Ascorbic and dehydroascorbic acids simultaneously quantified in biological fluids by liquid chromatography with...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Clinical Chemistry

Background:

• Ascorbic acid (AA) and its oxidized form, dehydroascorbic acid (DHA), are vital antioxidants.
• Accurate quantification of AA and DHA is crucial for assessing vitamin C status and related health conditions.
• Existing analytical methods may have limitations in sensitivity, specificity, or sample throughput.

Purpose of the Study:

• To develop and validate a high-performance liquid chromatographic (HPLC) method for the simultaneous separation and quantification of AA and DHA.
• To establish a sensitive and reliable analytical technique for measuring these analytes in biological matrices like plasma and urine.

Main Methods:

• Utilized a reversed-phase C18 column with an ion-pair reagent for separation.
• Employed post-column reaction with 4,5-dimethyl-o-phenylenediamine to form a fluorescent derivative of AA and DHA.
• Detected the fluorescent products using specific excitation (365 nm) and emission (440 nm) wavelengths.
• Isoascorbic acid (IA) was used as the internal standard for accurate quantification.

Main Results:

• Achieved distinct retention times for DHA (5.6 min), AA (15.5 min), and IA (19.9 min).
• Demonstrated good precision with between-day coefficients of variation (CVs) for AA in plasma at 9% (8 mg/L) and 7% (20 mg/L).
• Established low limits of detection: 10 ng for AA and 4 ng for DHA.
• Showed comparable accuracy for AA quantification against the methoxyaniline colorimetric method.

Conclusions:

• The developed HPLC method provides a robust and sensitive approach for quantifying ascorbic acid and dehydroascorbic acid.
• This method is suitable for routine analysis of vitamin C metabolites in clinical and research settings.
• The high sensitivity and accuracy support its application in assessing nutritional status and oxidative stress biomarkers.