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].

ASJC Scopus subject areas

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

Access to Document

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

@article{1b89d35d9a084d0698284bcc780f4104,

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~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].",

year = "2004",

month = oct,

doi = "10.1093/jat/28.7.553",

language = "English",

volume = "28",

pages = "553--562",

number = "7",

}

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.

UR - http://www.scopus.com/inward/record.url?scp=20844459313&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=20844459313&partnerID=8YFLogxK

U2 - 10.1093/jat/28.7.553

DO - 10.1093/jat/28.7.553

M3 - Article

C2 - 15516314

AN - SCOPUS:20844459313

SN - 0146-4760

VL - 28

SP - 553

EP - 562

JO - Journal of Analytical Toxicology

JF - Journal of Analytical Toxicology

IS - 7

ER -

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

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this