High-performance liquid chromatography of ciprofloxacin and its metabolites in serum, urine and sputum.

A versatile and sensitive method requiring no internal standard was developed for quantitating ciprofloxacin in serum, urine and sputum by high-performance liquid chromatography with fluorescence detection. Acetonitrile and chloroform were employed to remove protein and lipophilic substances from an aqueous, ciprofloxacin-containing sample layer. The proportions of acetonitrile and 0.1 M potassium phosphate, pH 2.5, in the mobile phase were varied to suit the purpose of the assay. For the routine determination of ciprofloxacin pharmacokinetics, isocratic 19% acetonitrile was used. A gradient from 15 to 35% acetonitrile was chosen to show the appearance of metabolites which formed during the biodisposition of ciprofloxacin. In the latter case urine samples were diluted for assay and protein was precipitated from serum samples with trichloroacetic acid. Four fluorescent metabolites were observed in all patient specimens, and with tandem ultraviolet detection two additional ultraviolet-absorbing metabolites were readily found in urine specimens.