Plasma indomethacin assay by reversed-phase ion pair high pressure liquid chromatography

A new rapid, selective and sensitive high pressure liquid chromatographic (HPLC) assay for indomethacin in plasma is described. The method involves precipitation of proteins with perchloric acid, followed by dichloromethane extraction using flurbiprofen as an internal standard. The organic solvent was evaporated and the residue dissolved in a water-methanol (2 + 3) phosphate buffer mixture with an apparent pH of 6.8. Aliquots of 100 microliters were injected automatically into the chromatograph. The separation of indomethacin was achieved on a reversed phase (C18, 10 micron) column with a mobile phase consisting of 65% (vol/vol) methanol in water solution of apparent pH 6.8 containing tetrabutylammonium hydrogensulfate as an ion pairing agent. Quantitation of indomethacin was performed by UV detection at 235 nm. At a 2.0 mg X l-1 concentration of indomethacin in plasma the analytical recovery was 82.9 +/- 3.4% (n = 7), the intra-day variability (CV) was 3.6% (n = 7) and the inter-day variability (CV) was 11.0% (n = 7). The calibration curve was linear (typical r-values greater than 0.990) in the range of plasma concentrations as usually found during indomethacin therapy (up to 6 mg X l-1). The limit of sensitivity is 0.025 mg X l-1. The metabolites O-desmethylindomethacin and O-desmethyldeschlorobenzoylindomethacin did not interfere with the method. The capacity of an analyst using this method with automated injection and peak integration is about forty samples in duplicate per day. The applicability of the method for pharmacokinetic studies is demonstrated.