Development and validation of a liquid chromatography-tandem mass spectrometry method for quantitation of indomethacin in rat plasma and its application to a pharmacokinetic study in rats.

A highly sensitive and rapid bioanalytical method has been developed and validated for the estimation of indomethacin in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves a simple liquid-liquid extraction of indomethacin and phenacetin (internal standard, IS) from rat plasma with acetonitrile. Chromatographic separation was achieved with 0.2% formic acid-acetonitrile (25:75, v/v) at a flow rate of 0.60 mL/min on an Atlantis dC18 column with a total run time 3.0 min. The MS/MS ion transitions monitored were 357.7 → 139.1 for indomethacin and 180.20 → 110.10 for IS. Method validation and pharmacokinetic study plasma analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.51 ng/mL and the linearity was observed from 0.51 to 25.5 ng/mL. The intra- and inter-day precisions were in the range of 1.00-10.2 and 5.88-9.80%, respectively. This novel method has been applied to an oral pharmacokinetic study in rats.