Simultaneous determination of flurbiprofen and its hydroxy metabolite in human plasma by liquid chromatography-tandem mass spectrometry for clinical application

Flurbiprofen (FLB) is one of the phenylalkanoic acid derivatives of non-steroidal anti-inflammatory drugs used for the management of pain and inflammation in patients with arthritis. We developed and validated a rapid and sensitive high-performance liquid chromatography analytical method utilizing tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of FLB and its major metabolite, 4′-hydroxyflurbiprofen (4′-OH-FLB), in human plasma. Probenecid was used as an internal standard (IS). After liquid-liquid extraction with methyl t-butyl ether, chromatographic separation of the two analytes was achieved using a reversed-phase Luna C18 column (2.0 mm × 50 mm, 5 μm particles) with a mobile phase of 10 mM ammonium formate buffer (pH 3.5)-methanol (15:85, v/v) and quantified by MS/MS detection in ESI negative ion mode. The flow rate of the mobile phase was 250 μl/min and the retention times of FLB, 4′-OH-FLB, and IS were 1.1, 0.8, and 0.9 min, respectively. The calibration curves were linear over a range of 0.01-10 μg/ml for FLB and 0.01-1 μg/ml for 4′-OH-FLB. The lower limit of quantifications using 100 μl of human plasma was 0.01 μg/ml for both analytes. The mean accuracy and precision for intra- and inter-run validation of FLB and 4′-OH-FLB were all within acceptable limits. The present HPLC-MS/MS method showed improved sensitivity for quantification of the FLB and its major metabolite in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.