High performance liquid chromatographic separation and determination of ciprofloxacin and its metabolites in urine

An improved HPLC procedure for separating ciprofloxacin and its four metabo lites, M1, M2, M3, M4 in urine, was developed. The procedure used is revers ed phase chromatography, the stationary phase being the C-18-column, follow ed by quantification using a UV detector. The mobile phase is a mixture of isopropanol, acetonitrile, tetrabutylammonium bromide (TBABr), heptanesulfo nic acid (HSA), and 0.05% triethylamine, (adjusted to pH=3.0 with phosphori c acid.) In the process of determining the optimum separation condition, th e effect of each ion-pairing reagent on the retention of all analytes was i nvestigated and explained as well. This method involves an off-line solid p hase extraction, using C-18 Sep-Pak cartridges for ciprofloxacin and its me tabolites in urine. The desorption condition is investigated by using diffe rent kinds of organic solvents, or a mixture of organic solvents and phosph ate buffer, adjusted to different values of pH. At the optimum desorption c ondition, all analytes show a recovery rate over 90%. Based on this optimum condition, this method was applied to pharmacokinetic study of solutes in urine sample.