Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine

A. F. Lehner, C. G. Hughes, W. Karpiesiuk, J. D. Harkins, L. Dirikolu, J. Bosken, F. Camargo, J. Boyles, A. Troppmann, W. E. Woods, T. Tobin

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


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.

Original languageEnglish
Pages (from-to)553-562
Number of pages10
JournalJournal of Analytical Toxicology
Issue number7
StatePublished - Oct 2004

Bibliographical note

Funding Information:
* 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. Supported by a grant from The Kentucky Racing Commission, Lexington, KY. t Author It:, whom correspondence should Lye addressed: Andreas E. tehner, 108 Gluck Equine Research Center, Dept. of Veterinary Science, University of Kentucky, Lexington, KY 40546-0099. E-mail:

ASJC Scopus subject areas

  • Analytical Chemistry
  • Environmental Chemistry
  • Toxicology
  • Health, Toxicology and Mutagenesis
  • Chemical Health and Safety


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