Fluorimetric determination of indomethacin in human serum by high-performance liquid chromatography coupled with post-column photochemical reaction with hydrogen peroxide.

A high-performance liquid chromatographic method involving post-column photochemical reaction and fluorimetric detection has been developed for the determination of indomethacin in serum. The bioanalytical set-up consisted of single-pump system for post-column photochemical reaction. The mobile phase consisted of 0.07 M phosphate buffer (pH 6.6)-acetonitrile (65:35, v/v) containing 180 mM hydrogen peroxide. The column effluent was irradiated with UV light to give fluorescence. The fluorimetric detection was monitored with excitation at 358 nm and emission at 462 nm. The calibration curve was linear over the range 0.05-30 micrograms/ml by injecting a volume of 10 microliters of indomethacin solution. The detection limit (signal-to-noise ratio = 5) for indomethacin was 10 ng/ml using a 100-microliters aliquot of deproteinized serum. The mean recovery from serum was 94.3%.