title: Immunoassay detection of drugs in racing horses: detection of ethacrynic acid and bumetanide in equine urine by ELISA.
abstract: We have raised antibodies and developed one-step enzyme-linked immunosorbent assays (ELISA) for the diuretics ethacrynic acid and bumetanide as part of a panel of pre- and post-race tests for high potency drugs in racing horses. These ELISA tests are rapid (completed within one hour), sensitive, and can be read by eye. The ELISA detects ethacrynic acid at a drug concentration for half-maximal inhibition (I-50) of about 2.5 ng/mL for the parent drug. After dosing horses intravenously with 5 mg ethacrynic acid per horse, the parent drug or its metabolites are detectable in urine for at least 8 hours. The bumetanide ELISA has an I-50 for the parent drug of about 2.0 ng/mL and will detect bumetanide or its metabolites for about 8 hours in urine after intravenous administration of a 1.7-mg dose per horse. Both antibodies are relatively specific for each drug and do not cross-react with other commonly used diuretics or other acidic compounds often found in post-race equine urine samples. Ethacrynic acid and bumetanide are potent diuretics suspected of being illegally substituted for furosemide in certain racing jurisdictions. Development of these rapid, sensitive, and simple tests for these agents will allow more effective pre- and post-race control of the use of these agents in racing horses. Both tests have recently uncovered several "positives" for these medications in a midwestern racing jurisdiction.
authors.affiliation: Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington 40546-0099.
authors.forename: S
authors.initials: S
authors.lastname: Stanley
authors.forename: T
authors.initials: T
authors.lastname: Wood
authors.forename: J P
authors.initials: JP
authors.lastname: Goodman
authors.forename: P A
authors.initials: PA
authors.lastname: Henry
authors.forename: W E
authors.initials: WE
authors.lastname: Woods
authors.forename: S L
authors.initials: SL
authors.lastname: Chang
authors.forename: H H
authors.initials: HH
authors.lastname: Tai
authors.forename: D
authors.initials: D
authors.lastname: Watt
authors.forename: S
authors.initials: S
authors.lastname: Kwiatkowski
authors.forename: J W
authors.initials: JW
authors.lastname: Blake
mesh_terms: D000818:Animals; D002034:Bumetanide / Q000652:urine*; D004300:Doping in Sports; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D004976:Ethacrynic Acid / Q000652:urine*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000662:veterinary; D006736:Horses / Q000652:urine*; D007275:Injections, Intravenous / Q000662:veterinary
chemical_list: D002034:Bumetanide; D004976:Ethacrynic Acid