Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine.
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.
Lehner
A F
AF
Maxwell H. Gluck Equine Research Center and the Department of Veterinary Science, University of Kentucky, Lexington, Kentucky 40546-0099, USA. alehner@uky.edu
Hughes
C G
CG
Karpiesiuk
W
W
Harkins
J D
JD
Dirikolu
L
L
Bosken
J
J
Camargo
F
F
Boyles
J
J
Troppmann
A
A
Woods
W E
WE
Tobin
T
T
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*
D000316:Adrenergic alpha-Agonists; D000578:Amidines; D014052:Toluidines; C025699:U 40481; C014983:amitraz