Semi-automated fluorometric determination of pyridoxal-5'-phosphate (vitamin B6) in whole blood by high-performance liquid chromatography (HPLC).

A sensitive and reliable method for the determination of vitamin B6 as pyridoxal-5'-phosphate (PLP) in whole blood is described. After acid extraction of PLP, a fully automated high-performance liquid chromatographic (HPLC) system is used to separate PLP from interfering compounds. PLP is detected fluorometrically as its semicarbazone. By calculating the concentration of PLP on-line with the aid of a computer, it is possible to run one hundred and twenty-five samples within forty-eight hours in routine analysis. The within-assay and the between-assay coefficients of variation of the analysis of PLP in whole blood were 3.3 and 4.7% respectively. The between-assay analytical recovery of PLP added to whole blood was 100.0 +/- 7.1% (mean +/- SD). Reference values of PLP in human blood were found in the range 55-110 nmol/l.