Immunoassay detection of drugs in racing horses. X. Detection of phenylbutazone in equine blood and urine by particle concentration fluoroimmunoassay and elisa

We have developed Particle Concentration Fluorescence Immunoassay (PCFIA) and Enzyme Linked Immunosorbent Assay (ELISA) tests for phenylbutazone as part of a panel of immunoassay tests for drugs in racing horses. Since phenylbutazone is a legal drug in most American racing jurisdictions, the primary use for this test is likely to be quantitation of plasma levels of PB. Our PCFIA test had an I-50 for phenylbutazone of about 1 μg/ml, close to the generally used 5 μg/ml plasma limit for phenylbutazone. Our ELISA test is substantially more sensitive to PB, with an I-50 for phenylbutazone of about 10 ng/ml. Both tests are based on our anti-phenylbutazone antibody, which shows limited cross-reactivity with oxyphenbutazone and does not cross-react significantly with other acidic drugs used in horses. Quantitation of phenylbutazone in equine blood and urine by these assays correlates well with gas chromatography/mass spectrometry quantitation of phenylbutazone. Both assays are sufficiently sensitive to detect the presence of phenylbutazone or its metabolites in equine urine for up to 48 hours, and in equine plasma for up to 24 hours after therapeutic doses of this drug. These tests are capable of providing rapid, sensitive, and economical methods for racing jurisdictions to detect and estimate plasma and/or urinary concentrations of phenylbutazone. Other uses include pre-race and pre-purchase screening for unauthorized blood levels of phenylbutazone and screening for phenylbutazone toxicity.