Determination of ofloxacin in human aqueous humour by high-performance liquid chromatography with fluorescence detection

A reversed-phase high-performance liquid chromatographic method is described for the determination of ofloxacin in human aqueous humour; the method involves fluroescence detection (excitation at 290 nm; emission at 500 nm) after direct injection of samples. The method utilized a 100 mm x 8 mm i.d, cartridge column packed with 4 mu m Novapak C-18 with a mobile phase methanol-acetonitrile-0.4 M citric acid (3:1:10, v/v/v) and a flow rate of 1 mi min(-1) at ambient temperature. The retention rimes for the internal standard pipemidic acid and for ofloxacin were 4.82 and 7.32 min respectively. The mean recovery (+/- ISD) from human aqueous humour was 103.24 +/- 4.45% for ofloxacin at 1 mu g ml(-1) (n = 6). The within-day and day-to-day RSDs at 0.1 mu g ml(-1) and 1 mu g ml(-1) were less than 6.71% (n = 6) and the lower limit of reliable determination corresponding to a signal-to-noise ratio of 2.5:1 was 20 ng ml(-1). The assay was shown to be suitable for measuring ofloxacin levels in human aqueous humour samples after topical, oral and intravenous administration.