Quantitation of norfloxacin, a new antibacterial agent in human plasma and urine by ion-pair reverse-phase chromatography

A specific and sensitive high-performance liquid chromatographic method for the analysis of norfloxacin in human plasma and urine is described. Norfloxacin was extracted from the sample matrix using dichloromethane under neutral conditions, followed by back extraction into dilute phosphoric acid for chromatographic analysis on a reverse-phase column with a mobile phase consisting of methanol, phosphate buffer, and ion-pairing reagent (pH 3.0) at a flow rate of 2.0 mL/min. The ability of this method to distinguish intact norfloxacin from its metabolites was demonstrated. The method is linear, quantitative, and reproducible for both plasma analysis (0.05-2.5 microgram/mL) and urinalysis (1.0-500 micrograms/mL) using peak area ratios (norfloxacin-internal standard) for quantitation. The stability of norfloxacin and its metabolites in dilute phosphoric acid was studied. To assess the presence of norfloxacin conjugates in the urine of dosed individuals, the effects of urine hydrolysis on drug quantitation were examined. Urine and plasma levels of norfloxacin at selected time points following the administration of single drug doses are presented.