High-throughput liquid chromatographic determination of rofecoxib in human plasma using a fully automated on-line solid-phase extraction system

A fully automated on-line solid-phase extraction method combined with high-performance liquid chromatography-fluorescence detection for the determination of rofecoxib in human plasma is described. A Spark Holland PROSPEKT system was used to perform all the automated on-line sample extraction and quantitation tasks, including sample loading, cartridge conditioning and washing, analyte elution, and start of a data acquisition system. Under postcolumn ultraviolet-irradiation conditions, the analytes were photochemically converted to highly fluorescent products and then monitored by a fluorescence detector. The assay was validated in the concentration range of 0.5-100 ng/mL using 0.25 mL of human plasma. The utility of this method was demonstrated by analysis of plasma samples from human subjects receiving oral doses of rofecoxib.