Development and Validation of an Improved LC Method for the Simultaneous Determination of Pirfenidone and Its Carboxylic Acid Metabolite in Human Plasma

A sensitive and specific assay based on liquid chromatography with ultraviolet detection was developed for the simultaneous determination of pirfenidone (PFD), a novel antifibrotic agent, and its carboxylic acid metabolite in human plasma. The carboxylic acid metabolite was further identified by mass spectrometric analysis. PFD, its carboxylic acid metabolite and the internal standard methyl-p-aminobenzoate were extracted from plasma by a simple one-step liquid-liquid extraction with ethyl acetate and subsequently separated on a Zorbax SB-C18 column with a mobile phase of trifluoroacetic acid-triethylamine-acetonitrile-water (0.1:0.15:28:71.75, v/v/v/v) and monitored at 314 nm. Extraction recovery was over 70% in plasma. The calibration curves were linear over the concentration range of 0.05-25 mu g mL(-1). The limit of detection (LOD) and lower limit of quantitation (LLOQ) in human plasma were 10 and 50 ng mL(-1), respectively. Intra- and inter-assay precision of the method were within 8.6%. The accuracy as expressed by the bias ranged between -4.5 and 4.0%. The method was successfully applied to determine pharmacokinetic parameters of PFD and its carboxylic acid metabolite after a single oral dose of 200 mg of PFD in healthy volunteers.