Liquid-chromatographic determination of vitamin K1 in plasma, with fluorometric detection

This assay for phylloquinone (vitamin K1) in plasma requires a single liquid-chromatographic step. Much smaller volumes of plasma (0.5-1.0 mL) are required than in previous assays. Before liquid chromatography, we purified crude lipid extracts by conventional chromatography on silica, then extracted the lipid fraction by dissolving it in an acidic mixture of hexane/acetonitrile (1/4 by vol) containing 70 mmol of zinc chloride per liter. The vitamin K1 was selectively extracted into acetonitrile after being converted to vitamin K1 hydroquinone by addition of zinc metal. This procedure removes greater than 99% of contaminating lipids. We injected the lipid extract directly onto a reversed-phase column after re-converting the vitamin K1 hydroquinone to vitamin K1. Vitamin K1 was quantified by comparison with the internal standard (dihydro-vitamin K1) and detected fluorometrically after post-column "on-line" reduction to the hydroquinone with zinc metal. The lower limit of detection for vitamin K1 in the final reversed-phase system was about 0.05 microgram/L plasma; CVs for replicates were less than 10%. The mean concentration of vitamin K1 in plasma from 22 healthy fasting adults was 0.55 (range 0.09-2.12) micrograms/L.