An Improved HPLC Method for the Determination of Furosemide in Plasma and Urine

Abstract

Abstract A sensitive HPLC method with minimal sample preparation and good reproducibility for the determination of furosemide in plasma and urine is described. Acidified plasma samples were extracted using CH2Cl2 containing desmethylnaproxen as internal standard (IS). Fresh urine samples were incubated with β-gluc-uronidase for 15 minutes and then treated with CH3CN containing IS. Chromatography was performed on a C18 column with 10 mcl sample injection, Mobile phases were: a) for plasma: 0.01 M NaH2PO4, pH 3.5 - CH3OH (65:35), and b) for urine: acetic acid, pH 3.5 - CHS3OH (60:40) at 3 ml/min and fluorescence detection at Ex 235/Em 389 nm. The plasma standard curve was linear from 0.01 to 15.0 mcg/ml and the urine from 0.5 to 200.0 mcg/ml. The within run CV's were 3,2% at 0.74 mcg/ml plasma and 2.0% at 10.7 mcg/ml urine. Recovery from plasma was 69.9% at 2.0 mcg/ml and 98.6% from urine at 5.0 mcg/ml. The stability of furosemide and its glucuronide were studied. Both methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.