J. Lipid Res.
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A more recent version of this article appeared on May 1, 2005

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J. Lipid Res., doi:10.1194/jlr.D400033-JLR200
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Submitted on November 15, 2004
Revised on January 24, 2005
Accepted on January 25, 2005

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

Dominic J. Harrington, Robin Soper, Christine Edwards, Geoffrey F. Savidge, Stephen J. Hodges, and Martin J. Shearer

The Haemophilia Reference Centre, St. Thomas' Hospital, London SE1 7EH

Corresponding Author: Domonic.Harrington{at}gstt.nhs.uk

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.


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This article has been cited by other articles:


Home page
 J. Nutr.Home page
D. J. Harrington, S. L. Booth, D. J. Card, and M. J. Shearer
Excretion of the Urinary 5C- and 7C-Aglycone Metabolites of Vitamin K by Young Adults Responds to Changes in Dietary Phylloquinone and Dihydrophylloquinone Intakes
J. Nutr., July 1, 2007; 137(7): 1763 - 1768.
[Abstract] [Full Text] [PDF]


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