HPLC analysis of ciprofloxacin and ciprofloxacin metabolites in body fluids.

An improved high-performance liquid chromatography (HPLC) procedure for the analysis of ciprofloxacin and three of its metabolites in plasma, serum and urine samples was developed. The previously published HPLC procedure described the isocratic separation of ciprofloxacin and three ciprofloxacin metabolites in urine samples on a polystyrene-divinylbenzene reverse-phase column followed by quantitation using a UV detector. The present procedure involved the same chromatographic separation, but is also applicable to the analysis of plasma and serum as well as urine samples, and quantitation was based on fluorometric detection after postcolumn induction of fluorescence instead of UV detection. The post-column induction of fluorescence was necessary because the M2 and M3 metabolites of ciprofloxacin have relatively weak native fluorescence, and induction enhanced the fluorometric signals of metabolites M2 and M3 forty-four-fold and eleven-fold, respectively. The observed enhancement of fluorescence may be attributed to the partial conversion by UV light of metabolites M2 and M3 to metabolite M1 which has intense native fluorescence. The lower quantitation limits of ciprofloxacin and metabolites M1, M2 and M3 were 0.05 micrograms ml-1, 0.01 micrograms ml-1, 0.05 micrograms ml-1, and 0.5 micrograms ml-1, respectively. All four analytes were quantitated using one isocratic elution of either plasma or serum supernatant after the precipitation of proteins or the isocratic chromatography of diluted urine samples.