Method Validation for the Determination of Furosemide in Plasma by Liquid- Liquid Extraction and High-Performance Liquid Chromatography With Fluorescence Detection

A practical reversed-phase high-performance liquid chromatography (HPLC) method was developed for 4-chloro-N-furfuryl-5-sulphamoylanthranilic 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 mg/mL. The detection limit was 0.001mg/mL and the quantification limit 0.003 mg/mL in plasma. The furosemide recovery from 1mL 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