Rapid high-performance liquid chromatographic assay for salicylic acid in plasma without solvent extraction

Abstract

The in vivo measurement of highly reactive free radicals, such as the hydroxyl radical (○OH), in humans is very difficult, if not impossible. Specific markers, based on the ability of ○OH to attack aromatic molecules and produce hydroxylated compounds, are under investigation. In vivo radical metabolism of salicylic acid produces two main hydroxylated derivatives: 2,3- and 2,5-dihydroxybenzoic acid (DHBA). The measurement of 2,3-DHBA, following oral administration of salicylic acid or its acetylated form (aspirin), is proposed for the assessment of in vivo oxidative stress. The intensity of oxidative stress is a function of the ratio of dihydroxylated derivatives to salicylic acid rather than the absolute dihydroxylated derivatives levels. Consequently, a simple, accurate, and sensitive assay of the salicylic acid level in plasma is needed to investigate the in vivo free radical production. In this work, a rapid and sensitive method is presented that is useful for the quantitation of salicylic acid in biological fluids. The methodology uses high-performance liquid chromatography with spectrophotometric detection for the identification and quantitation of salicylic acid without organic extraction. A detection limit of less than 5 μmol is achieved with spectrophotometric detector responses that are linear over at least 6 orders of magnitude. Plasma concentrations of salicylic acid determined by the present technique are reported following the administration of 1000 mg aspirin in 20 healthy subjects.