Ascorbic Acid Determination by Hydrophobic Liquid Chromatography of the Osazone Derivative. Application to the Analysis of Aqueous Humor

Abstract

The dinitrophenylhydrazine colorimetric method for the analysis of ascorbic acid has been converted to a high-performance liquid chromatography (HPLC) procedure. The bis-(dinitrophenyl) hydrazone resulting from the reaction of ascorbic acid and dinitrophenylhydrazine is separated on a reversed-phase system from other components in the reaction mixture and used for quantitation purposes. The specificity of the analysis is thus greatly improved and its sensitivity is brought down to the picomol level. The HPLC procedure described here is therefore specially suitable for the analysis of very small volume samples when high specificity and sensitivity are of prime importance. With this method, as in the original colorimetric procedure, it is also possible to specify the contribution of the reduced and oxidized forms of ascorbic acid to the total amount present in the sample. Basically, the same approach could be advantageously used to improve other colorimetric methods of analysis. Human aqueous humor samples taken from senile cataract patients at the time of surgery show variable but significant amounts of oxidized ascorbic acid. This oxidized traction, therefore, should not be neglected as is frequently done when the ascorbic acid system of the aqueous humor is studied.