[34] Determination of vitamin K compounds in plasma or serum by high-performance liquid chromatography using postcolumn chemical reduction and fluorimetric detection

Publisher Summary The high-performance liquid chromatography (HPLC) assays used for the determination of endogenous phylloquinone, phylloquinone 2,3-epoxide, menaquinones, and most recently 2',3'-dihydrophylloquinone have evolved from the postcolumn reduction and fluorimetric detection methodology. The analytical system utilized to reduce vitamin K compounds to their fluorescent hydroquinones consists of a postcolumn, dry chemical reactor containing zinc metal. Hydroquinones are produced by chemical reduction over zinc in the presence of zinc ions, which are provided by the mobile phase. This on-line reduction process forms the core of the chromatographic systems used for the determination of vitamin K. Although the column configuration, injector type, mobile-phase composition, and flow rates of analytical systems may vary, the online, postcolumn reduction is essentially the same. This chapter describes two assays based on postcolumn reduction, but with significant enhancements made in the sensitivity of analytical systems: (1) a simplified method for the determination of fasting plasma or serum concentrations of phylloquinone and (2) an assay for the simultaneous determination of endogenous phylloquinone and phylloquinone 2,3-epoxide in plasma or serum.