Simultaneous determination of l-ascorbic acid and dehydroascorbic acid in human plasma

Abstract

A method combining high-performance liquid chromatography with ultraviolet detection (HPLC-UV) was developed and validated for the accurate determination of L-ascorbic acid (AA) and dehydroascorbic acid (DHAA) concentrations in human plasma. In conventional HPLC-UV analysis, DHAA is indirectly measured by subtracting the native AA concentration from the total AA concentration. However, it is important to develop a direct method for the accurate and rapid analysis of AA and DHAA. Analyses were performed on a Primesep SB column (4.6 × 250 mm, particle size 5 μm), and the mobile phase consisted of 0.1% formic acid in water, 80%; and 0.08% formic acid in acetonitrile, 20%. The intra- and inter-day accuracies of the AA assay were 95.92–98.18% and 92.22–98.22%, respectively. The intra- and inter-day accuracies of the DHAA assay were 93.34–97.73% and 99.42–97.53%, respectively. The calibration curve was linear within the tested range of 2–100 μg mL⁻¹ for AA and 10–200 μg mL⁻¹ for DHAA. This HPLC method is a highly sensitive and reproducible analytical method for the simultaneous determination of ascorbic acid and DHAA in human plasma.