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

doi:10.1093/jat/28.7.553

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 journal › Article › peer-review

1 Scopus citations

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 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 language	English
Pages (from-to)	553-562
Number of pages	10
Journal	Journal of Analytical Toxicology
Volume	28
Issue number	7
DOIs	https://doi.org/10.1093/jat/28.7.553
State	Published - 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: [email protected].

Funding

* 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: [email protected].

Funders	Funder number
Dept. of Veterinary Science, University of Kentucky	KY 40546-0099
Kentucky Racing Commission

ASJC Scopus subject areas

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

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10.1093/jat/28.7.553

Cite this

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

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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 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.",

author = "Lehner, \{A. F.\} and Hughes, \{C. G.\} and W. Karpiesiuk and Harkins, \{J. D.\} and L. Dirikolu and J. Bosken and F. Camargo and J. Boyles and A. Troppmann and Woods, \{W. E.\} and T. Tobin",

note = "Funding Information: * Published as Article \#322 from the Equine Pharmacok\textasciitilde{}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: [email protected].",

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Lehner, AF, Hughes, CG, Karpiesiuk, W, Harkins, JD, Dirikolu, L, Bosken, J, Camargo, F, Boyles, J, Troppmann, A, Woods, WE & 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, vol. 28, no. 7, pp. 553-562. https://doi.org/10.1093/jat/28.7.553

TY - JOUR

T1 - Development of a method for the detection and confirmation of the alpha-2 agonist amitraz and its major metabolite in horse urine

AU - Lehner, A. F.

AU - Hughes, C. G.

AU - Karpiesiuk, W.

AU - Harkins, J. D.

AU - Dirikolu, L.

AU - Bosken, J.

AU - Camargo, F.

AU - Boyles, J.

AU - Troppmann, A.

AU - Woods, W. E.

AU - Tobin, T.

N1 - 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: [email protected].

PY - 2004/10

Y1 - 2004/10

N2 - 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.

AB - 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.

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

Abstract

Bibliographical note

Funding

ASJC Scopus subject areas

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