Analysis of biological fluids and animal hairs for beta-adrenergic agonists by liquid chromatography – mass-spectrometry

To control the excretion of beta-agonists used to accelerate the growth of muscle mass in animal husbandry, a method of determination of 20 compounds of this series in the cattle urine and blood, and 12 compounds in the cattle hairs samples by high-performance liquid chromatography – tandem mass spectrometry has been developed. The sample preparation of cattle urine included enzymatic hydrolysis in a phosphate buffer followed by purification using solid-phase extraction with a Copure C8/SCX sorbent. For the blood samples extraction of analytes by acetonitrile and acetic acid, followed by a phase separation and purification using C₁₈ and aluminum oxide were carried out. Hair samples were subjected to an alkaline hydrolysis, and the analytes were extracted using methyl tert butyl ether. The extract was then transferred to acetonitrile for further dispersive purification. The overall loss during sample preparation ranged from 27 to 85% for blood samples and from 2 to 80% for urine samples, depending on the compound being determined. Chromatographic separation was conducted in a gradient mode using an ACQUITY UPLC HSS C₁₈ column. Detection was performed in the selected reactions monitoring mode with registration of at least two product ions for each compound. The detection ranges of beta-agonists in urine were 0.25 – 20.0 μg/kg; 0.1 – 10.0 μg/kg in blood; 0.5 – 20.0 μg/kg in hairs depending on the analyte. For urine and blood analysis, the relative expanded uncertainty values ranged from 9 to 25%, while for hair analysis, they ranged from 9 to 30%.

1. Vulić A., Pleadin J., Perši N., et al. Accumulation of β-agonists clenbuterol and salbutamol in black and white mouse hair / J. Anal. Toxicol. 2011. Vol. 35. No. 8. P. 566 – 570. DOI: 10.1093/anatox/35.8.566

2. Liu J., Tang C., Long R., et al. The use of hair as a long-term indicator of low-dose β2 agonist treatments in cattle: Implications for growth-promoting purposes monitoring / Drug Test. Anal. 2019. Vol. 11. No. 6. P. 745 – 751. DOI: 10.1002/dta.2551

3. Zhang H., Chang Q., Wu X., et al. Rapid screening of β-agonists in animal hair by thermal desorption electrospray ionization mass spectrometry / J. AOAC Int. 2022. Vol. 105. No. 3. P. 812 – 821. DOI: 10.1093/jaoacint/qsab150

4. Decheng S., Xia F., Xiaoou S., et al. Application of hair analysis in the monitoring of abuse of β-agonists: a review / J. Chromatogr. A. 2025. Vol. 10. No. 1748. 465846. DOI: 10.1016/j.chroma.2025.465846

5. Chang K. C., Chang Y. T., Tsai C. E. Determination of ractopamine and salbutamol in pig hair by liquid chromatography tandem mass spectrometry / J. Food Drug Anal. 2018. Vol. 26. No. 2. P. 725 – 730. DOI: 10.1016/j.jfda.2017.09.005

6. Giannetti L., Ferretti G., Gallo V., et al. Analysis of beta-agonist residues in bovine hair: Development of a UPLC-MS/MS method and stability study / J. Chromatogr. B. 2016. Vol. 1036 – 1037. P. 76 – 83. DOI: 10.1016/j.jchromb.2016.09.041

7. Wan-Hua Y., Wen-Ting L., Qin F., et al. Determination of clenbuterol-like beta-agonist residues in hair / LC-GC North America. 2011. Vol. 29. No. 7. P. 600.

8. Zhang M. X., Li C., Wu Y. L. Determination of phenylethanolamine A in animal hair, tissues and feeds by reversed phase liquid chromatography tandem mass spectrometry with QuEChERS / J. Chromatogr. B. 2012. Vol. 900. P. 94 – 99. DOI: 10.1016/j.jchromb.2012.05.030

9. Di Stefano V., Pitonzo R., Giaccone V., et al. Analysis of β2-agonists in cattle hair samples using a rapid UHPLC-ESI-MS/MS method / Nat. Prod. Res. 2017. Vol. 31. No. 4. P. 482 – 486. DOI: 10.1080/14786419.2016.1190718

10. Garcia P., Paris A. C., Gil J., et al. Analysis of β-agonists by HPLC/ESI-MS(n) in horse doping control / Biomed. Chromatogr. 2011. Vol. 25. No. 1 – 2. P. 147 – 154. DOI: 10.1002/bmc.1562

11. Uzunov R., Hajrulai-Musliu Z., Stojkovski V., et al. Development and validation of LC-MS/MS method for determination of ten beta agonists in bovine urine / Kafkas Univ. Vet. Fak. Derg. 2019. Vol. 25. No. 1. P. 55 – 60. DOI: 10.9775/kvfd.2018.20324

12. Thorsten B., Peter F., Michal G. Quick screening of priority beta-agonists in urine using automated TurboFlow-LC/ Exactive mass spectrometry / Food Addit. Contam., Part A. 2011. Vol. 28. No. 10. P. 1352 – 1363. DOI: 10.1080/19440049.2011.619504

13. Nielen M. W., Lasaroms J. J., Essers M. L., et al. Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry / Anal. Bioanal. Chem. 2008. Vol. 391. No. 1. P. 199 – 210. DOI: 10.1007/s00216-007-1760-7

14. Josefsson M., Sabanovic A. Sample preparation on polymeric solid phase extraction sorbents for liquid chromatographic-tandem mass spectrometric analysis of human whole blood — a study on a number of beta-agonists and beta-antagonists / J. Chromatogr. A. 2006. Vol. 1120. No. 1 – 2. P. 1 – 12. DOI: 10.1016/j.chroma.2006.03.013

15. Salem A. A., Salama I. W., Al-Nassib S., et al. 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. 2017. Vol. 55. No. 8. P. 846 – 856. DOI: 10.1093/chromsci/bmx045

16. Sidler-Moix A. L., Mercier T., Decosterd L. A., et al. A highly sensitive LC-tandem MS assay for the measurement in plasma and in urine of salbutamol administered by nebulization during mechanical ventilation in healthy volunteers / Biomed. Chromatogr. 2012. Vol. 26. No. 5. P. 672 – 680. DOI: 10.1002/bmc.1718

17. Nguyen T. M. T., Nguyen T. K. L. Simultaneous determination of salbutamol and clenbuterol in human plasma using liquid chromatography coupled to tandem mass spectrometry / Pharm. Sci. Asia. 2019. Vol. 46. No. 2. P. 120 – 128. DOI: 10.29090/psa.2019.02.017.0025

18. Wu J., Ding C., Ge Q., et al. Simultaneous determination of ipratropium and salbutamol in rat plasma by LC-MS/MS and its application to a pharmacokinetic study / J. Chromatogr. B. 2011. Vol. 879. No. 30. P. 3475 – 3483. DOI: 10.1016/j.jchromb.2011.09.027

19. Zhang D., Teng Y., Chen K., et al. Determination of salbutamol in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry and its pharmacokinetic study / Biomed. Chromatogr. 2012. Vol. 26. No. 10. P. 1176 – 1182. DOI: 10.1002/bmc.2675

20. Thevis M., Schebalkin T., Thomas A., et al. Quantification of clenbuterol in human plasma and urine by liquid chromatography-tandem mass spectrometry / Chroma. 2005. Vol. 62. P. 435 – 439. DOI: 10.1365/s10337-005-0651-3