Assay of pyridoxal-5'-phosphate, pyridoxal and pyridoxic acid in biological material.

Abstract

The two vitamin B6-vitamers having an aldehyde function are oxidised to the corresponding acids and subjected to an HPLC separation on an RP 18 phase with a solvent consisting of 5% methanol in phosphate buffer at pH 3.5. The detection is carried out by fluorometry with excitation at 318 nm and emission at 418 nm. The peaks obtained correspond to pyridoxic acid 5'-phosphate and pyridoxic acid. Pyridoxal-5'-phosphate is determined as pyridoxic acid 5'-phosphate. Pyridoxal is determined as pyridoxic acid by subtracting the amount of pyridoxic acid already existing before oxidation.