High performance liquid chromatography for quantification of gatifloxacin in rat plasma following automated on-line solid phase extraction

An automated system using on-line solid phase extraction and HPLC with fluorimetric detection was developed and validated for quantification of gatifloxacin in rat plasma. The extraction was carried out using C18 cartridges (BondElut), with a high extraction yield. After washing, gatifloxacin was eluted from the cartridge with mobile phase onto a C18 HPLC column. The mobile phase consisted of a mixture of phosphoric acid (2.5 mM), methanol, acetonitrile and triethylamine (64.8:15:20:0.2, v/v/v/v, apparent pHapp. 2.8). All samples and standard solutions were chromatographed at 28 °C. The method developed was selective and linear for drug concentrations ranging between 20 and 600 ng/ml. Gatifloxacin recovery ranged from 95.6 to 99.7%, and the limit of quantification was 20 ng/ml. The intra and inter-assay accuracy were up to 94.3%. The precision determined not exceed 5.8% of the CV. High extraction yield up to 95% was obtained. Drug stability in plasma was shown in freezer at −20 °C up to 1 month, after three freeze-thaw cycles and for 24 h in the autosampler after processing. The assay has been successfully applied to measure gatifloxacin plasma concentrations in pharmacokinetic study in rats.