Determination of ethambutol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

A sensitive and selective HPLC method for the determination of ethambutol in human plasma and urine was developed. Ethambutol was extracted from basified plasma samples (0.2 ml) with diethyl ether, back-extracted into 0.01 M phosphoric acid and derivatized with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole. After 30 min at 80 degrees C and elimination of the reactive excess, the compound was determined by reversed-phase liquid chromatography. Urine was analysed for ethambutol after dilution 1:200 with distilled water and derivatization as described for plasma. Quantification in plasma and urine was achieved by fluorescence detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human plasma and urine was observed. The limit of quantification was 10 ng/ml in plasma and 10 mu g/ml in urine. The suitability of the method for in vivo samples was checked by analysis of plasma and urine samples drawn from healthy volunteers who had received a 1200-mg oral dose of the test compound.