High-performance liquid chromatography-fluorescence analysis for indomethacin and metabolites in biological fluids

A rapid and sensitive high-performance liquid chromatography-fluorescence method is described for the quantitative analysis of indomethacin and its metabolites in urine. A modified version is also shown for the detection of indomethacin in plasma. The method consists of a single extraction of an acid-buffered plasma sample or single extractions of two buffered aliquots in parallel of urine using ethyl acetate, followed by evaporation of the organic phase. Indomethacin (I) and the metabolite desmethylindomethacin (DMI) were deacylated to their fluorescent products, deschlorobenzoylindomethacin (DBI) and desmethyldeschlorobenzoylindomethacin (DMBI), respectively, prior to chromatography. The chromatographic phase utilized a reversed-phase C18-bonded column with a solvent system comprised of either 22.5% or 26% acetonitrile in 0.25% acetic acid. The elution times for indomethacin metabolites ranged from 12-26 min. The total DBI (including deacylated I) and DMBI (including deacylated DMI) in the extract were each determined using fluorometric detection, with excitation at 288 nm and emission at 390 nm (370 nm cutoff filter). An internal standard of indole-3-propionic acid was used for quantitation. The lower limit of sensitivity for I in plasma was 25 ng/ml.