High-performance liquid chromatography separation and quantitation of ofloxacin enantiomers in rat microsomes.

A sensitive, simple and accurate method for determination of enantiomers of ofloxacin in microsomal incubates was developed by chiral ligand-exchange RP-HPLC with fluorescence detection to examine stereoselective metabolism of ofloxacin in the glucuronidation process. The C18 stationary phase was used as analytical column. The solution of chiral mobile phase additive was made up of 6 mM L-phenylalamine mixed with 3 mM CuSO4 in water. Mobile phase consisted of the solution of chiral mobile phase additive-methanol (86:14). The fluorescence detector was operated at lambda(ex) 330 nm and lambda(em) 505 nm. The flow-rate of mobile phase was set at 1.0 ml/min. The achiral ODS column offers good separation of the two enantiomers in less than 25 min. The recovery of the assay was 97.9+/-6.1% (n=10) for S-ofloxacin and 99.6+/-6.0% (n = 10) for R-ofloxacin. The method provides a high sensitivity and good precision (RSD<10%). The LOD was 0.6 microM for both enantiomers and the LOQ was 5.70+/-0.45 microM (n=8) for S-ofloxacin and 5.66+/-0.47 microM (n=8) for R-ofloxacin. The standard curves showed excellent linearity over the concentration range 5.5-2078 microM for S-(-)-ofloxacin and R-(+)-ofloxacin. The enantioselective method developed has been applied to determine the stereoselectivity of glucuronidation metabolism of ofloxacin optical isomers in rat liver microsomes.