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

Abstract

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% HCl04 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 ,ug of the plasma per ml, with 0.01 absorbance units (full scale) and a signal-to-noise ratio of 3.In the past decade, pipemidic acid (4) has been found to possess higher antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, and Enterobacter and Citrobacter organisms (5, 12-14) than structurally similar agents, such as piromidic (11) and nalidixic acid (3), and thus has been applied to chemotherapy of parenchymatous urinary tract infections. The known chronic and acute toxicities of pipemidic acid are rather low (6-9); however, numerous pharmacokinetic studies have been conducted to determine the most effective concentrations in serum for clinical use. In 1975, Japanese workers (10) recommended a biological assay technique that uses the thin-layer cup plate of E. coli Kp strain as an indicator organism. More recently, French workers (M. Le Duff, D. Gibassier, P. A. Sado, and R. Le Verge, Abstr. C. R. Congr. Eur. Biopharm. Pharmacokinet. 1st, series 2, p. [508][509][510][511][512][513][514][515] 1981) have reported a high-pressure liquid chromatography procedure for determining the concentration of pipemidic acid in rabbit plasma. Since pipemidic acid has a very strong basic moiety in its molecular structure, conventional chromatographic procedures would not be adequate for pharmacological studies. This paper presents a new chromatographic technique which uses a silica gel (stationary phase) and reversed mobile-phase solvents for accurately determining pipemidic acid content in human plasma.MATERIALS AND METHODS Extraction and precolumn derivatization. A 2-ml plasma specimen was spiked with 20 ,ug of quinacrine per 20 ,ul of methanol solution, and the resulting mixture was acidified to ca. pH 4.5 with 100 ,ul of 2 N hydrochloric acid. Neutral and acidic contaminants were eliminated by washing the mixture with 2 ml of chloroform in a 10-ml test tube with a vortex mixer. The organic layer was eliminated, and the aqueous layer was diluted with 1 ml of distilled water. The aqueous mixture was adjusted at ca. pH 7.0 with 2 N so...