ASSAY OF VITAMIN-B6 IN HUMAN PLASMA WITH GRAPHITIC CARBON COLUMN

A high performance liquid chromatographic (HPLC) procedure for measuri ng pyridoxal-5'-phosphate (PLP) and certain forms of B6 Vitamers in pl asma is presented here. This HPLC procedure consisted of a single grap hitic carbon column with a fluorescence detector employing an isocrati c eluent (15% acetonitrile:1% perchloric acid: 0.05% sodium bisulfite) . The graphitic carbon column is useful in acidic eluent without deter iorization. The relatively low fluorescent intensity of PLP under acid ic conditions is improved by its derivatization with bisulfite in the eluent during chromatographic separation. Using this procedure, the de tection limit of PLP is 50 fmol, and an aliquot of 5-50 muL of human p lasma is required giving satisfactorily precise results within 5 min. We applied this method to the determination of PLP and certain B6 vita mers in human plasma after oral supplementation of pyridoxine.