Liquid chromatographic behavior of ascorbate on amine columns.

Area of Science:

• Analytical Chemistry
• Biochemistry

Background:

• High-performance liquid chromatography (HPLC) is a viable method for quantifying ascorbate.
• Separating ascorbic acid from its degradation products, dehydroascorbic acid and diketogulonic acid, at low concentrations is challenging.

Purpose of the Study:

• To assess the efficacy of prefilled amine columns from different manufacturers for separating low concentrations of [14C]ascorbic acid and its degradation products.
• To determine if adding nonlabeled ascorbic acid improves the separation and recovery of radiolabeled ascorbic acid.

Main Methods:

• Utilized prefilled amine HPLC columns from three manufacturers.
• Injected varying amounts of [14C]ascorbic acid and its degradation products.
• Tested the effect of adding nonlabeled ascorbic acid (0.5 mg/ml) to samples.

Main Results:

• Successful separation of [14C]ascorbic acid from its degradation products was achieved on some columns with as little as 30 pmol injected.
• On certain columns, 30-500 pmol of ascorbate resulted in unpredictable retention times for up to 80% of [14C]ascorbic acid.
• Inclusion of nonlabeled ascorbic acid improved [14C]ascorbic acid retention time predictability and dehydroascorbic acid peak definition.
• Nonlabeled ascorbic acid addition also led to more complete recovery of total injected [14C]activity.

Conclusions:

• Certain amine HPLC columns can effectively separate low concentrations of ascorbic acid and its degradation products.
• Supplementing samples with nonlabeled ascorbic acid is a beneficial strategy to enhance the accuracy and recovery of radiolabeled ascorbic acid quantification in biological and food samples.