Pyridoxal 5'-phosphate assay based on lucigenin chemiluminescence

The authors describe the first chemiluminescence (CL) based method for determination of pyridoxal 5'-phosphate (PLP). PLP is found to generate intense CL with lucigenin higher than that of the conventional lucigenin-H₂O₂ system by a factor of about 9.0. This new finding is used to be in a detection method for PLP via flow injection analysis (FIA). Response is linear in the 50 nM to 200 μM PLP concentration range with a correlation coefficient of 0.998, and the detection limit (at an S/N of 3) is 6.9 nM. The assay is highly selective over various amino acids, vitamins, sugars, coenzymes and metal ions cofactors. It exhibits advantages over the commonly employed HPLC methods in that it is rapid, more economic, eco-friendly and high throughput FIA detection of PLP without the need for toxic derivatization reagents, organic solvents, and HPLC instrumentation. The method was successfully applied to the determination of PLP in (spiked) human blood samples with recoveries in the range from 96.2-101.6% with % RSD < 4.0. The new system is also employed to determine lucigenin in the linear range of 0.3 to 100.0 μM with a correlation coefficient of 0.994 and the limit of detection is 0.04 μM. Graphical abstract Schematic of the chemiluminescent assay for pyridoxal 5'-phosphate (PLP). Lucigenin-PLP demonstrates 9-fold stronger chemiluminescence intensity than the lucigenin-H₂O₂ system. The detection limit of PLP is 6.9 nM. The method can detect PLP in human serum with good recoveries.