Ascorbate in plasma as measured by liquid chromatography and by dichlorophenolindophenol colorimetry

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Chemistry

Background:

• Accurate measurement of ascorbic acid (vitamin C) in biological samples is crucial for nutritional and clinical assessments.
• Understanding the forms of vitamin C present in plasma (ascorbic acid and dehydroascorbic acid) is important for accurate interpretation of results.

Purpose of the Study:

• To compare the accuracy and reliability of an automated colorimetric method with a high-performance liquid chromatography (HPLC) method for measuring ascorbic acid in human plasma.
• To quantify the levels of total ascorbate (ascorbic acid + dehydroascorbic acid) in plasma and assess the proportion of dehydroascorbic acid.

Main Methods:

• Plasma samples (n=125) were analyzed using an automated colorimetric assay (dichlorophenolindophenol) and HPLC with electrochemical detection.
• Total ascorbate was determined by HPLC following precolumn derivatization of ascorbic acid.
• Correlation analysis was performed to compare the results from the two measurement techniques.

Main Results:

• Both the colorimetric and HPLC methods yielded comparable results for ascorbic acid concentrations ranging from 1 to 20 mg/L (correlation coefficient r = 0.97).
• The study confirmed that plasma predominantly contains ascorbic acid, with only negligible amounts of dehydroascorbic acid detected.
• HPLC analysis with derivatization provided a comprehensive measure of total ascorbate.

Conclusions:

• The automated colorimetric method is a reliable and comparable alternative to HPLC for quantifying ascorbic acid in plasma within the tested concentration range.
• Plasma vitamin C levels are primarily attributable to ascorbic acid, indicating minimal oxidation to dehydroascorbic acid under typical conditions.
• These findings support the use of validated methods for accurate plasma vitamin C assessment in clinical and research settings.