Risto UZUNOV, Zehra HAJRULAI-MUSLIU, Velimir STOJKOVSKI, Elizabeta DIMITRIESKA-STOJKOVIC, Biljana STOJANOVSKA-DIMZOSKA, Pavle SEKULOVSKI, Dean JANKULOSKI

Development and Validation of LC-MS/MS Method for Determination of Ten Beta Agonists in Bovine Urine

The use of β-agonists in livestock production is prohibited in many countries because the residues of β-agonists in food pose a potential risk to human health. The present work describes the development and validation of the LC-MS/MS method for detection of ten β-agonists in bovine urine according to Commission Decision 2002/657/EC requirements. Linearity of the method resulted with coefficient of correlation >0.990. The decision limits (CCα) ranged from 0.127 ng/mL to 0.646 ng/mL, and the detection capability (CCβ) resulted in the range 0.140 ng/mL to 0.739 ng/mL. The observed recoveries in the fortified bovine urine samples were satisfactory at every fortification level with values from 73.67% to 118.80%. The coefficient of variation (CV, %) at three fortification levels for each β-agonist was in complete agreement with the requirements from Commission Decision 2002/657/EC. The CV for intraday precision varied from 1.619% to 15.472% and the CV for interday precision varied from 2.695% to 10.441%. From the obtained validation results the proposed method is an appropriate method for determination of β-agonists in bovine urine.

Sığır İdrarında 10 Beta Agonist Varlığının Belirlenmesi İçin LC-MS/MS Metodu Geliştirilmesi ve Validasyonu

Bir çok ülkede β-agonistlerin hayvancılıkta kullanılması yasaklanmıştır, çünkü gıda ürünlerinde bulunan β-agonist kalıntıları insan sağlığı için risk oluşturmaktadır. Bu çalışma, komisyon kararı 2002/657/EC ye uygun olarak, sığır idrarındaki 10 adet β-agonistin tespitini sağlayan metod gelişimini ve validasyonunu içermektedir. Doğrusallık korelasyon katsayısı >0.990 ile sonuçlanmıştır. Karar limitleri (CCα) 0.127 ng/ml ile 0.646 ng/ml arasında ve tespit kapasitesi (CCβ) 0.140 ng/mL ile 0.739 ng/ml arasında bir değer almıştır. Geri kazanım %73.67 ile %118.80 arasında bulunmuştur. Korelasyon varyasyonu (CV, %) her üç konsantrasyon seviyesi için Komisyon Kararı 2002/657/EC ‘ye tamamen uygun olarak yapılmıştır. Gün içi kesinlik CV değeri %1.619 ile %15.472 arasında ve günler arası CV kesinlik değeri %2.695 ile %10.441 arasında değişmektedir. Bu çalışmada elde edilen validasyon değerlerine göre geliştirilen metod sığır idrarında β-agonist tespiti için uygundur.

PDF

___

1. Wang X, Guo T, Wang S, Yuan J, Zhao R: Simultaneous determination of 11 β-agonists in human urine using high-performance liquid chromatography/ tandem mass spectrometry with isotope dilution. J Anal Toxicol, 39 (3): 213-218, 2015. DOI: 10.1093/jat/bku143

2. Pleadin J, Vulic A, Terzic S, Vahcic N, Sandor K, Perak E: Comparison of accumulation of clenbuterol and salbutamol residues in animal internal tissues, non-pigmented eyes and hair. J Anal Toxicol, 38 (9): 681-685, 2014. DOI: 10.1093/jat/bku081

3. Prezelj A, Obreza A, Pecar S: Abuse of clenbuterol and its detection. Curr Med Chem, 10 (4): 281-290, 2003. DOI: 10.2174/0929867033368330

4. Vulic A, Pleadin J, Perši N, Stojkovic R, Ivankovic S: Accumulation of β-agonists clenbuterol and salbutamol in black and white mouse hair. J Anal Toxicol, 35 (8): 566-570, 2011. DOI: 10.1093/anatox/35.8.566

5. Fesser ACE, Dickson LC, Macneil JD, Patterson JR, Lee S, Gedir R: Determination of β-agonists in liver and retina by liquid chromatographytandemmass spectrometry. J AOAC Int, 88 (1): 61-69, 2005.

6. Haasnoot W, Stouten P, Schilt R, Hooijerink D: A fast immunoassay for the screening of β-agonists in hair. Analyst, 123, 2707-2710, 1998. DOI: 10.1039/a805041d

7. De WaschK, De Brabander H, Courtheyn D: LC-MS-MS to detect and identify four beta-agonists andquantify clenbuterol in liver. Analyst, 123, 2701-2705, 1998. DOI: 10.1039/A805039B

8. Pleadin J, Vulic A, Persi N, Milic D, Vahcic N: Ractopamine and clenbuterol urinary residues in pigs as food-producing animals. Food Technol Biotechnol, 49 (4): 517-522, 2011.

9. Uzunov R, Hajrulai-Musliu Z, Ilieski V, Stojanovska-Dimzoska B, Dimitrieska-Stojkovic E, Sekulovski P, Stojkovski V: Validation methods for determination of β-agonists residues in feed. In, Proceedings 15th International Feed Technology Symposium Feed-to Food/Cost Feed for Health Joint Workshop (Ed): University of Novi Sad, Institute of Food Technology. 88-93, Novi Sad, Serbia, 2012.

10. Salleras L, Dominguez A, Mata E, Taberner JL, Moro I, Salva P: Epidemiologic study of an outbreak of clenbuterol poisoning in Catalonia, Spain. Public Health Rep, 110 (3): 338-342, 1995.

11. Martinez-Navarro JF: Food poisoning related to consumption of illicit β-agonist in liver. Lancet, 336: 1311, 1990.

12. Commission of the European Communities: 96/22/EC of 29 April 1996 concerning the prohibition on the use in stock farming of certain substances having a hormonal or thyrostatic action and of β-agonists, and repealing, Directives 81/602/ EEC, 88/146/EEC and 88/299/EEC, OJ L125 23/5/1996. OJEC, L125, 3-8, 1996.

13. Commission of the European Communities: Council Directive 96/23/EC of 29 April 1996 on measures to monitor certain substances and residues thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and Decisions 89/187EEC and 91/664/EEC. OJEC, L125, 10-32, 1996.

14. Commission of the European Communities: Commission Decision 2002/657/EC of 12th August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. OJEC, L221, 2002.

15. CRL guidance paper: CRLs view on state of the art analytical methods for national residue control plans, 4-5, 2007.

16. Nielen MWF, Lasaroms JJP, Essers ML, Oosterink JE, Meijer T, Sanders MB, Zuidema T, Stolker AAM: Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography electrospray ionization tandem mass spectrometry. Anal Bioanal Chem, 391 (1): 199-210, 2008. DOI: 10.1007/s00216-007- 1760-7

17. Van Hoof N, Schiltb R, Van Der Vlis E, Boshuis P, Van Baak M, Draaijer A, De Wasch K, Van De Wiele M, Van Hende J, Courtheyn D, De Brabander H: Detection of zilpaterol (Zilmax®) in calf urine and faeces with liquid chromatography-tandem mass spectrometry. Anal Chim Acta, 529 (1-2): 189-197, 2005. DOI: 10.1016/j.aca.2004.08.072

18. Liu X, He X, Moore C, Wang G, Coulter C: Highly sensitive and specific liquid chromatography–tandem mass spectrometry method for testing ractopamine in cow and sheep urine. J Anal Toxicol, 33 (6): 289- 293, 2009. DOI: 10.1093/jat/33.6.289

19. Giannettia L, Ferretti G, Galloa V, Neccia F, Giorgia A, Marinia F, Gennusoa E, Neria B: Analysis of beta-agonist residues in bovine hair: Development of aUPLC–MS/MS method and stability study. J Chromatogr B, 1036-1037, 76-83, 2016. DOI: 10.1016/j.jchromb.2016.09.041

20. Zhang LY, Chang BY, Dong T, He PL, Yang WJ, Wang ZY: Simultaneous determination of salbutamol, ractopamine, and clenbuterol in animal feeds by SPE and LC–MS. J Chromatogr Sci, 47 (4): 324-328, 2009. DOI: 10.1093/chromsci/47.4.324

21. Sakai T, Hitomi T, Sugaya K, Kai S, Murayama M, Maitani T: Determination method for ractopamine in swine and cattle tissues using LC/MS. Shokuhin Eiseigaku Zasshi, 48 (5): 144-147, 2007. DOI: 10.3358/ shokueishi.48.144