Assay of vitamin B6 in human plasma with graphitic carbon column

A high performance liquid chromatographic (HPLC) procedure for measuring pyridoxal-5'-phosphate (PLP) and certain forms of B6 vitamers in plasma is presented here. This HPLC procedure consisted of a single graphitic carbon column with a fluorescence detector employing an isocratic eluent (15% acetonitrile: 1% perchloric acid: 0.05% sodium bisulfite). The graphitic carbon column is useful in acidic eluent without deteriorization. The relatively low fluorescent intensity of PLP under acidic conditions is improved by its derivatization with bisulfite in the eluent during chromatographic separation. Using this procedure, the detection limit of PLP is 50 fmol, and an aliquot of 5-50 microL of human plasma is required giving satisfactorily precise results within 5 min. We applied this method to the determination of PLP and certain B6 vitamers in human plasma after oral supplementation of pyridoxine.