Ascorbate and dehydroascorbic acid as reliable biomarkers of oxidative stress: analytical reproducibility and long-term stability of plasma samples subjected to acidic deproteinization.

Lack of post-sampling stability of ascorbate and dehydroascorbic acid and failure to block their in vivo equilibrium have lowered their value as biomarkers of oxidative stress and limited the ability to further investigate their possible role in disease prevention. In the present article, analytic reproducibility was tested by repeated analysis of plasma aliquots from one individual over 4 years. The plasma was subjected to acidic deproteinization with an equal volume of 10% meta-phosphoric acid containing 2 mmol/L of EDTA and analyzed for ascorbate and dehydroascorbic acid by high-performance liquid chromatography with coulometric detection. In a parallel experiment, the stability of human plasma samples treated as above and stored at -80 degrees C for 5 years was tested in a cohort of 131 individuals. No degradation or shift in the equilibrium between ascorbate and dehydroascorbic acid was observed in either of the experiments. In conclusion, ascorbate and dehydroascorbic acid could be adequately preserved in plasma stored at -80 degrees C following acidic deproteinization with meta-phosphoric acid containing 2 mmol/L of EDTA.