Furosemide Concentrations in Serum and Urine, and Its Binding by Serum Proteins as Measured Fluorometrically

Abstract

Abstract We describe a modification of the fluorometric method of Häussler and Hajdú [Arzneim. Forsch. 14, 704 and 709 (1964)] for assay of furosemide in either serum or urine. A 1-ml sample, acidified to pH 2, is extracted with 5 ml of diethyl ether; 4 ml of the ether is back-extracted into 1 ml of phosphate buffer (pH 7.0, 0.1 mol/liter), and finally acidified with 1 ml of dilute HCl (0.6 mol/liter). A procedure for estimating blanks in urine was derived to correct for dilution caused by diuresis. Internal standards are used, and the "effective" extraction ratio is used to correct for the effects of quenching and extraction differences. In equilibrium, 93% of the drug is bound to serum proteins; 65% is tightly bound. Erythrocytes contain less than 5% of the drug. Quantum yield of fluorescence at pH 1 is 0.0496 for furosemide and is 0.0163 for 4-chloro-5-sulfamoylanthranilic acid. Furosemide fluorescence diminishes with increasing pH, while that of 4-chloro-5-sulfamoylanthranilic acid (a degradation product) increases.