Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

Abstract

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3′-3′-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5′-carboxy-3′-methyl-2′- pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting 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 postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 ≥ 0.999) and high sensitivity with an on-column detection limit of <3.5 fmol (<1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with 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-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K 2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status. Copyright © 2005 by the American Society for Biochemistry and Molecular Biology, Inc.