghai, China). Ammonium 129 6552025 65 acetate and sodium acetate were obtained from Tianjin 130 19 52 23 25 Chemical Reagents Company (Tianjin, China). Deionized

Please cite this article in press as: Yan K, et al., Rapid screening of toxic salbutamol, ractopamine, and clenbuterol in pork sample by high-performance liquid chromatographydUV method, Journal of Food and Drug Analysis (2016), http://dx.doi.org/10.1016/ j.jfda.2015.12.002 JFDA295_proof ■ 22 February 2016 ■ 3/7

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1 2.4. Linear range and limit of detection 2.6. Recovery 66 2 67 3 68 The linear correlation and dynamic range of b-agonist The recovery of the assay was determined by spiking known 4 69 detection were determined from calibration curves gener- amounts of standards into test samples. Nine b-agonist- 5 70 ated by serial analysis of b-agonist standards. A calibration spiked samples and nine control samples were compared. For 6 71 m m m b 7 curve for each compound was calculated based on peak areas pork samples, 2 g, 0.8 g, or 0.16 g of the three -agonists 72 m b 8 obtained by serially injecting 20 L of methanol-diluted so- were directly spiked into 2.0 g tissues. The extraction of - 73 9 lutions of b-agonist standards. Stock solutions of individual agonists was performed as described in the “Sample prepa- 74 10 b-agonists were serially diluted and spiked into matrix so- ration from tissue” section. The control samples were sub- 75 11 lution extracted from the pork. The lowest detection limits jected to the same procedure as test samples. 76 12 were determined as concentrations corresponding to three 77 13 times of noise (S/N ¼ 3). 78 14 79 15 3. Results 80 16 2.5. Precision 81 17 3.1. Method speciﬁcity 82 18 The precision of the method was evaluated using b-agonist- 83 19 spiked samples. Five extractions of the same b-agonist-spiked Complete baseline separation of three b-agonists was ach- 84 20 85 tissue sample were tested consecutively to determine ieved in < 20 minutes. The chromatogram of standards is 21 86 intraday relative standard deviations (RSDs). For interday shown in Figure 1A. The retention times for SAL, RAC, and CLB 22 87 b 23 precision, ﬁve extractions of the same -agonist-spiked tissue were 7.92 minutes, 13.09 minutes, and 16.88 minutes, 88 24 sample were tested in three sequences over 5 days. respectively. The optimized gradient is shown in Table 1. 89 25 90 26 91 27 92 28 93 29 94 30 95 31 96 32 97 33 98 34 99 35 100 36 101 37 102 38 103 39 104 40 105 41 106 42 107 43 108 44 109 45 110 46 111 47 112 48 113 49 114 50 115 51 116 52 117 53 118 54 119 55 120 56 121 57 122 58 123 59 124 60 125 61 126 62 127 63 128 e 64 Figure 1 Representative chromatograms for (A) standards, (B) blank samples, and (C) spiked sample. Mobile phase: eluent 129 65 A, 0.5% formic acid; eluent B, 0.5% formic acid in acetonitrile; and eluent C, 50 mmol/L ammonium acetate. The gradients are 130 shown in Table 1. CLB ¼ clenbuterol; RAC ¼ ractopamine; SAL ¼ salbutamol. Q17

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1 66 e b 2 Table 2 Linear correlation, ranges, and lower limit of detection of -agonists selected. 67 3 Standard Regression equation Coefficient (R2) Linear range (mg/L) Limita (mg/L) 68 4 69 SAL y ¼ 739.36x þ 9907.5 0.9970 0.2e25 0.02 5 70 RAC y ¼ 632.53x 10048 0.9992 0.4e50 0.05 6 71 CLB y ¼ 713.86x 12933 0.9901 0.4e50 0.05 7 72 8 CLB ¼ clenbuterol; RAC ¼ ractopamine; SAL ¼ salbutamol. 73 a ¼ 9 S/N 3. 74 10 75 11 76 12 Typical chromatograms of the blank and spiked samples are 90.1e82.5% and 79.2e88.4% for spiking, respectively, 0.4 mg/g 77 13 shown in Figures 1B and 1C. The b-agonists were not detected and 0.008 mg/g samples were obtained. 78 14 in blank samples, and no interference peaks were observed at 79 15 80 the retention time of b-agonist standards. All the spiked b- 3.5. Application to samples 16 81 agonist standards were well separated and detected with low- 17 82 abundance peaks from matrix partially overlapped with the b- 18 The method was used to analyze 142 pork samples from the 83 agonists. The quantitative results were still satisfactory 19 local Food and Drug Administration. The samples were 84 20 because interference from the overlapped peaks was minimal. randomly obtained from supermarkets, free markets, and 85 21 street vendors of the entire Shaanxi Province in China, ac- 86 22 3.2. Linear range and limit of detection cording to the local government monitoring plan. Five sam- 87 23 ples, collected from free markets, were detected to contain b- 88 24 89 Under optimized conditions, the linear ranges were 0.2e25 mg/ agonist residues. The SAL contents of two positive samples Q9 25 m m 90 L for SAL and 0.4e50 mg/L for RAC and CLB. The correlation were 0.14 g/kg and 1.13 g/kg. The RAC contents of two 26 91 > positive samples were 0.27 mg/kg and 0.87 mg/kg. The CLB 27 coefficients of calibration curves were 0.99. The lower limits 92 content of one positive sample was 0.65 mg/kg. The results 28 of detection were calculated based on a signal-to-noise ratio of 93 were consistent with that of the local Food and Drug Admin- 29 3, and the values varied from 0.02 mg/L to 0.05 mg/L (Table 2). 94 30 The method provides sensitive detection with a wide linear istration and showed that this method was able to screen 95 b 31 range. samples for -agonists routinely before performing confir- 96 32 matory tests. 97 33 98 34 3.3. Precision 99 35 4. Discussion 100 36 Five samples extracted from the same b-agonist-spiked tissue 101 37 (0.008 mg/g) were analyzed consecutively and also in different 102 SAL and CLB have a similar molecular structure except for 38 days (d ¼ 5, n ¼ 5). The precision was high for both intraday 103 different substituent groups on the aromatic ring, but have a 39 (RSD values 1.61e1.95%) and interday (RSD values 2.74e2.98%) 104 significant difference with RAC. These lead to their various 40 assays. The results are summarized in Table 3. Both inter- and 105 hydrophobicity scales, making them difficult to be separated 41 intraday RSDs are < 3%, indicating a high degree of assay 106 42 in a short liquid chromatography run. In this study, mixtures Q10 107 precision and reproducibility. 43 of acetonitrileeformic acideammonium acetate were evalu- 108 44 ated for their potential to separate b-agonists in a complex 109 45 3.4. Recovery matrix. When taking water and acetonitrile as the mobile 110 46 phase, all the peaks of b-agonist were severely trailing and 111 47 112 Different samples were selected for maximum variability with split into two or more peaks (Figure 2A). The possible reason 48 113 respect to provenance. Spiked samples at three levels (1.0 mg/ for multiple peaks, besides a hydrophobic interaction, is that 49 114 g, 0.4 mg/g, or 0.008 mg/g of tissue) were analyzed to evaluate three agonists in this mobile phase can be charged, leading to 50 115 the recovery (Table 4). Excellent recoveries of 84.9e91.9% for 51 an ion exchange interaction. Two different interactions lead 116 m 52 spiking 1.0 g/g samples and less satisfactory recoveries of to the split peaks. After addition of 0.05% formic acid (final 117 53 concentration), no trailing of CLB peak was observed and the 118 54 119 55 120 Table 3 e Analysis precisionsa for each b-agonist 56 121 compound. 57 Table 4 e Recovery of b-agonists from pork. 122 58 Standard Intraday RSD Interday RSD 123 ¼ ¼ Standard % Recovery 59 (%, n 5) (%, n 5) 124 Spiking Spiking Spiking 60 SAL 1.61 2.74 125 1.0 mg/g 0.4 mg/g 0.08 mg/g 61 RAC 1.83 2.85 126 62 CLB 1.95 2.98 SAL 91.9 ± 7.8 90.1 ± 7.1 88.4 ± 6.5 127 RAC 89.3 ± 5.4 88.3 ± 5.2 85.6 ± 4.8 63 CLB ¼ clenbuterol; RAC ¼ ractopamine; RSD ¼ relative standard 128 CLB 84.9 ± 4.7 82.5 ± 5.5 79.2 ± 5.9 64 deviation; SAL ¼ salbutamol. 129 65 a Spiking 0.008 mg/g in the tissue sample. CLB ¼ clenbuterol; RAC ¼ ractopamine; SAL ¼ salbutamol. 130

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1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 79 15 80 16 81 17 82 18 83 19 84 20 85 21 86 22 87 23 88 24 89 25 90 26 91 27 92 28 93 29 94 30 95 31 96 e b 32 Figure 2 Chromatograms of -agonist standards by different mobile phases. Mobile phase: (A) eluent A, water; eluent B, 97 33 acetonitrile; and eluent C, water; (B) eluent A, 0.05% formic acid; eluent B, 0.05% formic acid in acetonitrile; and eluent C, 98 34 0.05% formic acid; (C) eluent A, 0.5% formic acid; eluent B, 0.5% formic acid in acetonitrile; and eluent C, 0.5% formic acid. The 99 35 gradients are shown in Table 1. CLB ¼ clenbuterol; RAC ¼ ractopamine; SAL ¼ salbutamol. 100 36 101 37 102 38 103 39 104 40 105 peak became sharp, although the peaks corresponding to SAL additives that are soluble in solvents. In addition, organic Q12 41 106 42 Q11 and RAC were still split into two peaks (Figure 2B). On additives are compatible with mass spectrum and can be 107 e 43 increasing the formic acid concentration to 0.5%, the ion applied in the liquid chromatography mass spectrometry or 108 44 exchange interaction also became weak, and the peaks of gas chromatographyemass spectrometry. Compared with Q13 109 45 SAL and CLB both became sharper (Figure 2C). We continued this method, most of the published HPLCeUV and other 110 46 to increase formic acid concentration to improve the RAC methods need extra sample clean-up steps, such as solid- 111 47 peak shape, but as evident from the results, higher concen- phase extraction [24] and solid-phase microextraction [28]; 112 48 trations of formic acid had no further effect on it. Therefore, these complex methods need more sample preparation time 113 49 we added ammonium acetate into the mobile phase in order and may reduce the recovery. After the optimization of the 114 50 115 to improve the RAC peak shape. Ammonium acetate solution method, sensitivity and quantiﬁcation limits are slightly 51 116 was used as the third eluent. When the concentration of higher than the other HPLCeUV methods [26e28]. 52 117 ammonium acetate was 50 mmol/L, RAC also showed one 53 118 54 peak (Figure 1A). It is worth noting that ammonium acetate 119 55 caused broadening of the peaks of SAL and CLB, and 5. Conclusion 120 56 decreased their sensitivity. 121 57 Most of the reported HPLC methods for b-agonist separa- The extraction procedure for b-agonists did not need extra Q14 122 58 tion used inorganic acids or salts as an additive to improve the sample clean-up step, which reduced the sample preparation 123 59 peaks' resolution or shape. In this method, we used formic time. Moreover, as an additive in the mobile phase, formic 124 60 acid and ammonium acetate as the mobile phase additive, acid and ammonium acetate can improve the resolution and 125 61 126 which are easily soluble in organic solvents. When the pro- sensitivity of three b-agonists. Quantitative data showed good 62 127 portion of water in the mobile phase is low, inorganic acids or precision, linear range, and limit of detection. The method can 63 128 salts easily precipitate from the mobile phase; these very tiny 64 also be successfully applied to the 142 samples. Results sug- 129 65 precipitate particles can damage the pump head of HPLC in- gested that this method can be used as a screening method to 130 strument [29]. Hence, it is very important to use organic detect b-agonists.

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1 [12] Zhu G, Hu Y, Gao J, Zhong L. Highly sensitive detection of 66 2 Conflicts of interest clenbuterol using competitive surface-enhanced Raman 67 3 scattering immunoassay. Anal Chim Acta 2011;697:61e6. 68 4 Q15 The authors have no conflicts of interest to declare. [13] Dong JX, Li ZF, Lei HT, Sun YM, Ducancel F, Xu ZL, Boulain JC, 69 5 Yang JY, Shen YD, Wang H. Development of a single-chain 70 6 variable fragment-alkaline phosphatase fusion protein and a 71 7 sensitive direct competitive chemiluminescent enzyme 72 8 immunoassay for detection of ractopamine in pork. Anal 73 e 9 Q16 Acknowledgments Chim Acta 2012;736:85 91. 74 10 [14] Xie CH, Chen FJ, Yang TB. A high-affinity anti-salbutamol 75 11 The study was supported by grants from the National 863 monoclonal antibody: key to a robust lateral-flow 76 12 immunochromatographic assay. Anal Biochem 77 Program of China (Grant No. 2011AA02A101), National Natural e 13 2012;426:118 25. 78 Science Foundation of China (Grant Nos. 31200749 and [15] Li L, Du H, Yu H, Xu L, You T. Application of ionic liquid as 14 79 81102367), Natural Science Foundation (Grant Nos. additive in determination of three beta-agonists by capillary 15 80 2012JM4003 and 2011KTCL03-23) of the Science and Technol- electrophoresis with amperometric detection. 16 81 ogy Department, and “Special Research Foundation” (Grant Electrophoresis 2013;34:277e83. 17 82 No. 12JK0698) of the Education Department of Shaanxi [16] Wu C, Sun D, Li Q, Wu K. Electrochemical sensor for toxic 18 ractopamine and clenbuterol based on the enhancement 83 Province. 19 effect of graphene oxide. Sens Actuators B Chem 84 20 2012;168:178e84. 85 21 [17] Lin K, Hong C, Chen S. Simultaneous determination for toxic 86 22 87 references ractopamine and salbutamol in pork sample using hybrid 23 carbon nanotubes. Sens Actuators B Chem 2013;177:428e36. 88 24 [18] Liu M, Ning B, Qu L, Peng Y, Dong J, Gao N, Liu L, Gao Z. 89 25 Development of indirect competitive immunoassay for 90 26 [1] White DG, Rolph TP, Wagstaff AJ. The effects of salbutamol highly sensitive determination of ractopamine in pork liver 91 27 on blood pressure and heart rate in large white and Pietrain- samples based on surface plasmon resonance sensor. Sens 92 28 cross breeds of pig. J Vet Pharmacol Ther 1989;12:179e88. Actuators B Chem 2012;161:124e30. 93 29 [2] Marchant-Forde JN, Lay DJ, Marchant-Forde RM, [19] Jones DC, Dost K, Davidson G, George MW. The analysis of 94 30 McMunn KA, Richert BT. The effects of R-salbutamol on beta-agonists by packed-column supercritical fluid 95 31 behavior and physiology of finishing pigs. J Anim Sci chromatography with ultra-violet and atmospheric pressure 96 e 32 2008;86:3110 24. chemical ionisation mass spectrometric detection. Analyst 97 e 33 [3] Strydom PE, Frylinck L, Montgomery JL, Smith MF. The 1999;124:827 31. 98 [20] Zhang Y, Zhang Z, Sun Y, Wei Y. Development of an 34 comparison of three beta-agonists for growth performance, 99 carcass characteristics and meat quality of feedlot cattle. analytical method for the determination of beta2-agonist 35 100 Meat Sci 2008;81:557e64. residues in animal tissues by high-performance liquid 36 101 [4] Doerge DR, Churchwell MI, Holder CL, Rowe L, Bajic S. chromatography with on-line electrogenerated [Cu(HIO6)2] 37 e 102 Detection and confirmation of beta-agonists in bovine retina 5 -luminol chemiluminescence detection. J Agric Food Chem 38 103 using LC/APCI-MS. Anal Chem 1996;68:1918e23. 2007;55:4949e56. 39 [5] Kuiper HA, Noordam MY, Van Dooren-Flipsen MM, Schilt R, [21] Brambilla G, Fiori M, Rizzo B, Crescenzi V, Masci G. Use of 104 40 Roos AH. Illegal use of beta-adrenergic agonists: European molecularly imprinted polymers in the solid-phase 105 41 Community. J Anim Sci 1998;76:195e207. extraction of clenbuterol from animal feeds and biological 106 42 [6] Nicoli R, Petrou M, Badoud F, Dvorak J, Saugy M, Baume N. matrices. J Chromatogr B 2001;759:27e32. 107 43 Quantification of clenbuterol at trace level in human urine by [22] Blomgren A, Berggren C, Holmberg A, Larsson F, Sellergren B, 108 44 ultra-high pressure liquid chromatographyetandem mass Ensing K. Extraction of clenbuterol from calf urine using a 109 45 spectrometry. J Chromatogr A 2013;1292:142e50. molecularly imprinted polymer followed by quantitation by 110 46 [7] Guy PA, Savoy MC, Stadler RH. Quantitative analysis of high-performance liquid chromatography with UV detection. 111 47 clenbuterol in meat products using liquid J Chromatogr A 2002;975:157e64. 112 48 chromatographyeelectrospray ionisation tandem mass [23] Li Y, Fu Q, Liu M, Jiao Y, Du W, Yu C, Liu J, Chang C, Lu J. 113 49 spectrometry. J Chromatogr B 1999;736:209e19. Separation and enrichment of trace ractopamine in 114 50 [8] Suo DC, Zhao GL, Wang PL, Su XO. Simultaneous biological samples by uniformly-sized molecularly imprinted 115 e 51 determination of beta-agonists and psychiatric drugs in polymers. J Pharm Anal 2012;2:395 402. 116 e e 52 feeds by LC MS-MS. J Chromatogr Sci 2014;52:604 8. [24] Liu B, Yan H, Qiao F, Geng Y. Determination of clenbuterol in 117 53 [9] Gallo P, Brambilla G, Neri B, Fiori M, Testa C, Serpe L. porcine tissues using solid-phase extraction combined with 118 ultrasound-assisted dispersive liquid-liquid microextraction 54 Purification of clenbuterol-like beta2-agonist drugs of new 119 generation from bovine urine and hair by alpha1-acid and HPLCeUV detection. J Chromatogr B 2011;879:90e4. 55 120 glycoprotein affinity chromatography and determination by [25] Morales-Trejo F, Leon SV, Escobar-Medina A, Gutierrez- 56 121 gas chromatographyemass spectrometry. Anal Chim Acta Tolentino R. Application of high-performance liquid 57 122 2007;587:67e74. chromatography-UV detection to quantification of 58 123 [10] Bocca B, Di Mattia M, Cartoni C, Fiori M, Felli M, Neri B, clenbuterol in bovine liver samples. J Food Drug Anal 59 Brambilla G. Extraction, clean-up and gas 2013;21:414e20. 124 60 chromatographyemass spectrometry characterization of [26] Biosca YM, Baeza JJB, Ramis-Ramos G. Determination of 125 61 zilpaterol as feed additive in fattening cattle. J Chromatogr B clenbuterol in urine by azo-dye precolumn derivatization 126 62 2003;783:141e9. and micellar liquid chromatography. Chromatographia 127 63 [11] Shen L, He P. An electrochemical immunosensor based on 1997;44:145e50. 128 64 agarose hydrogel films for rapid determination of [27] Lawrence JF, Menard C. Determination of clenbuterol in beef 129 65 ractopamine. Electrochem Commun 2007;9:657e62. liver and muscle tissue using immunoaffinity 130

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1 chromatographic cleanup and liquid chromatography with simultaneous analysis of clenbuterol, salbutamol and 8 2 ultraviolet absorbance detection. J Chromatogr B ractopamine in pig samples. Biomed Chromatogr 9 3 1997;696:291e7. 2013;27:1775e81. 10 4 [28] Du W, Zhang S, Fu Q, Zhao G, Chang C. Combined solid- [29] Yan K, Zhu H, Dan N, Chen C. An improved method for the 11 5 phase microextraction and high-performance liquid separation and quantiﬁcation of major phospholipid classes 12 6 chromatography with ultraviolet detection for by LC-ELSD. Chromatographia 2010;72:815e9. 13 7 14