[25] Assay of K vitamins in tissues by high-performance liquid chromatography with special reference to ultraviolet detection

This chapter describes procedures that have been developed for the high-performance liquid chromatography (HPLC) assay of phylloquinone in plant and animal tissues using ultraviolet (UV) detection. The assay of phylloquinone with UV detection can have the following stages: (1) extraction of tissues, (2) preliminary purification of lipid extracts, and (3) high-performance liquid chromatography: (a) semipreparative adsorption HPLC, and (b) analytical reverse-phase HPLC. The factors that influence both the overall design of the assay and the choice of procedures for individual stages is considered in the chapter. Assays of phylloquinone by HPLC have been described with UV absorption and electrochemical and fluorometric detection. Because of the multiple chromatographic steps, many assays for phylloquinone have incorporated an internal standard, either a compound structurally related to vitamin K or tritium-labeled phylloquinone of high specific activity. Phylloquinone 2,3-epoxide and cis-2- chlorophylloquinone can be used as internal standards with UV detection and 2 ',3 ' -dihydrophylloquinone with electrochemical detection. The validity of an HPLC assay may be examined from several standpoints such as peak identity, precision, and recovery. In the assay of some plant tissues and of pharmacological levels of phylloquinone in plasma, the peak corresponding to phylloquinone can be seen at the semipreparative stage with a UV detector. This allows precise collection of the phylloquinone-containing fraction. For most tissues, however, the phylloquinone peak is masked and collection of the fraction must be carried out by reference to the retention of vitamin K standards.