DEVELOPMENT AND VALIDATION OF A LIQUID-CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF FUROSEMIDE, A DIURETIC, IN BOVINE-MILK

A rapid and sensitive reversed phase liquid chromatographic (LC) procedure was developed and validated for the quantitation of furosemide, a diuretic, in bovine milk. Whole milk was defatted by initial centrifugation at room temperature. The resulting skim milk was deproteinated with acetonitrile and centrifuged again. The acetonitrile from the supernatant was evaporated, and the remaining aqueous portion was directly analyzed by LC. The LC conditions employed include a Spherisorb 5 ODS 2 column, a phosphate/acetonitrile buffer (pH 3), and a fluorescence detector set at 272 and 410 nm excitation and emission wavelengths, respectively. The average recoveries of furosemide from milk fortified at 5, 10, and 20 ppb were 108, 91, and 85%, respectively, with corresponding CVs of 14, 8, and 6%. The method was validated by assaying milk obtained from a cow dosed intravenously with 500 mg of furosemide. The furosemide concentrations in 8 and 24 h milk samples were determined to be about 150 and 5 ppb, respectively. No furosemide was detected in 32 and 48 h samples.