Highly sensitive and simple liquid chromatographic determination in plasma of B-6 vitamers, especially pyridoxal 5'-phosphate

A simple and sensitive high-performance liquid chromatographic method for measuring the major vitamers of vitamin B-6, i.e. pyridoxal and pyridoxal 5'-phosphate (PLP), and 4-pyridoxic acid (4-PA) in plasma was developed. The vitamers and 4-PA from plasma were extracted with 0.8 mol/1 perchloric acid. The separation by HPLC is accomplished using an ODS reversed-phase column and a mobile phase of 0.1 mol/l potassium dihydrogen phosphate containing 0.1 mol/l sodium perchlorate, 0.5 g/1 sodium bisulfite adjusted to pH 3, at a flow-rate of 1.0 ml/min. The vitamers and 4-PA were eluted within 13 min and their concentration is determined with fluorometric detector (excitation, 300 nm; emission, 400 nn). In this method, PLP in plasma can be determined with high sensitivity using derivatization with sodium bisulfite in the mobile phase.