Determination of pyridoxal, pyridoxal 5'-phosphate and 4-pyridoxic acid in plasma of selected groups of the patients with a view to proper sample preparation using HPLC with fluorescence detection

Abstract

A method for the measurement of pyridoxal, pyridoxal 5'-phosphate and 4-pyridoxic acid using a high-performance liquid chromatography with fluorescence detection with regard to stability and recovery is described. For the separation, a reverse phase column LiChroCart 125-4, Purospher RP-18e, 5 μm, was used. The mixture of ethanol and 25 mmol/L Na2HPO4 (7:93, v/v), pH 6.0 was used as a mobile phase. The analytical performance of this method is satisfactory: the intra-assay and inter-assay coefficients of variation were below 10 %. Quantitative recoveries from spiked plasma samples were at intervals 93.0–101.8 %, except for pyridoxal (82.5–98.8 %). The limit of detection was 1.0 nmol/L for pyridoxal 5'-phosphate, 0.7 nmol/L for 4-pyridoxic acid and 1.1 nmol/L for pyridoxal. The preliminary reference range of pyridoxal 5'-phosphate, pyridoxal and 4-pyridoxic acid in a group of blood donors are 42.0–75.6 nmol/L, 2.6–12.8 nmol/L and 16.2–33.0 nmol/L, respectively.