The Use of Bamifylline as Internal Standard in the Reversed Phase HPLC Analysis of Mefenamic Acid in Pharmaceuticals and Small Volumes of Biological Fluids

The reversed-phase liquid chromatographic determination of mefenamic acid in pharmaceutical preparations and small volumes of biological fluids, blood serum (40 μl) and urine (100 μl) is developed. The biological fluids containing internal standard passed through C 18 cartridges, Bond Elut, for sample clean-up and subsequent separation of bamifylline and mefenamic acid from endogenous interfering compounds. HPLC analyses are performed with a Lichrosorb-RP 18, 10 μm, 250x4 mm I.D. column, employing gradient elution for the development of the chromatogram. The gradient system is formed with the following solutions: A = 0.05 M ammonium acetate and B = methanol. The chromatogram is started with solvent A containing 78% solvent B, followed by a sharp decrease from 78 to 72% in 5 min. The flow rate is 0.81 ml/min and the detection is achieved at 285 nm. The retention time is 3.90 min for mefenamic acid and 4.93 min for the internal standard, bamifylline. Thus the problems existing with caffeine in blood and urine samples from patients are overcomed The absolute detection limit is 1 pg while the quantitation limit is 1.36 ng and linearity is observed up to 120 ng. The method outlined in this paper is simple, rapid, sensitive, accurate and can be applied to tbe determination of mefenamic acid in pharmaceutical preparations (tablets, suppositories and suspensions) and biological fluids (blood serum and urine). Quantitation limits in blood serum and urine samles, were found to be 0.45 ng and 0.26 ng respectively, due to preconcentration on solid-phase cartridges. The proposed method can be readily utilised for clinical pharmacokinetic studies.