Indirect pulsed electrochemical detection following high-performance reversed-phase liquid chromatography.

For detection using pulsed electrochemical detection (PED), analytes must preadsorb to the working gold electrode. Indirect pulsed electrochemical detection (InPED) exploits this requirement by including a PED-active reagent in the mobile phase. The background signal provided by oxidation of this reagent is attenuated by the adsorption of analyte molecules to the electrode. In this paper, a method has been developed to allow the use of InPED in combination with high-performance reversed-phase liquid chromatography (RPLC). Biotin was used as a probe molecule to determine that the use of acetonitrile as the organic modifier in the mobile phase provided superior results to the use of methanol. In addition, a silica-based, high surface area C18 column (i.e., Varian Pursuit XRs) was found to give better results than a polymer-based reversed-phase column (i.e., Dionex IonPac NS1). Optimized experimental conditions were used todetermine lipoic acid, tiopronin, and penicillamine, obtaining detection limits of ≤1 μM (30 pmol injected). The analytical utility of RPLC-InPED was demonstrated by an assay of an over-the-counter-formulation containing lipoic acid.