Food Analytical Methods

, Volume 10, Issue 7, pp 2252–2261 | Cite as

Sensitive Determination of Toxic Clenbuterol in Pig Meat and Pig Liver Based on a Carbon Nanopolymer Composite

  • Chao-Zhi Lv
  • Yan Xun
  • Zhong Cao
  • Jing-Lei Xie
  • Dan Li
  • Gang Liu
  • Lei Yu
  • Ze-Meng Feng
  • Yu-Long Yin
  • Shu-Zhen Tan


The acidified single-walled carbon nanotubes (SWCNTs) were self-assembled on graphene oxide (GO) and then ultrasonically dispersed in a copolymer, Nafion solution, to form a GO/SWCNTs-Nafion polymer nanocomposite, which was employed to modify glassy carbon electrode (GCE). The surface morphological characteristics of different modified electrodes including bare GCE, GO-Nafion/GCE, and GO/SWCNTs-Nafion/GCE were imaged by scanning electron microscopy. For comparison, the differential pulse voltammetry and cyclic voltammetry behaviors were investigated, showing that the GO/SWCNTs-Nafion polymer composite has strong enhancement effect towards oxidation of clenbuterol (CLB). And the corresponding mechanism has been well discussed. During the reaction process, the anilino group of CLB molecule (1) was firstly oxidized to form a radical cation (2), exhibiting a characteristic oxidation peak (I) at 0.95 V, then two radical cations reacting via head-to-head coupling to form a diphenylamine intermediate (3), which was transformed into a CLB dimmer (4) through an azo bond by intramolecular electrons transferring under low potential, exhibiting a pair of reversible oxidation peak (II) and reduction peak (III). Under the optimum conditions, the composite modified electrode showed linear response to CLB in a concentration range of 1.0 × 10−8~6.0 × 10−6 mol/L with a detection limit of 6.0 × 10−9 mol/L. The modified electrode possessed good selectivity, reproducibility, and stability. In comparison with two routine analytical methods like ELISA kit and high-performance liquid chromatography (HPLC), the electrode can be successfully applied to determination of content of CLB in pig meat and pig liver samples with a recovery rate of 96.4~104.2%, suggesting a promising application in food security field.


Clenbuterol Graphene oxide Single-walled carbon nanotube Nafion polymer Pig meat Pig liver 



This work was financially supported by the projects of the National Natural Science Foundation of China (nos. 31527803, 21275022, and 21545010), STS Program of the Chinese Academy of Sciences (no. KFJ-SW-STS-173), and Scientific Research Project of Hunan Provincial Education Department of China (no. 14A012).

Compliance with Ethical Standards

Conflict of Interest

Chao-Zhi Lv declares that he has no conflict of interest. Yan Xun declares that she has no conflict of interest. Zhong Cao declares that he has no conflict of interest. Jing-Lei Xie declares that he has no conflict of interest. Dan Li declares that she has no conflict of interest. Gang Liu declares that he has no conflict of interest. Lei Yu declares that he has no conflict of interest. Ze-Meng Feng declares that he has no conflict of interest. Yu-Long Yin declares that he has no conflict of interest. Shu-Zhen Tan declares that she has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants performed by any of the authors. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Informed Consent

Not applicable.


  1. Chen D, Feng HB, Li JH (2012) Graphene oxide: preparation, functionalization, and electrochemical applications. Chem Rev 112(11):6027–6053CrossRefGoogle Scholar
  2. Chen Q, Fan LY, Zhang W, Cao CX (2008) Separation and determination of abused drugs clenbuterol and salbutamol from complex extractants in swine feed by capillary zone electrophoresis with simple pretreatment. Talanta 76(2):282–287CrossRefGoogle Scholar
  3. Chen XB, Wu YL, Yang T (2011) Simultaneous determination of clenbuterol, chloramphenicol and diethylstilbestrol in bovine milk by isotope dilution ultraperformance liquid chromatography–tandem mass spectrometry. J Chromatogr B 879(11):799–803CrossRefGoogle Scholar
  4. Cristino A, Ramos F, da Silveira MIN (2003) Control of the illegal use of clenbuterol in bovine production. J Pharmaceut Biomed 32(2):311–316CrossRefGoogle Scholar
  5. Du W, Zhang S, Fu Q, Zhao G, Chang C (2013) Combined solid-phase microextraction and high-performance liquid chromatography with ultraviolet detection for simultaneous analysis of clenbuterol, salbutamol and ractopamine in pig samples. Biomed Chromatogr 27(12):1775–1781CrossRefGoogle Scholar
  6. Fan L, Chen YQ, Zhang W, Cao CX (2013) Sensitive determination of illegal drugs of clenbuterol and salbutamol in swine urine by capillary electrophoresis with on-line stacking based on the moving reaction boundary. Anal Methods 5(11):2848–2853CrossRefGoogle Scholar
  7. Fan S, Miao H, Zhao Y, Chen H, Wu Y (2012) Simultaneous detection of residues of 25 β2-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass. J Agr Food Chem 60(8):1898–1905CrossRefGoogle Scholar
  8. Genetzky RM, Loparco FV (1985) Clinical efficacy of clenbuterol with COPD in horses. J Equine Vet Sci 5:320–323CrossRefGoogle Scholar
  9. Guo RX, Xu Q, Wang DY, Hu XY (2008) Trace determination of clenbuterol with an MWCNT-Nafion nanocomposite modified electrode. Microchim Acta 161(1–2):265–272CrossRefGoogle Scholar
  10. Hart JP, Smyth MR, Smyth WF (1981) Voltammetric determination of 2-3-and 4- chloroaniline in mixtures. Analyst 106(1259):146–152CrossRefGoogle Scholar
  11. He L, Su Y, Zeng Z, Liu Y, Huang X (2007) Determination of ractopamine and clenbuterol in feeds by gas chromatography–mass spectrometry. Anim Feed Sci Tech 132:316–323CrossRefGoogle Scholar
  12. Izquierdo-Lorenzo I, Sanchez-Cortes S, Garcia-Ramos JV (2010) Adsorption of beta-adrenergic agonists used in sport doping on metal nanoparticles: a detection study based on surface-enhanced Raman scattering. Langmuir 26(18):14663–14670CrossRefGoogle Scholar
  13. Jiang Y, Ni YN (2015) Automated headspace solid-phase microextraction and on-fiber derivatization for the determination of clenbuterol in meat products by gas chromatography coupled to mass spectrometry. J Sep Sci 38:418–425CrossRefGoogle Scholar
  14. Johansson MA, Hellenas KE (2004) Immunobiosensor determination of beta-agonists in urine using integrated immunofiltration clean-up. Int J Food Sci Tech 39(8):891–898CrossRefGoogle Scholar
  15. Juan C, Igualada C, Moragues F, León N, Manes J (2010) Development and validation of a liquid chromatography tandem mass spectrometry method for the analysis of β-agonists in animal feed and drinking water. J Chromatogr A 1217(39):6061–6068CrossRefGoogle Scholar
  16. Keskin S, Oezer D, Temizer A (1998) Gas chromatography-mass spectrometric analysis of clenbuterol from urine. J Pharmaceut Biomed 18:639–644CrossRefGoogle Scholar
  17. Lai YJ, Bai J, Shi XH, Zeng YB, Xian YZ, Hou J, Jin LT (2013) Graphene oxide as nanocarrier for sensitive electrochemical immunoassay of clenbuterol based on labeling amplification strategy. Talanta 107:176–182CrossRefGoogle Scholar
  18. Liang JJ, Liu HW, Huang CH, Yao CZ, Fu QQ, Li XQ, Cao DL, Luo Z, Tang Y (2015) Aggregated silver nanoparticles-based surface-enhanced Raman scattering enzyme-linked immunosorbent assay for ultrasensitive detection of protein biomarkers and small molecular. Anal Chem 87(11):5790–5796CrossRefGoogle Scholar
  19. Li CH, Luo W, Xu HY, Zhang Q, Xu H, Aguilar ZP, Lai WH, Wei H, Xiong YH (2013a) Development of an immunochromatographic assay for rapid and quantitative detection of clenbuterol in swine urine. Food Control 34(2):725–732CrossRefGoogle Scholar
  20. Li C, Wu YL, Yang T, Zhang Y, Huang-Fu WG (2010) Simultaneous determination of clenbuterol, salbutamol and ractopamine in milk by reversed-phase liquid chromatography tandem mass spectrometry with isotope dilution. J Chromatogr A 1217(50):7873–7877CrossRefGoogle Scholar
  21. Li KJ, Wang YJ, Zhang LL, Qin F, Guo XJ, Li FM (2013b) Simultaneous determination of trantinterol and its metabolites in rat urine and feces by liquid chromatography–tandem mass spectrometry. J Chromatogr B 934:89–96CrossRefGoogle Scholar
  22. Liu LJ, Pan HB, Du M, Xie WQ, Wang J (2010) Glassy carbon electrode modified with Nafion-Au colloids for clenbuterol electroanalysis. Electrochim Acta 55(24):7240–7245CrossRefGoogle Scholar
  23. Malucelli A, Ellendorff F, Meyer HH (1994) Tissue distribution and residues of clenbuterol, salbutamol, and terbutaline in tissues of treated broiler chickens. J Anim Sci 72:1555–1560Google Scholar
  24. Martinez-Navarro JF (1990) Food poisoning related to consumption of illicit β-agonist in liver. Lancet 336(8726):1311CrossRefGoogle Scholar
  25. Mazzanti G, Sott AD, Daniele C, Battinelli L, Brambilla G, Fiori M, Loizzo S, Loizzo A (2007) A pharmacodynamic study on clenbuterol-induced toxicity: β1 and β2 adrenoceptors involvement in guinea-pig tachycardia in an in vitro model. Food Chem Toxicol 45:1694–1699CrossRefGoogle Scholar
  26. McGrath GJ, O’Kane E, Smyth WF, Tagliaro F (1996) Investigation of the electrochemical oxidation of clenbuterol at a porous carbon electrode, and its application to the determination of this β-agonist in bovine hair by liquid chromatography with coulometric detection. Anal Chim Acta 322(3):159–166CrossRefGoogle Scholar
  27. Meyer JC, Geim AK, Katsnelson MI, Novoselov KS, Booth TJ, Roth S (2007) The structure of suspended graphene sheets. Nature 446(7131):60–63CrossRefGoogle Scholar
  28. Mitchell GA, Dunnavan G (1998) Illegal use of β-adrenergic agonists in the United States. J Anim Sci 76:208–211CrossRefGoogle Scholar
  29. Moane S, Barriera-Rodriguez JR, Miranda-Ordieres AJ, Tuiion-Blanco P, Smyth MR (1995) Electrochemical behaviour of clenbuterol at Nafion-modified carbon-paste electrodes. J Pharmaceut Biomed 14(1):57–63CrossRefGoogle Scholar
  30. Moane S, Smyth MR, O'Keeffe M (1996) Differential-pulse voltammetric determination of clenbuterol in bovine urine using a Nafion-modified carbon paste electrode. Analyst 121(6):779–784CrossRefGoogle Scholar
  31. Özkütük EB, Uğurağ D, Ersöz A, Say R (2016) Determination of clenbuterol by multiwalled carbon nanotube potentiometric sensors. Anal Lett 49(6):778–789CrossRefGoogle Scholar
  32. Parr MK, Opfermann G, Schänzer W (2009) Analytical methods for the detection of clenbuterol. Bioanalysis 1(2):437–450CrossRefGoogle Scholar
  33. Sarmah AK, Meyer MT, Boxall ABA (2006) A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 65(5):725–759CrossRefGoogle Scholar
  34. Tang YW, Lan JX, Gao X, Liu XY, Zhang DF, Wei LQ, Gao ZY, Li JR (2016) Determination of clenbuterol in pig and potable water samples by molecularly imprinted polymer through the use of covalent imprinting method. Food Chem 190:952–959CrossRefGoogle Scholar
  35. Vale A (2007) β2-Agonists. Medicine 35(11):597CrossRefGoogle Scholar
  36. Wang J (2008) Analy electrochemistry. Translated by Zhu YC, Zhang L, Chemical Industry Press, Beijing, pp 29–34Google Scholar
  37. Wang WY, Zhang YL, Wang JY, Shi X, Ye JN (2010) Determination of β-agonists in pig feed, pig urine and pig liver using capillary electrophoresis with electrochemical detection. Meat Sci 85(2):302–305CrossRefGoogle Scholar
  38. Wong CS, Chen YD, Chang JL, Zen JM (2015) Biomolecule-free, selective detection of clenbuterol based on disposable screen-printed carbon electrode. Electrochem Commun 60:163–167CrossRefGoogle Scholar
  39. Wu C, Sun D, Li Q, Wu KB (2012) Electrochemical sensor for toxic ractopamine and clenbuterol based on the enhancement effect of graphene oxide. Sensor Actuators B: Chem 168:178–184CrossRefGoogle Scholar
  40. Wu YL, Yang T, Shan JH, Huang-Fu WG (2010) Determination of residual clenbuterol enantiomers in swine urine by high performance liquid chromatography. Chinese J Anal Chem 38:833–837Google Scholar
  41. Yan FF, Zhang YC, Zhang S, Zhao JH, Liu SL, He LH, Feng XZ, Zhang HZ, Zhang ZH (2015) Carboxyl-modified graphene for use in an immunoassay for the illegal feed additive clenbuterol using surface plasmon resonance and electrochemical impedance spectroscopy. Microchim Acta 182:855–862CrossRefGoogle Scholar
  42. Zhai HY, Liu ZP, Chen ZG, Liang ZX, Su ZH, Wang SM (2015) A sensitive electrochemical sensor with sulfonated graphene sheets/oxygen-functionalized multi-walled carbon nanotubes modified electrode for the detection of clenbuterol. Sensor Actuators B: Chem 210:483–490CrossRefGoogle Scholar
  43. Zhang ML, Cao Z, He JL, Xue L, Zhou Y, Long S, Deng T, Zhang L (2012) A simple gold plate electrode modified with Gd-doped TiO2 nanoparticles used for determination of trace nitrite in cured food. Food Addit Contam A 29:1938–1946CrossRefGoogle Scholar
  44. Zhang ML, Huang DK, Cao Z, Liu YQ, He JL, Xiong JF, Feng ZM, Yin YL (2015) Determination of trace nitrite in pickled food with a nano-composite electrode by electrodepositing ZnO and Pt nanoparticles on MWCNTs substrate. LWT-Food Sci Technol 64(2):663–670CrossRefGoogle Scholar
  45. Zhang Q, Yang S, Zhang J, Zhang L, Kang P, Li J, Zhou H, Song XM (2011) Fabrication of an electrochemical platform based on the self-assembly of graphene oxide–multiwall carbon nanotube nanocomposite and horseradish peroxidase: direct electrochemistry and electrocatalysis. Nanotechnology 22(49):494010–494016CrossRefGoogle Scholar
  46. Zhang ZH, Duan FH, He LH, Peng DL, Yan FF, Wang MH, Zong W, Jia CX (2016) Electrochemical clenbuterol immunosensor based on a gold electrode modified with zinc sulfide quantum dots and polyaniline. Microchim Acta 183(3):1089–1097CrossRefGoogle Scholar
  47. Zhao C, Jin GP, Chen LL, Li Y, Yu B (2011) Preparation of molecular imprinted film based on chitosan/nafion/nano-silver/poly quercetin for clenbuterol sensing. Food Chem 129(2):595–600CrossRefGoogle Scholar
  48. Zhao L, Zhao J, Huangfu WG, Wu YL (2010) Simultaneous determination of melamine and clenbuterol in animal feeds by GC-MS. Chromatographia 72(3–4):365–368CrossRefGoogle Scholar
  49. Zhou J, Li Y, Liu Q, Fu GJ, Zhong ZZ (2013) Capillary electrophoresis of clenbuterol enantiomers and NMR investigation of the clenbuterol/carboxymethyl-β-cyclodextrin complex. J Chromatogr Sci 51(3):237–241CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Chao-Zhi Lv
    • 1
  • Yan Xun
    • 1
  • Zhong Cao
    • 1
  • Jing-Lei Xie
    • 1
  • Dan Li
    • 1
  • Gang Liu
    • 2
  • Lei Yu
    • 3
  • Ze-Meng Feng
    • 2
  • Yu-Long Yin
    • 2
  • Shu-Zhen Tan
    • 1
  1. 1.Hunan Provincial Engineering Research Center for Food Processing of Aquatic Biotic Resources, Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, School of Chemistry and Biological EngineeringChangsha University of Science and TechnologyChangshaChina
  2. 2.Institute of Subtropical Agriculture, Chinese Academy of SciencesChangshaChina
  3. 3.China Animal Disease Control CenterBeijingChina

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