Determination of Celecoxib, Rofecoxib, Sodium Diclofenac and Niflumic Acid in Human Serum Samples by HPLC with DAD Detection

A reverse-phase high-performance liquid chromatographic method has been developed for the separation and simultaneous determination of two COX-2 inhibitors, celecoxib and rofecoxib, in addition to two well-known non-steroidal anti-inflammatory drugs (NSAIDs), sodium diclofenac and niflumic acid in human serum samples. Good chromatographic separation was achieved using a C18 bonded silica column applying a gradient with acetronitrile and water, from 15 to 60% acetonitrile. The mobile phase contained 0.1% trifluoroacetic acid as an organic modifier. Detection was made using a diode array detector (DAD) and the analytical parameters were established at the wavelength maximum in the UV spectrum of each drug. Linearity was studied up to 100.0 mg L⁻¹. Calibration functions, quantification and detection limits, intra- and inter-day reproducibility and accuracy were estimated for each drug. Solid phase extraction was needed to separate and concentrate the drugs from human serum samples. The method was successfully applied to determine the drugs in human serum samples at levels of 1.0 mg L⁻¹.