High-pressure liquid chromatographic-reductive electrochemical detection analysis of serum trans-phylloquinone

A sensitive method for the determination of human serum trans-phylloquinone levels has been developed. Serum samples were extracted with hexane and subjected to preliminary separation on a silica semipreparative HPLC column with an 80% recovery as determined by the addition of [3H]phylloquinone. The portion of the eluate containing trans-phylloquinone was dried and injected into a muBondapak C-18 analytical HPLC column, and the concentration of the vitamin was determined by reductive electrochemical detection utilizing a glassy carbon electrode and an Ag/AgCl reference electrode. The presumed vitamin peak was confirmed as trans-phylloquinone by rechromatography at different HPLC conditions, hydrodynamic voltamography, and photodegradation. As little as 300 pg/ml trans-phylloquinone could be detected, and normal human serum concentrations were found to be in the range of 1 ng/ml.