HPLC Method for Plasma Vitamin K1: Effect of Plasma Triglyceride and Acute-Phase Response on Circulating Concentrations

The plasma concentration of vitamin K(1) (phylloquinone) is the most reliable index for assessing vitamin K status. Our aim was to analytically validate an HPLC method for quantifying phylloquinone in plasma and to examine the effect of plasma triglyceride concentration on the phylloquinone reference interval. We also examined the effect of acute-phase response on phylloquinone concentration in plasma. Phylloquinone was extracted from fasting plasma samples by deproteinization and C18 solid-phase extraction, separated by reversed-phase HPLC, and detected fluorometrically after postcolumn reduction with a platinum catalyst. We synthesized a novel internal calibrator, docosyl naphthoate. The recovery of phylloquinone was >90%. Between-run imprecision was 8.7%-9.0%, and within-run imprecision was 3.8%-7.0%. The linearity was up to 44.8 nmol/L, limit of detection 0.08 nmol/L, and limit of quantification 0.14 nmol/L. The correlation between plasma phylloquinone and triglyceride concentrations was r = 0.7 in the reference population. The 95% reference interval for the phylloquinone:triglyceride ratio was 0.20 to 2.20 nmol/mmol. Plasma concentrations of C-reactive protein were significantly increased, whereas triglyceride and phylloquinone but not the phylloquinone:triglyceride ratio were transiently decreased >50% after surgery. Phylloquinone population reference intervals should be expressed as a ratio of the triglyceride concentration. Phylloquinone concentrations in plasma are decreased in acute-phase response and, unless corrected for plasma triglyceride concentration, are unlikely to be a reliable index of vitamin K status.