Rapid and sensitive determination of dehydroascorbic acid in addition to ascorbic acid by reversed-phase...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Nutritional Science

Background:

• Vitamin C exists in two biologically active forms: l-ascorbic acid (AA) and l-dehydroascorbic acid (DHAA).
• Accurate quantification of both AA and DHAA is crucial for understanding vitamin C status and bioavailability.
• Existing methods may not efficiently or directly determine both forms simultaneously.

Purpose of the Study:

• To develop and validate an improved analytical procedure for the direct determination of both AA and DHAA.
• To enable sensitive and reproducible quantification of vitamin C forms in biological or food samples.

Main Methods:

• Utilized reversed-phase high-performance liquid chromatography (RP-HPLC) for separation of AA and DHAA.
• Implemented a post-column reaction system employing dithiothreitol (DTT) to reduce DHAA to AA.
• Optimized reaction conditions (50°C, 1-ml reaction coil) for complete DHAA to AA conversion.

Main Results:

• Achieved high recoveries for DHAA conversion to AA, ranging from 95-99%.
• Demonstrated sensitive spectrophotometric detection of both vitamin C forms at 267 nm.
• Obtained reproducible results with low relative standard deviations (2.4% for DHAA, 1.0% for AA) in rose hip sample analysis.

Conclusions:

• The developed RP-HPLC method provides a reliable and sensitive approach for direct determination of both AA and DHAA.
• This improved procedure facilitates accurate assessment of total vitamin C content in complex matrices.
• The method's robustness and reproducibility make it suitable for routine analysis in nutritional and food science research.