Simple microscale high-performance liquid chromatographic method for determination of furosemide in neonatal plasma

A simple microscale high-performance liquid chromatographic method using fluorescence detection for the quantitation of furosemide in neonatal plasma is described. Sample pre-treatment involved protein precipitation of 25 microliters of plasma with 100 microliters of acetonitrile. The mobile phase consisted of acetonitrile (460 ml) and 0.08 M orthophosphoric acid (540 ml) and was delivered at 1.1 ml/min. Calibration curves were linear from 0.1 to 25 micrograms/ml. Within-day and between-day imprecision (coefficient of variation) was 3.9-6.1, and 6.1-12.2%, respectively. Furosemide was eluted after 6.5 min and naproxen (internal standard) after 11.5 min. The assay was validated for application in neonatal plasma containing a wide range of albumin concentrations.