Amitraz (N′-(2,4-dimethylphenyl)-N-[[(2,4-dimethylphenyl)imino] methyl]-N-methyl-methanimidamide) is an alpha-2 adrenergic agonist used in veterinary medicine primarily as a scabicide- or acaricide-type insecticide. As an alpha-2 adrenergic agonist, it also has sedative/tranquilizing properties and is, therefore, listed as an Association of Racing Commissioners International Class 3 Foreign Substance, indicating its potential to influence the outcome of horse races. We identified the principal equine metabolite of amitraz as N-2,4-dimethylphenyl-N′-methylformamidine by electrospray ionization(+)-mass spectrometry and developed a gas chromatographic-mass spectrometric (GC-MS) method for its detection, quantitation, and confirmation in performance horse regulation. The GC-MS method involves derivatization with t-butyldimethylsilyl groups; selected ion monitoring (SIM) of m/z 205 (quantifier ion), 278, 261, and 219 (qualifier ions); and elaboration of a calibration curve based on ion area ratios involving simultaneous SIM acquisition of an internal standard m/z 208 quantifier ion based on an in-house synthesized d6 deuterated metabolite. The limit of detection of the method is approximately 5 ng/mL in urine and is sufficiently sensitive to detect the peak urinary metabolite at 1 h post dose, following administration of amitraz at a 75-mg/horse intraveneous dose.

Document Type


Publication Date


Notes/Citation Information

Lehner, A. F., Hughes, C. G., Karpiesiuk, W., Harkins, J. D., Dirikolu, L., Bosken, J., Camargo, F., Boyles, J., Troppmann, A., Woods, W. E., & Tobin, T. (2004). Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine. Journal of Analytical Toxicology, 28(7), 553-562. https://doi.org/10.1093/jat/28.7.553

Digital Object Identifier (DOI)


Funding Information

Supported by a grant from The Kentucky Racing Commission, Lexington, KY.

Related Content

Published as Article #322 from the Equine Pharmacok~gy, Therapeutics and Toxicology Program at the Maxwell H. Gluck Equine Research Center and the Department of Veterinary Science, University of Kentucky.

Published as Kentucky Agricultural Experiment Station Article # 03-14-040 with the approval of the Dean and Director, College of Agriculture and Kentucky Agricultural Experimental Station.