Determination of antipyrine metabolites in human plasma by solid-phase extraction and micellar liquid chromatography

Abstract

A rapid solid-phase extraction (SPE) procedure was developed for the quantitative isolation of three important antipyrine (dipyrone) metabolites from human plasma: 4-formylaminoantipyrine (FAA), 4-aminoantipyrine (AA) and 4-methylaminoantipyrine (MAA). Separation and quantitation were performed using micellar liquid chromatography (MLC) with a 0.1 mol l^–1 sodium dodecyl sulfate (SDS)–2.5% pentanol mobile phase and UV detection at 262 nm. The metabolites were well resolved in less than 5 min using an octadecyl silica-bonded stationary phase. The extraction procedure involved passing 0.3 ml of plasma sample through a disposable SPE cartridge packed with C₁₈ bonded porous silica. The adsorbed metabolites were removed from the cartridge with methanol. The eluent was evaporated to dryness and the residue was reconstituted with mobile phase and injected into the chromatographic system. The cartridge blank interferent peaks, the effects on reproducibility of sample loading in the cartridge and volume needed for desorption of metabolites were evaluated. The concentration of metabolites ranged between 2.4 and 4 µg ml^–1. The present procedure yields recoveries for the three metabolites ranging from 93 to 100%. The relative standard deviation (s_r) ranged between 1.2 and 13.6%. Limits of detection (LODs) were 10.5, 11.5 and 17.0 ng ml^–1 for FAA, AA and MAA, respectively.