Determination of pyridoxal 5′-phosphate in human serum by reversed-phase high-performance liquid chromatography combined with spectrofluorimetric detection of 4-pyridoxic acid 5′-phosphate as a derivative

Abstract

A very sensitive spectrofluorimetric method for the determination of pyridoxal 5′-phosphate (PLP) in human serum is described. The specificity is based on the selective oxidation of PLP to 4-pyridoxic acid 5′-phosphate with potassium cyanide. Separation of the highly fluorescent 4-pyridoxic acid 5′-phosphate is achieved by reversed-phase high-performance liquid chromatography. Specificity is improved by a careful choice of fluorescence filters, maximised at the excitation (325 nm) and emission (418 nm) wavelengths of 4-pyridoxic acid 5′-phosphate. The detection limit for the reaction is 0.22 ng ml^–1. For quantification, the serum is spiked with PLP before protein precipitation with 3.3%m/V trichloroacetic acid. The method can be used for the determination of PLP in serum, even in vitamin B₆ deficient patients. The mean value for human serum PLP from 30 healthy adults was found to be 14.6 ± 4.8 ng ml^–1(mean ± standard deviation).