Fluorometric Determination of Phylloquinone and Menaquinone-4 in Biological Materials using High Performance Liquid Chromatography

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Abstract
A sensitive and specific method for the determination of phylloquinone (PK) and menaquinone-4 (MK-4) in biological materials was developed. PK and MK-4 extracted from biological materials were separated by high performance liquid chromatography using Nucleosil C18 column with ethanol-water as a mobile phase. The separated PK and MK-4 were detected by fluorometry after reaction with ethanolic sodium borohydride in a reaction coil connected on-line to a chromatographic column. The method was applied to the determination of a very small quantity of PK or MK-4 in plasma and liver. Minimum detectable quantity of PK or MK-4 was 0.4 ng (about 1 pmol) and the coefficient of variation was about 2.0%. The proposed method is simpler, more specific and more sensitive than other conventional methods.