J. Agric. Food Chem. 1996, 44, 2455−2459
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Development of a Monoclonal Antibody-Based Immunoassay To Detect Furosemide in Cow’s Milk Larry H. Stanker,*,† Mark T. Muldoon,† Sandra A. Buckley,† Curtis Braswell,‡ Carol Kamps-Holtzapple,‡ and Ross C. Beier† Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 2881 F&B Road, College Station, Texas 77845-9594, and Dairy Cattle Science Center, Texas A&M University, College Station, Texas 77843
Furosemide is a potent diuretic drug used in both human and veterinary medicine. Highperformance 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. Keywords: Furosemide; immunoassay; residue detection; milk INTRODUCTION
The use of immunoassays as analytical tools to measure chemical residues in foods and environmental samples has increased greatly over the past several years. Recent reviews discuss the role of immunoassay in analytical chemistry and provide numerous examples of their application and ability to expand sampling capabilities (Hammock and Gee, 1995; Dixon-Holland, 1992; Morgan et al., 1992; Samarajeewa, 1990; Stanker, 1994). We previously reported on the development of a series of monoclonal antibodies to furosemide (Carlin et al., 1993). Furosemide is a potent diuretic used in both human and veterinary medicine for treatment of edema and ascites. Specifically, in veterinary medicine furosemide is used to treat dogs, cats, and horses for edema and it is used in cattle for the treatment of physiological parturient edema of the mammary gland (Darling, 1993). A 48 h withdrawal period is imposed both for milk taken from cows following the last treatment of furosemide (four milkings), and for cattle used for food (Code of Federal Regulations, 1994). High-performance liquid chromatography (HPLC) methods are commonly used for analysis of furosemide (Farthing et al., 1992; Singh et al., 1990; Voyksner et al., 1990; Singh et al., 1989; Uchino et al., 1984; Swezley et al., 1979). In addition to HPLC methods, immunoassays for detecting furosemide also have been reported. In an early study, Woods et al. (1988) used a polyclonal anti-furosemide antibody in a competition enzymelinked immunosorbent assay (cELISA) that was able to * Author to whom correspondence should be addressed [telephone (409) 260-9306; fax (409) 260-9377; e-mail
[email protected]]. † U.S. Department of Agriculture. ‡ Texas A&M University. S0021-8561(95)00362-1 CCC: $12.00
detect 20 ng/mL furosemide in equine blood. Tobin et al. (1988) reviewed the use of nonisotopic immunoassays for drug testing in horses. Recently, Singh et al. (1990) evaluated the performance of three methods for detecting and quantifying furosemide in equine serum and plasma: an HPLC method, a commercial ELISA testkit method, and a particle concentration fluorescence immunoassay kit (PCFIA). These authors concluded that the ELISA and PCFIA “lacked quantitative reproducibility” and were not suitable for quantitation of furosemide in equine plasma or for studying the pharmacokinetics of furosemide. In our initial report we described four monoclonal antibodies to furosemide, and the cross-reactivity of these antibodies was related to energy-minimized threedimensional molecular models of furosemide and a variety of other related compounds (Carlin et al., 1993). The antibodies were highly specific for furosemide. For example, monoclonal antibody FURO-73 was tested for binding to nine related compounds. Significant inhibition of antibody binding was observed only with furosemide (IC50 ) 0.02 ng/mL). This antibody weakly bound two other compounds, metolazone and furfuryl benzoate, with a cross reactivity of 0.05 and
