[Quantitative determination of furosemide in serum and urine by high-performance liquid chromatography with...

A high-performance liquid chromatographic method using electrochemical detection is described for the determination of furosemide in serum and urine. Serum proteins were precipitated with acetonitrile containing the internal standard and the clear supernatant was separated. Urine was diluted with water to 50 times. Furosemide was chromatographed on a reversed-phase column, 8MBC18 column, using 35% ethanol solution containing 5 mmol/L tetrabutylammonium phosphate (pH 7.50) as the mobile phase (1.0 ml/min) and detected at 0.90 volts. FD-Val-OH synthesized was used as internal standard in the furosemide assay. The retention times for furosemide and internal standard were 10 min and 15 min, respectively. The amount of furosemide was determined by measuring peak height ratio. The detection limit of furosemide was 16 and 9 ng per ml serum and urine respectively. The calibration curve was linear in the range from 0.25 ng/microliters to 5 ng/microliters (serum) and from 0.5 ng/microliters to 10 ng/microliters (urine). The recovery of furosemide was 100.5% (serum) and 100.6% (urine). Coefficient of variation was less than 4.6%.