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Clinical Chemistry 33: 1874-1878, 1987;
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Clinical Chemistry, Vol 33, 1874-1878, Copyright © 1987 by American Association for Clinical Chemistry

Ascorbic and dehydroascorbic acids simultaneously quantified in biological fluids by liquid chromatography with fluorescence detection, and comparison with a colorimetric assay

A Lopez-Anaya and M Mayersohn
College of Pharmacy, University of Arizona, Tucson 85721.

We describe a "high-performance" liquid-chromatographic method for separating and quantifying ascorbic acid (AA) and dehydroascorbic acid (DHA) in plasma and urine. We used a reversed-phase C18 column with an ion-pair reagent and detected the analytes by post-column reaction with 4,5-dimethyl-o-phenylenediamine to form a fluorescent derivative (measured at excitation and emission wavelengths of 365 and 440 nm, respectively). Isoascorbic acid (IA) is the internal standard. Retention times for DHA, AA, and IA are 5.6, 15.5, and 19.9 min, respectively. Between-day CVs for AA in plasma in concentrations of 8 and 20 mg/L were 9% and 7%, respectively. The limit of detection is 10 and 4 ng for AA and DHA, respectively. Results by the present method and the methoxyaniline colorimetric method for AA are comparably accurate.


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Copyright © 1987 by the American Association for Clinical Chemistry.