Fast HPLC method for the determination of ketoprofen in human plasma using a monolithic column and its application to...

A fast, specific, accurate, precise and reproducible high-performance liquid chromatography (HPLC) method with diode-array detector (DAD) was developed and validated for the determination of ketoprofen (CAS 22071-15-4) in human plasma using flubiprofen (CAS 5104-49-4) as an internal standard. The chromatographic separation was achieved on an onyx monolithic C18 (100 x 4.6 mm) analytical column with an isocratic mobile phase consisting of acetonitrile/potassium dihydrogen phosphate (KH2PO4) 0.01 M, (40:60, v/v) adjusted to pH 3.5. The flow was set at 5 ml x min(-1) and the wavelength at 254 nm. The total analysis time was less than 5 min. The ratio of peak area of analyte to internal standard was used for quantification. The limit of detection was defined as the ketoprofen concentration that produced a signal-to noise ratio greater than 3. The lower limit of quantification (LLOQ) was 10 ng x m(-1). At this level, the relative standard deviation (RSD) was lower than 13%. The calibration curve was linear over the concentration range 1-500 ng x ml(-1) with a minimum detectable limit of 10 ng x ml(-1). The coefficients of variation for the inter-day and intra-day assay were found to be less than 11%. The present method was successfully applied to the routine analysis of human plasma samples collected from healthy volunteers after dermal application of two topical formulations containing ketoprofen in order to assess the relative bioavailability and to demonstrate that the systemic bioavailability of ketoprofen administered topically is low enough to ensure a low incidence of gastrointestinal adverse events.