Sensitive and specific high-performance liquid chromatographic assay for 4′-hydroxyflurbiprofen and flurbiprofen in human urine and plasma

A high-performance liquid chromatographic assay has been developed for the simultaneous quantitation of flurbiprofen and its major metabolite, 4′-hydroxyflurbiprofen, in urine and plasma. No extraction procedure was necessary for analysis of these compounds, which reduced time involved in sample preparation. The analytes were separated on a Brownlee Spheri-5® C18 column with a mobile phase of acetonitrile–20 mM dibasic potassium phosphate pH 3 buffer (40:60, v/v). Fluorescence detection was utilized with an excitation wavelength of 260 nm and an emission wavelength of 320 nm, providing excellent sensitivity. The limit of quantitation for 4′-hydroxyflurbiprofen and flurbiprofen was 0.25 μg/ml in urine and 0.05 μg/ml and 0.25 μg/ml, respectively, in plasma. All components were eluted within 16 min. Intra-day, inter-day, freeze–thaw, and in process stability were tested for both compounds and the coefficient of variation was less than 14% in all cases. This method provides a sensitive and specific assay for the detection of flurbiprofen and 4′-hydoxyflurbiprofen in urine and plasma and is suitable for use in in vivo studies evaluating the regulation of CYP2C9 activity.