Fluorometric determination of indomethacin in serum by high performance liquid chromatography with in-line alkaline hydrolysis

Summary A fluorometric method for determining indomethacin in serum by reversed-phase high performance liquid chromatography has been developed. Deproteinized serum containing indomethacin was injected directly onto a C18-bonded vinyl alcohol copolymer column with an alkaline mobile phase (pH 10.0, 35% acetonitrile in phosphate buffer), and was detected fluorometrically (Ex. 298 nm and Em. 375 nm) via postcolumn in-line alkaline hydrolysis at high temperature (140°C). The calibration curve was linear over the range of 0.1–10.0 g/ml when injecting a volume of 10 l of deproteinized serum. The detection limit (signal-to-noise ratio=3) for indomethacin in serum was 10 ng/ml using a 20-l aliquot of deproteinized serum.