Simultaneous determination of levodopa and 3-O-methyldopa in human plasma by liquid chromatography with electrochemical detection

A simple and rapid assay is described for the simultaneous analysis of levodopa (l-DOPA) and 3-O-methyldopa (3-OMD) in human plasma samples, applying an ion-pair reversed-phase liquid chromatographic method with electrochemical detection, designed for clinical trials performed to study the effect of peripheral catechol-O-methyltransferase inhibitors on the metabolism of l-DOPA. After protein precipitation of 100 μl plasma sample aliquots with perchloric acid, the analytes are directly injected, separated within 10 min and simultaneously quantified down to 20 ng/ml by an electrochemical detector equipped with a dual-electrode system operating in redox mode eliminating effectively potential endogenous and exogenous interferences. The intra-assay precision for l-DOPA and 3-OMD was 1.34–6.54 and 3.90–5.50%, whereas the inter-assay precision was 2.09–7.69 and 4.16–9.90%, respectively. The recoveries were close to 90% for l-DOPA and almost 100% for 3-OMD. Satisfactory storage stability was achieved for up to 16 weeks at −70 °C by stabilizing plasma samples with antioxidants.