Determination of fenbufen and its metabolites in serum by reversed-phase high-performance liquid chromatography using solid-phase extraction and on-line post-column 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 consists of reversed-phase high-performance liquid chromatography and post-column irradiation with ultraviolet light and fluorescence detection. A highly selective chromatography separation was established on a cyanopropyl column at ambient temperature with a flow-rate of 0.5 ml/min. The analytes of interest were isolated from serum using a Bond-Elut CIN column with high recovery and selectivity. The fluorescence response of all three analytes upon UV irradiation was investigated. The post-column UV irradiation was optimized and the effect of irradiation time on the fluorescence response was determined for all three analytes. The 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.