Streamlined three step total vitamin C analysis by HILIC-UV for laboratory testing

Abstract

Objectives: In the clinical setting, the analysis and quantification of vitamin C (ascorbic acid) poses several challenges including analyte instability and poor retention by reverse phase HPLC systems. In this article we describe a rapid hydrophilic interaction chromatography ultraviolet method for the measurement of total vitamin C in plasma which overcomes these issues.

Methods: Ascorbic acid and the internal standard were separated under isocratic conditions using a Waters BEH-Amide column and a mobile phase containing 0.005 M potassium phosphate in 80% acetonitrile.

Results: The proposed method was validated and showed good precision (coefficient of variation <5%), accuracy (>99%), and analyte stability after extraction (>24 h).

Conclusions: The simple sample preparation allows full automation and rapid analytical run times of the assay and is therefore suitable for a high-throughput clinical chromatography laboratory.

Keywords: ascorbic acid; high performance liquid chromato-graphy-diode array detection (HPLC-DAD); high performance liquid chromatography-ultraviolet detection (HPLC-UV); hydrophilic interaction chromatography (HILIC); plasma; vitamin C.