Immunoassay detection of drugs in racing horses: detection of ethacrynic acid and bumetanide in equine urine by ELISA.

This research describes the development of a rapid and sensitive test using the enzyme-linked immunosorbent assays (ELISA) method to detect the diuretics ethacrynic acid and bumetanide in the urine of racing horses and provides evidence of its successful application.

Objective and Methodology

• The research aimed at identifying diuretics ethacrynic acid and bumetanide in the urine of race horses. These drugs are suspected to be illegally substituted for a legal drug called furosemide in order to improve performance in races. The researchers developed new tests based on enzyme-linked immunosorbent assays (ELISA), a common method used to detect compounds in biological samples.
• Antibodies were raised specifically against these substances and then used in the ELISA tests. The tests performed were fast (they could be completed in an hour), and the results could simply be read by eye.

Testing and Results

• The researchers tested the accuracy and efficiency of the ELISA method by injecting horses with specific amounts of ethacrynic acid and bumetanide. They then took urine samples from these horses and used their ELISA tests to determine the presence of these diuretics.
• The ELISA test could identify ethacrynic acid with a half-maximal inhibition concentration (I-50) of about 2.5 ng/mL. The drug could be detected in urine samples for up to 8 hours after administration.
• Similarly, the bumetanide ELISA test detected the drug with an I-50 of about 2.0 ng/mL and could identify bumetanide in urine up to 8 hours post-injection.

Specificity and Application

• These antibodies were found to be considerably specific, meaning they did not accidentally identify other diuretics or acidic compounds usually found in equine urine as the targeted drugs.
• The development of these tests provides an efficient method to control the illegal use of these substances pre- and post-race. In fact, this test has already found several instances of illegal drug use in a midwestern racing jurisdiction, illustrating the test’s practicality and effectiveness.