Food Analytical Methods

, Volume 11, Issue 9, pp 2561–2568 | Cite as

Sensitive Assay of Clenbuterol Residues in Beef by Ultra-High Performance Liquid Chromatography Coupled with Mass Spectrometry (UPLC-MS/MS) and Solid-Phase Extraction

  • Martha F. Medellín-Martínez
  • Israel Luna-Zavala
  • Maricela Martínez-Delgado
  • José T. Pérez-Urizar
  • Jorge A. Ramírez-Telles
  • Omar Patiño-Rodríguez


The use of clenbuterol (CLE) as a growth-promoting agent in livestock is banned in the USA, European Union, and Mexico. Nevertheless, different studies have reported the presence of this agent in meat products, potentially causing intoxication in consumers. In this study, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) is proposed for CLE determination in beef. A simple solid-phase extraction step was used as a cleanup sample method; the chromatographic separation was achieved with anAcquity UPLC HSS T3 column using a mobile-phase formic acid and methanol. We use a XEVO-TQS detector by positive-ion electrospray in the multiple reactions monitoring modes. The mass transitions monitored were m/z 277.08 → 202.95 for CLE and m/z 286.1 → 203.9 for clenbuterol-D9, used as an internal standard. The method was linear over the concentration range of 0.1–1.6 ng/g. Once validated, the method was applied to monitor CLE in beef samples collected at local markets of San Luis Potosí (central region of Mexico). Despite the ban on the use of CLE to grow cattle, we find levels of beta-agonist, with concentrations between 0.6 and 10.4 ng/g. Our method is ideal for routine analysis for quality control and food safety.


Beef Clenbuterol Food safety Ultra-performance liquid chromatography Mass spectrometry 



The authors acknowledge the technical support of team Dixpertia, especially QFB Gustavo Adolfo Meza-Galeana and QFB Adriana Flores-Moreno. This work was supported by the program of incentives for research and development (PEI), project number 178105-2012 Council of Science and Technology of México (CONACyT). We also thank CONACyT Mexico for the scholarship granted to Martha F. Medellín-Martínez for her Master studies.


This study was funded by the program of incentives for research and development (PEI), project number 178105-2012, Council of Science and Technology of México (CONACyT).

Compliance with Ethical Standards

Conflict of Interest

Martha F. Medellín-Martínez declares that she has no conflict of interest. Israel Luna-Zavala declares that he has no conflict of interest. Maricela Martínez-Delgado declares that she has no conflict of interest. José T. Pérez-Urizar declares that he has no conflict of interest. Jorge A. Ramírez-Telles declares that he has no conflict of interest. Omar Patiño-Rodríguez declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with animals performed by any of the authors. The meat samples were acquired directly from the certified supplier. For the study in local markets, the direct acquisition of meat with the local supplier was carried out.

Informed Consent

Not applicable.


  1. Andrée S, Jira W, Schwind KH, Wagner H, Schwägele F (2010) Review: chemical safety of meat and meat product. Meat Sci 86:38–48CrossRefGoogle Scholar
  2. Chávez LA, Díaz JA, Pérez B, Alarcón MA (2012) Trend in levels of clenbuterol in bovine samples from 2005 to 2010 in Guerrero, Mexico. Rev Mex Cienc Pecu 3:449–458Google Scholar
  3. Churchwell M, Twaddle N, Meeker L, Doerge D (2005) Improving LC-MS sensitivity through increases in chromatographic performance: comparisons of UPLC-ES/MS/MS to HPLC-ES/MS/MS. J Chromatogr B 825:134–143CrossRefGoogle Scholar
  4. Codex Alimentarius Commission (2011) Maximum residue limits for veterinary drugs in foods. CAC/MRL 2:7Google Scholar
  5. Daubert GP, Mabasa VH, Leung VW, Aaron C (2007) Acute clenbuterol overdose resulting in supraventricular tachycardia and atrial fibrillation. J Med Toxicol 3:56–60CrossRefGoogle Scholar
  6. De Wash K, De Brabander H, Courtheyn D (1998) LC-MS-MS to detect and identify four beta-agonists and quantify clenbuterol in liver. Analyst 123:2701–2705CrossRefGoogle Scholar
  7. Estrada-Montoya MC, González-Córdova AF, Torrescano G, Camou JP, Vallejo-Cordoba B (2008) Screening and confirmatory determination of clenbuterol residues in bovine and meat marketed in the northwest of Mexico. Ciencia y Tecnología Alimentaria 6:130–136CrossRefGoogle Scholar
  8. Geesink GH, Smulders FJ, van Laak HL, van Der Kolk JH, Wensing T, Breukink HJ (1993) Effects on meat quality of the use of clenbuterol in veal calves. J Animal Sci 71:1161–1170CrossRefGoogle Scholar
  9. Guy PA, Savoy MC, Stadler RH (1999) Quantitative analysis of clenbuterol in meat products using liquid chromatography-electrospray ionization tandem mass spectrometry. J Chromatogr B 736:209–219CrossRefGoogle Scholar
  10. Jiménez LA, Ramos JG, Sumano H, Fragoso H (2011) Sanitary surveillance on illegal use of clenbuterol and its intersectoral coordination in two states of Mexico. Vet México 42:11–23Google Scholar
  11. Johnson BJ, Smith SB, Chung KY (2014) Historical overview of the effect of β-adrenergic agonists on beef cattle production. Asian-Australas J Anim Sci 27:757–766CrossRefGoogle Scholar
  12. Juan C, Igualada C, Moragues F, León N, Mañes 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 1217:6061–6068CrossRefGoogle Scholar
  13. Kuiper HA, Noordam MY, van Dooren-Flipsen MM, Schilt R, Roos AH (1998) Illegal use of beta-adrenergic agonists: European Community. J Animal Sci 76:195–207CrossRefGoogle Scholar
  14. Le Bizec B, Pinel G, Antignac JP (2009) Options for veterinary drug analysis using mass spectrometry. J Chromatogr A 1216:8016–8034CrossRefGoogle Scholar
  15. Li C, Wu Y, Yang T, Zhang Y, Huang-Fu W (2010) Simultaneous determination of clenbuterol, salbutamol and ractopamine in milk by reversed-phase liquid chromatography tandem mass spectrometry with isotope dilution. J Chromatogr A 1217:7873–7877CrossRefGoogle Scholar
  16. Mitchell GA, Dunnavan G (1998) Illegal use of beta-adrenergic agonists in the United States. J Anim Sci 76:208–211CrossRefGoogle Scholar
  17. Morales-Trejo F, Vega-y León S, Escobar-Medina A, Gutiérrez-Tolentino R (2013) Application of high-performance liquid chromatography-UV detection to quantification of clenbuterol in bovine liver samples. J Food Drug Anal 21:414–420CrossRefGoogle Scholar
  18. NOM-061-ZOO 1999. Especificaciones zoosanitarias de los productos alimenticios para consumo animal. Available at: Retrieved: 19 October 2017
  19. Peña SD, Cordova-Izquierdo A, Uribe A, Michel AM (2008) Clenbuterol residues in bovine feed and meat. Res J Biol Sci 3:1444–1445Google Scholar
  20. Reig M, Toldrá F (2008) Veterinary drug residues in meat: concerns and rapid methods for detection. Meat Sci 78:60–67CrossRefGoogle Scholar
  21. Ren X, Zhang F, Chen F, Yang T (2009) Development of a sensitive monoclonal antibody-based ELISA for the detection of clenbuterol in animal tissues. Food Agric Immunol 20:333–344CrossRefGoogle Scholar
  22. Ricks CA, Dalrymple RH, Baker PK, Ingle DL (1984) Use of a β-agonist to alter fat and muscle deposition in steers. J Animal Sci 59:1247–1255CrossRefGoogle Scholar
  23. Salem AA, Wasfi I, Al-Nassib SS, Allawy Mohsin M, Al-Katheeri N (2017) Determination of some β-blockers and β2-agonists in plasma and urine using liquid chromatography-tandem mass spectrometry and solid phase extraction. J Chromatogr Sci 55:846–856CrossRefGoogle Scholar
  24. Shelver WL, Smith DJ (2000) Evaluation of commercial immunoassays for cross-reactivity to clenbuterol stereoisomers and bovine metabolites. Food Addit Contam 17:837–845CrossRefGoogle Scholar
  25. Smith DJ, Paulson GD (1997) Distribution, elimination, and residues of (14C) clenbuterol HCl in Holstein calves. J Anim Sci 75:454–461CrossRefGoogle Scholar
  26. Sumano H, Ocampo L, Gutiérrez L (2002) Clenbuterol and other β-agonists, are they an option for meat production or a threat for public health? Vet México 33:137–159Google Scholar
  27. Zheng H, Deng L, Lu X, Zhao S, Guo C, Mao J, Wang Y, Yang G, Aboul-Enein HY (2010) UPLC-ESI-MS-MS; determination of three β2-agonists in pork. Chromatographia 72:79–84CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Martha F. Medellín-Martínez
    • 1
  • Israel Luna-Zavala
    • 1
    • 2
  • Maricela Martínez-Delgado
    • 2
  • José T. Pérez-Urizar
    • 1
  • Jorge A. Ramírez-Telles
    • 1
  • Omar Patiño-Rodríguez
    • 2
    • 3
  1. 1.Facultad de Ciencias QuímicasUniversidad Autónoma de San Luis PotosíSan Luís PotosíMexico
  2. 2.DixpertiaInvestigación Biofarmacéutica y Farmacológica S.C.San Luis PotosíMexico
  3. 3.CONACyT-Instituto Politécnico NacionalCentro de Desarrollo de Productos Bióticos (CEPROBI)YautepecMexico

Personalised recommendations