Determination of Tolfenamic Acid in Human Plasma by HPLC

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Abstract
Tolfenamic acid is a potent prostaglandin synthetase inhibitor used clinically as non-steroidal anti-inflammatory and analgesic-antipyretic agent. A simple, sensitive, accurate, and precise reverse phase high performance liquid chromatographic method has been developed and validated for the quantitative determination of tolfenamic acid in small volumes of human plasma. The chromatographic separation of tolfenamic acid and the internal standard (phenylbutazone) was performed on a reversed phase, 5-μm C18 column (250 × 4 mm) using acetonitrile-10 mM phosphoric acid (60:40, v/v) as mobile phase with a flow rate of 1.1 ml/min and the chromatographic peaks were detected at 280 nm. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. Calibration curves were linear in the range 0.2–5.0 μg/ml with a squared correlation coefficient (r) of 0.999 or better and the detection limit was 50 ng/ml for 100-μl plasma samples. The method was not interfered with by other endogenous compounds or metabolites and one assay can be completed in 12 min. The within-day and between-day assay variation for three different concentrations was found to be less than 6% and the accuracy was nearly 100%.