Simultaneous Assay of Inorganic Phosphates and 2-Polyphosphate Esters of L-Ascorbate by High Performance Anion Exchange Chromatography

Abstract

High performance anion exchange chromatography with conductivity detection (HPAEC-CD) was used to separate and assay 2-mono-, 2-di-, and 2-triphosphate esters of L-ascorbic acid and four inorganic phosphates. The separation was performed on a Dionex AS-11 analytical column with a linear gradient of 20 mM to 80 mM aqueous sodium hydroxide in 10 min. The sensitivities (detection limits) were about 0.1–0.15 μg/mL (2–3 ng) for the inorganic phosphates and 0.2 μg/mL (4 ng) for each 2-phosphate ester of L-ascorbic acid, all calculated as free-acid forms. The method was applied to determine the major components in a commercial phosphorylated L-ascorbate.