|Thorson, Jennifer - North Dakota State University|
|Hammer, Carolyn - North Dakota State University|
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/24/2010
Publication Date: 4/14/2010
Publication URL: http://hdl.handle.net/10113/42957
Citation: Shelver, W.L., Thorson, J.F., Hammer, C.J., Smith, D.J. 2010. Depletion of Urinary Zilpaterol Residues in Horses as Measured by ELISA and UPLC-MS/MS. Journal of Agricultural and Food Chemistry. 58:4077-4083. DOI:10.1021/jf904253t.
Interpretive Summary: Zilpaterol is a growth promoter approved for use in cattle in Mexico, South Africa, and the United States. Because of its ability to improve feed efficiency, produce leaner meat, and enhance athletic performance, this agent has a potential to be used illegally. Zilpaterol use is forbidden in many countries and consequently it is necessary to have valid analytical methods capable of demonstrating the animal products are free from Zilpaterol. Since the beneficial effects of using growth promoters are also useful in species other than cattle, this study examined the zilpaterol levels in horses fed a supplement containing zilpaterol and the decline in zilpaterol levels after dosing was discontinued. The study demonstrated the Zilpaterol levels in urine dropped rapidly for the first five days but a slower elimination phase started around the 6th withdrawal day. Our analytical methods were able to detect the presence of zilpaterol in horse urine for up to 21 days after the zilpaterol feeding, showing them to be very powerful tools in detecting the illegal use of zilpaterol in horses.
Technical Abstract: Three horses were dosed with dietary zilpaterol and the urine concentration measured from withdrawal day 0 to withdrawal day 21. The analyses were carried out using both enzyme linked immunosorbent assay (ELISA) and an ultra-performance liquid chromatography with triple-quadrupole-tandem mass spectrometric detection (UPLC-MS/MS). The UPLC-MS/MS method was developed to provide rapid analysis with positive analyte identification by following three product ions and computing the two independent ion ratios. When urinary zilpaterol concentrations were between 0.2-2 ng/mL the ELISA had inter-day recoveries of 114-120% with the coefficients of variation (CV) less than 20%; intra-day recoveries were 79-110 % with CVs of less than 11%. For urinary zilpaterol concentrations of 0.4-40 ng/mL the UPLC-MS/MS method had inter-day recoveries of 94-104% with CVs of less than 8%; intraday recoveries were 97-102 % with CVs less than 7.5 %. Correlation analysis demonstrated that the ELISA and UPLC-MS/MS methods returned essentially the same results, especially at urinary zilpaterol concentrations below 2 ng/mL. Urinary excretion peaked rapidly after dosing at nearly 20,000 ng/mL, a value much higher than observed in other species. Peak urinary zilpaterol concentrations declined rapidly to approximately 2,000 ng/mL within 24 hours of peaking. After about 5 days, zilpaterol elimination slowed markedly, taking nearly 10 days for an order of magnitude decrease. The analytical methods were able to detect zilpaterol in the urine even at withdrawal day 21 demonstrating the sensitivity of each analytical method and the slow rate of zilpaterol depuration from horses.