Development of a monoclonal antibody-based immunoassay to detect furosemide in cow's milk

Furosemide is a potent diuretic drug used in both human and veterinary medicine. High-performance liquid chromatographic methods (HPLC) were developed to detect furosemide in blood and urine samples. Recently, immunoassay kits have appeared to measure furosemide; these were developed for the race horse industry where furosemide is used to treat epistaxis. In dairy cattle, furosemide is used for treatment of physiological parturient edema and there is a 48-h withdrawal period before milk from treated animals can be used. We report here the development of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detecting furosemide in milk. In addition, we report on the development of an HPLC method for detection of furosemide in milk that is an adaptation of published methods for its detection in serum and blood. Unlike the HPLC method, no sample preparation is necessary for the ELISA. Raw milk is added directly into the assay, or if needed, it is diluted with assay buffer. The immunoassay had a lower limit of quantification of 2 ppb and a lower limit of detection of approximately 0.5 ppb. Good correlations were observed between the HPLC and ELISA methods when samples with both incurred and spiked furosemide residues were analyzed.