DETERMINATION OF FENBUFEN AND ITS METABOLITES IN SERUM BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING SOLID-PHASE EXTRACTION AND ONLINE POSTCOLUMN ULTRAVIOLET-IRRADIATION AND FLUORESCENCE DETECTION

An improved analytical method for the detection and quantification of fenbufen and its two major metabolites is described. The assay consist s of reversed-phase high-performance liquid chromatography and post-co lumn irradiation with ultraviolet light and fluorescence detection. A highly selective chromatography separation was established on a cyanop ropyl column at ambient temperature with a flow-rate of 0.5 ml/min. Th e analytes of interest were isolated from serum using a Bond-Elut C-18 column with high recovery and selectivity, The fluorescence response of all three analytes upon UV irradiation was investigated. The post-c olumn UV irradiation was optimized and the effect of irradiation time on the fluorescence response was determined for all three analytes. Th e detection limits were 10 ng/ml for each analyte using 1 ml of serum, Linear calibration curves from 50 to 375 ng/ml for all three analytes show coefficients of determination of 0.99. Precision and accuracy of the method were within 3.9-6.5 and 5.1-7.4% for fenbufen, 3.5-6.4 and 4.9-6.3% for metabolite II (expressed as lactone III) and 5.4-7.4 and 2.6-7.4% for metabolite IV, respectively.