Simple in-line postcolumn oxidation and derivatization for the simultaneous analysis of ascorbic and dehydroascorbic...

Area of Science:

• Analytical Chemistry
• Food Science
• Biochemistry

Background:

• Accurate determination of Vitamin C (L-ascorbic acid and its isomers) is crucial for food quality and nutritional assessment.
• Existing analytical methods may lack specificity or require complex procedures for simultaneous analysis of ascorbic acid and its isomers.

Purpose of the Study:

• To develop and validate a novel, high-performance liquid chromatography (HPLC) method for the simultaneous determination of L-ascorbic acid (AA), isoascorbic acid (IAA), L-dehydroascorbic acid (DHAA), and isodehydroascorbic acid (IDHAA) in diverse food matrices.
• To establish a sensitive, specific, and reproducible analytical procedure suitable for routine food analysis.

Main Methods:

• Simultaneous separation of AA, IAA, DHAA, and IDHAA using HPLC.
• In-line oxidation of AA and IAA to DHAA and IDHAA on an activated charcoal column.
• Post-column derivatization of dehydroascorbic acids with 1,2-phenylenediamine to form fluorescent quinoxaline compounds.
• Fluorometric detection of the derivatized compounds.

Main Results:

• The HPLC method achieved good separation of LAA, LDHAA, and their diastereoisomers within approximately 10 minutes.
• A linear relationship was observed between peak area and concentration for all derivatives (0.1 to 100 µg/mL), with high recoveries (97-103%).
• The method demonstrated a low detection limit (0.1 mg/100 g) and was successfully applied to a wide range of foods, yielding results comparable to established methods.

Conclusions:

• The developed HPLC method offers a highly specific, simple, and reproducible approach for the simultaneous quantification of Vitamin C and its isomers in food.
• This analytical procedure is well-suited for serial determinations and provides reliable results for nutritional labeling and quality control.