Enzymatically validated liquid chromatographic method for the determination of ascorbic and dehydroascorbic acids in...

Area of Science:

• Analytical Chemistry
• Food Science
• Biochemistry

Background:

• Vitamin C, comprising ascorbic acid (AA) and dehydroascorbic acid (DHAA), is a vital nutrient.
• Accurate quantification of both AA and DHAA is crucial for assessing the nutritional value of fruits and vegetables.
• Existing methods may have limitations in separating and detecting both forms of vitamin C.

Purpose of the Study:

• To develop and validate a liquid chromatographic method for the simultaneous determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fruits and vegetables.
• To establish optimal conditions for the separation and detection of AA and DHAA.
• To validate the developed method against enzymatic assays.

Main Methods:

• High-performance liquid chromatography (HPLC) on a C18 column with a phosphate buffer mobile phase (0.2 M KH2PO4, pH 2.4).
• Pre-column reduction of DHAA to AA using dithiothreitol (DTT).
• Detection at 210 nm, with DHAA quantified indirectly by difference after reduction.

Main Results:

• Complete separation of AA and DHAA was achieved using the specified chromatographic conditions.
• Dithiothreitol effectively reduced DHAA to AA, with reaction times dependent on DTT concentration.
• Percentage recovery of vitamin C ranged from 81.7% to 105.9%.
• Comparative analysis with enzymatic assays demonstrated the method's validity.

Conclusions:

• The developed liquid chromatographic method provides a reliable means for quantifying total vitamin C (AA and DHAA) in food matrices.
• The method overcomes DHAA detection sensitivity issues by employing a reduction step.
• This technique is suitable for routine analysis of vitamin C content in fruits and vegetables.