Method validation for the determination of furosemide in plasma by liquid-liquid extraction and high-performance liquid chromatography with fluorescence detection

A practical re versed-phase high-perforinance liquid chromatography (HPLC) method was developed for 4-chloro-N-furfuryl-5-sulphamoylanthramlic acid (furosemide) determination in plasma. The plasma samples spiked with the drug in presence of propranolol hydrochloride as internal standard. were purified using a liquid-liquid extraction technique. The drugs were separated through a Kromasil 100-5C18 column with a mobile phase composed of acetonitrile and 0.02 M potassium dihydrogen phosphate (34 : 66 v/v) adjusted to pH 3.0 and quantified by fluorescence detection (excitation at 268 nm, emission at 410 nm). The calibration Curve was linear ranging between 0.005 and 1.500 mu g/mL. The detection limit was 0.001 mu g/mL and the quantification limit 0.003 mu g/mL in plasma. The furosemide recovery from 1 mL of plasma was between 99.8 and 101.6%. with a variation coefficient not higher than 4,85%. The method is sensitive enough for use in bioavailability and bioequivalence Studies after oral administration of 40 mg (a tablet) of furosemide.