Determination of pipemidic acid in plasma by normal-phase high-pressure liquid chromatography

An improved high-pressure liquid chromatography procedure for determining the concentration of pipemidic acid in human plasma was developed, which uses a normal-phase silica gel column and aqueous mobile solvent system similar to that used in reverse-phase chromatography. Precolumn derivatization of pipemidic acid was achieved by a methylation reaction with boron trifluoride in methanol after extraction from plasma with chloroform containing 4% ethanol. The percent recovery of pipemidic acid was 88.3 +/- 7.7, the variation of which became negligible when quinacrine was used as an internal standard for the determination. High-pressure liquid chromatography analysis was performed by conventional silica gel and mobile solvent mixtures containing 3% of 0.14% HClO4 solution, 19% methanol, and 78% chloroform. The UV detector was set at 265 nm. The detection limit of pipemidic acid methyl ester was as low as 10 ng of the injection amounts or 0.5 micrograms of the plasma per ml, with 0.01 absorbance units (full scale) and a signal-to-noise ratio of 3.