A fast, simple, and reliable hydrophilic interaction liquid chromatography method for the determination of ascorbic...

Area of Science:

• Analytical Chemistry
• Food Science
• Chromatography

Background:

• Accurate determination of total vitamin C requires resolving ascorbic acid (AA) and isoascorbic acid (IAA).
• Conventional reversed-phase systems struggle to resolve these polar molecules due to their low retention times.
• Existing methods may face challenges with matrix interferences and artifact formation.

Purpose of the Study:

• To develop a reliable, fast, and robust method for quantifying total vitamin C (AA and IAA).
• To resolve AA and IAA epimers and their oxidized forms in complex matrices.
• To validate the method for routine analysis in food and pharmaceutical products.

Main Methods:

• Hydrophilic interaction chromatography (HILIC) utilizing a TSKgel Amide-80 stationary phase.
• Isocratic elution for efficient separation of AA and IAA epimers.
• One-step extraction, stabilization, and reduction of vitamin C using phosphoric acid, EDTA, and tris(2-carboxyethyl)-phosphine.

Main Results:

• Successful resolution of AA and IAA epimers using HILIC.
• The method demonstrated compatibility with high concentrations of common reagents without affecting baseline or retention time.
• Validated for food products (juices, chestnut, ham) and multivitamin tablets with good precision, accuracy, and low LOQs (1.5 mg/L for AA, 3.7 mg/L for IAA).

Conclusions:

• The developed HILIC-DAD method is simple, fast (1 hour), accurate, and precise for routine total vitamin C determination.
• It offers advantages like IAA as an internal standard and minimal artifact formation.
• Suitable for analyzing diverse samples including fruit juices, processed foods, and supplements.