Determination of fenofibric acid in human plasma using automated solid-phase extraction coupled to liquid chromatography

The pharmacokinetic studies of fenofibrate require a rapid, selective and robust method to allow the determination of fenofibric acid, its active metabolite, in different biological matrixes (such as plasma, serum or urine). A new fully automated method for the determination of fenofibric acid in plasma has been developed, which involves the solid-phase extraction (SPE) of the analyte from plasma on disposable extraction cartridges (DECs) and reversed-phase HPLC with UV detection. The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with octadecyl silica was first conditioned with methanol and pH 7.4 phosphate buffer. A 0.8-ml volume of diluted plasma sample containing the internal standard (sulindac) was then applied on the DEC. The washing step was performed with the same buffer (pH 7.4). Finally, the analytes were successively eluted with methanol (1.0 ml) and 0.04 M phosphoric acid (1.0 ml). After a mixing step, 100 μl of the resultant extract was directly introduced into the HPLC system. The liquid chromatographic (LC) separation of the analytes was achieved on a Nucleosil RP-8 stationary phase (5 μm). The mobile phase consisted of a mixture of methanol and 0.04 M phosphoric acid (60:40, v/v). The analyte was monitored photometrically at 288 nm. The method developed was validated. In these conditions, the absolute recovery of fenofibric acid was close to 100% and a linear calibration curve was obtained in the concentration range from 0.25 to 20 μg/ml. The mean RSD values for repeatability and intermediate precision were 1.7 and 3.9% for fenofibric acid. The method developed was successfully used to investigate the bioequivalence between a micronized fenofibrate capsule formulation and a fenofibrate Lidose™ formulation.