Measurement of hydroxyl radical in rat blood vessel by microbore liquid chromatography and electrochemical detection: an on-line microdialysis study

Salicylic acid (0.5 mM) is used as a trapping reagent of hydroxyl radical, and the formed 2,3- and 2,5-dihydroxybenzoic acids were collected via an on-line microdialysis device from the blood vessels. This study revealed the use of a sensitive liquid chromatographic system with electrochemical detection for the determination of 2,3- and 2,5-dihydroxybenzoic acids. Mobile phase consisted of 0.1 M monochloroacetic acid, 10 mM EDTA, 0.5 mM sodium octylsulfate, 20% acetonitrile and 5% tetrahydrofuran in 1 l (pH 3.0 adjusted with 1 M NaOH), and the flow-rate of 0.05 ml/min were found to be optimum. Isocratic separation of these adducts on a microbore column (reversed-phase C₁₈, 150×1 mm I.D., 5 μm) was achieved within 10 min. The optimal applied potential of dihydroxybenzoic acids was set at 750 mV based on a hydrodynamic study. This method has the detection limits of 1.3 pmol/ml (or 0.2 ng/ml) for 2,3- and 2,5-dihydroxybenzoic acids in Ringer solution (at signal-to-noise ratio=3).