Improved Method for the Analysis of Ascorbic Acid in Plasma by High-Performance Liquid Chromatography with Electrochemical Detection

We improved the analytical method for the detection of ascorbic acid in plasma by high-performance liquid chromatography (HPLC) with an electrochemical detector (ECD) to be more selective and rapid than the protocol previously used. Main improvements are as follows. Applied potential of ECD to obtain the maximal response for ascorbic acid was +450mV versus Ag/AgCl, but it was reduced to+ 350mV. In that condition, uric acid did not respond to ECD, and only ascorbic acid was detected. EDTA contained in sample extrac-tion/stabilizing solution gave the peak after the ascorbic acid. The addition of EDTA (0.2mM) to the mobile phase eliminated the EDTA peak. These two improvements gave the chromatogram in which the peak that appeared from the plasma sample was only ascorbic acid, and shorten the sample run time. Ascorbic acid in plasma was unstable even though the plasma was treated with methanol/EDTA: it decreased from 1.5 h at 4°C. However, the treated sample, which was placed at -14°C until the analysis was performed, gave the reliable ascorbic acid value at least up to 6 h. The data obtained from the HPLC-ECD method was consistent with those from the hydrazine method.