title: Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine.
abstract: 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 d(6) 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 intravenous dose.
authors.affiliation: Maxwell H. Gluck Equine Research Center and the Department of Veterinary Science, University of Kentucky, Lexington, Kentucky 40546-0099, USA. alehner@uky.edu
authors.forename: A F
authors.initials: AF
authors.lastname: Lehner
authors.forename: C G
authors.initials: CG
authors.lastname: Hughes
authors.forename: W
authors.initials: W
authors.lastname: Karpiesiuk
authors.forename: J D
authors.initials: JD
authors.lastname: Harkins
authors.forename: L
authors.initials: L
authors.lastname: Dirikolu
authors.forename: J
authors.initials: J
authors.lastname: Bosken
authors.forename: F
authors.initials: F
authors.lastname: Camargo
authors.forename: J
authors.initials: J
authors.lastname: Boyles
authors.forename: A
authors.initials: A
authors.lastname: Troppmann
authors.forename: W E
authors.initials: WE
authors.lastname: Woods
authors.forename: T
authors.initials: T
authors.lastname: Tobin
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000493:pharmacokinetics / Q000652:urine*; D000578:Amidines / Q000138:chemical synthesis / Q000652:urine*; D000818:Animals; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006736:Horses / Q000378:metabolism* / Q000652:urine; D021241:Spectrometry, Mass, Electrospray Ionization; D015813:Substance Abuse Detection / Q000662:veterinary*; D013997:Time Factors; D014052:Toluidines / Q000493:pharmacokinetics / Q000652:urine*
chemical_list: D000316:Adrenergic alpha-Agonists; D000578:Amidines; D014052:Toluidines; C025699:U 40481; C014983:amitraz