ABSTRACT: We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their agyclone structures, 2-methyl-3-(3-3-carboxymethylpropyl)-1,4-naphthoquinone (5C-side-chain metabolite) and 2-methyl-3-(5-carboxy-3-methyl-2-pentenyl)-1,4-naphthoquinone (7C-side-chain metabolite), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid phase extraction (SPE) and the predominately conjugated vitamin K metabolites hydrolysed with methanolic HCl. The resultant carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by post-column coulometric reduction at an upstream electrode. The assay gave excellent linearity (r2 typically = 0.999) and high sensitivity with an on-column detection limit of <3.5 fmol (<1pg). The inter-assay precision was typically 10%. Metabolite recovery was compared to that of an internal standard (2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone), added to urine samples just before analysis. Using this methodology we confirmed that the 5C- and 7C-metabolite were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate non-invasive marker of total vitamin K status.
Harrington, D.J. and Soper, R. and Edwards, C. and Savidge, G.F. and Hodges, S.J. and Shearer, M.J. (2005) Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection. Journal of Lipid Research, 46 (5). pp. 1053-1060. ISSN 00222275.